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Collaboration between the innate immune receptors dectin-1, TLRs, and Nods
Abstract
Microbes are complex and present a wide variety of structures that phagocytes may recognize using innate immune receptors. Recognition triggers anti-microbial killing mechanisms and production of inflammatory cytokines and chemokines that orchestrate host defense. As a general rule, no single receptor is likely to be the sole mediator of activation of protective immune responses. Recent studies highlight the importance of collaboration between Toll-like receptors, the nucleotide oligomerization domain (Nod) proteins, and dectin-1 in regulating inflammatory responses. Studies on the molecular mechanisms of cross-talk and synergy between these receptors provide a framework in which to understand the importance of having multiple receptors recognize individual microbes.
... Clec7a, also known as Dectin-1, is a transmembrane receptor protein for β-1,3 and β-1,6 glucans, which is mainly expressed on macrophages, monocytes, microglia, neutrophils, most subsets of dendritic cells, and subpopulations of T-cells, B-cells, mast cells, and eosinophils [20]. As a pattern-recognition receptor, Clec7a has been found to recognize a broad range of microbial pathogens, including fungi and bacteria, which can cause autoimmune disorders and cancer [21]. It also plays a key role in regulating neuroinflammation in ischemic stroke, spinal cord injury and intracerebral hemorrhage [22][23][24]. ...
... A previous study of Rojewska group firstly observed the upregulation of Clec7a mRNA in both DRG and SC seven days after CCI-induced neuropathic pain using microarray and qPCR analyses [25], in line with our findings. Fu and colleagues indicated that the inhibition of Clec7a could ameliorate neuroinflammation after intracerebral hemorrhage in mice [21]; Deerhake and others also reported that Clec7a could limit autoimmune neuroinflammation in mice [26]. Consistently, our data demonstrated that intrathecal siRNA-mediated suppression of Clec7a effectively attenuated CCI-induced mechanical allodynia, implying a crucial role of Clec7a during neuropathic pain progression, which prompt us to investigate its underlying mechanisms. ...
Background
Growing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear.
Methods
A chronic constriction injury (CCI) rat model was constructed, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPβ-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro.
Results
Significant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPβ, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPβ knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector.
Conclusions
This pre-clinical investigation reveals that C/EBPβ-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.
... 20 As a pattern-recognition receptor, Clec7a has been found to recognize a broad range of microbial pathogens, including fungi and bacteria, which can cause autoimmune disorders and cancer. 21 It also plays a key role in regulating neuroin ammation in ischemic stroke, spinal cord injury and intracerebral hemorrhage. [22][23][24] A previous study of Rojewska group rstly observed the upregulation of Clec7a mRNA in both DRG and SC seven days after CCI-induced neuropathic pain using microarray and qPCR analyses, 25 in line with our ndings. ...
... [22][23][24] A previous study of Rojewska group rstly observed the upregulation of Clec7a mRNA in both DRG and SC seven days after CCI-induced neuropathic pain using microarray and qPCR analyses, 25 in line with our ndings. Fu and colleagues indicated that the inhibition of Clec7a could ameliorate neuroin ammation after intracerebral hemorrhage in mice; 21 Deerhake and others also reported that Clec7a could limit autoimmune neuroin ammation in mice. 26 Consistently, our data demonstrated that intrathecal siRNAmediated suppression of Clec7a effectively attenuated CCI-induced mechanical allodynia, implying a crucial role of Clec7a during neuropathic pain progression, which prompt us to investigate its underlying mechanisms. ...
Background
Growing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear.
Methods
A chronic constriction injury (CCI) rat model was established, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPβ-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro.
Results
Significant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPβ, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPβ knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector.
Conclusions
This pre-clinical investigation reveals that C/EBPβ-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.
... As shown in Figure 2, the TNF-α activation by heat-killed PC was partially, not completely, blocked by TLR2 inhibitor, suggesting signals other than TLR2 might exist. Therefore, PC might use multiple mechanisms of action through other possible pathways, which can compensate TLR2, 65,66 to prevent NTM-LD although the mechanism required further study. These results might suggest that PC or PC CPS may be further developed as a probiotic for ameliorating NTM-LD in immunocompromised patients. ...
The role of gut microbiota in host defense against nontuberculous mycobacterial lung disease (NTM-LD) was poorly understood. Here, we showed significant gut microbiota dysbiosis in patients with NTM-LD. Reduced abundance of Prevotella copri was significantly associated with NTM-LD and its disease severity. Compromised TLR2 activation activity in feces and plasma in the NTM-LD patients was highlighted. In the antibiotics-treated mice as a study model, gut microbiota dysbiosis with reduction of TLR2 activation activity in feces, sera, and lung tissue occurred. Transcriptomic analysis demonstrated immunocompromised in lung which were closely associated with increased NTM-LD susceptibility. Oral administration of P. copri or its capsular polysaccharides enhanced TLR2 signaling, restored immune response, and ameliorated NTM-LD susceptibility. Our data highlighted the association of gut microbiota dysbiosis, systematically compromised immunity and NTM-LD development. TLR2 activation by P. copri or its capsular polysaccharides might help prevent NTM-LD.
... Underhill has proposed several possibilities why the crosstalk has evolved; 1) To provide robust response against invading microbes. 2) Compensation against genetic diversity in host population, 3) Multiple receptors can facilitate a more tailored, specific response (136). The idea of "a more tailored, specific response" is very fascinating, because innate immune cells, which usually express TLRs predominantly, are rather considered promiscuous and relatively non-specific compared to adaptive immunity. ...
Immune system recognizes invading microbes at both pathogen and antigen levels. Toll-like receptors (TLRs) play a key role in the first-line defense against pathogens. Major functions of TLRs include cytokine and chemokine production. TLRs share common downstream signaling pathways with other receptors. The crosstalk revolving around TLRs is rather significant and complex, underscoring the intricate nature of immune system. The profiles of produced cytokines and chemokines via TLRs can be affected by other receptors. Integrins are critical heterodimeric adhesion molecules expressed on many different cells. There are studies describing synergetic or inhibitory interplay between TLRs and integrins. Thus, we reviewed the crosstalk between TLRs and integrins. Understanding the nature of the crosstalk could allow us to modulate TLR functions via integrins.
... In congruence with our results, a previous study conducted by our group showed that BALB/c mice receiving only ODN2088, the TLR9 inhibitor employed in our study, exhibited an increase in their IL-17A levels [16]. Furthermore, another study indicated that ODN2088 enhances IL-17A secretion in human helper T cells [22]. These observations could be attributed to possible crosstalk occurring between the TLR9 signaling pathway and other immune pathways triggered by various DNA sensors. ...
Epstein–Barr virus (EBV) DNA is known to be shed upon reactivation of latent EBV. Based on our previous findings linking Toll-like receptor-9 (TLR9) to an EBV DNA-driven surge in IL-17A production, we aimed to examine the therapeutic potential of TLR9 inhibition in EBV DNA-exacerbated arthritis in a collagen-induced arthritis (CIA) mouse model. C57BL/6J mice were administered either collagen, EBV DNA + collagen, EBV DNA + collagen + TLR9 inhibitor, or only the TLR9 inhibitor. After 70 days, paw thicknesses, clinical scores, and gripping strength were recorded. Moreover, affected joints, footpads, and colons were histologically scored. Furthermore, the number of cells co-expressing IL-17A, IFN-γ, and FOXP3 in joint sections was determined by immunofluorescence assays. Significantly decreased paw thicknesses, clinical scores, and histological scores with a significantly increased gripping strength were observed in the group receiving EBV DNA + collagen + TLR9 inhibitor, compared to those receiving EBV DNA + collagen. Similarly, this group showed decreased IL-17A+ IFN-γ+, IL-17A+ FOXP3+, and IL-17A+ IFN-γ+ FOXP3+ foci counts in joints. We show that inhibiting TLR9 limits the exacerbation of arthritis induced by EBV DNA in a CIA mouse model, suggesting that TLR9 could be a potential therapeutic target for rheumatoid arthritis management in EBV-infected individuals.
... In their resting state, DCs are considered to be immature but prepared to recognize and acquire antigens through numerous pattern-recognition receptors (PRRs). There are several groups of PRRs, including Toll-like receptors (TLRs), C-type lectins (CLRs), cytoplasmic retinoic acid-inducible gene-Ilike receptors (RLRs) and nucleotide oligomerization domain-like receptors (NLRs) (4)(5)(6). Upon PRRs activation, DCs undergo phenotypic and functional maturational changes (7). They include the upregulation of chemokine receptors (CCR7), adhesion molecules (CD54), co-stimulatory molecules (CD80 and CD86), immunoproteosomes and major histocompatibility complex (MHC)-MHC class I and II molecules, all important for DCs migration to the lymphoid tissues and activation of the immune response (8). ...
... In this frame, it has been demonstrated that TLR signalling is finely tuned by signals derived by C-type lectins, which bind carbohydrates expressed on cell surface of microbes to tailor immune responses towards either activation or tolerance. [31][32][33][34][35] Therefore, we have started to explore whether the two types of V. parvula LPS were also differently recognized by C-type lectins of the human innate immunity system. The four C-type lectins were chosen among those expressed in the gastrointestinal tract and whose activity span from inflammation to immune tolerance depending on several factors including the nature of the ligand. ...
Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota‐driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro‐inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol‐containing O‐antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid‐phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro‐inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible “immune modulating” action has been proposed for the peculiar O‐antigen portion.
... Epithelial cells are also particularly sensitive to the presence of a fungal toxin-candidalysin (Moyes et al., 2010). However, C. albicans developed different strategies to avoid and manipulate host immune responses (Marcos et al., 2016;Underhill, 2007). C. albicans, by modifying fungal responses to oxidative and nitrosative stress, protects fungal cells against toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by innate immune cells (Dantas et al., 2015;Miramón et al., 2012). ...
The oral cavity of humans is colonized by diversity of microbial community, although dominated by bacteria, it is also constituted by a low number of fungi, often represented by Candida albicans. Although in the vast minority, this usually commensal fungus under certain conditions of the host (e.g., immunosuppression or antibiotic therapy), can transform into an invasive pathogen that adheres to mucous membranes and also to medical or dental devices, causing mucosal infections. This transformation is correlated with changes in cell morphology from yeast‐like cells to hyphae and is supported by numerous virulence factors exposed by C. albicans cells at the site of infection, such as multifunctional adhesins, degradative enzymes, or toxin. All of them affect the surrounding host cells or proteins, leading to their destruction. However, at the site of infection, C. albicans can interact with different bacterial species and in its filamentous form may produce biofilms—the elaborated consortia of microorganisms, that present increased ability to host colonization and resistance to antimicrobial agents. In this review, we highlight the modification of the infectious potential of C. albicans in contact with different bacterial species, and also consider the mutual bacterial‐fungal relationships, involving cooperation, competition, or antagonism, that lead to an increase in the propagation of oral infection. The mycofilm of C. albicans is an excellent hiding place for bacteria, especially those that prefer low oxygen availability, where microbial cells during mutual co‐existence can avoid host recognition or elimination by antimicrobial action. However, these microbial relationships, identified mainly in in vitro studies, are modified depending on the complexity of host conditions and microbial dominance in vivo.
