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NLR, the nucleotide-binding domain leucine-rich repeat containing gene family
Abstract
The NLR (nucleotide-binding domain leucine-rich repeat containing) family is found in plants and animals, and serves as crucial regulators of inflammatory and innate immune response, though its functions are likely to extend greatly beyond innate immunity, and even beyond the immune system. This review discusses recent findings regarding the function of NLR proteins in the control of IL-1, NF-kappaB, and host response to pathogens including distinct forms of cell death. The review also covers recent advances regarding the biochemical nature of NLRs, its regulation by intracellular nucleotides and extracellular ATP, by the chaperone protein HSP90, and the ubiquitin ligase-associated protein SGT1. Its role in inflammation is linked to the formation of biochemical complexes such as the inflammasome, and its roles in cell death might be linked to the proposed formation of pyroptosome and necrosome.
... The NLR (nucleotide-binding domain leucine-rich repeat containing) proteins are a family of innate immune receptors that contribute to the regulation and induction of antimicrobial immune responses (Ye and Ting 2008). The genomic mining of evolutionary conserved regions with structural similarity and functional overlaps has led to the discovery of a large gene family encoding proteins with a characteristic arrangement of NLR. ...
... The genomic mining of evolutionary conserved regions with structural similarity and functional overlaps has led to the discovery of a large gene family encoding proteins with a characteristic arrangement of NLR. There is several number of NLR proteins in mammalian cells (Elinav et al. 2011;Ting et al. 2008). ...
... The NLRP3 is the key member of NLR proteins which actives in response to diverse stimuli. Both mtDNA and mitochondrial ROS (mtROS) directly induce the activation of NLRP3 inflammasome (Ye and Ting 2008). Mitochondrial dysfunction and programmed cell death lead to release of mtDNA into the cytosol. ...
Mitochondria dynamics have a pivotal role in many aspects of immune function. Viral infections affect mitochondrial dynamics and trigger the release of mitochondrial DNA (mtDNA) in host cells. Released mtDNA guides the immune response towards an inflammatory response against pathogens. In addition, circulating cell-free mtDNA (ccf-mtDNA) is considered an invaluable indicator for the prognosis and severity of infectious diseases. This study provides an overview of the role of mtDNA in the dynamics of the immune response to COVID-19. We focused on the possible roles of mtDNA in inducing the signaling pathways, and the inflammasome activation and regulation in SARS-CoV-2. Targeting mtDNA-related pathways can provide critical insights into therapeutic strategies for COVID-19.
... Co-immunoprecipitation (Co-IP) assays showed that Paxillin interacted with NLRP3, but not with ASC or pro-Caspase-1 (Fig. 1b). NLRP3 contains several prototypic domains, including a PYRIN domain, an NACHT domain, and seven LRR domains [19]. Paxillin was co-precipitated with NLRP3, NACHT, and LRR, but not with PYRIN (Fig. 1c). ...
... We thus performed membrane flotation assays on Optiprep gradients bottomloaded with lysate supernatants of TPA-differentiated THP-1 macrophages. Without treatments, NLRP3 floated from the bottom fractions (21)(22)(23)(24) into the membrane fractions (12)(13)(14)(15)(16)(17)(18)(19)(20), Paxillin floated from the bottom fractions (21)(22)(23)(24) into the membrane fractions (14)(15)(16)(17)(18)(19)(20), and P2X7R floated from the bottom fractions (21)(22)(23)(24) into the lighter fractions (15)(16)(17)(18)(19)(20), while Calnexin remained in the lighter membrane fraction (12)(13)(14)(15)(16)(17)(18) and Caspase-3 stayed in the bottom fractions (22-24) (Fig. 8b). Notably, after Triton X-100 treatment, NLRP3, Paxillin, Calnexin, and Caspase-3 remained in the bottom fractions (22)(23)(24), whereas P2X7R floated from the bottom fractions (22)(23)(24) into the lighter fractions (17-21) (Fig. 8c). ...
... We thus performed membrane flotation assays on Optiprep gradients bottomloaded with lysate supernatants of TPA-differentiated THP-1 macrophages. Without treatments, NLRP3 floated from the bottom fractions (21)(22)(23)(24) into the membrane fractions (12)(13)(14)(15)(16)(17)(18)(19)(20), Paxillin floated from the bottom fractions (21)(22)(23)(24) into the membrane fractions (14)(15)(16)(17)(18)(19)(20), and P2X7R floated from the bottom fractions (21)(22)(23)(24) into the lighter fractions (15)(16)(17)(18)(19)(20), while Calnexin remained in the lighter membrane fraction (12)(13)(14)(15)(16)(17)(18) and Caspase-3 stayed in the bottom fractions (22-24) (Fig. 8b). Notably, after Triton X-100 treatment, NLRP3, Paxillin, Calnexin, and Caspase-3 remained in the bottom fractions (22)(23)(24), whereas P2X7R floated from the bottom fractions (22)(23)(24) into the lighter fractions (17-21) (Fig. 8c). ...
Background
Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K⁺, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions.
Results
We show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K⁺ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages.
Conclusions
We have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.
... At the same time, we infected the PK-15 cells with SVA and used IgG, NLRP3, or SVA 3D as the IP primary antibody; we found 3D interacts with endogenous NLRP3 (Fig. 5B and Fig. S2B). NLRP3 consists of PYRIN, NACHT, and seven LRR domains (24). As shown in Fig. 5C to E and S2C-S2E, 3D directly interacted with the NACHT domain of NLRP3 but not LRR or PYRIN. ...
... Our research first identified the mechanisms of NLRP3 activation by SVA infection. As reported, RNA induces NF-k B activation through PRR recognition, which acts as "signal 1" to induce the pro-IL-1b and NLRP3 transcription (24). We found that SVA RNA can induce IL-1b production. ...
Inflammation refers to the response of the immune system to viral, bacterial, and fungal infections or other foreign particles in the body, which can involve the production of a wide array of soluble inflammatory mediators. The NLRP3 inflammasome is one of the best-characterized inflammasome leading to IL-1β production and maturation.
... NLRs are grouped into subfamilies based on their specific N-terminal protein-protein interaction domain: the caspase recruitment domain (CARD), the pyrin domain (PYD), the baculovirus inhibitor of apoptosis protein repeat (BIR) domain, or an acidic transactivation domain [1,2]. These N-terminal domains are involved in recruiting downstream effector molecules [2] and signal transduction. ...
... NLRs are grouped into subfamilies based on their specific N-terminal protein-protein interaction domain: the caspase recruitment domain (CARD), the pyrin domain (PYD), the baculovirus inhibitor of apoptosis protein repeat (BIR) domain, or an acidic transactivation domain [1,2]. These N-terminal domains are involved in recruiting downstream effector molecules [2] and signal transduction. Moreover, the N-terminal region of an additional subfamily, NLRX1, contains a mitochondrial targeting sequence that shares no homology to any other protein [3,4]. ...
NOD-like receptors (NLRs) are intracellular sensors of the innate immune system that recognize intracellular pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Little information exists regarding the incidence of positive selection in the evolution of NLRs of birds or the structural differences between bird and mammal NLRs. Evidence of positive selection was identified in four avian NLRs (NOD1, NLRC3, NLRC5, and NLRP3) using the maximum likelihood approach. These NLRs are under different selection pressures which is indicative of different evolution patterns. Analysis of these NLRs showed a lower percentage of codons under positive selection in the LRR domain than seen in the studies of Toll-like receptors (TLRs), suggesting that the LRR domain evolves differently between NLRs and TLRs. Modeling of human, chicken, mammalian, and avian ancestral NLRs revealed the existence of variable evolution patterns in protein structure that may be adaptively driven.
... The Nod-like receptor (NLR) family has multiple functions as signal sensors, including the regulation of inflammatory and the maintenance of homeostasis [10,11]. Among them, Nod-like receptor family CARD domain containing 3 (NLRC3) predominantly localizes in the cytoplasm, acting as a critical checkpoint to prevent inflammatory dysregulation [12]. ...
Acacetin, a flavonoid derived compound has been recognized for its diverse biological activities, such as anti-oxidative and anti-inflammatory effects. Acute lung injury (ALI) is a severe condition characterized by respiratory insufficiency and tissue damage, commonly triggered by pneumonia and severe sepsis. These conditions induce an inflammatory response via Toll-like receptor 4 (TLR4) signaling activation. This study explored acacetin's therapeutic potential against lipopolysaccharide (LPS) induced ALI in mice, focusing on its ability to modulate the NF-κB pathway via regulation of the Nod-like receptor family CARD domain containing 3 (NLRC3), a signal sensor that plays an important role in the regulation of inflammation and the maintenance of homeostasis. Our findings revealed that high-dose acacetin reduced the mortality rate of ALI mice, significantly ameliorated LPS-induced lung pathological changes, reduced lung edema, and decreased the expression of inflammatory mediators in lung tissues. This protective impact of acacetin appears to stem form its capacity to enhance NLRC3 expression, which, intern, can inhibit the activation of NF-κB and subsequently inhibit the production of inflammatory mediators. NLRC3 deficiency inhibits the protective effect of acacetin on ALI mice. Molecular docking also verified that acacetin tightly bound acacetin to NLRC3. Additionally, acacetin was found to influence macrophage recruitment dynamics via NLRC3, inhibiting the overactivation of NLRC3-NF-κB related pathways. Taken together, our results indicate that acacetin inhibited LPS-induced acute lung injury and macrophage overrecruitment to the lungs in mice by upregulating NLRC3.
... The Nod-like receptor (NLR) family has multiple functions as signal sensors, including the regulation of in ammatory and the maintenance of homeostasis [10,11]. As a member of NLR family, Nod-like receptor family CARD domain containing 3 (NLRC3) mainly exists in the cytoplasm and prevents the dysregulation of in ammatory response by acting as a checkpoint [12]. ...
Acute lung injury (ALI) is a severe condition characterized by respiratory insufficiency and tissue damage. Commonly caused by pneumonia and severe sepsis, which trigger an inflammatory response via Toll-like receptor 4 (TLR4) signaling activation. Nod-like receptor family CARD domain containing 3 (NLRC3), a member of the NLR family, modulates inflammation and immune responses by inhibiting NF-κB, activation in response to TLR4 activation. Dysregulation of NLRC3 has been linked to increased susceptibility to inflammatory diseases. In the context of ALI, overexpression of NLRC3 reduces lung inflammation, while its silencing exacerbates inflammation. Acacetin, a flavonoid from Agastache rugosa, exhibits anti-inflammatory properties and has been suggested to involve NLRC3 in its mechanism. Silencing NLRC3 abolishes the protective effect of acacetin on LPS-induced inflammation in macrophages. Moreover, NLRC3 negatively regulates TLR4 signaling, which is involved in lipopolysaccharide (LPS)-induced inflammation. Acacetin has been reported to inhibit TLR4 signaling in various cell types. Thus, acacetin's anti-inflammatory effects may be partly mediated by its modulation of NLRC3 expression and function. In this study, our objective was to investigate the potential targets and functional mechanisms of acacetin in combating ALI. We employed molecular docking technology to anticipate and authenticate the interaction between acacetin and NLRC3. The findings were subsequently validated using an ALI model and LPS-induced macrophage model.
