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A universal role for Myd88 in TLR/IL-1R-mediated signaling
Abstract
The MyD88 adapter protein links members of the toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of nuclear factor-kappaB and mitogen-activated protein kinases. Although originally identified as a myeloid-differentiation marker, MyD88 is now known to play an essential role in the innate immune response of insects and mammals. The generation of MyD88-deficient mice, as well as the identification of MyD88-related proteins and regulators of MyD88 signaling, has revealed new and important insights into the function of MyD88.
... Structurally, MyD88 is characterized with a N-terminal death domain and a C-terminal TIR domain (Akira and Takeda, 2004;Janssens and Beyaert, 2002). Death domain of MyD88 mediates the interaction with several members of interleukin-1 receptorassociated kinases (IRAKs), and its cytoplasmic TIR domain associates with other receptors or adaptors through homotypic domain interactions (Akira and Takeda, 2004;Chen et al., 2020;Janssens and Beyaert, 2002). ...
... Structurally, MyD88 is characterized with a N-terminal death domain and a C-terminal TIR domain (Akira and Takeda, 2004;Janssens and Beyaert, 2002). Death domain of MyD88 mediates the interaction with several members of interleukin-1 receptorassociated kinases (IRAKs), and its cytoplasmic TIR domain associates with other receptors or adaptors through homotypic domain interactions (Akira and Takeda, 2004;Chen et al., 2020;Janssens and Beyaert, 2002). Functionally, MyD88 is a central node linking TLR family members to IRAK family kinases. ...
... Functionally, MyD88 is a central node linking TLR family members to IRAK family kinases. Upon TLR stimulation, MyD88 interacts with IRAK4 and subsequently recruits IRAK1, leading to the activation of transcription factor nuclear factor-kappa B (NF-κB), which triggers the expression of proinflammatory cytokines (Akira and Takeda, 2004;Chen et al., 2020;Janssens and Beyaert, 2002). ...
As a universal adaptor used by most TLR members, the myeloid differentiation factor 88 (MyD88) plays essential roles in TLR-mediated inflammatory response of invertebrate and vertebrate animals, and functional features of MyD88 remain largely unknown in amphibians. In this study, a MyD88 gene named Xt-MyD88 was characterized in the Western clawed frog (Xenopus tropicalis). Xt-MyD88 and MyD88 in other species of vertebrates share similar structural characteristics, genomic structures, and flanking genes, suggesting that MyD88 is structurally conserved in different phyla of vertebrates ranging from fish to mammals. Moreover, Xt-MyD88 was widely expressed in different organs/tissues, and was induced by poly(I:C) in spleen, kidney, and liver. Importantly, over-expression of Xt-MyD88 triggered a marked activation of both NF-κB promoter and interferon-stimulated response elements (ISREs), implying that it may be play important roles in inflammatory responses of amphibians. The research represents the first characterization on the immune functions of amphibian MyD88, and reveals considerable functional conservation of MyD88 in early tetrapods.
... As a universal adaptor molecule that interacts with the cytoplasmic domains of toll-like receptors (TLRs) and IL-1Rs, MyD88 activates the signal transduction cascades of inflammatory signaling pathways (26,27). MyD88 is expressed ubiquitously in many types of tissues and consists of three main domains: an Nterminal death domain (DD), an intermediate domain (ID), and a C-terminal toll-interleukin-1 receptor (TIR) domain (27,28). ...
... The TIR domain interacts with other TIR domain-containing proteins, such as TLRs and IL-1Rs, while the DD interacts with IL-1Rassociated kinase 1 and 4 (IRAK1 and IRAK4) through homotypic DD interactions (27). The interaction between MyD88 and IRAK1/4 induces IRAK4-mediated IRAK1 phosphorylation, which in turn triggers activation of inflammatory signaling molecules in the NF-kB inflammatory signaling pathway (26)(27)(28)(29)(30). ...
... A number of studies have demonstrated that MyD88 plays different roles in multiple biological functions, including innate immune responses, carcinogenesis, autophagy, immune activation, learning activity, anxiety, and motor functions (26,(31)(32)(33)(34)(35)(36)(37). ...
Background
Inflammation, a vital immune response to infection and injury, is mediated by macrophage activation. While spleen tyrosine kinase (Syk) and myeloid differentiation primary response 88 (MyD88) are reportedly involved in inflammatory responses in macrophages, their roles and underlying mechanisms are largely unknown.
Methods
Here, the role of the MyD88-Syk axis and the mechanism by which Syk and MyD88 cooperate during macrophage-mediated inflammatory responses are explored using knockout conditions of these proteins and mutation strategy as well as flowcytometric and immunoblotting analyses.
Results
Syk rapidly activates the nuclear factor-kappa B (NF-κB) signaling pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the activation of the NF-κB signaling pathway is abolished in Syk−/− RAW264.7 cells. MyD88 activates Syk and Syk-induced activation of NF-κB signaling pathway in LPS-stimulated RAW264.7 cells but Syk-induced inflammatory responses are significantly inhibited in MyD88−/− RAW264.7 cells. MyD88 interacts with Syk through the tyrosine 58 residue (Y58) in the hemi-immunoreceptor tyrosine-based activation motif (ITAM) of MyD88, leading to Syk activation and Syk-induced activation of the NF-κB signaling pathway. Src activates MyD88 by phosphorylation at Y58 via the Src kinase domain. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) activation and Rac1-induced formation of filamentous actin (F actin) activate Src in LPS-stimulated RAW264.7 cells.
Conclusions
These results suggest that the MyD88-Syk axis is a critical player in macrophage-mediated inflammatory responses, and its function is promoted by an upstream Src kinase activated by Rac1-generated filamentous actin (F-actin).
... From the comparison, several genes with relatively substantial elevations in common for both groups (i.e., Wald-test statistics > 6.0) were shown: (i) innate immunity: Ifih1, Ifit2, Oas3, Parp9, Stat1, Stat2, Herc6, Parp14, Parp9, Znfx1, Casp1, and Tap1; (ii) adaptive immunity: Tap1 and Ccr1; (iii) mitochondrial innate immunity: Casp1, Myd88, Ifih1, and Znfx1; (iv) RAAS: Mlkl; (v) ISR: Nfe2l2 and Glrx. Among the gene list, some were reported in previous studies as follows: (i) Ifih1 was related to viral resistance in children [20]; (ii) Ifit2 was involved in resistance to viral infection such as influenza virus [21]; (iii) Znfx1 was associated with susceptibility to viral infections [22]; (iv) transcription of Tap1 could be rapidly upregulated in response to pro-inflammatory cytokines such as type-I IFN, IFN-γ, and TNF-α [23]; (v) Ccr1 could contribute to viral infection by activating inflammation in infection situations [24]; (vi) Myd88 was associated with IL-1 signaling [25]. ...
... Gene expressions associated with antiviral activity, including Znfx1, Adar, Ifi44, Ifih1, Ifit2, Ifitm3, and Isg20 [20][21][22]26,[45][46][47][48] were increased in common. Additionally, expressions of Myd88, which is involved in interleukin expression [25,49], and Irf7, implicated in IFN expression [50], were both increased. ...
The LPS-induced inflammation model is widely used for studying inflammatory processes due to its cost-effectiveness, reproducibility, and faithful representation of key hallmarks. While researchers often validate this model using clinical cytokine markers, a comprehensive understanding of gene regulatory mechanisms requires extending investigation beyond these hallmarks. Our study leveraged multiple whole-blood bulk RNA-seq datasets to rigorously compare the transcriptional profiles of the well-established LPS-induced inflammation model with those of several human diseases characterized by systemic inflammation. Beyond conventional inflammation-associated systems, we explored additional systems indirectly associated with inflammatory responses (i.e., ISR, RAAS, and UPR) using a customized core inflammatory gene list. Our cross-condition-validation approach spanned four distinct conditions: systemic lupus erythematosus (SLE) patients, dengue infection, candidemia infection, and staphylococcus aureus exposure. This analysis approach, utilizing the core gene list aimed to assess the model’s suitability for understanding the gene regulatory mechanisms underlying inflammatory processes triggered by diverse factors. Our analysis resulted in elevated expressions of innate immune-associated genes, coinciding with suppressed expressions of adaptive immune-associated genes. Also, upregulation of genes associated with cellular stresses and mitochondrial innate immune responses underscored oxidative stress as a central driver of the corresponding inflammatory processes in both the LPS-induced and other inflammatory contexts.
... This protein is associated with myeloid differentiation. Simultaneously, the numerical value "88" denotes the position of the activated gene within a list of five adaptors that interact with downstream signaling proteins associated with IL-1 and TLRs [62,63]. Type I transmembrane receptors known as TLRs possess external leucine-rich repeats, a transmembrane helix, and an internal Toll/IL-1 receptor domain (TIR) [63]. ...
... Simultaneously, the numerical value "88" denotes the position of the activated gene within a list of five adaptors that interact with downstream signaling proteins associated with IL-1 and TLRs [62,63]. Type I transmembrane receptors known as TLRs possess external leucine-rich repeats, a transmembrane helix, and an internal Toll/IL-1 receptor domain (TIR) [63]. TLRs are a subset of pattern recognition receptors (PRRs) that are typically triggered by pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular pattern molecules (DAMPs). ...
Innate lymphocytes, including microglial cells, astrocytes, and oligodendrocytes, play a crucial role in initiating neuroinflammatory reactions inside the central nervous system (CNS). The prime focus of this paper is on the involvement and interplay of neurons and glial cells in neurological disorders such as Alzheimer's Disease (AD), Autism Spectrum Disorder (ASD), epilepsy, and multiple sclerosis (MS). In this review, we explore the specific contributions of microglia and astrocytes and analyzes multiple pathways implicated in neuroinflammation and disturbances in excitatory and inhibitory processes. Firstly, we elucidate the mechanisms through which toxic protein accumulation in AD results in synaptic dysfunction and deregulation of the immune system and examines the roles of microglia, astrocytes, and hereditary factors in the pathogenesis of the disease. Secondly, we focus on ASD and the involvement of glial cells in the development of the nervous system and the formation of connections between neurons and investigates the genetic connections associated with these processes. Lastly, we also address the participation of glial cells in epilepsy and MS, providing insights into their pivotal functions in both conditions. We also tried to give an overview of seven different pathways like toll-like receptor signalling pathway, MyD88-dependent and independent pathway, etc and its relevance in the context with these neurological disorders. In this review, we also explore the role of activated glial cells in AD, ASD, epilepsy, and MS which lead to neuroinflammation. Even we focus on excitatory and inhibitory imbalance in all four neurological disorders as imbalance affect the proper functioning of neuronal circuits. Finally, this review concludes that there is necessity for additional investigation on glial cells and their involvement in neurological illnesses. Keywords: microglia; astrocytes; Alzheimer's Disease; autism spectrum disorder; epilepsy; multiple sclerosis; neuroinflammation; excitatory and inhibitory imbalance
... Previous data collected from other mollusc species showed hemocytes accumulation in the site of infection in order to phagocyte pathogens [12][13][14][15]36,60]. Indeed, they recognize, and subsequently engulf, pathogens through the specific membrane receptors TLRs [61,62]. Among different TLRs found throughout the animal kingdom [19,63] here we focused our attention on TLR4. ...
... Among different TLRs found throughout the animal kingdom [19,63] here we focused our attention on TLR4. This receptor is known to recognize components of Gram-negative bacterial membrane and, through the Myd88 pathways, to trigger several intracellular signalling events, including the production of pro-inflammatory cytokines [61,64,65] responsible of innate immune response orchestration. ...
Filter-feeding bivalves, such as the Mytilus species, are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. Mussels lack an adaptive immune system and hemocytes can recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to activate intracellular signaling pathways to trigger the antimicrobial effectors synthesis.
Among the areas of bivalve immunity that deserve study include the role of hemocyte subpopulations.
Since little information are available on immune responses at the tissue level to human pathogenic vibrios commonly detected in coastal waters involved in seafood-borne diseases, in this work, immunological parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vivo challenge with Vibrio splendidus.
The histological approach has been first used in order to identify the hemocytes recruitment at the infection site and the morphological change of muscular fibers.
In addition, using immunolabeling with specific antibody we detected the production of molecules involved in the inflammatory activated cascade: Toll-like receptors 4 (TLR4), the myeloid differentiation factor 88 (MyD88), the Allograft inflammatory factor-1 (AIF-1) and the ribonucleases RNASET2, belonging to the T2 family, that in vertebrates are involved in the recruitment and activation of macrophages.
Our results indicate the activation of TLR4 during bacterial infection preparatory to the recruitment of the MyD88 adapter with a putative role in recognition and intracellular signalling.
Furthermore, the data presented in this work suggest that challenging with Gram-negative bacteria causes a massive migration of AIF-1⁺ hemocytes and that the ribonuclease RNASET2 could play a key role in the recruitment of these activated hemocytes.
Our approach is useful for further understanding the complex molecular defence mechanisms of the host in invertebrates, especially in relation to the need to develop methods to evaluate the immunological response of bivalve molluscs used in aquaculture.