... Cellular defense mechanisms produced proinflammatory cytokines, and increase immune cells in the site of infection, and stimulate reactive oxygen species and antimicrobial peptides [119][120][121]. ...
T HIS review aims to summarize data on avian microbiota, its development, composition, effect, and factors that affect its diversity in the chicken gastrointestinal tract (GIT) to be available for students, practical poultry specialist, and researchers in the poultry industry. The GIT of chickens like other animals and human are harboring a diverse population or community of microorganisms, including bacteria (microbiota), fungi (mycobiota), protozoa, and viruses are in symbiotic to enhance vital activities and the health of birds.. On the other hand, a bird's cecum microbiota has a high complex composition and fewer characteristic features than crop and all intestinal parts. Microbiota starts to develop after hatching and gradually increased with age until the population reaches its balance. It can be affected by litter type, ration, as well as feed additives. The composition of poultry GIT microbiome was mainly investigated using microbiological culturing, while, molecular-based techniques provided more rapid and accurate characterization of the culture-able and un-culture-able members. The identification of intestinal microbiota helps in improving chickens' health and productivity programs. Therefore, GIT microbiota and mycobiota should be carefully investigated for meat, litter, aerosol, and processing plant contamination to ensure both food and personnel safety.
... Our results suggest that HK Lp WJL and isolated CW from Lp WJL can be sensed by the host and induce metabolic and hormonal changes, leading to improved growth kinetics during chronic undernutrition. To identify such host-sensing and signaling mechanism(s), we tested the signaling potential of HK strains and their CW in human embryonic kidney (HEK) cells stably transfected with a set of innate immune receptors previously reported to sense molecular determinants from bacteria CW (NOD1, NOD2, TLR2, and TLR4) (31,32 (26). We therefore extracted and analyzed the composition of the PGs from the two strains. ...
The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.
... Synthesis of TLR7 Agonist 1. 31 4-((6-Amino-2-butoxy-9H-purin-9-yl)methyl)benzoic acid (6). Compound 5 (1.737 g, 6.10 mmol) was suspended in dry n-butanol (60 mL). ...
The success of vaccination with subunit vaccines often relies on the careful choice of adjuvants. There is great interest in developing new adjuvants that can elicit a cellular immune response. Here, we address this challenge by taking advantage of the synergistic cross-talk between two pattern recognition receptors: nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). We designed two conjugated NOD2/TLR7 agonists, which showed potent immunostimulatory activities in human primary peripheral blood mononuclear cells and murine bone-marrow-derived dendritic cells. One of these, 4, also generated a strong antigen-specific immune response in vivo, with a Th1-polarized profile. Importantly, our study shows that novel NOD2/TLR7 agonists elicit sophisticated and fine-tuned immune responses that are inaccessible to individual NOD2 and TLR7 agonists.
... In line with this finding, several studies show that TLRs expression in the AT is elevated in obese individuals, with/without type 2 diabetes, and these alterations can be directly associated with BMI, cytokines/chemokines expression, and insulin resistance [38,40,45,61]. Being a PRR, dectin-1 synergizes with the TLR pathway to induce the expression of inflammatory cytokines, including TNF-α, IL-1β, and IL-12 [62][63][64]. In our study, an increase in the AT dectin-1 transcripts levels was associated with the TLR-downstream signaling molecules, including MyD88, IRAK1, and IRF5. ...
In obesity, macrophage activation and infiltration in adipose tissue (AT) underlie chronic low-grade inflammation-induced insulin resistance. Although dectin-1 is primarily a pathogen recognition receptor and innate immune response modulator, its role in metabolic syndromes remains to be clarified. This study aimed to investigate the dectin-1 gene expression in subcutaneous AT in the context of obesity and associated inflammatory markers. Subcutaneous AT biopsies were collected from 59 nondiabetic (lean/overweight/obese) individuals. AT gene expression levels of dectin-1 and inflammatory markers were determined via real-time reverse transcriptase-quantitative polymerase chain reaction. Dectin-1 protein expression was assessed using immunohistochemistry. Plasma lipid profiles were measured by ELISA. AT dectin-1 transcripts and proteins were significantly elevated in obese as compared to lean individuals. AT dectin-1 transcripts correlated positively with body mass index and fat percentage (r ≥ 0.340, p ≤ 0.017). AT dectin-1 RNA levels correlated positively with clinical parameters, including plasma C-reactive protein and CCL5/RANTES, but negatively with that of adiponectin. The expression of dectin-1 transcripts was associated with that of various proinflammatory cytokines, chemokines, and their cognate receptors (r ≥ 0.300, p ≤ 0.05), but not with anti-inflammatory markers. Dectin-1 and members of the TLR signaling cascade were found to be significantly associated, suggesting an interplay between the two pathways. Dectin-1 expression was correlated with monocyte/macrophage markers, including CD16, CD68, CD86, and CD163, suggesting its monocytes/macrophage association in an adipose inflammatory microenvironment. Dectin-1 expression was independently predicted by CCR5, CCL20, TLR2, and MyD88. In conclusion, dectin-1 may be regarded as an AT biomarker of metabolic inflammation in obesity.
... The mechanisms by which PRR pathways crosstalk has not been thoroughly clarified yet. The influential factors of PRR interactions may include the association between two or multiple signaling pathways and positive or negative effects of cross-talking on the cytokine productions and feedback loops after concomitant activation of different PRRs [149,238,250]. ...
For decades, vaccines have played a significant role in protecting public and personal health against infectious diseases and proved their great potential in battling cancers as well. This review focused on the current progress of therapeutic subunit vaccines for cancer immunotherapy. Antigens and adjuvants are key components of vaccine formulations. We summarized several classes of tumor antigens and bioinformatic approaches of identification of tumor neoantigens. Pattern recognition receptor (PRR)-targeting adjuvants and their targeted delivery platforms have been extensively discussed. In addition, we emphasized the interplay between multiple adjuvants and their combined delivery for cancer immunotherapy.
... This phenotype includes the expression of the cell surface receptors, which make these cells highly reactive to a variety of stimuli [2,8]. Several pattern recognition receptors have been described, including Toll-like receptors (TLRs), C-type lectins, cytoplasmic retinoic acid-inducible gene-I-like receptors, and nucleotide oligomerization domain-like receptors [9][10][11]. The evolutionarily conserved TLRs are the best characterized pattern recognition receptors, and they can initiate long-standing and protective immunity through presenting antigen motifs to the cells of the adaptive immune system. ...
Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.
... The histones and free nucleic acids from NETs are generally recognized by innate immune system through binding to damage-associated molecular pattern recognition receptors, such as Toll-like receptors (TLRs), cytosolic DNA sensors (CDSs), and Nod-like receptors (NLRs) [69]. Interacting with TLRs and NLRs, Dectin-1 also plays fundamental roles of regulating phagocytosis, ROS and cytokine production in myeloid cells, such as macrophage and neutrophil [61,70]. Moreover, the recognition for histones [54] and alarmin for neuroin ammation [71,72] broaden our understanding about Dectin-1 in innate immune system as well as CNS. ...
Background
Blood-brain barrier (BBB) disruption and leukocyte infiltration are two pathological features post traumatic brain injury (TBI). However, the role of circulating leukocytes in BBB disruption and the crosstalk between them are not fully elucidated. Neutrophil is the most abundant circulating cell type that migrates into brain tissue when TBI occurs instantly, while brain pericyte occupies a strategic position between circulating cell and interstitial space in BBB. Understanding their interactions is essential to provide insight into the intrinsic relationship and identify biological targets for TBI treatments. Methods
By analyzing brain tissues from TBI patients and mouse TBI model through immunohistochemical method and flow cytometry, we build the relationship between neutrophils, neutrophil extracellular traps (NETs) and brain pericyte. The components of NETs-related medium were investigated by proteomics and metabolomics to decipher the factors directly regulating pericytes. The molecular mechanisms were deeply explored by WB/CHIP/RT-PCR in primary brain pericyte/pericyte cell line MBVP treated with NETs-formed medium or specific NETs components. In mice TBI model, we also explored the possible therapeutic approaches for TBI treatment that targeting at the axis of neutrophil-NETs-pericyte. ResultsNETs formation is highly enhanced post TBI, inducing the appearance of CD11b expressing brain pericyte simultaneously. This novel CD11b ⁺ pericyte subset is characterized with increased permeability and pro-inflammatory profiles. Mechanistically, recognition of histones from NETs by Dectin-1 on pericyte contributes to CD11b induction in protein kinase C (PKC)-c-Jun-dependent manner. Transcription factor c-Jun directly binds to the promoter sequence of CD11b to enhance its expression in pericyte, conferring pericyte activation, BBB disruption and aggravated neutrophil infiltration post TBI. Either inhibiting NETs formation by Cl-Amidine or blocking Dectin-1 by Laminarin are both beneficial for decreasing neutrophil infiltration and brain pericyte activation post TBI. Conclusions
These results unfold that “neutrophil-NETs-pericyte” and “histones-Dectin-1-CD11b” are possible cellular and molecular mechanisms for building connection between BBB damage and neutrophil infiltration. Targeting at NETs formation and Dectin-1 are promising treatments for improving neurological outcomes of TBI patients.
... Innate immune cells use a variety of receptors to recognize invading pathogens and elicit appropriate immune responses. An increasing number of studies have shown that during host-pathogen interactions, two or more receptors often function simultaneously to orchestrate the overall immune responses [1][2][3][4][5][6][7] . This phenomenon is known as receptor crosstalk. ...
Many innate immune receptors function collaboratively to detect and elicit immune responses to pathogens, but the physical mechanisms that govern the interaction and signaling crosstalk between the receptors are unclear. In this study, we report that the signaling crosstalk between Fc gamma receptor (FcγR) and Toll-like receptor (TLR)2/1 can be overall synergistic or inhibitory depending on the spatial proximity between the receptor pair on phagosome membranes. Using a geometric manipulation strategy, we physically altered the spatial distribution of FcγR and TLR2 on single phagosomes. We demonstrate that the signaling synergy between FcγR and TLR2/1 depends on the proximity of the receptors and decreases as spatial separation between them increases. However, the inhibitory effect from FcγRIIb on TLR2-dependent signaling is always present and independent of receptor proximity. The overall cell responses are an integration from these two mechanisms. This study presents quantitative evidence that the nanoscale proximity between FcγR and TLR2 functions as a key regulatory mechanism in their signaling crosstalk.
... SSP4) shows a potential alteration to NOD2-dependent regulation of toll-like receptor (TLR)mediated NF-jB signaling that produces pro-inflammatory cytokines in response to the pathogen-associated molecular patterns such as lipopolysaccharide or muramyl dipeptide (MDP). This path is sparse and labeled medium confidence (orange) because the mechanism of interaction between NOD2 and TLRs is not known, nor is it clear how that interaction normally results in increased production of pro-inflammatory cytokines (Underhill, 2007). Although different models have been proposed to describe the mechanism, for none of these has the effect of the NOD2 1007fs variant been investigated (details are in the pop-up box of MM3). ...
Motivation:
Experimental findings on genetic disease mechanisms are scattered throughout the literature and represented in many ways, including unstructured text, cartoons, pathway diagrams, and network graphs. Integration and structuring of such mechanistic information greatly enhances its utility.