... It acts as the first line of defense, detecting the virus and bacteria through pattern recognition receptors (PRRs) and activating intracellular signaling cascades that promote the expression of proinflammatory cytokines, type I interferons, and other antiviral proteins that all coordinate the elimination of pathogens and infected cells. PRRs include Toll-like receptors, RIG-I-like receptors, nucleotide-binding domain and leucine-rich repeat-containing receptors, and C-type lectin receptors (CLRs) [3][4][5] . Furthermore, STING (also known as MITA, ERIS, MPYS or TMEM173) [6][7][8][9] has been shown to control a sensing pathway that is essential for the innate immune response against double-stranded DNA (dsDNA) viruses. ...
Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.
... Oxidative stress is one of the important aspects of the molecular pathogenesis of AF (61) and is also caused by exposure to AF risk factors and mitochondrial dysfunction (62)(63)(64). The mt-ROS activates the NLRP3 inflammasome and Cfc-mtDNA (65,66). Oxidative stress and mitochondrial dysfunction are enhanced by Ca 2+ overload-induced atrial tachycardia. ...
: Atrial fibrillation (AF), considered the most common tachyarrhythmia, is associated with severe complications, such as ischemic stroke and heart failure. Mitochondria are organelles that supply cells energy by converting various forms of molecules into adenosine triphosphate. Mitochondria has a double-stranded, independent DNA (mtDNA), the copy number of which in peripheral blood is associated with the oxidative stress status in the patient. Oxidative stress is also regarded as a potential factor contributing to AF. This study reviews the association between the cell-free circulating mitochondrial DNA (Cfc-mtDNA) in peripheral blood and AF. We searched for studies on AF and mtDNA; four investigations were included. The three of these studies revealed that the level of Cfc-mtDNA in serum appears to be associated with AF, but there are no similar results about Cfc-mtDNA and Per-AF. In addition, the age and gender of patients do not affect Cfc-mtDNA levels. However, further research is necessary in the future.
... In the nervous system, the microglial cells react to these signals, propagate and switch to different cellular states depending on the types of signals [29][30][31] . NOD-like receptor proteins (NLRPs) are one of the forms of sensors in the cell membrane that are activated upon ligand binding [32][33][34] . The NLRP3 in ammasome complex has been associated with in ammatory responses in the brain and inhibition or genetic deletion attenuates neuroin ammation and limits the extent of injury [35][36][37] . ...
Serum amyloid A (SAA) proteins increase dramatically in the blood following inflammation. Recently, SAAs are increased in humans following stroke and in ischemic animal models. However, the impact of SAAs on whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of SAA and SAA signaling in the ischemic brain. Wildtype and SAA deficient mice were exposed to middle cerebral artery occlusion and reperfusion, examined for the impact of infarct volumes, behavioral changes, inflammatory markers, TUNEL staining, and BBB changes. The underlying mechanisms were investigated using SAA deficient mice, transgenic mice and viral vectors. SAA levels were significantly increase following MCAo and mice deficient in SAAs showed reduced infarct volumes and improved behavioral outcomes. SAA deficient mice showed a reduction in TUNEL staining, inflammation and decreased glial activation. Mice lacking acute phase SAAs demonstrated a reduction in expression of the NLRP3 inflammasome and SAA/NLRP3 KO mice showed improvement. Restoration of SAA expression via SAA tg mice or adenoviral expression reestablished the detrimental effects of SAA. A reduction in BBB permeability was seen in the SAA KO mice and anti-SAA antibody treatment reduced the effects on ischemic injury. SAA signaling plays a critical role in regulating NLRP3-induced inflammation and glial activation in the ischemic brain. Blocking this signal will be a promising approach for treating ischemic stroke.
... Contrairement aux TLR liés à la membrane, qui détectent les PAMPs à la surface des cellules ou dans les endosomes, les NLR reconnaissent les molécules microbiennes dans le cytosol de l'hôte [419,420]. La famille NLR est composée de 23 membres chez l'homme, alors que le génome de la souris contient au moins 34 gènes de NLR [421]. Des homologues des NLR sont présents dans les plantes (gènes R) et les animaux, y compris des organismes phylogénétiquement plus primitifs tels que le poisson zèbre et l'oursin, bien qu'ils ne soient pas retrouvés chez les insectes ou les vers [422,423]. ...
Clostrioides difficile (C. difficile) est une cause majeure de diarrhée et de colites nosocomiales. Les infections à C. difficile (ICD) induisent une inflammation intestinale importante avec un taux de mortalité élevé. Les flagelles de C. difficile, pourraient jouer un rôle dans la pathogenèse en contribuant à la réponse inflammatoire de l'hôte et aux lésions de la muqueuse. En effet, la flagelline FliC de C. difficile interagit avec le récepteur de la réponse immunitaire innée TLR5 pour activer les voies de signalisation pro-inflammatoires de NF-kB et des MAPKs. Les flagelles des agents pathogènes intracellulaires peuvent activer la voie de l'inflammasome de la protéine 4 contenant le domaine CARD de la famille NLR (NLRC4). Notre objectif était d'étudier le rôle potentiel des flagelles de C. difficile sur l'activation de l'inflammasome NLRC4 in vitro. Nous avons montré que FliC de C. difficile, bactérie extracellulaire, est internalisée dans les cellules épithéliales et active l'inflammasome NLRC4. La microscopie confocale a montré l'internalisation de la GFP-FliC recombinante dans la lignée cellulaire intestinale et sa colocalisation avec NLRC4 et la pro-caspase-1. De plus, FliC active l'inflammasome caspase-1/NLRC4 et induit le clivage de la pro-caspase-1 en deux sous-unités, p20 et p10, l'activation de la gasdermine D (GSDMD), une surexpression des gènes pro-inflammatoires liées à l'inflammasome (interleukine (IL)-1β, IL-18 et IL-33), et la synthèse de l'IL-18. En outre, nous avons démontré un rôle d'I-kappa-B kinase alpha (IKK-α) sur l'expression des gènes d'interleukines liés à l'inflammasome dépendant de FliC. Ces résultats mettent en évidence le rôle des flagelles de C. difficile dans l'induction d'une réponse inflammatoire intestinale, via la voie de l'inflammasome NLRC4.
... These two host proteins are required for R protein-mediated inflammasome activation. Moreover, the function of HSP90 is not limited to R protein family members (Ye and Ting, 2008). Shirasu (2009) suggested that SGT1 is a co-chaperone of HSP90 and a component of a chaperone complex. ...
The necrotrophic phytopathogen Rhizoctonia solani (R. solani) causes disease in many plant species. This fungal genome encodes abundant small cysteine-rich (SCR)-secreted proteins in R. solani that may induce pathogenesis. To test their molecular functions, we introduced 10 SCR-secreted protein genes from R. solani into tobacco leaves via agroinfiltration. Consequently, we identified RsMf8HN, a novel SCR protein that triggers cell death and an oxidative burst in tobacco. RsMf8HN comprises 182 amino acids (aa), including a signal peptide (SP) of 17aa, and the protein has unique features: it is orthologous to an allergen protein Mal f 8 occurring in Malassezia species, and possesses a high glycine and serine content. RsMf8HN is coded in a genomic location along with its paralogues and a few other effector candidates. The elicitation of plant immunity by RsMf8HN was dependent on HSP90 and SGT1. RsMf8HN was translocated to multiple locations within the host cells: i.e., nuclei, chloroplasts, and plasma membranes. We confirmed the occurrence of in vivo cross-interactions of RsMf8HN with a rice molecule, the heavy metal-associated isoprenylated plant protein OsHIPP28, which is a protein related to the disease susceptibility factor Pi21. In summary, our results suggest that RsMf8HN is a potential effector that enables necrotrophic phytopathogens to interfere with host plant immunity.
... They play pivotal roles in response to stress signals and microbial molecules, which can activate inflammatory caspases that then induce cytokine production (IL-1β, IL-18) and pyroptotic cell death to enable certain inflammatory responses (Christgen et al., 2020). Recently, inflammasomes have been recognized for their functions as tumor suppressors or tumor promoters in the progression, initiation and regression of certain cancer types (Karki et al., 2017;Ye et al., 2022). Different inflammasomes may play diverse roles within the same cancer type. ...
Background
NOD-like receptor proteins (NLRs) are essential innate pattern recognition receptors that play fundamental roles in inflammation, and they have been associated with many cancers. NLRP13 (NLR Family Pyrin Domain Containing 13) belongs to the NLRP protein family, but its biological function is not as well defined as other related proteins in this family.
Methods and results
The expression profile and CpG-aggregated methylation status of the NLRP13 gene were determined using GENT2 and TCGA online platforms. Samples from patients with different tumor stages were used to evaluate the promoter methylation status of NLRP13 in cancer versus normal tissues in a tumor stage-dependent manner by using UALCAN server. We further studied NLRP13 expression in response to inflammatory and hypomethylating agents in two distinct cancer cell lines, HT-29 (human colon adenocarcinoma) and C6 (rat glioma cells), following the treatment with LPS and/or a hypomethylating agent, decitabine, to reveal NLRP13 methylation profiles in these cell lines.
Conclusions
Using bioinformatics tools, we found that NLRP13 expression is higher in healthy breast, head and neck, prostate tissues compared to tumor tissues, whereas its expression is significantly higher in colorectal adenocarcinoma tissue than in normal colon tissue. We also found that NLRP13 gene is mostly hypomethylated in cancer tissues compared to normal tissues, and its high expression is linked with worse overall survival depending on cancer type. Based on data from UALCAN database, NLRP13 promoter methylation is similarly decreased in many cancer patients depending on individual cancer stages. Protein-protein interactions (PPIs) data indicated that NLRP13 showed a confidence interaction with MEFV, NLRC4 and CASP1 proteins, and gene ontology terms associated with functional partners of NLRP13 were also identified in the current study. Our results further showed that NLRP13 expression is regulated by DNA methylation, and treatment with decitabine might effect NLRP13-mediated inflammatory responses in cancer since LPS-induced pro-inflammatory responses specified by IL-1β, IL-18 and caspase-1 levels change in certain cases in cancer cell lines following the pretreatment with this epigenetic modulator. Our study is the first to provide evidence for the differential expression and epigenetic regulation of the NLPR13 in multiple cancer types.
... Inflammasomes are multiprotein complexes, assembly of which is triggered by both microbial and endogenous danger signals and results in activation and cleavage of caspase-1. When activated, caspase-1 cleaves gasdermin D (23) to generate an N-terminal cleavage product that triggers inflammatory cell death (pyroptosis) and the release of pro-inflammatory cytokines interleukin-1b (IL-1b) and IL-18 (24). Excessive activation of the NLRP3 inflammasome has been shown to lead to diabetes, atherosclerosis, and obesity-induced insulin resistance (25). ...
Background
About 30% of women entering pregnancy in the US are obese. We have previously reported mitochondrial dysregulation and increased inflammation in the placentae of obese women. Vitamin D (VitD) is a major player in calcium uptake and was shown to modulate mitochondrial respiration and the immune/inflammation system. Studies show decreased VitD levels in obese individuals; however, the effect of maternal obesity on VitD metabolism and its association with placental function remains understudied.