... As a pivotal component of the innate immune system, MyD88 potentially participates in regulating the canonical TLR dependent activation of nuclear factor-kB and mitogenactivated protein kinases, which plays an important role in the host defense response against microbes (34). To date, MyD88s have been reported in a variety of aquatic animals (24,25,27); however, there is no experimental evidence regarding the presence of MyD88 in A. woodiana, one of the most important farmed freshwater shellfish in China. ...
... Sequence analysis showed that AwMyD88 contained a typical DD domain and a TIR domain, which is similar to other reported MyD88 proteins (21). Previous studies have demonstrated that the C-terminal TIR domain of MyD88 is responsible for its activation through TIR-TIR domain interactions with upstream TLRs (34,35). It was shown that MyD88's N-terminal death domain is essential for the recruitment and activation of downstream IRAK kinases to trigger immune response during pathogen challenge (35,36). ...
Myeloid differentiation factor 88 (MyD88) is a key adapter molecule in Toll-like receptor signal transduction that triggers downstream immune cascades involved in the host defense response to exogenous pathogens. However, the function of MyD88s in mollusks, especially in freshwater shellfish, remains poorly understood. In this study, a novel freshwater shellfish MyD88 (denoted AwMyD88) was characterized from Anodonta woodiana. The present AwMyD88 protein consists of 474 amino acids and contains a conserved a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain with three typical boxes. Quantitative real-time PCR (qRT-PCR) analysis showed that AwMyD88 was broadly expressed in all the examined tissues, and the highest expression level was observed in hemocytes of A. woodiana. When challenged with Aeromonas hydrophila and lipopolysaccharide (LPS), the mRNA expression levels of AwMyD88 were significantly induced in hemocytes of A. woodiana in vivo and in vitro. In addition, in vivo injection experiments revealed that MyD88 signaling pathway genes showed strong responsiveness to A. hydrophila challenge, and their expression levels were significantly upregulated in hemocytes. Knockdown of AwMyD88 reduced the transcript levels of immune related transcription factors (AwNF-κB and AwAP-1) and effectors (AwTNF, AwLYZ, AwDefense and AwAIF) during A. hydrophila infection. Moreover, subcellular localization analysis indicated that AwMyD88 was mainly localized to the cytoplasm in HEK293T cells. Finally, luciferase reporter assays revealed that AwMyD88 associates with AwTLR to activate the NF-κB and AP-1 signaling pathways in HEK293T cells. These results suggested that AwMyD88 might be involved in the host defense response to bacterial challenge, providing new insight into the immune function of the MyD88 signaling pathway in freshwater shellfish.
... One of the indirectly TRPV4-interacting proteins identified was Flightless 1 (Fli1), which binds to Myd88 48 and which in turn binds to activated IL-1R1. 49 To assess a possible role for Fli1 as a functional link between IL-1R1 and TRPV4, we immunoprecipitated TRPV4 and immunoblotted these immunoprecipitates for Fli1, but there was no enrichment of Fli1 ( Figure S1). We also stimulated Fli1-WT and knockdown (KND) fibroblasts 50 with IL-1β and measured ERK activation and [Ca 2+ ] i but neither of these measures were affected by Fli1 knockdown (Figure S2). ...
Ca²⁺ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL‐1β binding to its signaling receptor (IL‐1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca²⁺ flux across the plasma membrane. It is not known how IL‐1R1, which does not conduct Ca²⁺, generates Ca²⁺ signals in response to IL‐1. We examined whether TRPV4 mediates the Ca²⁺ fluxes required for ERK signaling in IL‐1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL‐1β showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL‐1 strongly increased [Ca²⁺]i. Pre‐incubation of cells with IL‐1 Receptor Antagonist blocked Ca²⁺ entry induced by IL‐1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non‐Ca²⁺‐conducting TRPV4 pore‐mutant or pre‐incubation with the TRPV4 inhibitor RN1734, blocked IL‐1‐induced Ca²⁺ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca²⁺ and ERK responses induced by IL‐1; these responses were absent in TRPV4‐null cells or cells expressing a non‐conducting TRPV4 pore‐mutant. Immunostained IL‐1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL‐1R1, TRPV4 associated with Src‐related proteins and Src co‐immunoprecipitated with TRPV4. Src inhibition reduced IL‐1‐induced Ca²⁺ responses. The functional linkage of TRPV4 with IL‐1R1 expands its repertoire of innate immune signaling processes by mediating IL‐1‐driven Ca²⁺ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.
... Interestingly, however, fibrogenesis may occur independently of inflammation and innate immune response. Re and co-workers determined lung response to silica in knockout mice for myeloid differentiation primary response gene 88 (MyD88), which links members of the toll-like receptor (TLR), nucleotide-binding oligomerization domain receptor (NLR), and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of NF-kappa B and mitogen-activated protein kinases [69]. They showed that MyD88 is crucial for the development of inflammation and granulomas following silica exposure, whereas pulmonary fibrosis in MyD88 −/− mice was associated with the accumulation of profibrotic regulatory T lymphocytes, IL-17-producing Th17 lymphocytes, and profibrotic cytokines such as TGF-β, IL-10, and PDGF-B) [68]. ...
Idiopathic pulmonary fibrosis (IPF), the most common and severe of the idiopathic interstitial pneumonias, is a chronic and relentlessly progressive disease, which occurs mostly in middle-aged and elderly males. Although IPF is by definition “idiopathic”, multiple factors have been reported to increase disease risk, aging being the most prominent one. Several occupational and environmental exposures, including metal dust, wood dust and air pollution, as well as various lifestyle variables, including smoking and diet, have also been associated with an increased risk of IPF, probably through interaction with genetic factors. Many of the predisposing factors appear to act also as trigger for acute exacerbations of the disease, which herald a poor prognosis. The more recent literature on inhalation injuries has focused on the first responders in the World Trade Center attacks and military exposure. In this review, we present an overview of the environmental and occupational causes of IPF and its pathogenesis. While our list is not comprehensive, we have selected specific exposures to highlight based on their overall disease burden.
... Under heat stress conditions, the expression levels of IL-1β, IL-6, TNF-α, NFKBIA, and MyD88 in the intestine of Xueshan chickens can be assessed through qPCR to provide an evaluation of intestinal immune characteristics [31][32][33][34][35]. These genes are important molecules closely related to immune response and inflammation regulation. ...
Simple Summary
The intestinal tract is an important part of the immune system and forms a congenital barrier against food antigens and pathogenic microorganisms. In poultry, heat stress can induce intestinal mucosal injury, damage intestinal tight junctions and microvillous structures, and trigger an inflammatory response and enterogenic infection. As an essential trace element, zinc has been shown to mitigate the adverse effects of heat stress on broilers. However, how the dietary supplementation of different sources and levels of zinc can improve the heat stress capacity of Chinese landraces remains unclear. Here, we investigated the effects of different levels of zinc sulfate (ZnS) and zinc proteinate with moderate chelation strength (Zn-Prot M) on the intestinal immune function under heat stress in Xueshan chickens, which comprise an important local breed in China.
Abstract
Heat stress can cause intestinal inflammation, impaired barrier integrity, and decreased immunity in poultry. While zinc is known to mitigate the adverse effects of heat stress, how the dietary supplementation of different sources and levels of it can improve the heat stress capacity of Chinese landraces remains unclear. This study investigated Xueshan chickens, which are an important local breed in China. The effects of different levels of ZnS and Zn-Prot M on their intestinal immune function under heat stress were compared. We found that different levels of ZnS and Zn-Prot M could effectively reduce the secretion level of IL-6 in the serum, and 60 mg/kg was optimal. Compared with ZnS, Zn-Prot M significantly increased duodenal villus height and VH/CD ratio. ZnS and Zn-Prot M effectively increased the villus height and villus width in the jejunum and ileum at 74 and 88 days old, with the 60 and 90 mg/kg groups outperforming other groups, and Zn-Prot M was more effective than ZnS. Both ZnS and Zn-Prot M significantly down-regulated TNF-α, IL-1β, and MyD88 in 102-day-old duodenum, and IL-1β, IL-6, and NFKBIA in jejunum and ileum at 74, 88, and 102 days old, with 60 mg/kg Zn-Prot M determined as optimal. In conclusion, our study demonstrates that Zn-Prot M is superior to ZnS in improving intestinal immunity in Xueshan chickens, and 60 mg/kg is the optimal addition dose.
... MyD88 signaling activates downstream molecules such as mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-B). [59,60] To determine whether TLR4 and its downstream molecule MyD88 are required for the immunoactivation effects of paclitaxel and LPS, the authors used primary macrophages from TLR4-deficient or MyD88-knockout mice. Macrophages from wild-type mice were shown to secrete tumor necrosis factor (TNF) and nitric oxide (NO) after treatment with paclitaxel, whereas macrophages from TLR4-deficient or MyD88-knockout mice produced significantly lower levels of these two soluble factors. ...
The immune system plays a crucial role in recognizing and eliminating pathogenic substances and malignant cells in the body. For cancer treatment, immunotherapy is becoming the standard treatment for many types of cancer and is often combined with chemotherapy. Although chemotherapeutic agents are often reported to have adverse effects, including immunosuppression, they can also play a positive role in immunotherapy by directly stimulating the immune system. This has been demonstrated in preclinical and clinical studies in the past decades. Chemotherapeutics can activate immune cells through different immune receptors and signaling pathways, depending on their chemical structure and formulation. In this review, the direct immunoactivation effects of chemotherapeutics and the possible mechanisms behind these effects are summarized and discussed. Finally, chemo‐immunotherapeutic combinations are prospected for the more effective and safer treatment of cancer.
... TIRAP is a sorting receptor that recruits MyD88, a signaling adaptor that bridges the activated response to a signaling cascade [47]. Most TLRs recruit MyD88 as a universal adapter for signaling, whereas TLR3 recruits another adapter protein, TRIF, to signaling pathway [48]. MyD88 interacts with members of the IL-1R-associated kinase (IRAK) family, including IRAK1, IRAK2, and IRAK4 [49,50], leading to the assembly of the myddosome, a protein complex that activates proinflammatory immune responses [51]. ...
CD14 is a co-receptor of Toll-like receptor (TLR)− 4, with a critical role in innate immune responses. CD14 recognizes bacterial lipopolysaccharides, pathogen-, and damage-associated molecular patterns, thereby facilitating inflammatory immune responses. In addition to its well-established association with TLR4, CD14 is also implicated in TLR4-independent signaling, which leads to the apoptotic death of differentiated dendritic cells and activation of the noncanonical inflammasome pathway. CD14 also has a role beyond that of the immune responses. It contributes to tissue homeostasis by promoting the clearance of various apoptotic cells via recognizing externalized phosphatidylinositol phosphates. CD14 also has context-dependent roles, particularly in barrier tissues that include the skin and gastrointestinal tract. For example, CD14+ dendritic cells in the skin can induce immunostimulatory or immunosuppressive responses. In the gastrointestinal system, CD14 is involved in producing inflammatory cytokines in inflammatory bowel disease and maintaining of intestinal integrity. This review focuses on the multifaceted roles of CD14 in innate immunity and its potential regulatory functions in barrier tissues characterized by rapid cell renewal. By providing insights into the diverse functions of CD14, this review offers potential therapeutic implications for this versatile molecule in immune modulation and tissue homeostasis.
... One of the best-known pathways that are linked to the activation of MyD88 is the NF-κB pathway, which is involved in the control of cell proliferation, immunity, and inflammatory responses. 59 The NF-κB pathway has long been considered a typical pro-inflammatory pathway based on its role in pro-inflammatory genes of cytokines, chemokines, and adhesion molecules expression. 60 Almost eight kinds of PFASs have been found to have varying degrees of NF-κB activation in different animal models. ...
Previous studies have reported the immunotoxicity of per- and polyfluoroalkyl substances (PFASs), but it remains a significant challenge to assess over 10,000 distinct PFASs registered in the distributed structure-searchable toxicity (DSSTox) database. We aim to reveal the mechanisms of immunotoxicity of different PFASs and hypothesize that PFAS immunotoxicity is dependent on the carbon chain length. Perfluorobutanesulfonic acid (PFBA), perfluorooctanoic acid (PFOA), and perfluorononanoic acid (PFNA) representing different carbon chain lengths (4-9) at environmentally relevant levels strongly reduced the host's antibacterial ability during the zebrafish's early-life stage. Innate and adaptive immunities were both suppressed after PFAS exposures, exhibiting a significant induction of macrophages and neutrophils and expression of immune-related genes and indicators. Interestingly, the PFAS-induced immunotoxic responses were positively correlated to the carbon chain length. Moreover, PFASs activated downstream genes of the toll-like receptor (TLR), uncovering a seminal role of TLR in PFAS immunomodulatory effects. Myeloid differentiation factor 88 (MyD88) morpholino knock-down experiments and MyD88 inhibitors alleviated the immunotoxicity of PFASs. Overall, the comparative results demonstrate differences in the immunotoxic responses of PFASs due to carbon chain length in zebrafish, providing new insights into the prediction and classification of PFASs mode of toxic action based on carbon chain length.