Results:
MecCog is a graphical framework for building integrated representations (mechanism schemas) of mechanisms by which a genetic variant causes a disease phenotype. A MecCog mechanism schema displays the propagation of system perturbations across stages of biological organization, using graphical notations to symbolize perturbed entities and activities, hyperlinked evidence tagging, a mechanism ontology, and depiction of knowledge gaps, ambiguities, and uncertainties. The web platform enables a user to construct, store, publish, browse, query, and comment on schemas. MecCog facilitates the identification of potential biomarkers, therapeutic intervention sites, and critical future experiments.
Availability:
The MecCog framework is freely available at http://www.meccog.org.
Supplementary information:
Supplementary data are available at Bioinformatics online.
... A) The top branch (SSP3àMM3 à SSP4) shows a potential alteration to NOD2-dependent regulation of Toll-like receptor (TLR) mediated NF-κB signaling that produces pro-inflammatory cytokines in response to the pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), or muramyl dipeptide (MDP). This path is sparse and labeled medium confidence (orange) because the mechanism of interaction between NOD2 and TLRs is not known, nor is it clear how that interaction normally results in increased production of pro-inflammatory cytokines (Underhill, 2007). Different models have been proposed to describe the mechanism: synergistic production of TNF-α by NOD2 and TLR4 (Wolfert et al., 2002); activation of the inflammasome by NOD2 via RICK to produce IL-1β from . ...
Motivation
Experimental findings on genetic disease mechanisms are scattered throughout the literature and represented in many ways, including unstructured text, cartoons, pathway diagrams, and network graphs. Integration and structuring of such mechanistic information will greatly enhance its utility.
Results
MecCog is a graphical framework for building integrated representations (mechanism schemas) of mechanisms by which a genetic variant causes a disease phenotype. A MecCog mechanism schema displays the propagation of system perturbations across stages of biological organization, using graphical notations to symbolize perturbed entities and activities, hyperlinked evidence tagging, a mechanism ontology, and depiction of knowledge gaps, ambiguities, and uncertainties. The web platform enables a user to construct, store, publish, browse, query, and comment on schemas. MecCog facilitates the identification of potential biomarkers, therapeutic intervention sites, and critical future experiments.
Availability and Implementation
The MecCog framework is freely available at http://www.meccog.org .
Contact
jmoult@umd.edu
Supplementary information
Supplementary material is available at Bioinformatics online.
... In many receptor-ligand interactions, ligand-induced receptor heterodimerization or co-localization precedes cellular signalling. [27][28][29] Herein, the confocal microscopy and co-immunoprecipitation studies suggest dectin-1-TLR2 co-localization induced by both β-glucans. We observed co-localization and clustering of dectin-1 and TLR2 in murine peritoneal macrophages stimulated by β-glucans, more with zymosan than barley β-glucan. ...
Toll‐like receptors (TLRs), TLR2 in particular, are shown to recognize various glycans and glycolipid ligands resulting in various immune effector functions. As barley β‐glucan and zymosan are the glycans implicated in immunomodulation, we examined whether these ligands interact with Dectin‐1, a lectin‐type receptor for glycans, and TLR2 and induce immune responses that can used against Leishmania infection in a susceptible host. The binding affinity of barley β‐glucan and zymosan with Dectin‐1 and TLR2 were studied in silico. Barley β‐glucan‐ and zymosan‐induced Dectin‐1 and TLR2 co‐localization was studied by confocal microscopy and co‐immunoprecipitation. These ligands induced signaling and effector functions were assessed by Western blot analyses and various immunological assays. Finally, the anti‐leishmanial potential of barley β‐glucan and zymosan was tested in L. donovani‐infected macrophages and in L. donovani‐infected BALB/c mice. Both barley β‐glucan and zymosan interacted with TLR2 and Dectin‐1, but with a much stronger binding affinity for the latter, and therefore induced co‐localization of these two receptors on BALB/c‐derived macrophages. Both ligands activated MyD88‐ and Syk‐mediated downstream pathways for heightened inflammatory responses in L. donovani‐infected macrophages. These two ligands induced T cell‐dependent host‐protection in L. donovani‐infected BALB/c mice. These results establish a novel modus operandi of β‐glucans through Dectin‐1 and TLR2 and suggest an immuno‐modulatory potential against infectious diseases.
... However, an unanticipated finding was a reduction or elimination of signal in THP1-Dual cells with TLR7 or TLR8 agonists (R848 and TL8-506, respectively) and in A549 cells with a STING agonist (2′3′-cGAMP). Perhaps the failure of the controls to elicit an immune response under these conditions implies cross-talk among innate immune system sentinels (40,41), whereby eliminating one signaling adaptor may have unintended consequences on other pathways (e.g., regulation of STING expression by RIG-I activation) (42). However, TLR7/8 agonist controls showed that TLR7 and TLR8 activate the NF-B pathway as expected in these cells ( fig. ...
Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N 1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.
... Complex interplays between TLRs and other PRRs is a common principle for pathogen recognition and discrimination [5,[15][16][17][18]. A high level of cross talk was recently reported between TLR2 and TLR8 [15]. ...
Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.
... β-glucan of G. lucidum is an immunomodulatory polysaccharide able to potential interact with pattern recognition receptors dectin-1. Dectin-1 is a c-type lectin, which can collaborate with TLR2 to activate response of innate immune in antigen-presenting cells [155][156][157]. Simultaneously inducing stability between pro-and antiinflammatory responses [158][159][160][161]. ...
... Pattern recognition receptors also recognize host signature molecules that are indicative of disease and cellular damage-damage-associated molecular patterns (Garg et al., 2010;Krysko et al., 2011). Pathogen recognition results in the activation of cellular defense mechanisms, production of secreted pro-inflammatory cytokines, recruitment of immune cells to the site of infection, and stimulate anti-microbicidal mechanisms, such as the production of reactive oxygen species and antimicrobial peptides (Carpenter and O'Neil, 2007;Lee and Kim, 2007;Underhill, 2007;Takeuchi and Akira, 2010). ...
The intestinal tract harbors a diverse community of microbes that have co-evolved with the host immune system. Although many of these microbes execute functions that are critical for host physiology, the host immune system must control the microbial community so that the dynamics of this interdependent relationship is maintained. To facilitate host homeostasis, the immune system ensures that the microbial load is tolerated, but anatomically contained, while remaining reactive to microbial invasion. Although the microbiota is required for intestinal immune development, immune responses regulate the structure and composition of the intestinal microbiota by evolving unique immune adaptations that manage this high-bacterial load. The immune mechanisms work together to ensure that commensal bacteria rarely breach the intestinal barrier and that any that do invade should be killed rapidly to prevent penetration to systemic sites. The communication between microbiota and the immune system is mediated by the interaction of bacterial components with pattern recognition receptors expressed by intestinal epithelium and various antigen-presenting cells resulting in activation of both innate and adaptive immune responses. Interaction between the microbial community and host plays a crucial role in the mucosal homeostasis and health status of the host. In addition to providing a home to numerous microbial inhabitants, the intestinal tract is an active immunological organ, with more resident immune cells than anywhere else in the body, organized in lymphoid structures called Peyer’s patches and isolated lymphoid follicles such as the cecal tonsils. Macrophages, dendritic cells, various subsets of T cells, B cells and the secretory immunoglobulin A (IgA) they produce, all contribute to the generation of a proper immune response to invading pathogens while keeping the resident microbial community in check without generating an overt inflammatory response to it. IgA-producing plasma cells, intraepithelial lymphocytes, and γδT cell receptor-expressing T cells are lymphocytes that are uniquely present in the mucosa. In addition, of the γδT cells in the intestinal lamina propria, there are significant numbers of IL-17-producing T cells and regulatory T cells. The accumulation and function of these mucosal leukocytes are regulated by the presence of intestinal microbiota, which regulate these immune cells and enhance the mucosal barrier function allowing the host to mount robust immune responses against invading pathogens, and simultaneously maintains immune homeostasis.
... Agonist MDP but Not Nod1 Agonist iE-DAP Combines with TLR4 Agonist LPS to Induce B Cell Viability and Proliferation and IgG2b Production. New functions in innate immune cells have been reported for the crosstalk between TLRs and NLRs [20][21][22]. There is a synergistic stimulation of human monocytes and dendritic cells by TLR4 and Nod1-and Nod2-activating agonists [23]. ...
Many studies have shown that Toll-like receptors (TLRs) and Nod-like receptors (NLRs) were expressed in B cells and their signaling affects B cell functions. Nonetheless, the roles played by these receptors in B cell antibody (Ab) production have not been completely elucidated. In the present study, we examined the effect of the Nod2 agonist muramyl dipeptide (MDP) in combination with the TLR4 agonist lipopolysaccharide (LPS), a well-known B cell mitogen, on B cell viability, proliferation, and activation, and Ab production by in vitro culture of purified mouse spleen resting B cells. MDP combined with LPS to reinforce B cell viability, proliferation, and activation. Moreover, MDP enhanced LPS-induced IgG2b production, germline γ 2b transcript (GLT γ 2b) expression, and surface IgG2b expression. In an experiment with Nod2- and TLR4-deficient mouse B cells, we observed that the combined effect of MDP and LPS is dependent on Nod2 and TLR4 receptors. Furthermore, the combined effect on B cell viability and IgG2b switching was not observed in Rip2-deficient mouse cells. Collectively, this study suggests that Nod2 signaling enhances TLR4-activated B cell proliferation, IgG2b switching, and IgG2b production.
... Dectin-1 is a Pattern Recognition Receptor (PRR) of innate immune system which is a type II transmembrane protein containing carbohydrate recognition domain (CRD) and immunoreceptor tyrosine-based activation motif (ITAM) at the extracellular C-terminal and intracellular N-terminal regions, respectively (Ariizumi, Shen et al. 2000, Adachi, Ishii et al. 2004. The CRD of Dectin-1 belongs to the C-type lectin-like domain (CTLD) subfamily and is considered as a non-classical Ctype lectin because it lacks the calcium binding site in its C-terminal domain and its ITAM motif does not contain the distal tyrosine residue (Tyr-3) owing only proximal tyrosine residue (Tyr-15) within the single YXXL sequence (Underhill, Rossnagle et al. 2005, Underhill 2007). Dectin-1 was first identified on dendritic cells but was later found to be expressed on monocytes, macrophages, neutrophils and a subset of T cells Gordon 2001, Taylor, Brown et al. 2002). ...
Stimulation of C-type lectin domain of human dectin-1 receptor by fungal β-glucans causes conformational changes in its cytoplasmic domain which initiates various cellular responses mediated by downstream signaling components. We aimed to build the three-dimensional structures of thecytoplasmic domain as well asC-type lectin domain of human Dectin-1along with their potential ligands through homology modeling.The overall three-dimensional fold of cytoplasmic domain was found to consist of mixed β-sheet whereas,in case of C-type lectin domain antiparallel β-sheets flanked byα-helices were observed. Protein-protein docking strategy was utilized to monitorkey interactions between cytoplasmic domainof dectin-1 receptor and PKCδ, as a prime regulator of Dectin-1 signaling. The interface was observed to have both hydrophilic and hydrophobic amino acid residues maintaining crucial contacts between the two proteins. The given three dimensional structural information can be implicated in structure-based drug designing to discover potential immunomodulators that can interfere with the immune responses and phagocytosis during inflammatory and infectious conditions.