Methods
Maternal and cord blood plasma and placental samples were collected upon C-section from normal-weight (NW, body mass index [BMI]<25) and obese (OB, BMI>30) women with uncomplicated pregnancies at term. We measured 25(OH)D 3 (calcidiol) levels in maternal and cord blood plasma using ELISA. We assessed the expression of CYP27B1, an activator of calcidiol, and Vitamin D receptor (VDR) in placentae from NW and OB, and women with gestational diabetes and preeclampsia. In addition, we examined the effects of VitD supplementation on mitochondrial function and inflammation in trophoblasts from NW and OB, using the Seahorse Bioanalyzer and Western blot, respectively.
Results
Vitamin D levels in blood from OB but not NW women and in cord blood from babies born to NW and OB women showed a significant inverse correlation with maternal pre-pregnancy BMI (r=-0.50, p <0.1 and r=-0.55, p =0.004 respectively). Cord plasma VitD levels showed a positive correlation with placental efficiency, i.e., the ratio between fetal and placental weight, as well as with maternal blood VitD levels (r=0.69 and 0.83 respectively, p <0.00). While we found no changes in CYP27B1 in OB vs. NW women, VDR expression were decreased by 50% ( p <0.03) independent of fetal sex. No changes in VDR expression relative to BMI-matched controls were observed in the placentae of women with gestational diabetes or preeclampsia. Cytotrophoblasts isolated from placentae of OB women showed a dose-dependent increase in VDR expression after 24-hour treatment with calcitriol (10 nM and 100 nM), an active form of VitD. Trophoblasts isolated from OB women and treated with calcitriol improved mitochondrial respiration ( p <0.05). We also found a two-fold increase in expression of the NLRP3 inflammasome and the pro-inflammatory cytokine IL-18 in trophoblasts isolated from placentae of OB women ( p <0.05), with IL-18 expression being reversed by calcitriol treatment (100 nM).
Conclusions
We show that VitD deficiency is at least partially responsible for mitochondrial dysfunction and increased inflammation in the placentae of obese women. Vitamin D supplementation could be beneficial in improving placental dysfunction seen in obese women.
... Both NOD1 and NOD2 might have played a role in recognizing periodontal pathogens, but the stimulatory activities of P. gingivalis are weaker than those of other periodontal pathogens [69]. In addition, the NLR family acts as an essential regulator of inflammatory and innate immune response, which can control IL-1, NF-κB, and host response to pathogens including distinct forms of cell death [70]. Depletion of NLRX1 can decrease F. nucleatum infection-activated NLRP3 in gingival epithelial cells (GECs). ...
Periodontitis is an oral chronic inflammatory disease and may cause tooth loss in adults. Oral epithelial cells provide a barrier for bacteria and participate in the immune response. Fusobacterium nucleatum (F. nucleatum) is one of the common inhabitants of the oral cavity and has been identified as a potential etiologic bacterial agent of oral diseases, such as periodontitis and oral carcinomas. F. nucleatum has been shown to be of importance in the development of diverse human cancers. In the dental biofilm, it exhibits a structural role as a bridging organism, connecting primary colonizers to the largely anaerobic secondary colonizers. It expresses adhesins and is able to induce host cell responses, including the upregulation of defensins and the release of chemokines and interleukins. Like other microorganisms, its detection is achieved through germline-encoded pattern-recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). By identification of the pathogenic mechanisms of F. nucleatum it will be possible to develop effective methods for the diagnosis, prevention, and treatment of diseases in which a F. nucleatum infection is involved. This review summarizes the recent progress in research targeting F. nucleatum and its impact on oral epithelial cells.
... 25 NLRCs contain the leucine-rich repeat (LRR), NACHT, and caspase activating and recruitment domain (CARD), whereas the LRR, NACHT and pyrin domain (PYD) domain exist in NLRPs. 26 ASC also contains PYD and CARD domain, and thus it can be recruited by NLRPs via PYD-PYD domain interaction, and ASC can further recruit and activate pro-caspase-1 through CARD domain. 27 NLRCs contain CARD domain, and thus they can directly recruit and activate pro-caspase-1 independent of ASC. ...
Pyroptosis is a form of programmed cell death, which is executed by gasdermin family proteins. Under the stimulation of pathogen- and/or damage-associated molecular patterns, pattern recognition receptors (PRRs) such as Nod like receptors could recruit apoptosis-associated speck-like protein containing a CARD (ASC) and pro-caspases to form inflammasomes and then activate caspases through various pathways. The activated caspases then cleave gasdermin family proteins, and N-terminal (NT) domains of gasdermins were released to form oligomeric pores, resulting in the increased membrane permeability, cell swelling, and final pyroptosis. During this process, caspases also promote the maturation and release of inflammatory cytokines such as IL-1β and IL-18, thus pyroptosis is also named inflammatory cell death. Unlike numerous gasdermin family proteins in mammals, only gasdermin E (GSDME) has been identified in fish. GSDME in fish can be cleaved by caspase-a/-b to release its NT domain and induce pyroptosis. Studies indicated that pyroptosis in fish mainly depends on NLR family pyrin domain-containing 3 (NLRP3) inflammasome. ASC and different caspase proteins also were identified in different fish species. The influences of pathogenic microorganism infection and environmental pollutants on fish pyroptosis were studied in recent years. Considering that fish living environment is affected by multiple factors such as water salinity, temperature, oxygen supply, and highly fluctuating food supply, the in-depth research about fish pyroptosis will contribute to revealing the mechanism of pyroptosis during evolution.
... Nucleotide-binding leucine-rich repeat-containing receptors (NLR), or receptors that respond to damage-associated molecular patterns (DAMPs), produced during tissue injury. 14,15 The most widely studied NLRs are inflammasome-forming NLRs, among which inflammasome complexes (NLRP1, NLRP3, NLRC4, and AIM2) have been well identified in the process of inflammation. 16,17 Upon activating DAMPs, these NLRs promote the release of proinflammatory cytokines such as IL-1β and IL-18. ...
Background
Postoperative cognitive dysfunction (POCD) is associated with worsened prognosis especially in aged population. Clinical and animal studies suggested that electroacupuncture (EA) could improve POCD. However, the underlying mechanisms especially EA’s regulatory role of inflammasomes remain unclear.
Methods
The model of POCD was established by partial hepatectomy surgery in 18‐month mice with or without postoperative EA treatment to the Baihui acupoint (GV20) for 7 days. Cognitive functions were assessed by Morris water maze test, and proinflammatory cytokines IL‐1β and IL‐6 and microglia activity were assayed by qPCR, ELISA, or immunohistochemistry. Tight junction proteins, NLRP3 inflammasome and downstream proteins, and NF‐κB pathway proteins were evaluated by western blotting.
Results
EA markedly preserved cognitive dysfunctions in POCD mice, associated with the inhibition of neuroinflammation as evidenced by reduced microglial activation and decreased IL‐1β and IL‐6 levels in brain tissue. EA also preserved hippocampal neurons and tight junction proteins ZO‐1 and claudin 5. Mechanistically, the activation of NLRP3 inflammasome and NF‐κB was inhibited by EA, while NLRP3 activation abolished EA’s treatment effects on cognitive function.
Conclusion
EA alleviates POCD‐mediated cognitive dysfunction associated with ameliorated neuroinflammation. Mechanistically, EA’s treatment effects are dependent on NLRP3 inhibition.
... The PYD domain mediates the interaction with ASC, leading to inflammasome assembly, and the NOD domain has ATPase activity, which can promote the autooligomerization of NLRP3. The LRR domain is an essential part of NLRP3 inflammasome activation and interacts with ligands to regulate NLRP3 activity, but its specific function remains to be clarified (25)(26)(27)(28). NLRP3 can be activated in vivo by a variety of stimuli, such as reactive oxygen species (ROS), adenosine triphosphate (ATP), PM 2.5, bacterial, and fungal components (29)(30)(31). ...
Objective:
This paper briefly reviews the pathological characteristics and regulatory mechanism of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and summarizes the relationship between it and rheumatoid arthritis (RA) as a means to improve its therapeutic potential and clinical application.
Background:
RA is a systemic inflammatory disease with a high incidence rate. The early diagnosis and treatment of the disease is difficult, and the current treatment effect of most patients is not significant and accompanied by serious infection risk. Inflammation is an immune protective mechanism in the body. Inflammasome is an intracellular multi-body protein that stimulates the inflammatory response [inducing the release of pro-inflammatory cytokine interleukin (IL)-1β and IL-18] and promotes the death of thermophiles. The NLRP3 inflammatory bodies are assembled from NLRP3, apoptosis-associated speck-like protein (ASC), and pro-caspase-1. Previous studies have enriched our understanding of the activation mechanism of NLRP3 inflammasome, and animal model data suggests that it plays an important role in autoimmune diseases, including RA.
Methods:
Literatures about inflammation and RA were extensively reviewed to analyze and discuss.
Conclusions:
Especially, we focused on the role of NLRP3 inflammasome in the pathogenesis of RA and the potential of NLRP3 inflammasome or their derivatives in the treatment of RA, which enriched the treatment strategies of inflammatory diseases.
... Members of the nucleotide-binding and leucine-rich repeat (NB-LRR) protein family have been found to serve as crucial regulators of inflammatory and innate immune responses in animals and plants, respectively (Ye and Ting 2008). The majority of plant disease resistance (R) proteins conferring resistance to bacterial, fungal, oomycete or viral pathogens encode proteins belonging to the NB-LRR protein family (Dangl and Jones 2001). ...
Key message
Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves.
Abstract
To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a “Kranz-like” anatomy in leaves that includes decreased interveinal spacing close to that in C 4 plant species. As C 4 photosynthesis has evolved from C 3 photosynthesis independently in multiple lineages, the genes required to facilitate C 4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 ( CVS1 ), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.
... Plant disease resistance genes share common structural features among distantly related species of plants. The most common features are nucleotide binding site and the leucine-rich repeat sequence (NBS-LRR) domains (Ye and Ting 2008;Luo et al. 2012). Isolation and identification of disease resistance genes can be time-consuming and expensive, but it is more straightforward to isolate Sugar Tech (Jan-Feb 2022) 24(1):354-368 363 ...
Currently, genome analysis has become a routine component of molecular breeding of major crops. Today’s commercial sugarcane hybrids are the products of breeding over one hundred years with the starting clones produced by crossing two founding species (Saccharum officinarum and S. spontaneum) in India and Indonesia. Current sugarcane varieties have a highly complex and large genome with 100–130 chromosomes. Despite the complexity and size of the genome, considerable progress has recently been made in sugarcane genomics, including the sequencing of a haploid S. spontaneum, AP85-441 and sugarcane cultivar hybrids R570 and SP80-3280. AP85-441 genome is assembled to chromosome level and allele-defined. Significant progress also has been made in genetic research of important agronomic traits. Here, we outline these advances in order to provide a reference for future sugarcane genomics and genetics research. © 2021, The Author(s), under exclusive licence to Society for Sugar Research & Promotion.