... Tis phenomenon can be used to determine whether a compound binds to TLR7. Dimerization results in association with the adaptor protein myeloid diferentiation primary response 88 (MyD88) and initiates signaling cascades that induce the transcription of infammatory mediators [29]. Te signaling pathways of TLR7 activate three transcriptional factors: NF-κB, AP-1, and IRF7. ...
Toll-like receptor 7 (TLR7) is an attractive target for developing immune modulators to enhance innate immunity against ssRNA virus infections, including hepatitis C and COVID-19. Ten 3-(5-hydroxyphenyl)-5-phenyl-2-pyrazolines were tested using TLR7 reporter cells, overexpressing TLR7 and the NF-κB-inducible SEAP reporter gene to discover a novel TLR7 agonist enhancing innate immunity. Of these, 2-(3-(2-hydroxynaphthalen-1-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (compound 6) showed the best TLR7 agonistic activity, and further experiments were carried out to study the immune-modulatory capability of compound 6. Treatment with compound 6 rapidly induced phosphorylation of IRAK4, IKKα/β, IκBα, and p65/RelA in THP1 monocytic cells. In addition, it increased the expression of NF-κB-regulated innate cytokines, such as TNFα and IL1β, in THP1 monocytic cells. These data suggest that compound 6 induces an innate immune response by agonizing TLR7 activity in THP1 human monocytic cells. Therefore, compound 6 can be used as an innate immune modulator to develop antiviral agents and vaccine adjuvants.
... Knockout of MyD88 or TRAF6 eliminated the potentiation effects of IL-1b but not TNF-a, PMA, EGF, or VEGF ( Figures S4E-S4H), which is consistent with the fact that IL-1R activates NF-kB via MyD88 and TRAF6, while TNFR, GF-Rs, and PKC do not require these two to activate NF-kB. 27,28 We then wondered whether activation of the noncanonical NF-kB pathway would also affect STING signaling. To this end, we knocked out p52 (NF-kB2; a major component of non-canonical NF-kB signaling) on RAW264.7 cells and stimulated the cells with DMXAA. ...
It is widely known that stimulator of interferon genes (STING) can trigger nuclear factor κB (NF-κB) signaling. However, whether and how the NF-κB pathway affects STING signaling remains largely unclear. Here, we report that Toll-like receptor (TLR)-, interleukin-1 receptor (IL-1R)-, tumor necrosis factor receptor (TNFR)-, growth factor receptor (GF-R)-, and protein kinase C (PKC)-mediated NF-κB signaling activation dramatically enhances STING-mediated immune responses. Mechanistically, we find that STING interacts with microtubules, which plays a crucial role in STING intracellular trafficking. We further uncover that activation of the canonical NF-κB pathway induces microtubule depolymerization, which inhibits STING trafficking to lysosomes for degradation. This leads to increased levels of activated STING that persist for a longer period of time. The synergy between NF-κB and STING triggers a cascade-amplified interferon response and robust host antiviral defense. In addition, we observe that several gain-of-function mutations of STING abolish the microtubule-STING interaction and cause abnormal STING trafficking and ligand-independent STING autoactivation. Collectively, our data demonstrate that NF-κB activation enhances STING signaling by regulating microtubule-mediated STING trafficking.
... Inflammation was a major pathway activated in CD11b + myeloid clusters from IL-1rn-KO versus WT mice, with the exceptions of G1 neutrophils that showed no changes and type I monocytes that showed a reduced inflammatory signature (Supplementary Fig. S6A). Among the differentially expressed genes responsible for these enriched signatures, we found upregulation of several major pro-inflammatory genes (Hif1a, Csf2rb, Myd88, Cxcr2, Lmo4) [40][41][42][43][44] as well as downregulation of anti-inflammatory genes (Nfkbia, Cebpb) 45,46 . A few genes that control inflammation (Lyn) 47 were upregulated in CD11b + myeloid clusters from IL-1rn-KO versus WT mice ( Supplementary Fig. S6B). ...
Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression. Enhanced IL-1β signaling pathway causes hematopoietic stem cell (HSC) to differentiate into myeloid cells and contributes to malignant hematopoiesis. Here the authors reveal that HSC differentiation is controlled by balanced levels of IL-1 receptor antagonist (IL-1rn) and IL-1β under steady-state, and that IL-1rn protects against pre-leukemic myelopoiesis by repressing IL-1β signaling.
... Moreover, the expression of IRG1 was elevated when TLR4 was overexpressed in macrophages [39]. Myeloid Differentiation Primary Response Gene 88 (Myd88) encodes a cytosolic adapter protein that plays a central role in the innate and adaptive immune response and functions as an essential signal transducer in the interleukin-1 and Toll-like receptor signaling pathways [40]. Hoshino K et al. demonstrated that TLR4 signaling induced IRG1 gene expression in MyD88-dependent and Myd88-independent pathways, and TLR9 induced IRG1 in a MyD88-dependent manner [34]. ...
Endogenous small molecules are metabolic regulators of cell function. Itaconate is a key molecule that accumulates in cells when the Krebs cycle is disrupted. Itaconate is derived from cis-aconitate decarboxylation by cis-aconitate decarboxylase (ACOD1) in the mitochondrial matrix and is also known as immune-responsive gene 1 (IRG1). Studies have demonstrated that itaconate plays an important role in regulating signal transduction and posttranslational modification through its immunoregulatory activities. Itaconate is also an important bridge among metabolism, inflammation, oxidative stress, and the immune response. This review summarizes the structural characteristics and classical pathways of itaconate, its derivatives, and the compounds that release itaconate. Here, the mechanisms of itaconate action, including its transcriptional regulation of ATF3/IκBζ axis and type I IFN, its protein modification regulation of KEAP1, inflammasome, JAK1/STAT6 pathway, TET2, and TFEB, and succinate dehydrogenase and glycolytic enzyme metabolic action, are presented. Moreover, the roles of itaconate in diseases related to inflammation and oxidative stress induced by autoimmune responses, viruses, sepsis and IRI are discussed in this review. We hope that the information provided in this review will help increase the understanding of cellular immune metabolism and improve the clinical treatment of diseases related to inflammation and oxidative stress.
... PD-L1 surface expression is weak or absent on WM tumor B-cells. However soluble PD-L1 serum levels are increased in WM patients and PD-L1 is upregulated by IL6 (55). ...
La Maladie de Waldenström (MW), est un lymphome B indolent incurable. Ce lymphome, d’un point de vue plus général, se différencie des autres lymphomes indolents, par l’expansion d’un clone lymphoplasmocytaire de localisation médullaire quasi constante avec sécrétion d’une IgM monoclonale. On observe une différenciation lymphoplasmocytaire ainsi qu’une infiltration des cellules tumorales dans la moelle osseuse. Il est admis que ce lymphome est généralement précédé par une phase asymptomatique au cours de laquelle seule l’IgM monoclonale est détectée dans le sérum (MGUS IgM). La découverte récente de la mutation L265P de MYD88 activatrice de NF-κB dans plus de 90% des cas a démontré que la MW est génétiquement distincte des autres lymphomes indolents avec sécrétion d’IgM.MYD88 est une protéine adaptatrice impliquée dans l’immunité innée (action des TLR et des récepteurs à l’IL-1). Elle est impliquée dans l’inflammation qui favorise la cancérogénèse. Des mutations oncogéniques activatrices ont récemment été identifiées comme la mutation L265P. MYD88 L265P joue également un rôle intrinsèque dans la signalisation constitutive de la voie NF-κB, en se liant via le domaine TIR des TLRs. Dans ce contexte il est apparu comme fondamental, compte tenu de l’implication de MYD88 muté dans la signalisation de cette voie, d’évaluer les conséquences de la surexpression de MYD88 muté in vitro ainsi que ces fonctions in vivo en termes de signalisation, d’inflammation et de transformation. Pour cela nous avons mené une étude in vitro à l’aide d’un modèle cellulaire EREB2.5 inductible à l’oestradiol. Cette lignée cellulaire à la particularité de pouvoir réprimé de façon réversible la voie NF-κB en absence d’oestradiol. Il devient alors possible d’étudier spécifiquement l’effet de l’expression de la mutation MYD88 L265P sur les voies de signalisation situées en aval de la voie NF-κB. Une étude in vivo a été mené sur un modèle des souris Knock-in dans lesquelles les allèles sauvages de MYD88 sont remplacés par des formes mutantes L265P de la protéine mutée. A l’aide de ce modèle nous avons pu démontrer que l'activation continue de MYD88 est capable de favoriser l'expansion précoce des cellules IgM lymphoplasmocytaire et des plasmocytes avec, d'abord, une hyper Ig polyclonale sérique puis un pic d'Ig monoclonale. Les pics d'Ig étaient constamment associés à des lymphomes à cellules B partageant des caractéristiques avec la MW. Ici, nous avons montré pour la première fois que la différenciation des lymphocytes B lymphoplasmocytaires IgM est au coeur du potentiel de transformation de MYD88L252P.
... The MyD88-dependent pathway mediates the antibacterial and antiviral responses in D. melanogaster and other invertebrates. MyD88 is an intracellular adaptor protein involved in the Toll signalling pathway and activation of the NF-κB transcription factor ( Figure 1) [53]. ...
In recent years, the scientific community's interest in T. molitor as an insect model to investigate immunity and host-pathogen interactions has considerably increased. The reasons for this growing interest could be explained by the peculiar features of this beetle, which offers various advantages compared to other invertebrates models commonly used in laboratory studies. Thus, this review aimed at providing a broad view of the T. molitor immune system in light of the new scientific evidence on the developmental/tissue-specific gene expression studies related to microbial infection. In addition to the well-known cellular component and humoral response process, several studies investigating the factors associated with T. molitor immune response or deepening of those already known have been reported. However, various aspects remain still less understood, namely the possible crosstalk between the immune deficiency protein and Toll pathways and the role exerted by T. molitor apolipoprotein III in the expression of the antimicrobial peptides. Therefore, further research is required for T. molitor to be recommended as an alternative insect model for pathogen host interaction and immunity studies.
... Mb) overlaps with a haptoglobin concentration quantitative trait locus detected at 0 and 10 dpv in 16 week-old PRRS virus vaccinated pigs [56]. The MYD88 gene encodes an adaptor protein that is involved in the toll-like receptor/IL-1R receptor signalling pathway [59]. Activation of the latter induces the nuclear factor kappa (NF-kB) and mitogenactivated protein kinase (MAPK) signalling pathways, which are essential for the innate immune response [60]. ...
Background
There is a growing interest to decipher the genetic background of resilience and its possible improvement through selective breeding. The objective of the present study was to provide new insights into the genetic make-up of resilience in growing pigs by identifying genomic regions and candidate genes associated with resilience indicators. Commercial Duroc pigs were challenged with an attenuated Aujeszky vaccine at 12 weeks of age. Two resilience indicators were used: deviation from the expected body weight at 16 weeks of age given the growth curve of non-vaccinated pigs (∆BW) and the increase in acute-phase protein haptoglobin at four days post-vaccination (∆HP). Genome-wide association analyses were carried out on 445 pigs, using genotypes at 41,165 single nucleotide polymorphisms (SNPs) and single-marker and Bayesian multiple-marker regression approaches.
Results
Genomic regions on pig chromosomes 2, 8, 9, 11 (∆BW) and 8, 9, 13 (∆HP) were found to be associated with the resilience indicators and explained high proportions of their genetic variance. The genomic regions that were associated explained 27 and 5% of the genetic variance of ∆BW and ∆HP, respectively. These genomic regions harbour promising candidate genes that are involved in pathways related to immune response, response to stress, or signal transduction ( CD6 , PTGDR2 , IKZF1 , RNASEL and MYD88 ), and growth ( GRB10 and LCORL ).
Conclusions
Our study identified novel genomic regions that are associated with two resilience indicators (∆BW and ∆HP) in pigs. These associated genomic regions harbour potential candidate genes involved in immune response and growth pathways, which emphasise the strong relationship between resilience and immune response.
... Subsequently, TRAF6 becomes ubiquitinated (Ub) and activates TAK1 (TGF-β-activated kinase 1), the latter phosphorylates and activates the IκB kinase (IKK) complex. IKK phosphorylates IκB, an inhibitor of nuclear factor kappa B (NF-κB), which allows NF-κB to be rapidly activated and transferred to nucleus, promoting expression of related genes by binding to κB (Janssens and Beyaert, 2002;Wu et al., 2018). NF-κB is a multifunctional dimeric transcription factor that coordinates cell proliferation and closely related to cancer development and progression (Yang et al., 2017) (Figure 1B). ...
Tissues undergo constant remodeling to maintain architecture during growth, in normal physiology and in response to disease. Interactions of the host with commensals and pathogens may affect immune responses and tissue remodeling, including for example through the generation of neo-epitopes and resulting in damage-associated molecular patterns. Roles for the microbiome, viriome, parasites, and fungi in host-pathogen interactions and in homeostasis is a current topic with considerable interest regarding effects relating to the gut-brain axis, chronic disease, cancer, dysbiosis, host metabolism, and drug metabolism.
This E-book contains state-of-the-art primary research studies and review articles from international experts and diverse leading groups in the field to further current understanding of the contributions of commensals and pathogens in tissue remodeling in physiological and pathophysiological processes of the host.