... This observation suggests that ODN2088, while inhibiting TLR9 and the MyD88 pathway, can stimulate cytokine release by activating other mediators. This observation may be due to pattern recognition receptor (PRR) cross talk, as proposed by Underhill; hence, innate immune receptors can collaborate either positively or negatively in regulating the immune response against a pathogen (47). Research studies conducted in the last few years demonstrated the existence of multiple receptors that detect DNA viruses in the cytosol. ...
We have previously demonstrated that Epstein-Barr virus DNA increases the production of the pro-inflammatory cytokine IL-17A in mice. This property may contribute to the established association between EBV and autoimmune diseases. The objective of the current study was to elucidate mechanisms through which EBV DNA modulates IL-17A levels in mice. To determine whether endosomal Toll-Like Receptors (TLRs) played a role in this pathway, the expression of TLR3, 7 or 9 was assessed by real-time RT-PCR in mouse spleens after injection of EBV DNA. Moreover, specific inhibitors were used for these TLRs in mouse Peripheral Blood Mononuclear Cells (PBMCs) cultured with EBV DNA and in mice injected with this viral DNA; IL-17A levels were then assessed using Enzyme Linked Immunosorbent Assay. The expression of the endosomal receptors TLR3, 7, and 9 was increased in mice injected with EBV DNA. When mouse immune cells were cultured with EBV DNA and a TLR3, 7 or 9 inhibitor or when mice were injected with the viral DNA along with either of these inhibitors, a significant decrease in IL-17A levels was detected. Therefore, endosomal TLRs are involved in the EBV DNA-mediated triggering of IL-17A production in mice. Targeting these receptors in EBV positive subjects with autoimmunity may be useful pending investigations assessing whether they play a similar role in humans.
IMPORTANCE The Epstein-Barr virus is a pathogen that causes persistent infection with potential consistent viral DNA shedding. The enhancement of production of pro-inflammatory cytokines by viral DNA itself may contribute to autoimmune disease development or exacerbation. In this project we identified that endosomal Toll-like receptors are involved in triggering pro-inflammatory mediators in response to viral DNA. Pathways and receptors involved may serve as future therapeutic targets in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.
... Lastly, we examined the protein expression of pattern recognition receptor, dectin-1 (Clec7a) 23 , which showed reduced RNAseq expression in both sham and 4d CCI-injured juvenile whole blood compared to respective adult mice samples. Dectin-1, a c-type lectin, plays a key role in innate immunity through Toll-like receptor and Nod protein interactions to regulate phagocytosis, ROS and cytokine production 24 . Immune cells were isolated from the spleen and peripheral whole blood of naïve juvenile and adult mice then immuno-stained for Ly6G and dectin-1 protein. ...
The peripheral immune system is a major regulator of the pathophysiology associated with traumatic brain injury (TBI). While age-at-injury influences recovery from TBI, the differential effects on the peripheral immune response remain unknown. Here, we investigated the effects of TBI on gene expression changes in murine whole blood using RNAseq analysis, gene ontology and network topology-based key driver analysis. Genome-wide comparison of CCI-injured peripheral whole blood showed a significant increase in genes involved in proteolysis and oxidative-reduction processes in juvenile compared to adult. Conversely, a greater number of genes, involved in migration, cytokine-mediated signaling and adhesion, were found reduced in CCI-injured juvenile compared to CCI-injured adult immune cells. Key driver analysis also identified G-protein coupled and novel pattern recognition receptor (PRR), P2RY10, as a central regulator of these genes. Lastly, we found Dectin-1, a c-type lectin PRR to be reduced at the protein level in both naïve neutrophils and on infiltrating immune cells in the CCI-injured juvenile cortex. These findings demonstrate a distinct peripheral inflammatory profile in juvenile mice, which may impact the injury and repair response to brain trauma.
... Our results showed that salecan significantly activated the expression of Dectin1 in the jejunum under pathological condition, indicating that Dectin1 can recognize salecan in the jejunum of mice. TLRs, Dectin1, and other receptors collaborated together to modulate the innate immunity [44], which plays a role in the development of gastrointestinal mucositis. We showed that salecan can modulate the expression of TLRs and Dectin1 in the intestine of mice with MTX-induced mucositis. ...
Purpose
Methotrexate (MTX) is a widely used cancer chemotherapy agent. The efficacy of MTX is often limited by serious side effects, such as intestinal mucositis. The aim of this study was to evaluate the protective effect of water-soluble β-glucan salecan on MTX-induced intestinal toxicity in mice.
Methods
Intestinal mucositis was induced in C57BL/6 mice by intraperitoneal injection of MTX for two consecutive days. Mice were orally administrated with saline or salecan for 6 days before MTX injection and continued to the end of the study. Several histological and biochemical parameters were measured in the jejunum.
Results
Orally administration of salecan improved the severity of intestinal mucositis in a dose-dependent manner, as evidenced by the well-maintained mucosal architecture and body weight in salecan-treated groups. Salecan treatment inhibited MTX-induced oxidative stress and effectively scavenged free radicals both in vitro and in vivo. Metabolomics analysis revealed that salecan treatment reversed the intestinal metabolic profiling changes in mice with MTX-induced mucositis. Salecan treatment modulated the innate immunity through the regulation of TLR and Dectin1 expression in the jejunum, thus protecting mice from MTX-induced intestinal damage.
Conclusions
Salecan has potential advantages in the treatment of MTX-induced intestinal mucositis, and its protective effect is mainly attributed to its antioxidant and immunomodulatory properties.
... β-glucan of G. lucidum is an immunomodulatory polysaccharide able to potential interact with pattern recognition receptors dectin-1. Dectin-1 is a c-type lectin, which can collaborate with TLR2 to activate response of innate immune in antigen-presenting cells [155][156][157]. Simultaneously inducing stability between pro-and antiinflammatory responses [158][159][160][161]. ...
... Tlrs are associated with the activation of the innate immune system by recognizing pathogen-associated molecular patterns (39). Proinflammatory effects have been described for Tlrs previously (34), whereas other studies implied protective effects of activated Tlr2, Tlr7 and Tlr8 in mouse models of asthma (35,36,40). ...
The current study analyzed gene expression profiles of the endolymphatic sac (ES) in rats and identified expressed genes, present in the human and rat ES, to reveal key hubs for inflammatory responses. Microarray data (accession no. E-MEXP-3022) were obtained from the European Bioinformatics Institute database, including three biological replicates of ES plus dura tissues and three replicates of pure dura tissues form rats. Differentially expressed genes (DEGs) were screened using the Linear Model for Microarray data method and a protein-protein interaction (PPI) network was constructed using data from the Search Tool for the Retrieval of Interacting Genes/Proteins database followed by a module analysis via Clustering with Overlapping Neighborhood Expansion. Function enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery online tool. A total of 612 DEGs were identified, including 396 upregulated and 216 downregulated genes. Gene ontology term enrichment analysis indicated DEGs were associated with cell adhesion, including α5-integrin (Itga1) and secreted phosphoprotein 1 (Spp1); T cell co-stimulation, including C-C chemokine ligand (Ccl)21 and Ccl19; and the toll-like receptor signaling pathway, including toll-like receptor (Tlr)2, Tlr7 and Tlr8. These conclusions were supported by Kyoto Encyclopedia of Genes and Genomes pathway analyses revealing extracellular matrix-receptor interaction, including Itga1 and Spp1; leukocyte transendothelial migration, includingclaudin-4 (Cldn4); and malaria, including Tlr2. The hub roles of Itga1, Cd24 and Spp1 were revealed by calculating three topological properties of the PPI network. Ccl21, Ccl19 and Cldn4 were demonstrated to be crucial following significant module analysis according to the corresponding threshold, which revealed they were enriched in inflammation pathways. Tlr7, Tlr2, granzyme m and Tlr8 were common genes associated with inflammatory responses in rat and human ES. In conclusion, abnormal expression of the aforementioned inflammation-associated genes may be associated with the development of autoimmune inner ear diseases.
... Several studies have shown that responses to bacterial and viral pathogens are not exclusively dependent on activation of individual TLRs, but results from complex TLR-TLR interactions (Underhill, 2007;Delaloye et al., 2009;Slater et al., 2010;Negishi et al., 2012). Nevertheless, the effects of engagement of more than one innate immune receptor in close temporal proximity are not well studied. ...
Innate immune signaling is essential to mount a fast and specific immune response to pathogens. Monocytes and macrophages are essential cells in the early response in their capacity as ubiquitous phagocytic cells. They phagocytose microorganisms or damaged cells and sense pathogen/damage-associated molecular patterns (PAMPs/DAMPs) through innate receptors such as Toll-like receptors (TLRs). We investigated a phenomenon where co-signaling from TLR2 and TLR8 in human primary monocytes provides a distinct immune activation profile compared to signaling from either TLR alone. We compare gene signatures induced by either stimulus alone or together and show that co-signaling results in downstream differences in regulation of signaling and gene transcription. We demonstrate that these differences result in altered cytokine profiles between single and multi-receptor signaling, and show how it can influence both T-cell and neutrophil responses. The end response is tailored to combat extracellular pathogens, possibly by modifying the regulation of IFNβ and IL12-family cytokines.
... TLR3 and TLR9 both localize to intracellular compartments and recognize C. albicans 626 nucleic acids and chitin, respectively, contributing to a protective cytokine response to the 627 fungus(Nahum et al., 2011; Wagener et al., 2014). Interestingly, Dectin1 can collaborate with 628 plasmalemmal TLRs (TLR2 or TRL4) to enhance signaling and augment cytokine production 629(Ferwerda et al., 2008; Netea et al., 2006;Underhill, 2007) and a similar synergy may apply to 630 endomembrane TLRs. Indeed, Dectin1 recognition is required for TLR9 localization to the C.631 albicans phagosome and TLR9-dependent gene expression (Khan et al., 2016). ...
Candida albicans
hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae.
Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro‐inflammatory and immunomodulatory responses depending on their chemical structures. Here we report about the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol‐containing O‐antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whether a pro‐inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible “immune modulating” action has been proposed for the peculiar O‐antigen portion.
The law and mechanism of the interaction between polysaccharides and pattern recognition receptors (PRRs) has been unclear. Herein, three glucomannans with different structures were selected to explore the universal mechanism for PRRs to recognize glucomannans. Screening results showed that the silence of TLR4 but not TLR2 severely blocked the production of inflammatory cytokines and the transduction of signal pathways. In-depth results revealed that the participation of myeloid differentiation protein 2 (MD2) and CD14 and the dimerization of the TLR4-MD2 complex were required for glucomannan-activated TLR4 signal transduction. Mannose receptor (MR) was also engaged in glucomannan-induced respiratory burst, endocytosis, and inflammatory signaling pathways in a spleen tyrosine kinase-dependent manner. The internalization of glucomannans into the cytoplasm by MR directly initiated complex intracellular signaling cascades. Finally, molecular docking characterized the binding energy and binding sites between glucomannans and multiple receptors from other perspectives. The essence of glucomannans recognized by PRRs was the non-covalent interaction of multiple receptors and the subsequent transmission of the signal cascade was triggered in a multi-channel and cooperative manner. As a result, the hypothesis that "Innate immune receptors co-recognition of polysaccharides initiates multi-pathway synergistic immune response" was proposed to outline these meaningful phenomena.