... TLRs and NLRs are patternrecognition receptors E539 that recognize 2 types of ligands: pathogenassociated molecu lar patterns (PAMPs), linked to various microbes, and damageassociated molecular patterns (DAMPs), produced during tissuebased injury. [12][13][14] After microbial recognition, both TLRs and NLRs activate signalling pathways that lead to the formation of cytokines and chemokines. 15 PAMPs and DAMPs include proteins from bacterial mem branes, intracellular proteins (such as heatshock proteins) and protein products (such as adenosine triphosphate [ATP], urea and nucleic acid). ...
Background:
Abnormalities of inflammation have been implicated in the pathophysiology of depression and suicide, based on observations of increased levels of proinflammatory cytokines in the serum of people who were depressed and died by suicide. More recently, abnormalities in cytokines and innate immunity receptors such as toll-like receptors have also been observed in the postmortem brains of people who were depressed and died by suicide. In addition to toll-like receptors, another subfamily of innate immunity receptors known as NOD-like receptors containing pyrin (NLRPs) are the most widely present NOD-like receptors in the central nervous system. NLRPs also form inflammasomes that play an important role in brain function. We studied the role of NLRPs in depression and suicide.
Methods:
We determined the protein and mRNA expression of NLRP1, NLRP3 and NLRP6 and the components of their inflammasomes (i.e., adaptor molecule apoptosis-associated speck-like protein [ASC], caspase1, caspase3, interleukin [IL]-1β and IL-18) postmortem in the prefrontal cortex of people who were depressed and died by suicide, and in healthy controls. We determined mRNA levels using quantitative polymerase chain reaction, and we determined protein expression using Western blot immunolabelling.
Results:
We found that the protein and mRNA expression levels of NLRP1, NLRP3, NLRP6, caspase3 and ASC were significantly increased in people who were depressed and died by suicide compared to healthy controls.
Limitations:
Some people who were depressed and died by suicide were taking antidepressant medication at the time of their death.
Conclusion:
Similar to toll-like receptors, NLRP and its inflammasomes were upregulated in people who were depressed and died by suicide compared to healthy controls. Innate immunity receptors in general - and NLRPs and inflammasomes in particular - may play an important role in the pathophysiology of depression and suicide.
... Three PSGs (IP6K3, MAGIX and FAM149B1) are found to be associated with the nervous system, synapse formation and structural plasticity, as well as motor skills and coordination (Feng and Zhang 2009;Crocco et al. 2016;Shaheen et al. 2019). Three further PSGs (DUSP19, TNLG2B, and LRRC4) are related to the immune system (Ye and Ting 2008;Premzl 2016;Xie et al. 2020) and HEMK1 regulates methylation processes (Ishizawa et al. 2008). ...
Species of the mustelid subfamily Guloninae inhabit diverse habitats on multiple continents, and occupy a variety of ecological niches. They differ in feeding ecologies, reproductive strategies and morphological adaptations. To identify candidate loci associated with adaptations to their respective environments, we generated a de novo assembly of the tayra (Eira barbara), the earliest diverging species in the subfamily, and compared this with the genomes available for the wolverine (Gulo gulo) and the sable (Martes zibellina). Our comparative genomic analyses included searching for signs of positive selection, examining changes in gene family sizes, as well as searching for species-specific structural variants (SVs). Among candidate loci that appear to be associated with phenotypic traits, we observed many genes related to diet, body condition and reproduction. For the tayra, which has an atypical gulonine reproductive strategy of aseasonal breeding, we observe species-specific changes in many pregnancy-related genes. For the wolverine, a circumpolar hypercarnivore that must cope with seasonal food scarcity, we observed many specific changes in genes associated with diet and body condition. Despite restricting some of our analyses to single-copy orthologs present in all three study species, we observed many candidate loci that may be linked to species traits related to environment-specific challenges in their respective habitats.
... In the VIGS assays, cell death by RsSCR10 was dependent on Hsp90 (Figure 6), a host protein required for R protein-mediated inflammasome activation. However, the function of Hsp90 is not limited to R protein family members (Ye and Ting, 2008). Shirasu (2009) reported that SGT1 was a co-chaperone of Hsp90, and the two molecules form a chaperone complex. ...
The necrotrophic phytopathogen Rhizoctonia solani ( R. solani ) is a fungus that causes disease in a wide range of plant species. Fungal genomes encode abundant, small cysteine-rich (SCR) secreted proteins, and the probable importance of these to pathogenesis has been highlighted in various pathogens. However, there are currently no reports of an R. solani SCR-secreted protein with evidential elicitor activity. In this study, the molecular function of 10 SCR-secreted protein genes from R. solani was explored by agroinfiltration into Nicotiana benthamiana ( N. benthamiana ) leaves, and a novel SCR protein RsSCR10 was identified that triggered cell death and oxidative burst in tobacco. RsSCR10 comprises 84 amino acids, including a signal peptide (SP) of 19 amino acids that is necessary for RsSCR10 to induce tobacco cell death. Elicitation of cell death by RsSCR10 was dependent on Hsp90 but not on RAR1, proving its effector activity. Two cysteine residues have important effects on the function of RsSCR10 in inducing cell death. Furthermore, RsSCR10 showed cross-interaction with five rice molecules, and the inferred functions of these rice proteins suggest they are instrumental in how the host copes with adversity. Overall, this study demonstrates that RsSCR10 is a potential effector that has a critical role in R. solani AG1 IA-host interactions.
... The major PPRs, including TLRs, detect and capture pathogens on the cell surface or within endosomes, while NLRs are cytoplasmic receptors and detect their ligands in the cytosol, thereby providing another level of cell protection [5]. ...
The innate immune system plays a critical role in the early detection of pathogens, primarily by relying on pattern-recognition receptor (PRR) signaling molecules. Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic receptor that recognizes invading molecules and danger signals inside the cells. Recent studies highlight the importance of NOD2′s function in maintaining the homeostasis of human body microbiota and innate immune responses, including induction of proinflammatory cytokines, regulation of autophagy, modulation of endoplasmic reticulum (ER) stress, etc. In addition, there is extensive cross-talk between NOD2 and the Toll-like receptors that are so important in the induction and tuning of adaptive immunity. Polymorphisms of NOD2′s encoding gene are associated with several pathological conditions, highlighting NOD2′s functional importance. In this study, we summarize NOD2′s role in cellular signaling pathways and take a look at the possible consequences of common NOD2 polymorphisms on the structure and function of this receptor.
... Nucleotidebinding oligomerization domain (NOD)-like receptors (NLRs) are the vital constituents of the immune system, which are considered to play an important role in the development of inflammatory disorders. NLRs comprise of a class of proteins that primarily aid in the regulation of immune system (Ye and Ting 2008;Kanneganti et al. 2006;Sutterwala et al. 2006). NLRs induce inflammation by recognizing danger signals such as endogenous agents, which are formed during the inflammatory phase (Sutterwala et al. 2006;Mariathasan et al. 2006;Duewell et al. 2010). ...
Inflammasome activity plays a vital role in various non-microbial disease states correlated with prolonged inflammation. NLRP3 inflammasome function and IL-1β formation are augmented in obesity and several obesity-linked metabolic disorders (i.e. diabetes mellitus, hypertension, hepatic steatosis, cancer, arthritis, and sleep apnea). Also, several factors are associated with the progression of diseases viz. increased plasma glucose, fatty acids, and β-amyloid are augmented during obesity and activate NLRP3 inflammasome expression. Prolonged NLRP3 stimulation seems to play significant role in various disorders, though better knowledge of inflammasome regulation and action might result in improved therapeutic tactics. Numerous compounds that mitigate NLRP3 inflammasome expression and suppress its chief effector, IL-1β are presently studied in clinical phases as therapeutics to manage or prevent these common disorders. A deep research on the literature available till date for inflammasome in obesity was conducted using various medical sites like PubMed, HINARI, MEDLINE from the internet, and data was collected simultaneously. The present review aims to examine the prospects of inflammasome as a major progenitor in the progression of obesity via directing their role in regulating appetite.
... Invading pathogens are sensed by pattern recognition receptors (PRRs) in the host cell (1,2). PRRs include Toll-like receptors (3), RIG-I-like receptors (4), and nucleotide-binding domain and leucine-rich repeat-containing receptors (5), C-type lectin receptors (CLRs) (6). They bind microbial molecules such as CpG DNA, viral RNAs, and lipopolysaccharides. ...
DNA is present in the nucleus and mitochondria of eukaryotic cells. There are, however, certain instances in which DNA emerges in the cytosol. The two major sources of cytosolic DNA are self DNA that is leaked out from the nucleus or mitochondria, and non-self DNA from DNA viruses. The cytosolic DNA triggers the host immune response. Recent studies have identified two key molecules, cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING) in this immune response. STING is an endoplasmic reticulum (ER) protein. After STING binding to cGAMP, STING exits the ER and translocates to the Golgi, where STING triggers the type I interferon- and proinflammatory responses through the activation of interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB). STING also activates other cellular responses including cell senescence, autophagy, and cell death. In this review, we focus on emerging issues regarding the regulation of STING by membrane traffic, with a particular focus on the retrograde membrane traffic from the Golgi to the ER. The retrograde membrane traffic is recently shown by us and others to be critical for silencing the STING signaling pathway and the defect in this traffic underlies the pathogenesis of the COPA syndrome, a monogenic autoinflammatory disease caused by missense mutations of coatomer protein complex subunit α (COP-α).
... NACHT and WD Repeat Domain Containing proteins belong to immunity proteins [22]. Originally discovered in fish, Nwd1 had orthologs in rodents and human, too [23]. ...
(1) Background: Preoptic region of hypothalamus is responsible to control maternal behavior, which was hypothesized to be associated with gene expressional changes. (2) Methods: Transcriptome sequencing was first applied in the preoptic region of rat dams in comparison to a control group of mothers whose pups were taken away immediately after parturition and did not exhibit caring behavior 10 days later. (3) Results: Differentially expressed genes were found and validated by quantitative RT-PCR, among them NACHT and WD repeat domain containing 1 (Nwd1) is known to control androgen receptor (AR) protein levels. The distribution of Nwd1 mRNA and AR was similar in the preoptic area. Therefore, we focused on this steroid hormone receptor and found its reduced protein level in rat dams. To establish the function of AR in maternal behavior, its antagonist was administered intracerebroventricularly into mother rats and increased pup-directed behavior of the animals. (4) Conclusions: AR levels are suppressed in the preoptic area of mothers possibly mediated by altered Nwd1 expression in order to allow sustained high-level care for the pups. Thus, our study first implicated the AR in the control of maternal behaviors.
... The NLRP3 gene encodes for the protein NLRP3, which is part of the cytoplasmatic nucleotid-binding domain, a family member of the intracellular "NOD like" receptor (NLR) [39]. NLRP3 nucleates an intracellular multi-molecular complex, called the NLRP3 inflammasome [40]. ...