... MYD88 associates in a TIR-mediated interaction with the intracellular portion of the receptors and with the cytoplasmic serine-threonine kinase IL-1R activated kinase 4 (IRAK4) through the DD, which in turn associates with and phosphorylates the kinases IRAK1 and IRAK2 in the so called "Myddosome" (11). The E3-ubiquitin ligase Tumor Necrosis Factor Receptor associated factor 6 (TRAF6) is then recruited and in turn binds to TAK1-binding protein 2 (TAB2) and activates the TGFb activated kinase 1 (TAK1), thereby triggering the downstream activation of the nuclear factor of activated B cells (NF-kB) transcription factor and mitogen activated kinase (MAPK) signaling pathways (12,13). LPL/ WM cells harboring the MYD88 L265P mutation in the TIR domain display a constitutively active NF-kB pathway. ...
Lymphoplasmacytic lymphoma (LPL) is a rare subtype of B cell-derived non-Hodgkin lymphoma characterized by the abnormal growth of transformed clonal lymphoplasmacytes and plasma cells. This tumor almost always displays the capability of secreting large amounts of monoclonal immunoglobulins (Ig) of the M class (Waldenström Macroglobulinemia, WM). The clinical manifestations of WM/LPL may range from an asymptomatic condition to a lymphoma-type disease or may be dominated by IgM paraprotein-related symptoms. Despite the substantial progresses achieved over the last years in the therapy of LPL/WM, this lymphoma is still almost invariably incurable and exhibits a propensity towards development of refractoriness to therapy. Patients who have progressive disease are often of difficult clinical management and novel effective treatments are eagerly awaited. In this review, we will describe the essential clinical and pathobiological features of LPL/WM. We will also analyze some key aspects about the current knowledge on the mechanisms of drug resistance in this disease, by concisely focusing on conventional drugs, monoclonal antibodies and novel agents, chiefly Bruton’s Tyrosine Kinase (BTK) inhibitors. The implications of molecular lesions as predictors of response or as a warning for the development of therapy resistance will be highlighted.
... It is well established that Nuclear factor-κB (NF-κB) regulates pro- inflammatory cytokines including TNF-α, IL-1β and IL-6. Moreover, myeloid differentiation primary response protein 88 (MyD88) is interlinked with IL-1 and TLR family to the downstream activation of NF-κB [69][70][71][72]. In our study, the levels of TNF-α and IL-1β were markedly reduced in splenocytes upon 14 days oral administration of opuntiol/ opuntioside and opuntiol-coated NPs in arthritic rats. ...
Rheumatoid arthritis (RA) is a chronic autoimmune disease of synovial inflammation and joint destruction. This study reports anti-arthritic potential of opuntioside-I opuntiol, and its gold and silver nanoparticles (NPs) against Complete Freund's Adjuvant (CFA)-induced arthritic rats. The mechanistic studies were performed targeting TLRs (TLR-2 and TLR-4) and cytokines (IL-1β and TNF-α) expressions to validate their anti-inflammatory and immuno-modulatory response. The nano-formulations were successfully characterized employing Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) analysis. Opuntiol and opuntioside (OP and OPG: 10, 50 and 100 mg/kg) and opuntiol-coated silver and gold NPs (OP-AgNPs and OP-AuNPs: 0.5, 1 and 3 mg/kg) treatments in arthritic rat have shown minimal arthritic score exhibiting mild to moderate articular changes and tissue swelling in ankle joints. Radiographic examination reveals significant reduction in synovitis with improvement in joints degen-arative changes in the presence of aforementioned treatments. Likewise, histology of rat ankle joints depicted comparatively lesser influx of inflammatory cells and diminished granulamatous inflammation. Moreover, treatment groups suppressed protein and mRNA expressions of TLRs (TLR-2 and TLR-4) and cytokines (IL-1β and TNF-α) levels were also significantly declined in the presence of OPG, OP and its NPs comparing to arthritic control. This investigation concludes, the tested compounds and nano-formulations successfully restored the disease progression in CFA-induced arthritic rat owing to their immunomodulatory and anti-inflammatory potentials and can be considered for RA targeted therapy to address the utmost challenges of the disease.
... TLRs play a critical role in the recognition of damage-associated molecular patterns (DAMPs), which stimulates a regenerative response to trauma [12]. After recognition of DAMPs, TLRs signal through the adaptor protein Myd88, resulting in the secretion of pro-inflammatory cytokines that trigger immune cell recruitment [13]. To investigate the role of TLR signaling in the aging response to injury and, due to the central role of MyD88 in this process, we next analyzed the callus of young and middle-aged mice for MyD88 expression. ...
Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.
... The type I Transmembrane receptors (TLRs) contain intercellular Toll/IL1 signaling domain and leucine-rich repeats. The molecules that are termed as pathogen-associated molecular patterns (PAMP) include lipoproteins, LPS, and flagellin with bacterial and viral genetic material are the significant legend for this receptor (Janssens and Beyaert, 2002). TLRs can initialize the signaling pathways that enhance the inflammatory mediator's expression, cell adhesion molecules, and chemokines after molecular exposure of these molecules (Lai and Gallo, 2008). ...
Autophagy is an important cellular self-digestion and recycling pathway that helps in maintaining cellular homeostasis. Dysregulation at various steps of the autophagic and endolysosomal pathway has been reported in several neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington disease (HD) and is cited as a critically important feature for central nervous system (CNS) proteostasis. Recently, another molecular target, namely transcription factor EB (TFEB) has been explored globally to treat neurodegenerative disorders. This TFEB, is a key regulator of autophagy and lysosomal biogenesis pathway. Multiple research studies suggested therapeutic potential by targeting TFEB to treat human diseases involving autophagy-lysosomal dysfunction, especially neurodegenerative disorders. A common observation involving all neurodegenerative disorders is their poor efficacy in clearing and recycle toxic aggregated proteins and damaged cellular organelles due to impairment in the autophagy pathway. This dysfunction in autophagy characterized by the accumulation of toxic protein aggregates leads to a progressive loss in structural integrity/functionality of neurons and may even result in neuronal death. In recent years TFEB, a key regulator of autophagy and lysosomal biogenesis, has received considerable attention. It has emerged as a potential therapeutic target in numerous neurodegenerative disorders like AD and PD. In various neurobiology studies involving animal models, TFEB has been found to ameliorate neurotoxicity and rescue neurodegeneration. Since TFEB is a master transcriptional regulator of autophagy and lysosomal biogenesis pathway and plays a crucial role in defining autophagy activation. Studies have been done to understand the mechanisms for TFEB dysfunction, which may yield insights into how TFEB might be targeted and used for the therapeutic strategy to develop a treatment process with extensive application to neurodegenerative disorders. In this review, we explore the role of different transcription factor-based targeted therapy by some natural compounds for AD and PD with special emphasis on TFEB.
... Verbascoside has been reported to prevent IκBα degradation which keeps NF-κB in its inactive state in the cytoplasm of cells [27,37]. Therefore, MyD88 upon activation by upper regulatory signals initiates apoptosis pathway through FAS-associated death domain protein (FADD) which consequently affects procaspase-8 and apoptosis cascade [38]. Verbascoside can activate HIPK2 [9] which in turn directly inhibits NF-κB to promote apoptosis [10]. ...
Background
Despite significant advancements in breast cancer therapy, novel drugs with lower side effects are still being demanded. In this regard, we investigated the anti-cancer features of verbascoside in 4 T1 mouse mammary tumor cell.
Methods
First, MTT assay was performed with various concentrations (ranging between 5 to 200 μM) of verbascoside and IC50 was calculated. Then the expression of Bax , Bcl-2 , and caspase-3 was evaluated in treated 4 T1 cells. In addition, we investigated the expression of TLR4 , MyD88 , and NF-κB to ascertain the underlying mechanism of the anti-proliferative feature of verbascoside. Also, flow cytometry followed by double PI and Annexin V was conducted to confirm the apoptosis-inducing effect of verbascoside.
Results
Our results from MTT assay showed verbascoside inhibits proliferation of 4 T1 cancer cells (IC50 117 μM) while is safe for normal HEK293T cells. By qRT-PCR, we observed that verbascoside treatment (100, 117 and, 130 μM) increases the expression of caspase-3 and Bax while reduces the expression of Bcl-2 . Also, verbascoside (100, 117 and, 130 μM) increased the expression of TLR4 only at 130 μM dose and the expression of MyD88 whereas reduced the expression of NF-κB at mRNA level. Flow cytometry analysis also confirmed verbascoside induces apoptosis in 4 T1 cells at 117 μM.
Conclusion
Taken together, our data showed verbascoside is a safe natural compound for normal cells while has apoptosis-inducing feature through TLR4 axis on 4 T1 cells.
... After pathogens invasion, microbial products signal through TLRs on tissue-resident mast cells and macrophages, activate these cells to produce proinflammatory cytokines, which coordinates the recruitment of leukocytes together with the antimicrobial function [31]. MYD88 acts as a signaling transductor of TLRs (not only of TLR2), by which is recruited [33]. The lower abundance during the week after dry-off of TLR2 and MYD88 might be a proxy of the suppression of the immune system activity in this phase. ...
Dairy cows at dry-off undergo several management and physiological changes, resulting in alterations in plasma biomarkers of inflammation, oxidative stress, and immune system. High milk yield at the end of lactation exacerbates these responses. The underlying mechanism of these changes has yet to be elucidated. We hypothesized altered leukocyte gene expression after dry-off and different responses in cows with different milk yield. Thirteen Holstein dairy cows were sampled at the turn of dry-off to investigated whole blood leukocyte gene expression and were grouped according to the average milk yield during the last week of lactation: low (< 15 kg/d) and high milk yield (> 15 kg/d). Blood samples were collected in PAXgene tubes (Preanalytix, Hombrechtikon, Switzerland) at -7, 7, and 34 days from dry-off (DFD) to measure mRNA abundance of 37 genes. Normalized gene abundance data were subjected to MIXED model ANOVA (SAS Institute Inc., Cary, NC). Compared with -7 DFD, at 7 DFD RNA abundance of lipoxygenase genes (ALOX5, ALOX15) and myeloperoxidase (MPO) increased, and that of the antioxidant gene (SOD2) decreased. Meanwhile, genes related to recognition and immune mediation (CD16, MYD88, TLR2), migration and cell adhesion (CX3CR1, ITGAL, ITGB2, TLN1), and the antimicrobial gene MMP9 were downregulated at 7 or 34 DFD, whereas the antimicrobial IDO1 gene was upregulated. Compared with low-producing cows, cows with high milk yield at dry-off cows had upregulated expression of the pro-inflammatory cytokines IL8 and IL18 and a greater reduction in transcript abundance of the toll-like receptor (TLR) recognition-related gene TLR2. Overall, the dry-off confirmed to be a phase of intense changes, triggering an inflammatory response and somewhat suppressing leukocyte immune function. In cows with high milk yield during the week before dry-off, the inflammatory response was exacerbated.
... Activation of PRR in corneal epithelial cells causes downstream activation of myeloid differentiation primary response protein 88 (MyD88)-dependent and independent pathways (Deguine and Barton, 2014). MyD88 is the master adaptor protein for all TLRs (except TLR3), leading to activation of pro-inflammatory cascade through nuclear factor-κB (NF-κB) and the mitogen-associated protein kinases (MAPKs) (Janssens and Beyaert, 2002). In bacterial keratitis, activation of MyD88 releases pro-inflammatory chemokines, leading to rapid phagocyte activation and recruitment within the cornea and significantly elevated levels of AMPs, and increased resistance of corneal epithelial cells against adhesion of invading bacteria (Gao et al., 2013;Huang et al., 2006;Tam et al., 2011;Yu and Hazlett, 2006). ...
Bacterial keratitis continues to be one of the leading causes of corneal blindness in the developed as well as the developing world, despite swift progress since the dawn of the “antibiotic era”. Although we are expeditiously developing our understanding about the different causative organisms and associated pathology leading to keratitis,
extensive gaps in knowledge continue to dampen the efforts for early and accurate diagnosis, and management in these patients, resulting in poor clinical outcomes. The ability of the causative bacteria to subdue the therapeutic challenge stems from their large genome encoding complex regulatory networks, variety of unique virulence factors, and rapid secretion of tissue-damaging proteases and toxins. In this review article, we have provided an overview of the established classical diagnostic techniques and therapeutics for keratitis caused by various bacteria. We have extensively reported our recent in-roads through
novel tools for accurate diagnosis of mono- and poly-bacterial corneal infections. Furthermore, we outlined the recent progress by our group and others in understanding the sub-cellular genomic changes that lead to antibiotic resistance in these organisms. Finally, we discussed in detail, the novel therapies and drug delivery systems in development for the efficacious management of bacterial keratitis.
... TLRs are able to recognize different microbial structures/patterns, thus intervening in the early immune responses to several etiologic agents . Their activity is often mediated by MyD88, which acts as an adapter involved in signal transduction (Janssens and Beyaert, 2002). ...