Innate immune cells recognize and elicit responses against pathogens by integrating signals from different types of cell-surface receptors. How the receptors interact in the membrane to enable their signaling crosstalk is poorly understood. Here, we reveal the nanoscale organization of TLR2 and Dectin-1, a receptor pair known to cooperate in regulating antifungal immunity, through their synergistic signaling crosstalk at macrophage cell membranes. Using super-resolution single-molecule localization microscopy, we show that discrete noncolocalized nanoclusters of Dectin-1 and TLR2 are partially overlapped during their synergistic crosstalk. Compared to when one type of receptor is activated alone, the simultaneous activation of Dectin-1 and TLR2 leads to a higher percentage of both receptors being activated by their specific ligands and consequently an increased level of tyrosine phosphorylation. Our results depict, in nanoscale detail, how Dectin-1 and TLR2 achieve synergistic signaling through the spatial organization of their receptor nanoclusters.
Small molecule adjuvants are attractive for enhancing broad protection and durability of immune responses elicited by subunit vaccines. Covalent attachment of an adjuvant to an immunogen is particularly attractive because it simultaneously delivers both entities to antigen presenting cells resulting in more efficient immune activation. There is, however, a lack of methods to conjugate small molecule immune potentiators to viral glycoprotein immunogens without compromising epitope integrity. We describe herein a one‐step enzymatic conjugation approach for the covalent attachment of small molecule adjuvants to N‐linked glycans of viral glycoproteins. It involves the attachment of an immune potentiator to CMP‐Neu5AcN3 by Cu(I)‐catalyzed azide‐alkyne 1,3‐cycloaddition followed by sialyltransferase‐mediated transfer to N‐glycans of a viral glycoprotein. The method was employed to modify a native‐like HIV envelope trimer with a Toll‐like receptor 7/8 agonist. The modification did not compromise Env‐trimer recognition by several broadly neutralization antibodies. Electron microscopy confirmed structural integrity of the modified immunogen.
Innate immune cells recognize and elicit responses against pathogens by integrating signals from different types of cell-surface receptors. How the receptors interact in the membrane to enable their signaling crosstalk is poorly understood. Here, we reveal the nanoscale organization of TLR2 and Dectin-1, a receptor pair known to cooperate in regulating antifungal immunity, through their synergistic signaling crosstalk at macrophage cell membranes. Using super-resolution single-molecule localization microscopy, we show that discrete non-colocalized nanoclusters of Dectin-1 and TLR2 are partially overlapped during their synergistic crosstalk. Compared to when one type of receptor is activated alone, the simultaneous activation of Dectin-1 and TLR2 leads to a higher percentage of both receptors being activated by their specific ligands, and consequently an increased level of tyrosine phosphorylation. Our results depict, in nanoscale detail, how Dectin-1 and TLR2 achieve synergistic signaling through the spatial organization of their receptor nanoclusters.
Diabetic retinopathy (DR) is the most frequent microvascular complication of diabetes mellitus (DM) and a leading cause of blindness worldwide. Evidence has shown that DR is an inflammatory disease with hyperglycemia playing a causative role in the development of its main features, including inflammation, cellular apoptosis, neurodegeneration, oxidative stress, and neovascularization. Toll-like receptors (TLRs) are a well-known family of pattern recognition receptors (PRRs) responsible for the initiation of inflammatory and immune responses. TLR4 identifies both endogenous and exogenous ligands and is associated with various physiological and pathological pathways in the body. While the detailed pathophysiology of DR is still unclear, increasing data suggests a crucial role for TLR4 in the development of DR. Due to hyperglycemia, TLR4 expression increases in diabetic retina, which activates various pathways leading to DR. Considering the role of TLR4 in DR, several studies have focused on the association of TLR4 polymorphisms and risk of DR development. Moreover, evidence concerning the effect of microRNAs in the pathogenesis of DR, through their interaction with TLR4, indicates the determinant role of TLR4 in this disease. Of note, several agents have proven as effective in alleviating DR through the inhibition of the TLR4 pathway, suggesting new avenues in DR treatment. In this review, we provided a brief overview of the TLR4 structure and biological function and a more comprehensive discussion about the mechanisms of TLR4 activation in DR. Furthermore, we summarized the relationship between TLR4 polymorphisms and risk of DR and the relationship between microRNAs and TLR4 in DR. Finally, we discussed the current progress in designing TLR4 inhibitors, which could be helpful in DR clinical management.
RNA viruses, including the coronavirus, develop a unique strategy to evade the host immune response by interrupting the normal function of cytosolic retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors (RLRs). RLRs rapidly detect atypical nucleic acids, thereby triggering the antiviral innate immune signaling cascade and subsequently activates the interferons transcription and induction of other proinflammatory cytokines and chemokines. Nonetheless, these receptors are manipulated by viral proteins to subvert the host immune system and sustain the infectivity and replication potential of the virus. RIG‐I senses the single‐stranded, double‐stranded, and short double‐stranded RNAs and recognizes the key signature, a 5′‐triphosphate moiety, at the blunt end of the viral RNA. Meanwhile, the melanoma differentiation‐associated gene 5 (MDA5) is triggered by longer double stranded RNAs, messenger RNAs lacking 2′‐O‐methylation in their 5′‐cap, and RNA aggregates. Therefore, structural insights into the nucleic‐acid‐sensing and downstream signaling mechanisms of these receptors hold great promise for developing effective antiviral therapeutic interventions. This review highlights the critical roles played by RLRs in viral infections as well as their ligand recognition mechanisms. In addition, we highlight the crosstalk between the toll‐like receptors and RLRs and provide a comprehensive overview of RLR‐associated diseases as well as the therapeutic potential of RLRs for the development of antiviral‐drugs. Moreover, we believe that these RLR‐based antivirals will serve as a step toward countering the recent coronavirus disease 2019 pandemic.
β-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of β-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.
Dendritic cells (DCs) play a primary role in antigen presentation to CD4⁺ and CD8⁺ T cells and induce acquired immune response against cancer cells. Therefore, determining positive modulators of DC activation to improve therapeutic approaches for cancer immunotherapy is greatly needed. In this study, we investigated the effect of maitake α-glucan YM-2A, isolated from Grifola frondosa, on the maturation and function of DCs and its adjuvant effect on a tumor-associated antigen (TAA)-loaded DC vaccine against murine tumor. We showed that YM-2A induced morphological changes and increased cell-surface maturation markers and cytokine production in DCs. In a mixed lymphocyte reactions assay, YM-2A-treated DCs increased proliferation and production of IFN-γ by allogeneic CD4⁺ and CD8⁺ T cells. These results indicate that YM-2A phenotypically and functionally activates DCs. Furthermore, YM-2A-treated TAA-loaded DC vaccine significantly reduced tumor growth and improved survival in two murine tumor models, CT-26 tumor-bearing BALB/c mice and B16 melanoma-bearing C57BL/6 mice. YM-2A-treated TAA-loaded DC vaccine increased splenic IFN-γ producing CD4⁺ and CD8⁺ T cells in CT-26 tumor-bearing BALB/c mice. Antibody neutralization studies indicated that YM-2A-induced DC maturation is mediated, in part, by the Dectin-1-dependent pathway. Overall, YM-2A-treatment with a TAA-loaded DC vaccine could be an excellent candidate for immunotherapy against cancer.
Nucleic acid-based pattern recognition receptor agonists are effective adjuvants and immunotherapeutic agents. Rather than single applications, ligand combinations could synergistically potentiate immune responses by elevating cytokine and chemokine production via triggering multiple signaling pathways. However, short half-lives of such labile ligands due to nuclease attack and limited cellular uptake due to their structure significantly hamper their in vivo performances. More importantly, simultaneous delivery and activity presentation of protein antigen and nucleic acid ligands critically limit the clinical development of these constructs. In this work, we approached this problem by co-encapsulating a model antigen ovalbumin along with TLR9 and STING ligands within liposomes, a well-established drug delivery system that enables payload stability and enhanced cellular activity upon internalization. Moreover, by loading dual ligands we postulated to achieve heightened Th-1 immune response that would yield pronounced protective vaccine efficacy. We show that, pH-sensitive liposomes co-encapsulating CpG ODN and cGAMP induced synergistic innate immune response by elevating type I and type II interferon levels. Most importantly, this vaccine formulation led to ~70% regression of established melanoma tumor. pH-sensitive liposomal vaccine administration elevated IgG2c/IgG1 antibody ratio, indicative of augmented OVA-specific Th1-biased immunity. Importantly, while the frequency of tumor-specific IFN-γ producing CD8+ T-cells was significantly increased, the M2-type anti-inflammatory macrophage levels were decreased in the tumor bed. In conclusion, our strategy induces reversal of immunosuppressive tumor microenvironment, while enhancing effective anti-tumor immune-response. We propose that this could be coupled with standard therapies during combating tumor eradication.
The myocardial inflammatory response is a consequence of myocardial infarction (MI), which may deteriorate cardiac remodeling and lead to dysfunction in the heart post-MI. Dectin-1 is a c-type lectin, which has been shown to regulate innate immune responses to pathogens. However, the role of Dectin-1 in the heart diseases remains largely unknown. In this study, we aimed to investigate the effects of Dectin-1 on cardiac remodeling post-MI. We found that cardiac Dectin-1 mRNA and protein expressions were significantly elevated in C57BL/6 mice after MI. In vitro, hypoxia induced cardiomyocyte injury in parallel with increased Dectin-1 protein expression. Knockdown of Dectin-1 remarkably attenuated cardiomyocyte death under hypoxia and lipopolysaccharide (LPS) stimulation. In vivo administration of adeno-associated virus serotype 9 mediated silencing of Dectin-1, which significantly decreased cardiac fibrosis, dilatation, and improved cardiac function in the mice post-MI. At the molecular level, downregulation of Dectin-1 dramatically suppressed up-regulation of nuclear factor-κB (NF-κB), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and the inflammatory genes involved in fibrogenesis and cardiac remodeling after MI. Furthermore, treatment with BAY11-7082, an inhibitor of NF-κB, repressed the activation of NF-κB, and attenuated LPS induced elevation of NLRP3 and cell death in cardiomyocytes. Collectively, upregulation of Dectin-1 in cardiomyocytes post-MI contributes to cardiac remodeling and cardiac dysfunction at least partially by activating NF-κB and NLRP3. This study identified Dectin-1 as a promising therapeutic target for ischemic heart disease.