The cryopyrin-associated periodic syndromes (CAPS) are usually caused by heterozygous NLRP3 gene variants, resulting in excessive inflammasome activation with subsequent overproduction of interleukin (IL)-1β. The CAPS spectrum includes mild, moderate, and severe phenotypes. The mild phenotype is called familial cold autoinflammatory syndrome (FCAS), the moderate phenotype is also known as Muckle–Wells syndrome (MWS), and the neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic cutaneous articular syndrome (CINCA) describes the severe phenotype. The CAPS phenotypes display unspecific and unique clinical signs. Dermatologic, musculoskeletal, ocular, otologic, and neurologic disease symptoms combined with chronic systemic inflammation are characteristic. Nevertheless, making the CAPS diagnosis is challenging as several patients show a heterogeneous multi-system clinical presentation and the spectrum of genetic variants is growing. Somatic mosaicisms and low-penetrance variants lead to atypical clinical symptoms and disease courses. To avoid morbidity and to reduce mortality, early diagnosis is crucial, and a targeted anti-IL-1 therapy should be started as soon as possible. Furthermore, continuous and precise monitoring of disease activity, organ damage, and health-related quality of life is important. This review summarizes the current evidence in diagnosis and management of patients with CAPS.
... Inflammasomes are endowed with intracellular PRR receptors (or sensors) that recognize cell stress-linked DAMPs or pathogen-derived PAMPs. Every type of inflammasome has its own distinct receptors [200][201][202][203]. According to their N-terminus features, NLRs are classified into four subfamilies, i.e., NLRA, NLRB, NLRC, and NLRP. ...
Fibrillar aggregates and soluble oligomers of both Amyloid-β peptides (Aβs) and hyperphosphorylated Tau proteins (p-Tau-es), as well as a chronic neuroinflammation are the main drivers causing progressive neuronal losses and dementia in Alzheimer's disease (AD). However, the underlying pathogenetic mechanisms are still much disputed. Several endogenous neurotoxic ligands, including Aβs, and/or p-Tau-es activate innate immunity-related danger-sensing/pattern recognition receptors (PPRs) thereby advancing AD's neuroinflammation and progression. The major PRR families involved include scavenger, Toll-like, NOD-like, AIM2-like, RIG-like, and CLEC-2 receptors, plus the calcium-sensing receptor (CaSR). This quite intricate picture stresses the need to identify the pathogenetically topmost Aβ-activated PRR, whose signaling would trigger AD's three main drivers and their intra-brain spread. In theory, the candidate might belong to any PRR family. However, results of preclinical studies using in vitro nontumorigenic human cortical neurons and astrocytes and in vivo AD-model animals have started converging on the CaSR as the pathogenetically upmost PRR candidate. In fact, the CaSR binds both Ca 2+ and Aβs and promotes the spread of both Ca 2+ dyshomeostasis and AD's three main drivers, causing a progressive neurons' death. Since CaSR's negative allosteric modulators block all these effects, CaSR's candidacy for topmost pathogenetic PRR has assumed a growing therapeutic potential worth clinical testing.
... 178 Since Hsp90 can work as an immune sensor and assist antigen presentation, it may function in the same way in EBV infection. 177,[179][180][181][182] The function of HSP90 family on cell transformation during retrovirus infection Several HSPs functions as oncoproteins to promote cellular transformation. Hsp90 participates in the HTLV-1-induced cellular transformation. ...
Stress proteins (SPs) including heat-shock proteins (HSPs), RNA chaperones, and ER associated stress proteins are molecular chaperones essential for cellular homeostasis. The major functions of HSPs include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. Regarded as a double-edged sword, HSPs also cooperate with numerous viruses and cancer cells to promote their survival. RNA chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnRNPs), which are essential factors for manipulating both the functions and metabolisms of pre-mRNAs/hnRNAs transcribed by RNA polymerase II. hnRNPs involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, RNP assembly and stabilization, RNA export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. Dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., Parkinson’s diseases, Alzheimer disease), stroke and infectious diseases. In this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. As SPs also attract a great interest as potential antiviral targets (e.g., COVID-19), we also discuss the present progress and challenges in this area of HSP-based drug development, as well as with compounds already under clinical evaluation.
... Co-immunoprecipitation (Co-IP) assays revealed that Paxillin interacted with 88 NLRP3, but not with ASC or pro-Caspase-1 ( Fig 1B). NLRP3 contains several prototypic domains, 89 including a PYRIN domain, an NACHT domain, and seven LRR domains (Ye and Ting, 2008). 90 Paxillin was co-precipitated with NLRP3, NACHT, and LRR, but not with PYRIN ( Fig 1C). ...
The stimulation of P2X7 receptor by extracellular ATP leads to activation of NLRP3 inflammasome and release of pro-inflammatory cytokines. Here, we reveal a distinct mechanism by which Paxillin promotes ATP-induced activation of P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we reveal that UPS13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from cytosol to plasma membrane and facilitates P2X7-Paxillin interaction and Paxillin-NLRP3 association, resulting in the formation of P2X7-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as we as in human PBMCs and THP-1-differentiated macrophages. Thus, Paxillin plays key roles in ATP-induced activation of P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7-Paxillin-NLRP3 complex.
... We propose that the cGAS-STING-NLRP3 axis is essential for host defense against HSV-1 infection. NACHT-associated domain (NAD), and Leucine rich repeats (LRR) [16]. Next, the domain of NLRP3 involved in the interaction with STING was determined by evaluating the plasmids encoding NLRP3, PYRIN, NACHT, or LRR ( Fig 1D) as described previously [17]. ...
One of the fundamental reactions of the innate immune responses to pathogen infection is the release of pro-inflammatory cytokines, including IL-1β, processed by the NLRP3 inflammasome. The stimulator of interferon genes (STING) has the essential roles in innate immune response against pathogen infections. Here we reveal a distinct mechanism by which STING regulates the NLRP3 inflammasome activation, IL-1β secretion, and inflammatory responses in human cell lines, mice primary cells, and mice. Interestingly, upon HSV-1 infection and cytosolic DNA stimulation, STING binds to NLRP3 and promotes the inflammasome activation through two approaches. First, STING recruits NLRP3 and facilitates NLRP3 localization in the endoplasmic reticulum, thereby facilitating the inflammasome formation. Second, STING interacts with NLRP3 and attenuates K48- and K63-linked polyubiquitination of NLRP3, thereby promoting the inflammasome activation. Collectively, we demonstrate that the cGAS-STING-NLRP3 signaling is essential for host defense against HSV-1 infection.
... To evaluate the effects of xenobiotic compounds on homeostatic balance, we selected a total of 10 genes of interest. The selected genes were associated with known pathways related to xenobiotic metabolism or as established biomarkers of homeostatic balance and apoptotic activities [23][24][25][26][27]. As the reference genes, 2 genes were used, AHR and GAPDH, which were both commonly used housekeeping genes. ...
Simple Summary: Eggs are one of the most affordable and nutritious animal proteins available, and with increasing human population, there is an increased demand for production. As feed is the main expense in poultry production, novel protein sources and feed additives need to be evaluated for their benefits for poultry health and performance. In this study, we evaluated the standard soybean-based diets against an alternate source-cottonseed meal, in the context of prebiotic addition. Prebiotics putatively improves health and production. We assessed the homeostatic and immune balance by assaying the expression of select marker genes. We find that the inclusion of yeast cell wall products as prebiotic alters homeostatic balance. Particularly, the upregulation of apoptosis-a normal cell process-suggests that these products may promote homeostatic balance. Abstract: The ingredients of poultry feeds are chosen based on the least-cost formulation to meet nutritional requirements. However, this approach can lead to the introduction of anti-nutritional ingredients in the feed. The objective of this study was to evaluate the impacts of two diets (with or without prebiotic) on homeostatic genes in the liver and spleen of laying hens. Hy-Line Brown layers were raised either on a soybean meal or cottonseed meal-based diets with and without an added prebiotic (yeast cell wall), totaling four experimental diets. A total of 120, 63-week old layers were housed individually in a wire cage system. We investigated differences in the expression of select homeostatic marker genes in the liver and spleen of hens from each treatment. We then used the ∆∆CT and generalized linear models to assess significance. Results show that the inclusion of prebiotic yeast cell-wall (YCW) increased the expression of the BAK gene in the liver tissue for both the soybean meal (SBM) and cottonseed meal (CSM) diets. For splenic tissue, the combination of YCW with the CSM diet increased the POR gene over six log2 fold. Altogether, our results suggest altered homeostasis, which can have consequences for health and performance.
... Key steps in inflammation involve recognition of the inducers, signal transduction, release of pro-inflammatory molecules, activation of the effectors of inflammation, and resolution of the inflammation (6). The inducers are recognized by a range of receptors, such as toll-like receptors and nucleotidebinding domain and leucine-rich-repeat-containing receptors, leading to the activation and nuclear translocation of the transcription factor NF-κB (7)(8)(9). This induces the expression of a number of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, IL-8, IL-12, and Tumor Necrosis Factor-α (TNF-α). ...
Syndecans are transmembrane proteoglycans with heparan and chondroitin sulfate chains attached to their extracellular domain. Like many proteoglycans, they interact with a large number of ligands, such as growth factors, adhesion receptors, soluble small molecules, proteinases, and other extracellular matrix proteins to initiate downstream signaling pathways. Syndecans play a major role in inflammation, mainly by regulating leukocyte extravasation and cytokine function. At the same time, syndecans can undergo cytokine mediated changes in their expression levels during inflammation. The function of syndecans during inflammation appears to depend on the stage of inflammation, sulfation of heparan/chondroitin sulfate chains, the rate of ectodomain shedding and the solubility of the ectodomains. From the current literature, it is clear that syndecans are not only involved in the initial recruitment of pro-inflammatory molecules but also in establishing a balanced progression of inflammation. This review will summarize how cell surface and soluble syndecans regulate multiple aspects of inflammation.
Objective
Parry-Romberg syndrome (PRS) is an acquired disease characterized by progressive unilateral atrophy of the facial skin, subcutaneous tissue, muscle, and bone. There are various hypotheses to try to explain the occurrence of the disease, but the specific etiology and pathogenesis remain unclear. This study aimed to explore the potential molecular pathogenesis of the disease by using next-generation RNA-sequencing technology.
Methods
The authors collected oral mucosal tissue from the affected side and the healthy side from 3 patients with PRS. Tissue samples were subjected to RNA extraction, whole transcriptome sequencing, and bioinformatics analysis. Differentially expressed genes were obtained from both groups of samples and then analyzed for functional enrichment.
Results
A total of 186 differentially expressed genes were screened from the 2 groups of samples. Compared with the healthy side, several immune-related genes, including immunoglobulin kappa variable (IGKV)2D-28, IGKV1D-33, IGKV1-33, and NLRP10, were significantly upregulated in the affected tissue. In addition, the differential genes were significantly enriched in metabolic pathways including pancreatic secretion, protein and fat digestion, and absorption.
Conclusions
The authors described the gene expression differences between the affected and healthy tissues of patients with PRS for the first time. Immune responses may play a role in the pathogenesis of PRS.