Coxiella burnetii is an obligate intracellular Gram-negative bacterium and the causative agent of a worldwide zoonosis known as Q fever. The pathogen invades monocytes and macrophages, replicating within acidic phagolysosomes and evading host defenses through different immune evasion strategies that are mainly associated with the structure of its lipopolysaccharide. The main transmission routes are aerosols and ingestion of fomites from infected animals. The innate immune system provides the first host defense against the microorganism, and it is crucial to direct the infection towards a self-limiting respiratory disease or the chronic form. This review reports the advances in understanding the mechanisms of innate immunity acting during C. burnetii infection and the strategies that pathogen put in place to infect the host cells and to modify the expression of specific host cell genes in order to subvert cellular processes. The mechanisms through which different cell types with different genetic backgrounds are differently susceptible to C. burnetii intracellular growth are discussed. The subsets of cytokines induced following C. burnetii infection as well as the pathogen influence on an inflammasome-mediated response are also described. Finally, we discuss the use of animal experimental systems for studying the innate immune response against C. burnetii and discovering novel methods for prevention and treatment of disease in humans and livestock.
... IL-1β/IL-1R and LPS/TLR4mediated NF-κB signaling share a similar signaling pathway. Receptor IL-1R as well as TLR4 recruit the adaptor protein MyD88 after binding to ligand IL-1 or LPS, respectively, then MyD88 forms complex with IL-1R-associated kinases (IRAKs), including IRAK1, IRAK2, and IRAK4, and Tumor necrosis factor receptor-associated factor 6 (TRAF6; Janssens and Beyaert, 2002). Once the MyD88 complex is activated, TRAF6 servers as an E3 ubiquitin ligase to catalyze the synthesis of K63-linked polyubiquitin chains conjugated to itself or other proteins or as free ubiquitin chains (Shi and Sun, 2018). ...
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a RING domain ubiquitin ligase that plays an important role in nuclear factor-κB (NF-κB) signaling by regulating activation of the TAK1 and IKK complexes. However, the molecular mechanisms that regulate TRAF6 E3 activity remain unclear. Here, we found that ZDHHC11, a member of the DHHC palmitoyl transferase family, functions as a positive modulator in NF-κB signaling. ZDHHC11 overexpression activated NF-κB, whereas ZDHHC11 deficiency impaired NF-κB activity stimulated by IL-1β, LPS, and DNA virus infection. Furthermore, Zdhhc11 knockout mice had a lower level of serum IL6 upon treatment with LPS and D-galactosamine or HSV-1 infection than control mice. Mechanistically, ZDHHC11 interacted with TRAF6 and then enhanced TRAF6 oligomerization, which increased E3 activity of TRAF6 for synthesis of K63-linked ubiquitination chains. Collectively, our study indicates that ZDHHC11 positively regulates NF-κB signaling by promoting TRAF6 oligomerization and ligase activity, subsequently activating TAK1 and IKK complexes.
... The lack of MyD88 molecule completely eliminated the effect of BCG in tumor volume reduction. MyD88 drives a wide role as an adapter molecule for TLRs and also interleukin-1 receptor (IL-1R) family 70 . The importance of MyD88 to the inflammasome complex activation can be related to the first signal after TLRs induction or downstream IL-1R signaling 71 . ...
Bacillus Calmette-Guerin (BCG) is the only FDA approved first line therapy for patients with nonmuscle invasive bladder cancer. The purpose of this study is to better understand the role of innate immune pathways involved in BCG immunotherapy against murine bladder tumor. We first characterized the immunological profile induced by the MB49 mouse urothelial carcinoma cell line. MB49 cells were not able to activate an inflammatory response (TNF-α, IL-6, CXCL-10 or IFN-β) after the stimulus with different agonists or BCG infection, unlike macrophages. Although MB49 cells are not able to induce an efficient immune response, BCG treatment could activate other cells in the tumor microenvironment (TME). We evaluated BCG intratumoral treatment in animals deficient for different innate immune molecules (STING−/−, cGAS−/−, TLR2−/−, TLR3−/−, TLR4−/−, TLR7−/−, TLR9−/−, TLR3/7/9−/−, MyD88−/−, IL-1R−/−, Caspase1/11−/−, Gasdermin-D−/− and IFNAR−/−) using the MB49 subcutaneous mouse model. Only MyD88−/− partially responded to BCG treatment compared to wild type (WT) mice, suggesting a role played by this adaptor molecule. Additionally, BCG intratumoral treatment regulates cellular infiltrate in TME with an increase of inflammatory macrophages, neutrophils and CD8+ T lymphocytes, suggesting an immune response activation that favors tumor remission in WT mice but not in MyD88−/−. The experiments using MB49 cells infected with BCG and co-cultured with macrophages also demonstrated that MyD88 is essential for an efficient immune response. Our data suggests that BCG immunotherapy depends partially on the MyD88-related innate immune pathway.
... Among the numerous TIR domain-containing adaptors, MyD88 is the common adaptor recruited by all TLRs except TLR3 (Janssens and Beyaert, 2002). TIRAP is a unique adapter in TLR2 and TLR4 signaling and is associated with MyD88 for NF-κB activation (Horng et al., 2002). ...
The Toll/interleukin-1 receptor (TIR) domain is a structural unit responsible for the assembly of signal protein complexes in Toll-like receptor (TLR) and interleukin-1 receptor signaling pathways. TIR domain homologs are found in a considerable number of bacteria and enhance bacterial infection and survival in host organisms. However, whether TIR domain homologs exist in Aeromonas hydrophila, a ubiquitous waterborne bacterium in aquatic environments, remains poorly understood. In this study, a TIR domain protein (TcpAh) was identified from A. hydrophila JBN2301. TIR domain of TcpAh is highly homologous to the counterpart domains in TLRs and myeloid differentiation factor 88 (MyD88). The zebrafish infected with mutant A. hydrophila with tcpAh deletion had a remarkably lower mortality than those infected with the wild-type strain. This result suggests that TcpAh is a crucial virulence factor for A. hydrophila infection. TcpAh exhibited a strong ability to associate with MyD88, tumor necrosis factor receptor-associated factor 3 (TRAF3) and TRAF-associated NF-κB activator-binding kinase 1 (TBK1) in TIR–TIR, TIR–Death domain (DD), and other alternative interactions. This finding suggests that TcpAh extensively interferes with MyD88 and TIR domain-containing adapter inducing interferon (IFN)-β (TRIF) signaling pathways downstream of TLRs. Consequently, CD80/86 expression was suppressed by TcpAh via attenuating TLR-stimulated NF-κB activation, which ultimately led to the impairment of the major costimulatory signal essential for the initiation of adaptive humoral immunity against A. hydrophila infection. We believe that this study is the first to show a previously unrecognized mechanism underlying A. hydrophila evades from host antibacterial defense by intervening CD80/86 signal, which bridges innate and adaptive immunity. The mechanism will benefit the development of therapeutic interventions for A. hydrophila infection and septicemia by targeting TcpAh homologs.
... TLRs dimerize upon binding their cognate ligand (Fig. 2B), leading to conformational changes that allow for the recruitment of adapter molecules and initiating signaling cascades that ultimately induce transcription of inflammatory mediators [27]. In the case of TLR7/8, dimerization initiates the TIR signaling cascade, which results in association with the adaptor protein myeloid differentiation primary response 88 (MyD88) at its carboxy terminus [28]. MyD88 also has an N-terminal death domain that recruits and associates with similar death domains present on two serine-threonine protein kinases: IL-1-receptor associated kinase (IRAK4) and IRAK1 [29] (Fig. 2C). ...
Imidazoquinoline derivatives (IMDs) and related compounds function as synthetic agonists of Toll-like receptors 7 and 8 (TLR7/8) and one is FDA approved for topical antiviral and skin cancer treatments. Nevertheless, these innate immune system-activating drugs have potentially much broader therapeutic utility; they have been pursued as antitumor immunomodulatory agents and more recently as candidate vaccine adjuvants for cancer and infectious disease. The broad expression profiles of TLR7/8, poor pharmacokinetic properties of IMDs, and toxicities associated with systemic administration, however, are formidable barriers to successful clinical translation. Herein, we review IMD formulations that have advanced to the clinic and discuss issues related to biodistribution and toxicity that have hampered the further development of these compounds. Recent strategies aimed at enhancing safety and efficacy, particularly through the use of bioconjugates and nanoparticle formulations that alter pharmacokinetics, biodistribution, and cellular targeting, are described. Finally, key aspects of the biology of TLR7 signaling, such as TLR7 tolerance, that may need to be considered in the development of new IMD therapeutics are discussed.
... The accessory protein, MD-2, physically associates with TLR4 to form an active TLR4/MD-2 complex that functions as a signaling receptor on the macrophage surface [69]. Main upstream transduction events activated by LPS-TLR4/MD-2 binding also involve the adaptor molecule MyD88 [70]. A defective expression of TLR4 [71,72], MD-2 [69] or MyD88 [73] reduces the macrophage response to LPS. ...
Background:
Pulmonary macrophages are a highly heterogeneous cell population distributed in different lung compartments.
Methods:
We separated two subpopulations of macrophages from human lung parenchyma according to flotation over density gradients.
Results:
Two-thirds 65.4% of the lung macrophages have a density between 1.065 and 1.078 (high-density macrophages: HDMs), and the remaining one-third (34.6) had a density between 1.039 and 1.052 (low-density macrophages: LDMs). LDMs had a larger area (691 vs. 462 μm2) and cell perimeter (94 vs. 77 μm) compared to HDMs. A significantly higher percentage of HDMs expressed CD40, CD45, and CD86 compared to LDMs. In contrast, a higher percentage of LDMs expressed the activation markers CD63 and CD64. The release of TNF-α, IL-6, IL-10 and IL-12 induced by lipopolysaccharide (LPS) was significantly higher in HDMs than in LDMs.
Conclusion:
The human lung contains two subpopulations of macrophages that differ in buoyancy, morphometric parameters, surface marker expression and response to LPS. These subpopulations of macrophages probably play distinct roles in lung inflammation and immune responses.
The silkworm, a crucial model organism of the Lepidoptera, offers an excellent platform for investigating the molecular mechanisms underlying the innate immune response of insects toward pathogens. Over the years, researchers worldwide have identified numerous immune‐related genes in silkworms. However, these identified silkworm immune genes are not well classified and not well known to the scientific community. With the availability of the latest genome data of silkworms and the extensive research on silkworm immunity, it has become imperative to systematically categorize the immune genes of silkworms with different database IDs. In this study, we present a meticulous organization of prevalent immune‐related genes in the domestic silkworm, using the SilkDB 3.0 database as a reliable source for updated gene information. Furthermore, utilizing the available data, we classify the collected immune genes into distinct categories: pattern recognition receptors, classical immune pathways, effector genes and others. In‐depth data analysis has enabled us to predict some potential antiviral genes. Subsequently, we performed antiviral experiments on selected genes, exploring their impact on Bombyx mori nucleopolyhedrovirus replication. The outcomes of this research furnish novel insights into the immune genes of the silkworm, consequently fostering advancements in the field of silkworm immunity research by establishing a comprehensive classification and functional understanding of immune‐related genes in the silkworm. This study contributes to the broader understanding of insect immune responses and opens up new avenues for future investigations in the domain of host–pathogen interactions.
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
Background
This study aimed to investigate the contribution of myeloid differentiation primary‐response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis ‐induced experimental periodontitis.
Methods
Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate‐resistant acid phosphatase (TRAP), the receptor activator of nuclear factor‐kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age‐ and sex‐matched homozygous littermates (wild‐type [WT, Myd88 +/+ ] and Myd88 −/− on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed.
Results
P. gingivalis ‐infected Myd88 −/− mice showed alleviated bone loss, TRAP ⁺ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3 ⁺ CD4 ⁺ T cells in infected Myd88 −/− CLNs and a higher frequency of RORγt ⁺ CD4 ⁺ T cells in infected WT mice was noted. Increased IL‐10 and IL‐17a expressions in gingival tissue at D14–D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88 −/− mice. The Myd88 −/− mice exhibited characteristic increases in gram‐positive species and species having probiotic properties, while gram‐negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88 −/− mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection.
Conclusions
MyD88 plays an important role in inflammation‐induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis ‐induced experimental periodontitis.
It is known that interactions between nociceptors and dendritic cells (DCs) can modulate immune responses in barrier tissues. However, our understanding of the underlying communication frameworks remains rudimentary. Here, we show that nociceptors control DCs in three molecularly distinct ways. First, nociceptors release the calcitonin gene-related peptide that imparts a distinct transcriptional profile on steady-state DCs characterized by expression of pro-interleukin-1β and other genes implicated in DC sentinel functions. Second, nociceptor activation induces contact-dependent calcium fluxes and membrane depolarization in DCs and enhances their production of proinflammatory cytokines when stimulated. Finally, nociceptor-derived chemokine CCL2 contributes to the orchestration of DC-dependent local inflammation and the induction of adaptive responses against skin-acquired antigens. Thus, the combined actions of nociceptor-derived chemokines, neuropeptides, and electrical activity fine-tune DC responses in barrier tissues.