Vulvovaginal candidiasis (VVC) is one of the most common infections in women. The purpose of this study was to evaluate the ability of a water-soluble β-glucan salecan to protect against Candida albicans (C. albicans) vaginal infection. The model was reproduced with intravaginal inoculation of yeast blastoconidia in pseudoestrus mice. We found that mice that received salecan (0.5 mg per mouse) after infection had 85% fewer CFU than infected mice given saline. Compared with the C. albicans group, salecan reduced the migration of polymorphonuclear neutrophils (PMNs) in the vagina, decreased mRNA levels of cytokines IL23, IL22, IL17a, and IL17f, anti-candidal genes S100a8 and S100a9 and C. albicans pattern recognition receptor Dectin1. The analysis for vaginal microbial community composition at different taxa levels revealed that the bacterial flora composition in the vagina of the salecan-treated mice was similar to that of the uninfected mice, and distinguished from the infected mice. The vaginal lavages from the salecan treated group had more Enterococcus and its metabolite lactate. Our results suggest salecan might be a potential therapeutic agent for vaginal infection of C. albicans.
Candida albicans can enhance the invasion of oral epithelial cells by Porphyromonas gingivalis, although the fungus is not a periodontal pathogen. In this study, we investigated whether C. albicans augments proinflammatory cytokine production by mouse macrophage-like J774.1 cells incubated with synthetic bacterial components. Mouse macrophage-like J774.1 cells, mouse primary splenocytes, human THP-1 cells, and A549 cells were pretreated with or without heat-killed C. albicans (HKCA) or substitutes for C. albicans cell wall components in 96-well flat-bottomed plates. Cells were then washed and incubated with Pam3CSK4, a Toll-like receptor (TLR) 2 ligand, or lipid A, a TLR4 ligand. Culture supernatants were analyzed by ELISA for secreted IL-6, MCP-1, TNF-α, and IL-8. HKCA augmented TLR ligand-induced proinflammatory cytokine production by J774.1 cells, mouse splenocytes, and THP-1 cells, but not A549 cells. However, IL-6, MCP-1, and TNF-α production induced by Pam3CSK4 or lipid A was not augmented when cells were pretreated with curdlan, a dectin-1 ligand, or mannan, a dectin-2 ligand. In contrast, pretreatment of cells with TLR ligands upregulated the production of IL-6 and TNF-α, but not MCP-1, induced by Pam3CSK4 or lipid A. The results suggest that C. albicans augments synthetic bacterial component-induced cytokine production by J774.1 cells via the TLR pathway, but not the dectin-1 or dectin-2 pathway.
The prevailing concept regarding the immunological function of immunoglobulin A (IgA) is that it binds to and neutralizes pathogens to prevent infection at mucosal sites of the body. However, recently, it has become clear that in humans IgA is also able to actively contribute to the initiation of inflammation, both at mucosal and non-mucosal sites. This additional function of IgA is initiated by the formation of immune complexes, which trigger Fc alpha Receptor I (FcαRI) to synergize with various other receptors to amplify inflammatory responses. Recent findings have demonstrated that co-stimulation of FcαRI strongly affects pro-inflammatory cytokine production by various myeloid cells, including different dendritic cell subsets, macrophages, monocytes, and Kupffer cells. FcαRI-induced inflammation plays a crucial role in orchestrating human host defense against pathogens, as well as the generation of tissue-specific immunity. In addition, FcαRI-induced inflammation is suggested to be involved in the pathogenesis of various chronic inflammatory disorders, including inflammatory bowel disease, celiac disease, and rheumatoid arthritis. Combined, IgA-induced inflammation may be used to either promote inflammatory responses, e.g. in the context of cancer therapy, but may also provide new therapeutic targets to counteract chronic inflammation in the context of various chronic inflammatory disorders.
Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans We describe a new TAK1-TPL2-MKK1-ERK1/2 pathway in macrophages, which is activated by Dectin-1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper-inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ-null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans.
A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-α production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-α in response to LPS. LPS-induced activation of both NF-κB and c-Jun NH2-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.
Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1beta release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.
Macrophages are permissive hosts to intracellular pathogens, but upon activation become microbiocidal effectors of innate and cell-mediated immunity. How the fate of internalized microorganisms is monitored by macrophages, and how that information is integrated to stimulate specific immune responses is not understood. Activation of macrophages with interferon (IFN)-gamma leads to rapid killing and degradation of Listeria monocytogenes in a phagosome, thus preventing escape of bacteria to the cytosol. Here, we show that activated macrophages induce a specific gene expression program to L. monocytogenes degraded in the phago-lysosome. In addition to activation of Toll-like receptor (TLR) signaling pathways, degraded bacteria also activated a TLR-independent transcriptional response that was similar to the response induced by cytosolic L. monocytogenes. More specifically, degraded bacteria induced a TLR-independent IFN-beta response that was previously shown to be specific to cytosolic bacteria and not to intact bacteria localized to the phagosome. This response required the generation of bacterial ligands in the phago-lysosome and was largely dependent on nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor known to respond to bacterial peptidoglycan fragments. The NOD2-dependent response to degraded bacteria required the phagosomal membrane potential and the activity of lysosomal proteases. The NOD2-dependent IFN-beta production resulted from synergism with other cytosolic microbial sensors. This study supports the hypothesis that in activated macrophages, cytosolic innate immune receptors are activated by bacterial ligands generated in the phagosome and transported to the cytosol.
Immunostimulant activities of muramyl dipeptide (enhancement of specific immune responses and of nonspecific resistance to infection) were retained by its N-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester derivative, although very large amounts administered intravenously, or even by the very sensitive intracerebroventricular route, did not elicit fever in the rabbit. This analog also appeared to be devoid of other secondary effects which have been observed after administration of muramyl dipeptide.
After in vitro exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP), cultured resident mouse peritoneal macrophages were primed to display enhanced generation of superoxide anion (O2-) in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Priming with LPS (1 microgram/ml) produced a sevenfold enhancement of PMA-stimulated O2- generation; priming was detected within 30 min and persisted for at least 4 d. Exposure to MDP (1 muM) primed the macrophages to double their O2- release; the response was first observed after 4 h and persisted for at least 3 d. The priming response was not observed with stereoisomers of MDP, which are inactive as adjuvants. LPS and MDP appeared to work directly on the macrophages rather than indirectly by interacting with adherent lymphocytes: (a) Addition of nonadherent cell populations that contained lymphocytes had no effect on the response. (b) The response was normal with cells from nude mice, which lack mature T lymphocytes. (c) Macrophages from C3H/HeJ mice, whose B lymphocytes fail to respond to LPS, were weak in their response to priming LPS; the addition of normal (C3Heb/FeJ) nonadherent cells had no effect on this weak response. (d) The macrophage-like cell line J774.1 also showed enhanced O2--generating capacity after a 4-h exposure to LPS or MDP. The O2--generating capacity of macrophages primed with LPS in vitro was equivalent to that previously observed with cells elicited in vivo by injection of LPS or activated by infection with Bacille Calmette-Guérin. The data suggest that previous exposure to bacterial products could prime macrophages to respond with increased production of toxic oxygen metabolites on contact with invading microorganisms or tumor cells.
Agents that deplete cells of K+ without grossly disrupting the plasma membrane were found to stimulate the cleavage of pro-interleukin (IL)-1 beta to mature IL-1 beta. Agents examined in this study included staphylococcal alpha-toxin and gramicidin, both of which selectively permeabilize plasma membranes for monovalent ions, the ionophores nigericin and valinomycin, and the Na+/K+ ATPase inhibitor ouabain. K+ depletion by brief hypotonic shock also triggered processing of pro-IL-1 beta. The central role of K+ depletion for inducing IL-1 beta maturation was demonstrated in cells permeabilized with alpha-toxin: processing of pro-IL-1 beta was totally blocked when cells were suspended in medium that contained high K+, but could be induced by replacing extracellular K+ with Na+, choline+ or sucrose. To test whether K+ flux might also be important in physiological situations, monocytes were stimulated with lipopolysaccharide (LPS) for 1-2 h to trigger pro-IL-1 beta synthesis, and transferred to K(+)-rich medium. This maneuver totally suppressed IL-1 beta maturation. Even after 16 h, however, removal of K+ from the medium resulted in rapid processing and export of IL-1 beta. Ongoing export of mature IL-1 beta from cells stimulated with LPS for 2-6 h could also be arrested by transfer to K(+)-rich medium. Moreover, a combination of two K+ channel blockers inhibited processing of IL-1 beta in LPS-stimulated monocytes. We hypothesize that K+ movement and local K+ concentrations directly or indirectly influence the action of interleukin-1 beta-converting enzyme (ICE) and, possibly, of related intracellular proteases.
Lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages produce large quantities of interleukin (IL)-1 beta but in the absence of a secondary stimulus little of this cytokine is proteolytically processed to its mature biologically active state and externalized. The potassium-proton ionophore nigericin and ATP are known to promote the maturation and release of IL-1 beta from LPS-stimulated cells. We investigated the mechanisms by which these agents act in an attempt to understand requirements of the post-translational processing. Like nigericin, the ionophores A204 and lasalocid induced the release and maturation of IL-1 beta. The electrogenic potassium ionophore valinomycin, however, did not stimulate these post-translational events. Addition of nigericin or lasalocid to LPS-stimulated cells produced a rapid intracellular acidification; A204, however, did not alter pH, indicating that an acidification was not necessary for activation of IL-1 beta maturation. Macrophages treated with ATP became rounded and swollen, and after 30 min of treatment their appearance was comparable with cells treated with nigericin. Post-translational maturation and release of IL-1 beta began immediately after ATP addition. The majority of the 17-kDa mature IL-1 beta produced within the first 30 min of treatment was recovered extracellularly; in contrast, during this same time period the 35-kDa IL-1 beta precursor and the cytoplasmic marker enzyme lactate dehydrogenase and the lysosomal enzyme beta-N-acetylglucosaminidase remained cell-associated. ATP, therefore, promoted both the proteolytic maturation of IL-1 beta and the release of the biologically active species in the absence of cell lysis. Longer incubations with ATP caused cytolysis as judged by the release of the cytoplasmic enzymes. ADP was less active than ATP at initiating the post-translational maturation and release of IL-1 beta and AMP, GTP, and UTP were totally inactive, ATP, nigericin, A204, and lasalocid promoted a rapid and complete loss of the potassium analog 86Rb+ from cells that were preloaded with this cation; valinomycin-treated cells released only a portion of the radiolabeled cation. Agents that promoted the maturation and release of IL-1 beta from LPS-stimulated macrophages, therefore, shared an ability to mobilize intracellular potassium. Macrophages treated with ATP or nigericin in medium that contained KCl rather than NaCl failed to proteolytically activate and to release IL-1 beta. These data suggest that ATP and nigericin induce a net decrease in intracellular levels of K+ which is necessary for activation of the post-translational maturation of IL-1 beta.
In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.
Life-threatening infections caused by the encapsulated fungal pathogen Cryptococcus neoformans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs. Intricate host-organism interactions make the full understanding of pathogenicity and virulence of C. neoformans difficult. We discuss the current knowledge of the characteristics C. neoformans must possess to enter the host and establish progressive disease: basic growth requirements and virulence factors, such as the polysaccharide capsule; shed products of the organism; melanin production; mannitol secretion; superoxide dismutase; proteases; and phospholipases.