ZUSAMMENFASSUNG
Die Cryopyrin-assoziierten periodischen Syndrome (CAPS) umfassen ein klinisches Spektrum autoinflammatorischer Phänotypen unterschiedlicher Schweregrade. Hierzu zählen das familiäre autoinflammatorische Kälte-Syndrom (FCAS), das Muckle-Wells-Syndrom (MWS) und das chronisch infantile neurologische kutane und artikuläre Syndrom (CINCA), das auch als neonatale Multisystem-Entzündungserkrankung (NOMID) bekannt ist. Die Ursache für CAPS liegt meist in pathogenen NLRP3-Varianten, die zu erhöhter Aktivität des Inflammasoms, Überproduktion von Interleukin-1β und Entzündungen führen. Die Klinik umfasst erhöhte Entzündungsmarker, Müdigkeit, Fieber, Hautausschläge sowie Muskel-, Skelett- und ZNS-Symptome, Hörverlust und Sehstörungen. Die diagnostische Herausforderung liegt in sich überlappenden Phänotypen, somatischen Mutationen und Varianten mit geringer Ausprägung oder unklarer Bedeutung. Diagnose und Klassifikationskriterien unterstützen bei der Diagnosestellung und helfen bei der Definition von Studienkohorten. Für die wirksame Behandlung stehen biologische Therapien zur Verfügung, wovon Interleukin (IL)-1-Inhibitoren für die Behandlung zugelassen sind.
We present the first results of the proof-of-concept phase 2a study of oral NLRP3 inflammasome inhibitor in subjects with cryopyrin-associated periodic syndromes (CAPS). Three adult subjects with a confirmed diagnosis of CAPS were enrolled and administered 50 mg of ZYIL1 twice daily for 7 days. A total of 5 treatment-emergent adverse events (TEAEs) were reported in 2 subjects. All 5 TEAEs were mild in severity and considered unrelated to the study drug. At steady state, the average plasma concentration and trough concentration ranged from 2.5 to 4.2 and 1.4 to 2.5 μg/mL, respectively. Inflammatory markers and disease activity (physician and patient global assessment score) decreased notably 12 hours post-last dose.
Background:
Nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) was the first nucleotide-binding region receptor (NLR) proposed to be expressed and function only in the reproductive system. Recent evidence suggests that NLRP9 is also capable of playing a role in infectious and inflammatory diseases.
Results:
and conclusions: In this study, we examined the expression of NLRP9 in various tissues of piglets and IPEC-J2 cells. The results showed that high expression of NLRP9 mRNA and protein were detected in both intestine of piglets and IPEC-J2 cells. Both LPS and poly I:C significantly up-regulated NLRP9 protein levels in the IPEC-J2 cells. Besides, poly I:C upregulated the level of transcriptional elements NF-κB, IRF3, IRF7, ISG15, ISG56, OAS1, and IFNB1. Furthermore, interference with the NLRP9 gene in the presence of poly I:C strongly downregulated the expression of all the above genes. Moreover, we demonstrated for the first time that NLRP9 acts in combination with VIM (Vimentin). These results suggested that NLRP9 may participate in the antiviral innate immune by binding to VIM in the porcine intestine. The findings provide preliminary insights into the molecular mechanisms involved in the regulation of mucosal immunity in the porcine intestine by NLRP9.
Along with the emergence of green plants on this planet one billion years ago, the nucleotide binding site leucine-rich repeat (NLR) gene family originated and diverged into at least three subclasses. Two of them, with either characterized N-terminal toll/interleukin-1 receptor (TIR) or coiled-coil (CC) domain, serve as major types of immune receptor of effector-triggered immunity (ETI) in plants, whereas the one having a N-terminal Resistance to powdery mildew8 (RPW8) domain, functions as signal transfer component to them. In this review, we briefly summarized the history of identification of diverse NLR subclasses across Viridiplantae lineages during the establishment of NLR category, and highlighted recent advances on the evolution of NLR genes and several key downstream signal components under the background of ecological adaption.
Autophagy-related 4B (ATG4B) is found to exert a vital function in viral replication, although the mechanism through which ATG4B activates type-I IFN signaling to hinder viral replication remains to be explained, so far. The current work revealed that ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. In addition, ATG4B overexpression inhibited PEDV replication in both Vero cells and LLC-PK1 cells. On the contrary, ATG4B knockdown facilitated PEDV replication. Moreover, ATG4B was observed to hinder PEDV replication by activating type-I IFN signaling. Further detailed analysis revealed that the ATG4B protein targeted and upregulated the TRAF3 protein to induce IFN expression via the TRAF3-pTBK1-pIRF3 pathway. The above data revealed a novel mechanism underlying the ATG4B-mediated viral restriction, thereby providing novel possibilities for preventing and controlling PEDV.
Innate immune system plays a critical role in early detection of pathogen and inflammation-associated diseases. Detection of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs) triggers the activation of inflammatory responses by the immune cells. Macrophage plays a critical role in innate immunity. Detection of pathogen by macrophages leads to activation of innate and adaptive immune response for neutralization and clearance of the pathogen. Salmonella typhimurium, an intracellular bacterium can infect and multiply in the cytosol of macrophages. NLR family CARD domain-containing protein 4 (NLRC4), a cytosolic PRR, has a vital role in detection of the infection. NLRC4 can detect flagellin of S. typhimurium which results in its clearance by pyroptosis. Clearance of S. typhimurium via pyroptosis is dependent on NLRC4- mediated activation of caspase-1, and independent of interleukin-1β(IL-1β) and IL-18. NLRC4 dependant activation of caspase-1 provides protection against a large number of translocated virulence factors. This method of innate immune detection permits the macrophage to discriminate virulent from avirulent bacteria. In this review we discuss that how S. typhimurium is detected and cleared by the innate immune system.
Multiple organ dysfunction syndrome (MODS) represents one of the prominent cause of mortality and morbidity in critically ill patients. The understanding of MODS pathophysiology has progressively advanced over time; however, several aspects remain uncertain. Numerous different insults may represent the initial triggers of the immune system. However, MODS is characterized by the loss of the normal homeostasis between the pro- and anti-inflammatory system resulting in an uncontrolled activation of the inflammatory response. The mechanism and cellular pathophysiology involved in the transition from a physiological inflammatory response to MODS remain unclear. In this chapter, we aimed to provide an overview of the current knowledge regarding the pathophysiology of MODS, to describe the organ-specific mechanism of dysfunction, to highlight the potential nosocomial hit, to outline possible predictor of MODS and to review some of promising new target treatment.
The nucleotide oligomerization domain (NOD)-like receptor (NLR) is a relatively conserved receptor family involved in natural immunity that plays a key role in the resistance to pathogen invasion and regulation of the innate immune response. Lethenteron reissneri (lamprey) is a representative species of existing ancient jawless vertebrates. Studies of the evolutionary relationship of immune system-related molecules in lampreys can provide an important reference for the origin and evolution of innate immunity. However, the characterization and evolutionary patterns of the NLR family remain unclear in the lamprey genome. Based on the genome database of L. reissneri, we identified nine NLR genes, characterized their functional domains and chromosomal positions, and constructed a network comprising the results of gene structure and gene-collinearity analyses. Comparative genomics studies suggest that Lr-NODa and Lr-NODb most likely share the common ancestor of NOD1 and NOD2 in jawed vertebrates, and that Lr-NODb may have been generated by lamprey-specific tandem duplication of Lr-NODa. Additionally, phylogenetic analysis of the NLRC subfamily suggests that Lr-NLRC3a has ancestral traits and may be derived from the common ancestor of another vertebrate NLRC subfamily. Further analysis of the formation of the NLRC subfamily has shown that exon shuffling, domain recombination, and chromosome rearrangement play important roles in its structural evolution. Furthermore, real-time quantitative polymerase chain reaction shows that most NLR genes in lamprey are highly expressed in the immune tissues of the heart, gill, and supraneural body, with these genes also showing significant responses to polyinosinic-polycytidylic acid infection. These results indicate that NLR genes are involved in the immune protection of L. reissneri and provide an important theoretical foundation for studies of the functional evolution of vertebrate NLRs involved in the innate immune system.
Immunotherapies are disease management strategies that specifically target or modulate immune system components. As countries continue to grapple with a slew of new and resurgent diseases, the most current global health concern being the SARS-CoV2 pandemic, infectious diseases continue to pose a substantial threat to human health, as evidenced by the recent SARS-CoV2 pandemic. While significant progress has been achieved in diagnosing and understanding the etiology of infectious diseases, current antimicrobial chemotherapy has been shown to be ineffective against a large number of current pathogens. Emerging and re-emerging microorganisms create new infectious diseases, have developed resistance to available antibacterial drugs, or are unable to be treated due to a lack of treatment options or are generally ineffective due to underlying host immunological impairment. The HIV epidemic and advancements in cancer, transplantation, and autoimmune disease treatment have increased the number of people with a weakened immune system. As a result, novel approaches to infectious diseases treatment are required. A variety of immunotherapeutic strategies are currently being examined as viable alternative therapeutics for infectious diseases as a result, which has resulted in remarkable advances in the understanding of pathogen–host immunity interactions. The growth of multidrug-resistant bacteria, sometimes known as superbugs, is leading to the emergence of diseases that are becoming increasingly difficult to treat. Gram-negative bacteria and mycobacteria are among the most serious medical problems of the twenty-first century. Because bacteria have evolved strategies to subvert the immune response, even bacteria that are sensitive to antibiotics can persist in the presence of these drugs. As a result, a diverse variety of effective treatment options or adjuncts to conventional antimicrobial therapy are urgently needed to minimize the burden of disease posed by antibiotic resistance. As indicated by the recent invention and success of drugs such as monoclonal antibodies (mAbs), immunotherapies already offer a great deal of promise for overcoming these constraints while also pushing the boundaries of medicine. While the majority of immunotherapeutic strategies have their origins in cancer treatment, recent advances, such as those in the treatment of HIV/AIDS and tuberculosis as well as zika virus, malaria, and most recently COVID-19 strengthen the role that immunotherapeutic approaches play in disease control. Finally, the complete specificity, safety, and affordability of immunotherapies will have an effect on their general adoption. Various immunotherapies, including vaccines (both prophylactic and therapeutic), monoclonal antibodies, pathogens, recombinant proteins, cellular immunotherapies, and cytokines are being developed, and their application in the treatment of a wide range of diseases is changing our approach to treating these diseases. In this chapter we will explore recent breakthroughs in the field of bacterial infection treatment and emphasize present and future perspectives on the use of immunotherapies to treat bacterial infections. Recent advances in the field of bacterial infection prevention and treatment will also be discussed in this chapter, as will present and future perspectives on the use of immunotherapies to combat bacterial infections in the context of infectious diseases.
The NOD-like receptor X1 (NLRX1) is a member of highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family (known as NLR), that localizes to the mitochondrial outer membrane and regulate the innate immunity by interacting with mitochondrial antiviral-signaling protein (MAVS). As one of cytoplasmic PRRs, NLRX1 plays key roles for pathogen recognition, autophagy and regulating of subsequent immune signaling pathways. In this study, we identified the nlrx1 in turbot as well as its expression profiles in mucosal surfaces following bacterial infection. In our results, the full-length nlrx1 transcript consists of an open reading frame (ORF) of 4,886 bp encoding the putative peptide of 966 amino acids. The phylogenetic analysis revealed the SmNlrx1 showed the closest relationship to Cynoglossus semilaevis. In addition, the Nlrx1 mRNA expression could be detected in all the examined tissues, with the most abundant expression level in head kidney, and the lowest expression level in liver. Moreover, Nlrx1 showed similar expression patterns following Vibrio anguillarum and Streptococcus iniae infection, that were both significantly up-regulated following challenge, especially post S. iniae challenge. Finally, fluorescence microscopy unveiled that the SmNlrx1 localized to mitochondria in HEK293T by N-terminal mitochondrial targeting sequence. Characterization of Nlrx1 might have an important implication in bioenergetic adaptation during metabolic stress, oncogenic transformation and innate immunity and will probably contribute to the development of novel intervention strategies for farming turbot.