HIV-1 reservoir shows high variability in size and activity among virally suppressed individuals. Differences in the size of the viral reservoir may relate to differences in plasma protein concentrations. We tested whether plasma protein expression levels are associated with levels of cell-associated (CA) HIV-1 DNA and RNA in 211 virally suppressed people living with HIV (PLHIV). Plasma concentrations of FOLR1, IL1R1, MICA, and FETUB showed a positive association with CA HIV-1 RNA and DNA. Moreover, SNPs in close proximity to IL1R1 and MICA genes were found to influence the levels of CA HIV-1 RNA and DNA. We found a difference in mRNA expression of the MICA gene in homozygotes carrying the rs9348866-A allele compared to the ones carrying the G allele (p
Background: Cerebral ischemia-reperfusion injury (CIRI) inevitably occurs after vascular recanalization treatment for ischemic stroke. The accompanying inflammatory cascades have a major impact on outcome and regeneration after ischemic stroke. Evidences have demonstrated that TLR/MyD88/NF-κB signaling contributes to CIRI. This study aimed to investigate the druggability of MyD88 in the central nervous system (CNS) and the neuroprotective and anti-neuroinflammatory effects of the MyD88 inhibitor TJ-M2010-5 on CIRI.
Methods: A middle cerebral artery occlusion (MCAO) model was used to simulate CIRI in mice. BV-2 cells were stimulated with oxygen glucose deprivation/reoxygenation (OGD/R) or lipopolysaccharide, and SH-SY5Y cells were induced by OGD/R in vitro . Neurological deficit scores and cerebral infarction volumes were evaluated. Immunofluorescence staining was performed to measure neuronal damage and apoptosis in the brain. The anti-neuroinflammatory effect of TJ-M2010-5 was evaluated by analyzing the expression of inflammatory cytokines, activation of microglia, and infiltration of peripheral myeloid cells. The expression of proteins of the MyD88/NF-κB and ERK pathway was detected by Simple Western. The concentrations of TJ-M2010-5 in the blood and brain were analyzed by liquid chromatography-mass spectrometry.
Results: The cerebral infarction volume decreased in mice treated with TJ-M2010-5, with the most prominent decrease being approximately 80% of the original infarction volume. Neuronal loss and apoptosis were reduced following TJ-M2010-5 treatment. TJ-M2010-5 inhibited the infiltration of peripheral myeloid cells and the activation of microglia. TJ-M2010-5 also downregulated the expression of inflammatory cytokines and inhibited the MyD88/NF-κB and ERK pathway. Furthermore, TJ-M2010-5 showed good blood-brain barrier permeability and no neurotoxicity.
Conclusion: TJ-M2010-5 has an excellent therapeutic effect on CIRI as a novel CNS drug candidate by inhibiting excessive neuroinflammatory responses.
The hemocytes of invertebrates are composed of different cell subsets with different morphologies and structures. Different cell subsets have different immune functions, which play an important role in innate immune response against pathogens. However, the understanding of the classification of Apostichopus japonicus coelomocytes and the molecular basis of immune function of different cell subsets is very limited. In this study, two coelomocyte subpopulations of A. japonicus were isolated by Percoll density gradient centrifugation. They were identified from their morphological and structural characteristics, namely, spherical cells with a size of 10–12 μm spherical in shape and a large number of small granules inside; lymphocyte-like cells with a size of 4–5 μm spherical or oval in shape, and 1–3 filopodia. Functionally, the phagocytic capacity and lysosomal activity in spherical cells were significantly greater than those in lymphocyte-like cells. The results suggest that spherical cells may play a more critical role in the immune responses. Meanwhile, transcriptome sequencing analysis was performed to further clarify the functional differences between the two cell subsets. The data indicated significantly different gene expression patterns in them. Spherical cells tend to participate in immune defense, whereas lymphocyte-like cells tend to participate in energy metabolism. In addition, lymphocyte-like cells may convert oxidative phosphorylation to glycolysis by changing the manner of energy metabolism to quickly adapt to the energy demand of external stimuli. Spherical cells may respond to LPS stimulation through phagocytosis, and their response time is slower than that of lymphocyte-like cells. The expression of genes involved in endocytosis, phagocytosis, and lysosomal and humoral immunity in spherical cells was significantly higher than that in lymphocyte-like cells. These data provide valuable information for understanding the molecular basis of cellular and humoral immunity in A. japonicus.
Inflammation is an indispensable immune response that allows survival during damage or infection and sustains tissue homeostasis at various detrimental conditions. Inflammation is originated at the cost of the temporary tissue malfunction, contributing to the pathogenesis of diseases in homeostatic alterations. Macrophages are a major component of the mononuclear phagocyte system consisting of bone marrow cells, blood monocytes and tissue macrophages. The monocytes in blood are migrated into the tissues where macrophages are transformed. Macrophages play a fundamental role in all stages of the inflammatory responses. In this book chapter, we will discuss the key roles of macrophages in the inflammation based on an immune-modulation with the secretion of various cytokines and growth factors, Toll-like receptors, and update of the recent researches in terms of mathematical and systematical biology approaches.
Interleukin-1 (IL-1) is a key player in the immune response to pathogens due to its role in promoting inflammation and recruiting immune cells to the site of infection. In tuberculosis (TB), tight regulation of IL-1 responses is critical to ensure host resistance to infection while preventing immune pathology. In the mouse model of Mycobacterium tuberculosis infection, both IL-1 absence and overproduction result in exacerbated disease and mortality. In humans, several polymorphisms in the IL1B gene have been associated with increased susceptibility to TB. Importantly, M. tuberculosis itself has evolved several strategies to manipulate and regulate host IL-1 responses for its own benefit. Given all this, IL-1 appears as a promising target for host-directed therapies in TB. However, for that to succeed, more detailed knowledge on the biology and mechanisms of action of IL-1 in vivo, together with a deep understanding of how host-M. tuberculosis interactions modulate IL-1, is required. Here, we discuss the most recent advances in the biology and therapeutic potential of IL-1 in TB as well as the outstanding questions that remain to be answered.
Urinary tract infections (UTIs) are commonly caused by uropathogenic E. coli (UPEC), which uses different mechanisms to invade and damage hosts. NLRP3 inflammasome activation is a double-edged sword, although beneficial in the clearance of dysfunctional cells or pathogens but results in severe pathologies, if unchecked. NLRP3 inflammasome plays a critical role in UPEC mediated inflammatory cell death during UPEC infection, though the effects of its activation are not clearly understood yet. Inflammatory response usually involves Signal-I (MyD88, TRIF and NF-κB signaling) and Signal-II (Inflammasome oligomerization) which is induced by the pathogen associated molecular patterns during infection. We observed that, pro-IL-1β and mature IL-1β remained unchanged on inhibition of MyD88 and TRIF dependent signaling in CFT073 insulted THP1m. However, inhibition of NF-κB pathway resulted in concomitant reduction in both pro- and cleaved forms of IL-1β in cellular extracts but there was no change in IL-1 β secretion. Interestingly, inhibition of NLRP3 completely abolished the maturation and release of cleaved form of IL-1β. Additionally, NLRP3 mediated mitochondrial dysfunction (increased mitoROS and mitochondrial hyperpolarization) resulting in cell death was induced in CFT073 insulted THP-1m, which was rescued upon inhibition of NLRP3 activation. Therefore, we have concluded that UPEC uses a different signal-I pathway (other than MyD88 and TRIF), while NLRP3 inflammasome is the major signal-II for the induction of inflammatory response. Further this study signifies the importance of NLRP3 mediated mitochondrial dysfunction in CFT073 induced inflammatory cell death of macrophages, which could be rescued through inhibition of NLRP3 activation.
Bacterial infections of the cornea, or bacterial keratitis (BK), are notorious for causing rapidly fulminant disease and permanent vision loss, even among treated patients. In the last sixty years, dramatic upward trajectories in the frequency of BK have been observed internationally, driven in large part by the commercialization of hydrogel contact lenses in the late 1960s. Despite this worsening burden of disease, current evidence-based therapies for BK – including broad-spectrum topical antibiotics and, if indicated, topical corticosteroids – fail to salvage vision in a substantial proportion of affected patients. Amid growing concerns of rapidly diminishing antibiotic utility, there has been renewed interest in urgently needed novel treatments that may improve clinical outcomes on an individual and public health level. Bridging the translational gap in the care of BK requires the identification of new therapeutic targets and rational treatment design, but neither of these aims can be achieved without understanding the complex biological processes that determine how bacterial corneal infections arise, progress, and resolve. In this chapter, we synthesize the current wealth of human and animal experimental data that now inform our understanding of basic BK pathophysiology, in context with modern concepts in ocular immunology and microbiology. By identifying the key molecular determinants of clinical disease, we explore how novel treatments can be developed and translated into routine patient care.
Les Céphalopodes comme la seiche Sepia officinalis, malgré une durée de vie courte, sont retrouvés dans les océans depuis des millions d’années. Chez ces animaux atypiques, peu de pathologies ont été observées révélant la présence d’un système immunitaire efficace mais peu étudié qui repose sur des processus innés. L’étude du système immunitaire de la seiche a été menée au niveau du corps blanc, organe hématopoïétique ; des cellules circulantes, les hémocytes et au niveau de la peau, première barrière avec le milieu extérieur. Au niveau du corps blanc, l’étude transcriptomique et protéomique a mis en évidence la présence de facteurs en lien avec l’hématopoïèse dont des membres de la voie de signalisation JAK-STAT. Des facteurs immunitaires ont également été identifiés révélant une possible multifonctionnalité du corps blanc. La réponse immunitaire face à Vibrio splendidus a pu être appréhendées à partir d’une analyse transcriptomique comparative sur les hémocytes. Toutefois cette dernière n’ayant pas permis d’identifier clairement des peptides antimicrobiens, une analyse in silico originale a été développée permettant de sélectionner cinq peptides candidats dont trois ont révélé une activité antibactérienne ciblée contre des bactéries du genre Vibrio. Enfin, une étude au niveau de la peau et de son mucus a été initiée. Cette étude par des approches -omiques a permis l’identification de facteurs en lien avec la reconnaissance des pathogènes et la réponse immunitaire. Par ailleurs, douze souches ont pu être isolées et identifiées au niveau du microbiome cutané. L’ensemble de ces résultats représente un apport majeur concernant le système immunitaire chez la seiche permettant d’initier des études fonctionnelles lors d’une infection ou en fin de vie. Ces études permettraient de comprendre le mode d’action des facteurs immunitaires identifiés, l’implication de chaque entité dans la réponse immunitaire ou dans la mise en place et la maintenance du microbiome.
The Toll-mediated signaling cascade using the NF-κB pathway has been shown to be essential for immune responses in adult Drosophila, and we recently reported that a human homolog of the Drosophila Toll protein induces various immune response genes via this pathway. We now demonstrate that signaling by the human Toll receptor employs an adaptor protein, MyD88, and induces activation of NF-κB via the Pelle-like kinase IRAK and the TRAF6 protein, similar to IL-1R-mediated NF-κB activation. However, we find that Toll and IL-1R signaling pathways are not identical with respect to AP-1 activation. Finally, our findings implicate MyD88 as a general adaptor/regulator molecule for the Toll/IL-1R family of receptors for innate immunity.
Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFκB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFκB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.
Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two
vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TIR) domain,
a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity
and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically
with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NFκB, and A52R
potently blocked both IL-1- and TLR4-mediated NFκB activation. MyD88 is a TIR domain-containing adapter molecule known to
have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of
MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R
are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.
The idea of using simple, genetically tractable host organisms to study the virulence mechanisms of pathogens dates back at least to the work of Darmon and Depraitere [1]. They proposed using the predatory amoeba Dictyostelium discoideum as a model host, an approach that has proved to be valid in the case of the intracellular pathogen Legionella pneumophila [2]. Research from the Ausubel laboratory has clearly established the nematode Caenorhabditis elegans as an attractive model host for the study of Pseudomonas aeruginosa pathogenesis [3]. P. aeruginosa is a bacterium that is capable of infecting plants, insects and mammals. Other pathogens with a similarly broad host range have also been shown to infect C. elegans [3,4]. Nevertheless, the need to determine the universality of C. elegans as a model host, especially with regards pathogens that have a naturally restricted host specificity, has rightly been expressed [5]. We report here that the enterobacterium Salmonella typhimurium, generally considered to be a highly adapted pathogen with a narrow range of target hosts [6], is capable of infecting and killing C. elegans. Furthermore, mutant strains that exhibit a reduced virulence in mammals were also attenuated for their virulence in C. elegans, showing that the nematode may constitute a useful model system for the study of this important human pathogen.
MyD88 has a modular organization, an N-terminal death domain (DD) related to the cytoplasmic signaling domains found in many members of the tumor necrosis factor receptor (TNF-R) superfamily, and a C-terminal Toll domain similar to that found in the expanding family of Toll/interleukin-1-like receptors (IL-1R). This dual domain structure, together with the following observations, supports a role for MyD88 as an adapter in IL-1 signal transduction; MyD88 forms homodimers in vivo through DD-DD and Toll-Toll interactions. Overexpression of MyD88 induces activation of the c-Jun N-terminal kinase (JNK) and the transcription factor NF-kappaB through its DD. A point mutation in MyD88, MyD88-lpr (F56N), which prevents dimerization of the DD, also blocks induction of these activities. MyD88-induced NF-kappaB activation is inhibited by the dominant negative versions of TRAF6 and IRAK, which also inhibit IL-1-induced NF-kappaB activation. Overexpression of MyD88-lpr or MyD88-Toll (expressing only the Toll domain) acted to inhibit IL-1-induced NF-kappaB and JNK activation in a 293 cell line overexpressing the IL-1RI. MyD88 coimmunoprecipitates with the IL-1R signaling complex in an IL-1-dependent manner.