Chronic granulomatous disease (CGD) is an inherited disorder of phagocyte function in which defective superoxide production results in deficient microbicidal activity. CGD patients suffer from recurrent, life-threatening infections, and nearly half develop chronic gastrointestinal (GI) complications (colitis, gastric outlet obstruction, or perirectal abscess) and/or autoimmune/rheumatologic disorders (AIDs). To identify genetic modifiers of disease severity, we studied a cohort of 129 CGD patients, in whom seven candidate genes (myeloperoxidase [MPO], mannose binding lectin [MBL], Fcgamma receptors IIa, IIIa, IIIb, TNF-alpha, and IL-1 receptor antagonist), each containing a physiologically relevant polymorphism predicted to influence the host inflammatory response, were selected for analysis. Genotypes of MPO (P = 0.003) and FcgammaRIIIb (P = 0.007) were strongly associated with an increased risk for GI complications, while an FcgammaRIIa (P = 0.05) genotype was suggestive for an association. Patients with all three associated genotypes had the highest risk for GI complications (P < 0.0001). The risk of AIDs was strongly associated with variant alleles of MBL (P = 0.01) and weakly associated with an FcgammaRIIa genotype (P = 0.04). Patients with variant forms of both MBL and FcgammaRIIa had the highest risk of developing an AID (P = 0.003).
We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1 beta by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1 beta and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1 beta processing was not due to perturbation of the export machinery for pro-IL-1 beta and IL-1 beta or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1 beta processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1 beta maturation.
Nod1 is an Apaf-1-like molecule composed of a caspase-recruitment domain (CARD), nucleotide-binding domain, and leucine-rich
repeats that associates with the CARD-containing kinase RICK and activates nuclear factor κB (NF-κB). We show that self-association
of Nod1 mediates proximity of RICK and the interaction of RICK with the γ subunit of the IκB kinase (IKKγ). Similarly, the
RICK-related kinase RIP associated via its intermediate region with IKKγ. A mutant form of IKKγ deficient in binding to IKKα
and IKKβ inhibited NF-κB activation induced by RICK or RIP. Enforced oligomerization of RICK or RIP as well as of IKKγ, IKKα,
or IKKβ was sufficient for induction of NF-κB activation. Thus, the proximity of RICK, RIP, and IKK complexes may play an
important role for NF-κB activation during Nod1 oligomerization or trimerization of the tumor necrosis factor α receptor.
BCL10/CLAP is an activator of apoptosis and NF-kappaB signaling pathways and has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue. Although its role in apoptosis remains to be determined, BCL10 likely activates NF-kappaB through the IKK complex in response to upstream stimuli. The N-terminal caspase recruitment domain (CARD) of BCL10 has been proposed to function as an activation domain that mediates homophilic interactions with an upstream CARD-containing NF-kappaB activator. To identify upstream signaling partners of BCL10, we performed a mammalian two-hybrid analysis and identified CARD9 as a novel CARD-containing protein that interacts selectively with the CARD activation domain of BCL10. When expressed in cells, CARD9 binds to BCL10 and activates NF-kappaB. Furthermore, endogenous CARD9 is found associated with BCL10 suggesting that both proteins form a pre-existing signaling complex within cells. CARD9 also self-associates and contains extensive coiled-coil motifs that may function as oligomerization domains. We propose here that CARD9 is an upstream activator of BCL10 and NF-kappaB signaling.
Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.
The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.
Oxygen metabolites generated bymyeloperoxidase (MPO) and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase contribute
to microbial killing by phagocytes. To compare the importance of the 2 enzymes for host defense, MPO-deficient (MPO−/−) mice and NADPH-oxidase-deficient mice with chronic granulomatous disease (CGD mice) were intraperitoneally infected with
3 different doses of Candida albicans, and their infection severity was analyzed. CGD mice had increased mortality and exhibited increased tissue fungal burden
in a dose-dependent manner, whereas normal mice showed no symptoms. Of interest, at the highest dose, the mortality of MPO−/− mice was comparable to that of CGD mice, but at the lowest dose, it was the same as that of normal mice. At the middle dose,
the number of fungi disseminated into various organs of the MPO−/− mice was comparable to that of the CGD mice at day 6 of infection, but it was significantly lower at day 14. These results
suggest that MPO and NADPH-oxidase are equally important for early host defense against a large inoculum of Candida.
Although the basis for the high mortality rate for patients with mixed bacterial infections is likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP) with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-alpha (TNF-alpha) secretion. Although incubation of cells with MDP alone produced minimal TNF-alpha, it caused significant expression of TNF-alpha mRNA. These findings suggest that the majority of TNF-alpha mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous incubation of cells with MDP and either LPS or PGN resulted in TNF-alpha mRNA expression that approximated the sum of the amounts expressed in response to MDP, LPS, and PGN individually. These findings indicate that the apparent synergistic effect of MDP on TNF-alpha production induced by either LPS or PGN is due to removal of a block in translation of the mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its effect on the production of TNF-alpha by LPS and PGN were determined to be independent of CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts through receptor(s) other than those primarily responsible for transducing the effects of LPS and PGN. Successful treatment of patients having mixed bacterial infections is likely to require interventions that address the mechanisms involved in responses induced by a variety of bacterial cell wall components.
Zymosan is a beta-glucan- and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a beta-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the beta-glucan receptor mediating this activity. To address the role of the recently described beta-glucan receptor, Dectin-1, we generated a novel anti-Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the beta-glucan-dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte beta-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of beta-glucans for therapeutic drug design.
We recently identified dectin-1 (betaGR) as a major beta-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate beta-glucans. Using a novel mAb (2A11) raised against betaGR, we show here that the receptor is not dendritic cell-restricted as first reported, but is broadly expressed, with highest surface expression on populations of myeloid cells (monocyte/macrophage (Mphi) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the betaGR, but at lower levels. Alveolar Mphi, like inflammatory Mphi, exhibited the highest surface expression of betaGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mphi expressed much lower levels of betaGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mphi suggested the existence of an additional beta-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mphi. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mphi mannose receptor and complement receptor 3 in this process. These observations imply the existence of an additional mannan-dependent receptor involved in the recognition of zymosan by resident peritoneal Mphi.
The mononuclear phagocyte system (MPS) was defined as a family of cells comprising bone marrow progenitors, blood monocytes, and tissue macrophages. In this review, we briefly consider markers for cells of this lineage in the mouse, especially the F4/80 surface antigen and the receptor for macrophage colony-stimulating factor. The concept of the MPS is challenged by evidence that there is a separate embryonic phagocyte lineage, the blurring of the boundaries between macrophages and other cells types arising from phenotypic plasticity and transdifferentiation, and evidence of local renewal of tissue macrophage populations as opposed to monocyte recruitment. Nevertheless, there is a unity to cells of the MPS suggested by their location, morphology, and shared markers. We discuss the origins of macrophage heterogeneity and argue that macrophages and antigen-representing dendritic cells are closely related and part of the MPS.
Generation of oxidative products by phagocytic cells is known to be an important host defense mechanism directed toward killing of invading microorganisms. The importance of two major oxidant-producing enzymes, myeloperoxidase (MPO) and NADPH-oxidase, in in vivo fungicidal action was directly compared in genetically engineered mice. Both MPO-deficient (MPO-/-) and NADPH-oxidase-deficient (X-linked chronic granulomatous disease [X-CGD]) mice showed increased susceptibility to pulmonary infections with Candida albicans and Aspergillus fumigatus compared with normal mice, and the X-CGD mice exhibited shorter survivals than MPO-/- mice. This increased mortality of X-CGD mice was associated with a 10- to 100-fold increased outgrowth of the fungi in their organs during the first 6 days. These results suggest that superoxide (O2-) produced by NADPH-oxidase is more important than hypochlorous acid (HOCl) produced by MPO, although both oxidative products obviously contribute to the host defense against pulmonary infection with those fungi. We also observed that MPO-/-/X-CGD double knockout mice showed comparable levels of susceptibility to the X-CGD mice against C. albicans and A. funigatus, indicating that MPO is unable to play a role in host defense in the absence of NADPH-oxidase. This strongly suggests that hydrogen peroxide, the precursor of HOCl, is solely derived from O2- produced by NADPH-oxidase.
Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.
The ability of fungal-derived beta-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-alpha, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-alpha in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-alpha in response to fungi, a critical step required for the successful control of these pathogens.
It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracycline-regulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.
The adaptive immune system can generate distinct classes of responses, but the mechanisms that determine this are poorly understood. In this study, we demonstrate that different Toll-like receptor (TLR) ligands induce distinct dendritic cell (DC) activation and immune responses in vivo. Thus, Escherichia coli LPS (TLR-4 stimulus), activates DCs to produce abundant IL-12(p70), but little IL-10, and stimulates Th1 and Tc1 responses. In contrast, Pam-3-cys (TLR-2 stimulus) elicits less IL-12(p70), but abundant IL-10, and favors Th2 and T cytotoxic 2 (Tc2) responses. These distinct responses likely occur via differences in extracellular signal-regulated kinase signaling in DCs. Thus, Pam-3-cys induces enhanced extracellular signal-regulated kinase signaling, compared with LPS, resulting in suppressed IL-12(p70) and enhanced IL-10 production, as well as enhanced induction of the transcription factor, c-Fos. Interestingly, DCs from c-fos(-/-) mice produce more IL-12(p70), but less IL-10, compared with control DCs. Therefore, different TLR ligands induce distinct cytokines and signaling in DCs, and differentially bias Th responses in vivo.
In higher metazoans, phagocytosis is essential in host defense against microbial pathogens and in clearance of apoptotic cells. Both microbial and apoptotic cells are delivered on a common route from phagosomes to lysosomes for degradation. Here, we found that activation of the Toll-like receptor (TLR) signaling pathway by bacteria, but not apoptotic cells, regulated phagocytosis at multiple steps including internalization and phagosome maturation. Phagocytosis of bacteria was impaired in the absence of TLR signaling. Two modes of phagosome maturation were observed, constitutive and inducible; their differential engagement depended on the ability of the cargo to trigger TLR signaling.
Blocking the activity of IL-1 beta has entered the clinical arena of treating autoimmune diseases. However, a successful outcome of this approach requires a clear definition of the mechanisms controlling IL-1 beta release. These are still unclear as IL-1 beta, lacking a secretory signal peptide, follows a nonclassical pathway of secretion. Here, we analyze the molecular mechanism(s) undergoing IL-1 beta processing and release in human monocytes and provide a unifying model for the regulated secretion of the cytokine. Our data show that in a first step, pro-caspase-1 and endotoxin-induced pro-IL-1 beta are targeted in part to specialized secretory lysosomes, where they colocalize with other lysosomal proteins. Externalization of mature IL-1 beta and caspase-1 together with lysosomal proteins is then facilitated by extracellular ATP. ATP triggers the efflux of K(+) from the cell, followed by Ca(2+) influx and activation of three phospholipases: phosphatidylcholine-specific phospholipase C and calcium-independent and -dependent phospholipase A(2). Whereas calcium-independent phospholipase A(2) is involved in processing, phosphatidylcholine-specific phospholipase C and calcium-dependent phospholipase A(2) are required for secretion. Dissection of the events that follow ATP triggering allowed to demonstrate that K(+) efflux is responsible for phosphatidylcholine-specific phospholipase C induction, which in turn allows the rise in intracellular free calcium concentration required for activation of phospholipase A(2). This activation is ultimately responsible for lysosome exocytosis and IL-1 beta secretion.