Overdose of acetaminophen (APAP), an antipyretic drug, is an important cause of liver injury. However, the mechanism in the rat model remains undetermined. We analyzed APAP-induced hepatotoxicity using rats based on M1/M2-macrophage functions in relation to damage-associated molecular patterns (DAMPs) and autophagy. Liver samples from six-week-old rats injected with APAP (1000 mg/kg BW, ip, once) after 15 h fasting were collected at hour 10, and on days 1, 2, 3, and 5. Liver lesions consisting of coagulation necrosis and inflammation were seen in the affected centrilobular area on days 1 and 2, and then, recovered with reparative fibrosis by day 5. Liver exudative enzymes increased transiently on day 1. CD68+ M1-macrophages increased significantly on days 1 and 2 with increased mRNAs of M1-related cytokines such as IFN-g and TNF-α, whereas CD163+ M2-macrophages appeared later on days 2 and 3. Macrophages reacting to MHC class II and Iba1 showed M1-type polarization, and CD204+ macrophages tended to be polarized toward M2-type. At hour 10, interestingly, HMGB1 (representative DAMPs) and its related signals, TLR-9 and MyD88, as well as LC3B+ autophagosomes began to increase. Collectively, the pathogenesis of rat APAP hepatotoxicity, which is the first, detailed report for a rat model, might be influenced by macrophage functions of M1 type for tissue injury/inflammation and M2-type for anti-inflammatory/fibrosis; particularly, M1-type may function in relation to DAMPs and autophagy. Understanding the interplayed mechanisms would provide new insight into hepato-pathogenesis and contribute to the possible development of therapeutic strategies.
The first breath taken by newborns after birth initiates the transition from fetal to neonatal life. Successful transition is dependent on the establishment of effective gas exchange in the lungs. This exchange takes place in the alveoli, the terminal units of the lung. The embryonic lung undergoes branching morphogenesis to form a vast network of branched airways and subsequent formation and multiplication of alveoli by septation during the late stage of fetal lung development. These developmental processes are regulated by diverse crosstalk between the airway epithelium and surrounding mesenchyme, which are highly coordinated by transcriptional factors, growth factors, and extracellular matrix. Bronchopulmonary dysplasia is a chronic lung disease of premature infants that results from a developmental arrest of the immature lung caused by injurious stimuli such as mechanical ventilation, oxygen exposure, and intrauterine or postnatal infections. This chapter provides a brief overview of normal lung developmental processes, the key signaling pathways and proposed models in regulating lung budding, branching morphogenesis, alveolarization and vascular development, and the mechanism by which injury from mechanical ventilation and oxygen exposure modulates some of these key pathways, thus affecting neonatal lung development in the context of prematurity.
The airway epithelium represents a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli including microbes and allergens. However, lung epithelial cells are increasingly recognised as active effectors of microbial defence, contributing to both innate and adaptive immune function in the lower respiratory tract. These cells express an ample repertoire of pattern‐recognition receptors with specificity for conserved microbial and host motifs. Modern molecular techniques have uncovered the complexity of the lower respiratory tract microbiome. The interaction between the microbiota and the airway epithelium is key to understanding how stable immune homeostasis is maintained. Loss of epithelial integrity following exposure to infection can result in the onset of inflammation in susceptible individuals and may culminate in lung disease. Here we discuss the current knowledge regarding the molecular and cellular mechanisms by which the pulmonary epithelium interacts with the lung microbiome in shaping immunity in the lung. Specifically, we focus on the interactions between the lung microbiome and the cells of the conducting airways in modulating immune cell regulation and how defects in barrier structure and function may culminate in lung disease. Understanding these interactions is fundamental in the search for more effective therapies for respiratory diseases.
To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.
Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease(1). Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways(2,3). Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1 beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1 beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappa B and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1 beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.
Prior genetic and physical mapping has shown that the Naip gene cluster on mouse chromosome 13D1-D3 contains a gene, Lgn1 , that is responsible for determining the permissivity of ex vivo macrophages to Legionella pneumophila replication. We have identified differences in the structure of the Naip array among commonly used inbred mouse strains, although these gross structural differences do not correlate with differences in L. pneumophila permissiveness. A physical map of the region employing clones of the C57BL/6J haplotype confirms that there are fewer copies of Naip in this strain than are in the physical map of the 129 haplotype. We have also refined the genetic location of Lgn1 , leaving only Naip2 and Naip5 as candidates for Lgn1 . Our genetic map suggests the presence of two hotspots of recombination within the Naip array, indicating that the 3′ portion of Naip may be involved in the genomic instability at this locus.
[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF240489 – AF240530 .]
For over 50 years immunologists have based their thoughts, experiments, and clinical treatments on the idea that the immune
system functions by making a distinction between self and nonself. Although this paradigm has often served us well, years
of detailed examination have revealed a number of inherent problems. This Viewpoint outlines a model of immunity based on
the idea that the immune system is more concerned with entities that do damage than with those that are foreign.
In inbred mouse strains, permissiveness to intracellular replication of Legionella pneumophila is controlled by a single locus (Lgn1), which maps to a region within distal Chromosome 13 that contains multiple copies of the gene baculoviral IAP repeat-containing 1 (Birc1, also called Naip; refs. 1-3). Genomic BAC clones from the critical interval were transferred into transgenic mice to functionally complement the Lgn1-associated susceptibility of A/J mice to L. pneumophila. Here we report that two independent BAC clones that rescue susceptibility have an overlapping region of 56 kb in which the entire Lgn1 transcript must lie. The only known full-length transcript coded in this region is Birc1e (also called Naip5).
Salmonella enterica, the causative agent of food poisoning and typhoid fever, induces programmed cell death in macrophages, a process found to be dependent on a type III protein secretion system, and SipB, a protein with membrane fusion activity that is delivered into host cells by this system. When expressed in cultured cells, SipB caused the formation of and localized to unusual multimembrane structures. These structures resembled autophagosomes and contained both mitochondrial and endoplasmic reticulum markers. A mutant form of SipB devoid of membrane fusion activity localized to mitochondria, but did not induce the formation of membrane structures. Upon Salmonella infection of macrophages, SipB was found in mitochondria, which appeared swollen and devoid of christae. Salmonella-infected macrophages exhibited marked accumulation of autophagic vesicles. We propose that Salmonella, through the action of SipB, kills macrophages by disrupting mitochondria, thereby inducing autophagy and cell death.
Shigella-induced macrophage cell death is an important step in the induction of acute inflammatory responses that ultimately lead
to bacillary dysentery. Cell death was previously reported to be dependent upon the activation of caspase-1 via interaction
with IpaB secreted by intracellular Shigella, but in this study, we show that Shigella infection of macrophages can also induce cell death independent of caspase-1 or IpaB activity. Time-lapse imaging and electron
microscopic analyses indicated that caspase-1-dependent and -independent cell death is morphologically indistinguishable and
that both resemble necrosis. Analyses of Shigella mutants or Escherichia coli using co-infection with Listeria suggested that a component common to Gram-negative bacteria is involved in inducing caspase-1-independent cell death. Further
studies revealed that translocation of bacterial lipid A into the cytosol of macrophages potentially mediates cell death.
Notably, cell death induced by cytosolic bacteria was TLR4-independent. These results identify a novel cell death pathway
induced by intracellular Gram-negative bacteria that may play a role in microbial-host interactions and inflammatory responses.
A wide variety of pathogenic microorganisms have been demonstrated to cause eukaryotic cell death, either as a consequence of infecting host cells or by producing toxic products. Pathogen-induced host cell death has been characterized as apoptosis in many of these systems. It is increasingly being recognized that cell death with some of the features of apoptosis may result from a variety of molecular pathways and that experimental techniques used to identify cell death often do not distinguish among these mechanisms. We propose that a clear understanding of the diversity of processes mediating cell death has been obscured by the simplicity of the nomenclature system commonly employed to describe eukaryotic cell death. This review presents a perspective on eukaryotic cell death and discusses experimental techniques used to study these processes.
Nods are cytosolic proteins that contain a nucleotide-binding oligomerization domain (NOD). These proteins include key regulators of apoptosis and pathogen resistance in mammals and plants. A large number of Nods contain leucine-rich repeats (LRRs), hence referred to as NOD-LRR proteins. Genetic variation in several NOD-LRR proteins, including human Nod2, Cryopyrin, and CIITA, as well as mouse Naip5, is associated with inflammatory disease or increased susceptibility to microbial infections. Nod1, Nod2, Cryopyrin, and Ipaf have been implicated in protective immune responses against pathogens. Together with Toll-like receptors, Nod1 and Nod2 appear to play important roles in innate and acquired immunity as sensors of bacterial components. Specifically, Nod1 and Nod2 participate in the signaling events triggered by host recognition of specific motifs in bacterial peptidoglycan and, upon activation, induce the production of proinflammatory mediators. Naip5 is involved in host resistance to Legionella pneumophila through cell autonomous mechanisms, whereas CIITA plays a critical role in antigen presentation and development of antigen-specific T lymphocytes. Thus, NOD-LRR proteins appear to be involved in a diverse array of processes required for host immune reactions against pathogens.
The CATERPILLER (CLR, also NOD and NLR) proteins share structural similarities with the nucleotide binding domain (NBD)-leucine-rich repeat (LRR) superfamily of plant disease-resistance (R) proteins and are emerging as important immune regulators in animals. CLR proteins contain NBD-LRR motifs and are linked to a limited number of distinct N-terminal domains including transactivation, CARD (caspase activation and recruitment), and pyrin domains (PyD). The CLR gene, Monarch-1/Pypaf7, is expressed by resting primary myeloid/monocytic cells, and its expression in these cells is reduced by Toll-like receptor (TLR) agonists tumor necrosis factor (TNF) alpha and Mycobacterium tuberculosis. Monarch-1 reduces NFkappaB activation by TLR-signaling molecules MyD88, IRAK-1 (type I interleukin-1 receptor-associated protein kinase), and TRAF6 (TNF receptor (TNFR)-associated factor) as well as TNFR signaling molecules TRAF2 and RIP1 but not the downstream NFkappaB subunit p65. This indicates that Monarch-1 is a negative regulator of both TLR and TNFR pathways. Reducing Monarch-1 expression with small interference RNA in myeloid/monocytic cells caused a dramatic increase in NFkappaB activation and cytokine expression in response to TLR2/TLR4 agonists, TNFalpha, or M. tuberculosis infection, suggesting that Monarch-1 is a negative regulator of inflammation. Because Monarch-1 is the first CLR protein that interferes with both TLR2 and TLR4 activation, the mechanism of this interference is significant. We find that Monarch-1 associates with IRAK-1 but not MyD88, resulting in the blockage of IRAK-1 hyperphosphorylation. Mutants containing the NBD-LRR or PyD-NBD also blocked IRAK-1 activation. This is the first example of a CLR protein that antagonizes inflammatory responses initiated by TLR agonists via interference with IRAK-1 activation.