Non-avian dinosaurs are mostly medium to large-sized animals, and to date all known mature specimens are larger than the most primitive bird, Archaeopteryx. Here we report on a new dromaeosaurid dinosaur, Microraptor zhaoianus gen. et sp. nov., from the Early Cretaceous Jiufotang Formation of Liaoning, China. This is the first mature non-avian dinosaur to be found that is smaller than Archaeopteryx, and it eliminates the size disparity between the earliest birds and their closest non-avian theropod relatives. The more bird-like teeth, the Rahonavis-like ischium and the small number of caudal vertebrae of Microraptor are unique among dromaeosaurids and improve our understanding of the morphological transition to birds. The nearly completely articulated foot shows features, such as distally positioned digit I, slender and recurved pedal claws, and elongated penultimate phalanges, that are comparable to those of arboreal birds. The discovery of these in non-avian theropods provides new insights for studying the palaeoecology of some bird-like theropod dinosaurs.
Transcription regulatory elements have been analyzed in upstream sequences of an Interleukin-6 (II-6) primary response gene,
MyD88. MyD88 2.3 kb mRNA is strongly and persistently induced in the course of myeloleukemic M1 cells differentiation with
II-6. MyD88 cDNA sequences were found in a region of 12 kb of mouse genomic DNA. Using II-6 treated M1 cell RNAs, two transcription
start sites have been localized, ∼100 bp upstream from the 5′ end of the cloned cDNA. We sequenced 1.4 kb of 5′ genomic DNA
including the first exon. In 5′ of mRNA transcription start site, MyD88 nucleotidic sequence is 85% identical to 5′ complementary
sequences of the rat 3′-ketoacetyl CoA thiolase gene, over 1.2 kb. A DNA element conferring II-6-inducible transcription to
reporter genes, and localized 30 bp upstream of MyD88 first RNA start site, contains overlapping binding sites for cytokine
activated transcription factors Stat and for the Interferon Regulatory Factor-1 and -2 (IRF-1 and IRF-2). In vitro binding assays showed that attachment of Stat factors to this element early in II-6 treatment requires tyrosine kinase activation.
IRF1, an activator of transcription, is also induced to bind to this sequence at later times. A model of persistent activation
of MyD88 gene through these two types of factors is proposed.
Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.
The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.
Mutagenized human 293 cells containing an interleukin-1 (IL-1)-regulated herpes thymidine kinase gene, selected in IL-1 and
gancyclovir, have yielded many independent clones that are unresponsive to IL-1. The four clones analyzed here carry recessive
mutations and represent three complementation groups. Mutant A in complementation group I1 lacks IL-1 receptor-associated
kinase (IRAK), while the mutants in the other two groups are defective in unknown components that function upstream of IRAK.
Expression of exogenous IRAK in I1A cells (I1A-IRAK) restores their responsiveness to IL-1. Neither NFκB nor Jun kinase is
activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required
for both. To address the role of the kinase activity of IRAK in IL-1 signaling, its ATP binding site was mutated (K239A),
completely abolishing kinase activity. In transfected I1A cells, IRAK-K239A was still phosphorylated upon IL-1 stimulation
and, surprisingly, still complemented all the defects in the mutant cells. Therefore, IRAK must be phosphorylated by a different
kinase, and phospho-IRAK must play a role in IL-1-mediated signaling that does not require its kinase activity.
Activation of NF-kappaB as a consequence of signaling through the Toll and IL-1 receptors is a major element of innate immune responses. We report the identification and characterization of a novel intermediate in these signaling pathways that bridges TRAF6 to MEKK-1. This adapter protein, which we have named ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), is specific for the Toll/IL-1 pathways and is a regulator of MEKK-1 processing. Expression of wild-type ECSIT accelerates processing of MEKK-1, whereas a dominant-negative fragment of ECSIT blocks MEKK-1 processing and activation of NF-kappaB. These results indicate an important role for ECSIT in signaling to NF-kappaB and suggest that processing of MEKK-1 is required for its function in the Toll/IL-1 pathway.
Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.
The atypical protein kinase C (aPKC)-interacting protein, p62, has previously been shown to interact with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). Here we demonstrate that the inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this we show that p62 selectively interacts with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, we demonstrate that the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6.
MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH(2)-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1(-/-) mice are defective in eyelid closure, a TGFalpha-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1(-/-) mice are inhibited in their migration relative to MEKK1(+/+) fibroblasts. MEKK1 is required for JNK but not NF-kappaB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFalpha or IL-1 regulation of JNK or NF-kappaB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-kappaB in response to selected stress stimuli.
The innate immune system uses Toll family receptors to signal for the presence of microbes and initiate host defense. Bacterial lipoproteins (BLPs), which are expressed by all bacteria, are potent activators of Toll-like receptor-2 (TLR2). Here we show that the adaptor molecule, myeloid differentiation factor 88 (MyD88), mediates both apoptosis and nuclear factor-kappaB (NF-kappaB) activation by BLP-stimulated TLR2. Inhibition of the NF-kappaB pathway downstream of MyD88 potentiates apoptosis, indicating that these two pathways bifurcate at the level of MyD88. TLR2 signals for apoptosis through MyD88 via a pathway involving Fas-associated death domain protein (FADD) and caspase 8. Moreover, MyD88 binds FADD and is sufficient to induce apoptosis. These data indicate that TLR2 is a novel 'death receptor' that engages the apoptotic machinery without a conventional cytoplasmic death domain. Through TLR2, BLP induces the synthesis of the precursor of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). Interestingly, BLP also activates caspase 1 through TLR2, resulting in proteolysis and secretion of mature IL-1beta. These results indicate that caspase activation is an innate immune response to microbial pathogens, culminating in apoptosis and cytokine production.
IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-alpha or IL-12 to induce IFN-gamma. Here we show that in human NK and T cells IFN-alpha and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-alpha enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-alpha or IL-12 enhanced IL-18-induced NF-kappaB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-alpha and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.
Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.
MyD88 is an adaptor protein that is involved in interleukin-1 receptor (IL-1 R)- and Toll-like receptor (TLR)-induced activation of NF-kappaB [1-3]. It is composed of a C-terminal Toll/IL-IR homology (TIR) domain and an N-terminal death domain (DD), which mediate the interaction of MyD88 with the IL-1R/TLR and the IL-1R-associated kinase (IRAK), respectively. The interaction of MyD88 with IRAK triggers IRAK phosphorylation, which is essential for its activation and downstream signaling ability [4, 5]. Both domains of MyD88 are separated by a small intermediate domain (ID) of unknown function. Here, we report the identification of a splice variant of MyD88, termed MyD88s, which encodes for a protein lacking the ID. MyD88s is mainly expressed in the spleen and can be induced in monocytes upon LIPS treatment. Although MyD88s still binds the IL-1R and IRAK, it is defective in its ability to induce IRAK phosphorylation and NF-kappaB activation. In contrast, MyD88s behaves as a dominant-negative inhibitor of IL-1- and LPS-, but not TNF-induced, NF-kappaB activation. These results implicate the ID of MyD88 in the phosphorylation of IRAK. Moreover, the regulated expression and antagonistic activity of MyD88s suggest an important role for alternative splicing of MyD88 in the regulation of the cellular response to IL-1 and LPS.
IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-α or IL-12 to induce IFN-γ. Here we show that in human NK and T cells IFN-α and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-α enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-α or IL-12 enhanced IL-18-induced NF-κB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-α and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.
TRAF6 is a signal transducer that activates IκB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS)1-4. IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and INK.
Recent progress in research into programmed cell death has resulted in the identification of the principal protein domains involved in this process. The evolution of many of these domains can be traced back in evolution to unicellular eukaryotes or even bacteria, where the domains appear to be involved in other regulatory functions. Cell-death systems in animals and plants share several conserved domains, in particular the family of apoptotic ATPases; this allows us to suggest a plausible, even if still incomplete, scenario for the evolution of apoptosis.
Differentiation inducible leukemic as well as normal myeloid precursors treated with physiological myeloid differentiation inducer have been used to explore the immediate early genetic response of cells to terminal differentiation and growth arrest stimuli. cDNA clones of 12 distinct genes, referred to as MyD genes, which are activated in the absence of protein synthesis following induction of myeloid differentiation and growth arrest have been isolated. Sequence analysis of both ends of MyD cDNA clones, and analysis of MyD gene expression following induced differentiation of M1D+ and normal myeloid precursors, has shown that the immediate early genetic response of myeloid cells to the induction of terminal differentiation is complex. This complex response involves a variety of genes, some of which are known and others unknown, including: transient induction of ICAM-1, a gene encoding for a ligand to a cell surface adhesion receptor; stable induction of Jun-B, a gene encoding for a nuclear transcription factor; and increased expression of histone genes which encode for terminal differentiation histone variants. These findings demonstrate that terminal differentiation and growth arrest immediate early response genes encode for at least three distinct types of gene products, which may play a role to reprogram the transcriptional activity of proliferating and non-differentiated cells towards their conversion into terminally differentiated nonproliferating cells.
Loss of maternal function of the tube gene disrupts a signaling pathway required for pattern formation in Drosophila, causing cells throughout the embryo to adopt the fate normally reserved for those at the dorsal surface. Here we demonstrate that tube mutations also have a zygotic effect on pupal morphology and that this phenotype is shared by mutations in Toll and pelle, two genes with apparent intracellular roles in determining dorsoventral polarity. We then describe the isolation of a functionally full-length tube cDNA identified in a phenotypic rescue assay. The tube mRNA is expressed maximally early in embryogenesis and again late in larval development, corresponding to required periods of tube activity as defined by distinct maternal and zygotic loss-of-function phenotypes in tube mutants. Sequence analysis of the cDNA indicates that the tube protein contains five copies of an eight-residue motif and shares no significant sequence similarity with known proteins. These results suggest that tube represents a class of protein active in signal transduction at two stages of development.
To better understand the immediate early genetic response of myeloid cells to terminal differentiation and growth inhibitory stimuli, complementary DNA clones of myeloid differentiation primary response (MyD) genes have recently been isolated. In this study, a set of known (junB, c-jun, ICAM-1, H1(0), and H3.3 histone variants) and novel (MyD88, MyD116) MyD genes were used as immediate early molecular markers to further dissect the primary genetic response of myeloid cells to various differentiation and growth inhibitory stimuli. Expression of all of these MyD genes was highly induced in autonomously replicating differentiation inducible M1D+ myeloblasts following induction of terminal differentiation and growth inhibition by interleukin 6. Expression of all MyD genes except MyD88 was induced upon inhibition of M1D+ cell growth and induction of early, but not late, differentiation markers by interleukin 1 and lipopolysaccharide. In sharp contrast, only expression of H1(0) and H3.3 histone variants was increased following inhibition of M1D+ cell growth by interferon beta or gamma, which did not induce any differentiation associated properties. No increase in the expression of any of these MyD genes was seen in a clone of WEHI-3B D- myelomonocytic cells following stimulation with interleukin 6, which neither induced it for differentiation nor inhibited its growth. 12-O-Tetradecanoylphorbol-13-acetate, known to be a potent inducer of jun expression in many cell types, failed to induce high or stable expression of junB and c-jun in M1D+ cells, where it did not induce differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
We report here the full length cDNA sequence and the deduced amino-acid sequence of MyD88, a novel myeloid differentiation primary response gene activated in M1D+ myeloid precursors, following induction of terminal differentiation and growth arrest by IL6. Detectable levels of MyD88 RNA were observed in myeloid precursor enriched murine bone-marrow, but not in several other non-myeloid murine tissues.
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Within the Drosophila embryo, tube and the protein kinase pelle transduce an intracellular signal generated by the transmembrane receptor Toll. This signal directs import of the rel-related protein dorsal into ventral and ventrolateral nuclei, thereby establishing dorsoventral polarity. We show by immunolocalization that tube protein associates with the plasma membrane during interphase. We also find that tube sequences required for signaling interact with pelle in a yeast two-hybrid assay. We demonstrate that fusion of the pelle catalytic domain to the transmembrane receptor torso is sufficient to induce ventral fates; this activity is independent of Toll or tube. Lastly, we find that fusion of the tube protein to torso also induces ventral fates, but only in the presence of functional pelle. We propose a model wherein tube activates pelle by recruiting it to the plasma membrane, thereby propagating the axis-determining signal.
The interleukin-1 receptor in mammals and the product of the Toll gene in Drosophila are related transmembrane receptors, involved in the activation of transcription factors of the rel family. Whereas the interleukin-1 receptor mediates the effects of interleukin-1 in the immune system, Toll is part of the system that determines dorsoventral polarity in the Drosophila embryo, although Toll may also have a function in the immune response in the fly. Here, I demonstrate that the open reading frame of MyD88, a gene induced in myeloid differentiation, is related to the cytoplasmic domains of the interleukin-1 receptor and the Toll gene product. The three related proteins define a family of signal transmitters, the original function of which may be to mediate responses in the immune system.