The phagocytosis of pathogens is a critical event in host defense, not only for clearance of the invading microorganism, but also for the subsequent immune response. We have examined Dectin-1, a proinflammatory nonopsonic receptor for beta-glucans, and show that it mediates the internalization of beta-glucan-bearing ligands, including yeast particles. Although requiring tyrosine phosphorylation and the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like motif, uptake mediated by Dectin-1 was different from any previously reported phagocytic receptor and was not dependent on Syk-kinase in macrophages. Furthermore, intracellular trafficking of this receptor was influenced by the nature of the beta-glucan ligand, which has significance for the biologic activity of these immunomodulatory carbohydrates.
-Glucan is one of the most abundant polysaccharides in fungal pathogens, yet its importance in antifungal immunity is unclear. Here we show that deficiency of dectin-1, the myeloid receptor for -glucan, rendered mice susceptible to infection with Candida albicans. Dectin-1-deficient leukocytes demonstrated significantly impaired responses to fungi even in the presence of opsonins. Impaired leukocyte responses were manifested in vivo by reduced inflammatory cell recruitment after fungal infection, resulting in substantially increased fungal burdens and enhanced fungal dissemination. Our results establish a fundamental function for -glucan recognition by dectin-1 in antifungal immunity and demonstrate a signaling non–Toll-like pattern-recognition receptor required for the induction of protective immune responses.
Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide-binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro-inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc-L-Ala-γ-D-Glu-meso-diaminopimelic acid, MtriDAP) and NOD2 (MurNAc-L-Ala-D-isoGln, MDP; MurNAc-L-Ala-γ-D-Glu-L-Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro-inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro- and anti-inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub-active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll-like receptor 4 to instruct the onset of adaptive immune responses.
Mammalian Toll-like receptors (TLRs) play a critical role in detection of invading pathogens as well as triggering of subsequent
inflammatory and immune responses. Each TLR recognizes distinct microbial components and activates different signaling pathways
by selective utilization of adaptor molecules. The signaling via TLRs is delivered from the cell surface and/or the endosome.
Recently, the intracytoplasmic detection system of microbes has been identified in mammals as well. Peptidoglycan breakdown
products and double-stranded RNA are sensed by NOD family and RNA helicase domain containing proteins, respectively. Thus,
mammals make use of both receptor-type and intracellular proteins as detectors of invading pathogens.
The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably “gain of function” to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.
We described elsewhere that the synergistic antitumor activity of endotoxic extracts from Re mutants of gram-negative bacteria and trehalose mycolate against guinea pig syngeneic line 10 tumor was abrogated after peptide substances accompanying these extracts had been removed. This activity could be restored by combining peptide-free endotoxin either with cell wall skeleton from Bacillus Calmette-Guérin, a polymeric mycolic acid-arabinogalactan-mucopeptide complex, or with a combination of two separate components, trehalose dimycolate and N-acetylmuramyl-L-alanyl-(L-seryl)-D-isoglutamine (MDP). We report here that when a combination of endotoxin (150 microgram) and a mixture of MDP (150 microgram) and trehalose dimycolate (150 microgram) was inoculated into established dermal tumors, a significant number of the animals died, presumably of endotoxic shock. All surviving animals suffered severe but temporary lethargy. When administered alone intradermally in the dose levels tested, none of the components caused severe lethargy or lethality. The lethal effects of 159 microgram of MDP also occurred in combination with relatively weak endotoxic products, such as Pseudomonas vaccine (Pseudogen), and these effects did not depend upon the presence of malignant tissue. Guinea pigs inoculated i.v. were even more susceptible inasmuch as the addition of as little as 6 microgram of MDP to 150 microgram of Pseudogen, itself not lethal, caused the death of 80% of the animals.
The neutrophils and monocytes of a patient with disseminated candidiasis were found to lack detectable levels of the lysosomal enzyme myeloperoxidase (MPO), although they had normal levels of other granule-associated enzymes. Leukocytes from one of the patient's sisters also lacked detectable MPO; leukocytes from his four sons contained approximately one-third of mean normal peroxidase levels. Neither the patient nor his affected relatives had experienced frequent or unusual bacterial infections. The phagocytic activity of the patient's MPO-deficient neutrophils was intact, and the cells displayed normal morphologic and metabolic responses to phagocytosis. In contrast to normal leukocytes which killed 30.5+/-7.3% of ingested Candida albicans in 1 hr, however, the patient's neutrophils killed virtually none. His leukocytes also failed to kill the strain of C. albicans recovered from his lesions, as well as other Candida species. These MPO-deficient neutrophils killed Serratia marcescens and Staphylococens aureus 502A at an abnormally slow rate, requiring 3-4 hr to achieve the bactericidal effect attained by normal leukocytes after 45 min. No other abnormalities in his cellular or humoral immune responses were demonstrated. These findings suggest that hereditary MPO deficiency is transmitted as an autosomal recessive characteristic, that the homozygous state conveys enhanced susceptibility to disseminated candidiasis, and that MPO is necessary for candidacidal activity in human neutrophils. Although lending support to the suggested bactericidal role of MPO in leukocytes, the data indicate that alternative bactericidal mechanisms, effective in the absence of MPO, are functionally dominant in the human neutrophil.
Candida albicans and Saccharomyces cerevisiae switch from a yeast to a filamentous form. In Saccharomyces, this switch is controlled by two regulatory proteins, Ste12p and Phd1p. Single-mutant strains, ste12/ste12 or phd1/phd1, are partially defective, whereas the ste12/ste12 phd1/phd1 double mutant is completely defective in filamentous growth and is noninvasive. The equivalent cph1/cph1 efg1/efg1 double mutant in Candida (Cph1p is the Ste12p homolog and Efg1p is the Phd1p homolog) is also defective in filamentous growth, unable to form hyphae or pseudohyphae in response to many stimuli, including serum or macrophages. This Candida cph1/cph1 efg1/efg1 double mutant, locked in the yeast form, is avirulent in a mouse model.
Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.
Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.
Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.
DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.
Candidiasis is a common infection of the skin, oral cavity and esophagus, gastrointestinal tract, vagina and vascular system of humans. Although most infections occur in patients who are immunocompromised or debilitated in some other way, the organism most often responsible for disease, Candida albicans, expresses several virulence factors that contribute to pathogenesis. These factors include host recognition biomolecules (adhesins), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), secreted aspartyl proteases and phospholipases. Additionally, 'phenotypic switching' is accompanied by changes in antigen expression, colony morphology and tissue affinities in C. albicans and several other Candida spp. Switching might provide cells with a flexibility that results in the adaptation of the organism to the hostile conditions imposed not only by the host but also by the physician treating the infection.
The carbohydrate polymers known as beta-1,3-d-glucans exert potent effects on the immune system - stimulating antitumour and antimicrobial activity, for example - by binding to receptors on macrophages and other white blood cells and activating them. Although beta-glucans are known to bind to receptors, such as complement receptor 3 (ref. 1), there is evidence that another beta-glucan receptor is present on macrophages. Here we identify this unknown receptor as dectin-1 (ref. 2), a finding that provides new insights into the innate immune recognition of beta-glucans.
Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-kappaB, whereas dominant negative Rip2 inhibited NF-kappaB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-gamma production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.
We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.
Host recognition of bacterial pathogens is a critical component of the immune response. Intracellular bacterial pathogens are able to evade the humoral immune system by residing within the host cell. Here we show the existence of an innate host surveillance mechanism in macrophages that specifically distinguishes bacteria in the cytosol from bacteria in the vacuole. Recognition of Gram-positive and Gram-negative bacterial products by this surveillance system results in transcription of the ifnb gene. The activation of cytosol-specific signaling is associated with translocation of NF-kappaB into the nucleus and phosphorylation of the p38 mitogen-activated protein (MAP) kinase. Activation of the p38 kinase is required for the induction of gene expression by the cytosolic surveillance pathway. Our studies suggest that infection by intracellular bacterial pathogens results in an immune response distinct from that of infection by extracellular bacterial pathogens.
The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.
The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.
Caspases not only play an essential role during apoptotic cell death, but a subfamily of them-the inflammatory caspases-are associated with immune responses to microbial pathogens. Activation of inflammatory caspases, such as caspase-1 and caspase-5, occurs upon assembly of an intracellular complex, designated the inflammasome. This results in the cleavage and activation of the proinflammatory cytokines IL-1beta and IL-18. Mutations in one of the scaffold proteins of the inflammasome, NALP3/Cryopyrin, are associated with autoinflammatory disorders underscoring the importance of regulating inflammatory caspase activation.
Innate and adaptive immunity depends critically on host recognition of pathogen-associated molecules. Toll-like receptors (TLRs) are key mediators of pathogen surveillance at the cell or phagocytic vacuole surface. However, mechanisms underlying recognition of pathogens in other cellular compartments remain unclear, and responses elicited by cytosolic challenge are poorly characterized. We therefore used mouse cDNA microarrays to investigate gene expression triggered by infection of bone marrow-derived macrophages with cytosol- and vacuole-localized Listeria monocytogenes (Lm), a model cytosolic pathogen. The resulting gene expression program included two basic categories of induced genes: an "early/persistent" cluster consistent with NF-kappaB-dependent responses downstream of TLRs, and a subsequent "late response" cluster largely composed of IFN-responsive genes (IRGs). The early/persistent cluster was observed upon infection with WT, heat-killed, or mutant Lm lacking listeriolysin O, the pore-forming hemolysin that promotes escape from phagocytic vacuoles. However, the IRG cluster depended on entry of WT Lm into the cytosol. Infection with listeriolysin O-expressing, cytosolic Bacillus subtilis (Bs) strikingly recapitulated the expression profile associated with WT Lm, including IRG induction. IRG up-regulation was associated with MyD88-independent induction of IFN-beta transcription and activity. Whereas Staphylococcus aureus (Sa) lipoteichoic acid treatment confirmed that many late-response genes could also be stimulated through TLRs, our study identified a cytosol-specific transcriptional program independent of TLR signaling through MyD88. Further characterization of cytosolic surveillance pathway(s) and their points of convergence with TLR- and IFN-dependent pathways will enhance our understanding of the means by which mammals detect and respond to pathogens.
Activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokine IL-1beta is triggered upon assembly of the inflammasome complex. It is generally believed that bacterial lipopolysaccharides (LPS) are activators of the inflammasome through stimulation of Toll-like receptor 4 (TLR4). Like TLRs, NALP3/Cryopyrin, which is a key component of the inflammasome, contains Leucine-Rich-Repeats (LRRs). LRRs are frequently used to sense bacterial components, thus raising the possibility that bacteria directly activate the inflammasome. Here, we show that bacterial peptidoglycans (PGN), but surprisingly not LPS, induce NALP3-mediated activation of caspase-1 and maturation of proIL-1beta. Activation is independent of TLRs because the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs, is the minimal-activating structure. Macrophages from a patient with Muckle-Wells syndrome, an autoinflammatory disease associated with mutations in the NALP3/Cryopyrin gene, show increased IL-1beta secretion in the presence of MDP. The activation of the NALP3-inflammasome by MDP may be the basis of the potent adjuvant activity of MDP.