MyD88 was first characterized as a myeloid differentiation primary response gene in mice, activated in M1 myeloleukemic cells following interleukin-6 (IL-6) induced growth arrest and terminal differentiation. Analysis of expressed sequence tags (ESTs) from activated dendritic cell libraries led to the indentification of cDNAs encoding the human homolog (hMyD88). The original description of MyD88 as a 243 aa protein may reflect a truncated mouse cDNA since the 2682 nt hMyD88 cDNA predicts a 296 aa cytoplasmic protein. Consistent with this proposal is the detection of a 33 kDa protein in human heart, kidney and liver tissue. The expression pattern of MyD88 is also more widespread than originally believed: a 2.6 kb hMyD88 mRNA species was found to be constitutively expressed in many adult human tissues; in addition MyD88 expression was observed in monocyte, T, B, NK and dendritic cells. The MyD88 protein has a modular structure composed of an N-terminal 'death domain' (DD) similar to the intracellular segments of TNF receptor 1 (TNFR1) and FAS and a C-terminal region related to the signaling domains of vertebrate interleukin-1 receptors (IL-1R) and the Drosophila morphogen Toll. This intriguing structural framework may endow MyD88 with unique signaling capabilities.
The Toll-Dorsal pathway in Drosophila and the interleukin-1 receptor (IL-1R)-NF-kappa B pathway in mammals are homologous signal transduction pathways that mediate several different biological responses. In Drosophila, genetic analysis of dorsal-ventral patterning of the embryo has defined the series of genes that mediate the Toll-Dorsal pathway. Binding of extracellular ligand activates the transmembrane receptor Toll, which requires the novel protein Tube to activate the cytoplasmic serine/threonine kinase Pelle. Pelle activity controls the degradation of the Cactus protein, which is present in a cytoplasmic complex with the Dorsal protein. Once Cactus is degraded in response to signal, Dorsal is free to move into the nucleus where it regulates transcription of specific target genes. The Toll, tube, pelle, cactus, and dorsal genes also appear to be involved in Drosophila immune response. Because the IL-1R-NF-kappa B pathway plays a role in vertebrate innate immunity and because plant homologues of the Toll-Dorsal pathway are important in plant disease resistance, it is likely that this pathway arose before the divergence of plants and animals as a defense against pathogens.
Murine MyD88, an RNA with homology both to the interleukin-1 receptor signaling domain and to 'death-domains', is rapidly upregulated during differentiation of the myeloleukemic cell line M1. We have cloned the human homologue of murine MyD88 and re-evaluated the murine sequence. The open reading frame for both species encodes a 296 amino acid protein, which for murine MyD88 is 53 amino acids longer than originally published. Human MyD88 cDNA is encoded by 5 exons, and maps to chromosome 3p21.3-p22 by fluorescence in situ hybridization (FISH). Overexpression of the death domain region leads to transcriptional activation of the IL-8 promoter.
The myeloid differentiation (MyD) marker MyD88 was initially characterized as a primary response gene, upregulated in mouse M1 myeloleukemic cells in response to differentiation induced by interleukin-6. Subsequent analysis revealed that MyD88 possesses a unique modular structure, which consists of an N-terminal "death domain," similar to the intracellular segments of TNF receptor 1 and Fas, and a C-terminal region related to the cytoplasmic domains of the Drosophila morphogen Toll and vertebrate interleukin-1 receptors. In this report we describe the cloning and gene structure of mouse MyD88. The complete coding sequence of mouse MyD88 spans five exons, with the first exon encoding the complete death domain. Zooblot analysis revealed that MyD88 is an evolutionarily conserved gene. MyD88 was localized to the distal region of mouse chromosome 9 by interspecific backcross mapping. The human homolog (hMyD88) was mapped to chromosome 3p22-p21.3 by PCR analysis of a human chromosome 3 somatic cell hybrid mapping panel. Northern blot analysis revealed widespread expression of MyD88 in many adult mouse tissues, and RT-PCR studies detected MyD88 mRNA in T and B cell lines and differentiating embryonic stem cells. The broad expression pattern demonstrates that mouse MyD88 expression is not restricted to cells of myeloid lineage as was originally believed.
The interleukin-1 receptor (IL-1R) signaling pathway leads to nuclear factor kappa B (NF-kappaB) activation in mammals and is similar to the Toll pathway in Drosophila: the IL-1R-associated kinase (IRAK) is homologous to Pelle. Two additional proximal mediators were identified that are required for IL-1R-induced NF-kappaB activation: IRAK-2, a Pelle family member, and MyD88, a death domain-containing adapter molecule. Both associate with the IL-1R signaling complex. Dominant negative forms of either attenuate IL-1R-mediated NF-kappaB activation. Therefore, IRAK-2 and MyD88 may provide additional therapeutic targets for inhibiting IL-1-induced inflammation.
IL-1 is a proinflammatory cytokine that signals through a receptor complex of two different transmembrane chains to generate multiple cellular responses, including activation of the transcription factor NF-kappaB. Here we show that MyD88, a previously described protein of unknown function, is recruited to the IL-1 receptor complex following IL-1 stimulation. MyD88 binds to both IRAK (IL-1 receptor-associated kinase) and the heterocomplex (the signaling complex) of the two receptor chains and thereby mediates the association of IRAK with the receptor. Ectopic expression of MyD88 or its death domain-containing N-terminus activates NF-kappaB. The C-terminus of MyD88 interacts with the IL-1 receptor and blocks NF-kappaB activation induced by IL-1, but not by TNF. Thus, MyD88 plays the same role in IL-1 signaling as TRADD and Tube do in TNF and Toll pathways, respectively: it couples a serine/threonine protein kinase to the receptor complex.
MyD88, originally isolated as a myeloid differentiation primary response gene, is shown to act as an adaptor in interleukin-1 (IL-1) signaling by interacting with both the IL-1 receptor complex and IL-1 receptor-associated kinase (IRAK). Mice generated by gene targeting to lack MyD88 have defects in T cell proliferation as well as induction of acute phase proteins and cytokines in response to IL-1. Increases in interferon-gamma production and natural killer cell activity in response to IL-18 are abrogated. In vivo Th1 response is also impaired. Furthermore, IL-18-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK) is blocked in MyD88-/- Th1-developing cells. Taken together, these results demonstrate that MyD88 is a critical component in the signaling cascade that is mediated by IL-1 receptor as well as IL-18 receptor.
Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-kappaB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-kappaB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-gamma response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.
Toll and Toll-related proteins play an important role in antibacterial innate immunity in insect, plants, and mammals. We present the first comprehensive phylogenetic analyses of Toll-related genes from both insects and mammals. Drosophila melanogaster contains Toll and a highly homologous gene, Tehao. The protein, Dm Tehao, comprises 795 amino acid residues and its cytoplasmic domain shares a striking 61% identity with Dm Toll. Two Toll homologues were found in another dipteran of medical importance, Anopheles gambiae, a vector for human malaria. One Toll-like gene each was identified from Aedes aegypti and Glossina palpalis palpalis, vectors for yellow fever and trypanosomiasis, respectively. Phylogenetic analyses revealed separate clustering of Toll and related proteins from insects and mammals, suggesting independent evolution of the Toll family of proteins and of innate immunity in arthropods and vertebrates. These results also provide new avenues to understanding the function of Toll proteins in insect innate immunity against bacteria, fungi, and protozoans.
Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFkappaB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFkappaB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.
Interleukin-1 (IL-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of IL-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (IL-1Rs), IL-1RI and IL-1RAcP, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.
The TAK1 MAPKKK mediates activation of JNK and NF-KB in the IL-1-activated signaling pathway. Here we report the identification of TAB2, a novel intermediate in the IL-1 pathway that functionally links TAK1 to TRAF6. Expression of TAB2 induces JNK and NF-kappaB activation, whereas a dominant-negative mutant TAB2 impairs their activation by IL-1. IL-1 stimulates translocation of TAB2 from the membrane to the cytosol where it mediates the IL-1-dependent association of TAK1 with TRAF6. These results define TAB2 as an adaptor linking TAK1 and TRAF6 and as a mediator of TAK1 activation in the IL-1 signaling pathway.
The innate immune system evolved to recognize conserved microbial products, termed pathogen-associated molecular patterns (PAMPs), which are invariant among diverse groups of microorganisms. PAMPs are recognized by a set of germ-line encoded pattern recognition receptors (PRRs). Among the best characterized PAMPs are bacterial lipopolysaccharide (LPS), peptidoglycan (PGN), mannans, and other constituents of bacterial and fungal cell walls, as well as bacterial DNA. Recognition of bacterial DNA is the most enigmatic of these, as it depends on a particular sequence motif, called the CpG motif, in which an unmethylated CpG present in a particular sequence context accounts for a potent immunostimulatory activity of CpG DNA. Receptor(s) of the innate immune system that mediate recognition of CpG DNA are currently unknown. Here, we report that recognition of CpG DNA requires MyD88, an adaptor protein involved in signal transduction by the Toll-like receptors (TLRs), essential components of innate immune recognition in both Drosophila and mammals [1,2]. Signaling induced by CpG DNA was found to be unaffected in cells deficient in TLR2 or TLR4, suggesting that some other member of the Toll family mediates recognition of bacterial DNA.
We describe three novel genes, encoding members of the Toll-like receptor (Tlr) family (TLR7, TLR8, and TLR9). These Tlr family members, unlike others reported to date, were identified within a genomic database. TLR7 and TLR8 each have three exons, two of which have coding function, and lie in close proximity to one another at Xp22, alongside a pseudogene. The remaining gene (TLR9) resides at 3p21.3 (in linkage with the MyD88 gene), and is expressed in at least two splice forms, one of which is monoexonic and one of which is biexonic, the latter encoding a protein with 57 additional amino acids at the N-terminus. The novel Tlrs comprise a cluster as nearest phylogenetic neighbors. Combining all sequence data related to Toll-like receptors, we have drawn several inferences concerning the phylogeny of vertebrate and invertebrate Tlrs. According to our best estimates, mammalian TLRs 1 and 6 diverged from a common mammalian ancestral gene 95 million years ago. TLR4, which encodes the endotoxin sensor in present-day mammals, emerged as a distinct entity 180 million years ago. TLRs 3 and 5 diverged from a common ancestral gene approximately 150 million years ago, as did Tlr7 and Tlr8. Very likely, fewer Tlrs existed during early vertebrate evolution: at most three or four were transmitted with the primordial vertebrate line. Phylogenetic data that we have adduced in the course of this work also suggest the existence of a Drosophila equivalent of MyD88, and indicate that the plasma membrane protein SIGIRR is close functional relative of MyD88 in mammals. Finally, a single present-day representative of the Toll-like proteins in Drosophila has striking cytoplasmic domain homology to mammalian Tlrs within the cluster that embraces TLRs 1, 2, 4, and 6. This would suggest that an ancestral (pre-vertebrate) Tlr may have adopted a pro-inflammatory function 500 million years ago.
The Toll/Dorsal pathway regulates dorsoventral axis formation in the Drosophila embryo. We had previously obtained evidence that a homologous pathway exists in Xenopus, however, its role during normal frog development had not been established. Here we report the cloning of Xenopus MyD88 (XMyD88), whose mammalian homologs are adaptor proteins linking Toll/IL-1 receptors and IRAK kinases. We show that in the frog embryo overexpression of a dominant-negative form of XMyD88 blocked Toll receptor activity, specifically inhibited axis formation and reduced expression of pivotal organizer genes. The observed stage-dependency of interference suggests a function for maternal XMyD88 soon after fertilization. We conclude that XMyD88 activity is required for normal Spemann organizer formation, implying an essential role for maternal Toll/IL-1 receptors in Xenopus axis formation.
TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.
Toll-like receptors (TLRs) and the interleukin-1 receptor superfamily (IL-1Rs) are integral to both innate and adaptive immunity for host defence. These receptors share a conserved cytoplasmic domain, known as the TIR domain. A single-point mutation in the TIR domain of murine TLR4 (Pro712His, the Lps(d) mutation) abolishes the host immune response to lipopolysaccharide (LPS), and mutation of the equivalent residue in TLR2, Pro681His, disrupts signal transduction in response to stimulation by yeast and gram-positive bacteria. Here we report the crystal structures of the TIR domains of human TLR1 and TLR2 and of the Pro681His mutant of TLR2. The structures have a large conserved surface patch that also contains the site of the Lps(d) mutation. Mutagenesis and functional studies confirm that residues in this surface patch are crucial for receptor signalling. The Lps(d) mutation does not disturb the structure of the TIR domain itself. Instead, structural and functional studies indicate that the conserved surface patch may mediate interactions with the down-stream MyD88 adapter molecule, and that the Lps(d) mutation may abolish receptor signalling by disrupting this recruitment.
Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor-kappa B (NF-kappa B) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain. Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF-kappa B transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of I kappa B alpha degradation. Thus Rac1 controls a second, I kappa B-independent, pathway to NF-kappa B activation and is essential in innate immune cell signaling via TLR2.