Instead of the immunoglobulin-type antigen receptors of jawed vertebrates, jawless fish have variable lymphocyte receptors (VLRs), which consist of leucine-rich repeat (LRR) modules. Somatic diversification of the VLR gene is shown here to occur through a multistep assembly of LRR modules randomly selected from a large bank of flanking cassettes. The predicted concave surface of the VLR is lined with hypervariable positively selected residues, and computational analysis suggests a repertoire of about 10(14) unique receptors. Lamprey immunized with anthrax spores responded with the production of soluble antigen-specific VLRs. These findings reveal that two strikingly different modes of antigen recognition through rearranged lymphocyte receptors have evolved in the jawless and jawed vertebrates.
A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.
Development of the acute and chronic inflammatory responses known as gout and pseudogout are associated with the deposition of monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals, respectively, in joints and periarticular tissues. Although MSU crystals were first identified as the aetiological agent of gout in the eighteenth century and more recently as a 'danger signal' released from dying cells, little is known about the molecular mechanisms underlying MSU- or CPPD-induced inflammation. Here we show that MSU and CPPD engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active interleukin (IL)-1beta and IL-18. Macrophages from mice deficient in various components of the inflammasome such as caspase-1, ASC and NALP3 are defective in crystal-induced IL-1beta activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasome-deficient mice or mice deficient in the IL-1beta receptor (IL-1R). These findings provide insight into the molecular processes underlying the inflammatory conditions of gout and pseudogout, and further support a pivotal role of the inflammasome in several autoinflammatory diseases.
The pathogenesis of Bacillus anthracis, the bacterium that causes anthrax, depends on secretion of three factors that combine to form two bipartite toxins. Edema toxin, consisting of protective antigen (PA) and edema factor (EF), causes the edema associated with cutaneous anthrax infections, whereas lethal toxin (LeTx), consisting of PA and lethal factor (LF), is believed to be responsible for causing death in systemic anthrax infections. EF and LF can be transported by PA into the cytosol of many cell types. In mouse macrophages, LF can cause rapid necrosis that may be related to the pathology of systemic infections. Inbred mouse strains display variable sensitivity to LeTx-induced macrophage necrosis. This trait difference has been mapped to a locus on chromosome 11 named Ltxs1 (refs. 7,8). Here we show that an extremely polymorphic gene in this locus, Nalp1b, is the primary mediator of mouse macrophage susceptibility to LeTx. We also show that LeTx-induced macrophage death requires caspase-1, which is activated in susceptible, but not resistant, macrophages after intoxication, suggesting that Nalp1b directly or indirectly activates caspase-1 in response to LeTx.
Baculovirus inhibitor of apoptosis repeat-containing 1 (Birc1) proteins have homology to several germline-encoded receptors of the innate immune system. However, their function in immune surveillance is not clear. Here we describe a Birc1e-dependent signaling pathway that restricted replication of the intracellular pathogen Legionella pneumophila in mouse macrophages. Translocation of bacterial products into host-cell cytosol was essential for Birc1e-mediated control of bacterial replication. Caspase-1 was required for Birc1e-dependent antibacterial responses ex vivo in macrophages and in a mouse model of Legionnaires' disease. The interleukin 1beta converting enzyme-protease-activating factor was necessary for L. pneumophila growth restriction, but interleukin 1beta was not required. These results establish Birc1e as a nucleotide-binding oligomerization-leucine-rich repeat protein involved in the detection and control of intracellular L. pneumophila.
Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens.
To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1beta secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.
NOD (nucleotide-binding oligomerization domain) proteins are members of a family that includes the apoptosis regulator APAF1 (apoptotic protease activating factor 1), mammalian NOD-LRR (leucine-rich repeat) proteins and plant disease-resistance gene products. Several NOD proteins have been implicated in the induction of nuclear factor-kappaB (NF-kappaB) activity and in the activation of caspases. Two members of the NOD family, NOD1 and NOD2, mediate the recognition of specific bacterial components. Notably, genetic variation in the genes encoding the NOD proteins NOD2, cryopyrin and CIITA (MHC class II transactivator) in humans and Naip5 (neuronal apoptosis inhibitory protein 5) in mice is associated with inflammatory disease or increased susceptibility to bacterial infections. Mammalian NOD proteins seem to function as cytosolic sensors for the induction of apoptosis, as well as for innate recognition of microorganisms and regulation of inflammatory responses.
Gram-negative bacteria that replicate in the cytosol of mammalian macrophages can activate a signaling pathway leading to caspase-1 cleavage and secretion of interleukin 1beta, a powerful host response factor. Ipaf, a cytosolic pattern-recognition receptor in the family of nucleotide-binding oligomerization domain-leucine-rich repeat proteins, is critical in such a response to salmonella infection, but the mechanism of how Ipaf is activated by the bacterium remains poorly understood. Here we demonstrate that salmonella strains either lacking flagellin or expressing mutant flagellin were deficient in activation of caspase-1 and in interleukin 1beta secretion, although transcription factor NF-kappaB-dependent production of interleukin 6 or the chemokine MCP-1 was unimpaired. Delivery of flagellin to the macrophage cytosol induced Ipaf-dependent activation of caspase-1 that was independent of Toll-like receptor 5, required for recognition of extracellular flagellin. In macrophages made tolerant by previous exposure to lipopolysaccharide, which abrogates activation of NF-kappaB and mitogen-activated protein kinases, salmonella infection still activated caspase-1. Thus, detection of flagellin through Ipaf induces caspase-1 activation independently of Toll-like receptor 5 in salmonella-infected and lipopolysaccharide-tolerized macrophages.
Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. After infection of human macrophages, the Legionella-containing phagosome (LCP) avoids fusion with the lysosome allowing intracellular replication of the bacterium. In macrophages derived from most mouse strains, the LCP is delivered to the lysosome resulting in Legionella degradation and restricted bacterial growth. Mouse macrophages lacking the NLR protein Ipaf or its downstream effector caspase-1 are permissive to intracellular Legionella replication. However, the mechanism by which Ipaf restricts Legionella replication is not well understood. Here we demonstrate that the presence of flagellin and a competent type IV secretion system are critical for Legionella to activate caspase-1 in macrophages. Activation of caspase-1 in response to Legionella infection also required host Ipaf, but not TLR5. In the absence of Ipaf or caspase-1 activation, the LCP acquired endoplasmic reticulum-derived vesicles, avoided fusion with the lysosome, and allowed Legionella replication. Accordingly a Legionella mutant lacking flagellin did not activate caspase-1, avoided degradation, and replicated in wild-type macrophages. The regulation of phagosome maturation by Ipaf occurred within 2 h after infection and was independent of macrophage cell death. In vivo studies confirmed that flagellin and Ipaf play an important role in the control of Legionella clearance. These results reveal that Ipaf restricts Legionella replication through the regulation of phagosome maturation, providing a novel function for NLR proteins in host defense against an intracellular bacterium.
P2X(7) receptors are ATP-gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). It has not been known what this dye-uptake path is, or whether it is involved in downstream signalling to IL-1beta release. Here, we identify pannexin-1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1beta induced by P2X(7) receptor activation.
Many plant-associated microbes are pathogens that impair plant growth and reproduction. Plants respond to infection using a two-branched innate immune system. The first branch recognizes and responds to molecules common to many classes of microbes, including non-pathogens. The second responds to pathogen virulence factors, either directly or through their effects on host targets. These plant immune systems, and the pathogen molecules to which they respond, provide extraordinary insights into molecular recognition, cell biology and evolution across biological kingdoms. A detailed understanding of plant immune function will underpin crop improvement for food, fibre and biofuels production.
The intracellular Nod-like proteins or receptors are a family of sensors of intracellularly encountered microbial motifs and 'danger signals' that have emerged as being critical components of the innate immune responses and of inflammation in mammals. Several Nod-like receptors, including Nod1, Nod2, NALP3, Ipaf and Naip, are strongly associated with host responses to intracellular invasion by bacteria or the intracellular presence of specific bacterial products. An additional key function of Nod-like receptors is in inflammatory conditions, which has been emphasized by the identification of several different mutations in the genes encoding Nod1, Nod2 and NALP3 that are associated with susceptibility to inflammatory disorders. Those and other issues related to the Nod-like receptor family are discussed here.
Pannexin-1 is a recently identified membrane protein that can act as a nonselective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X7 receptor (P2X7R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1beta (IL-1beta) in response to P2X7R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X7R to provide the conduit for dye uptake and downstream signaling to processing and release of IL-1beta. We have pursued this hypothesis by measuring dye uptake and IL-1beta processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1beta release via the same mechanism. The experiments were carried out over time periods during which no lactate dehydrogenase was released from cells to examine only noncytolytic pathways. P2X7R activation evoked dye uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1-independent phase. Maitotoxin-evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1beta processing and release in response to all three stimuli. Thus, although pannexin-1 is required for IL-1beta release in response to maitotoxin, nigericin, and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.
Mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene are associated with a spectrum of autoinflammatory diseases, including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and chronic infantile neurologic, cutaneous, articular syndrome, also known as neonatal-onset multisystem inflammatory disease. CIAS1 encodes cryopyrin, a protein that localizes to the cytosol and functions as pattern recognition receptor. Cryopyrin also participates in nuclear factor-kappaB regulation and caspase-1-mediated maturation of interleukin 10. In this study, we showed that disease-associated mutations in CIAS1 induced rapid cell death of THP-1 monocytic cells. The features of cell death, including 7-AAD staining, the presence of cellular edema, and early membrane damage resulting in lactate dehydrogenase (LDH) release, indicated that it was more likely to be necrosis than apoptosis, and was effectively blocked with the cathepsin B-specific inhibitor CA-074-Me. CA-074-Me also suppressed induced by disease-associated mutation lysosomal leakage and mitochondrial damage. In addition, R837, a recently identified activator of cryopyrin-associated inflammasomes, induced cell death in wild type CIAS1-transfected THP-1 cells. These results indicated that monocytes undergo rapid cell death in a cathepsin B-dependent manner upon activation of cryopyrin, which is also a specific phenomenon induced by disease-associated mutation of CIAS1.
CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-kappaB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-kappaB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.
Autoimmune and autoinflammatory diseases involve interactions between genetic risk factors and environmental triggers. We searched for a gene on chromosome 17p13 that contributes to a group of epidemiologically associated autoimmune and autoinflammatory diseases. The group includes various combinations of generalized vitiligo, autoimmune thyroid disease, latent autoimmune diabetes in adults, rheumatoid arthritis, psoriasis, pernicious anemia, systemic lupus erythematosus, and Addison's disease.
We tested 177 single-nucleotide polymorphisms (SNPs) spanning the 17p13 linkage peak for association with disease and identified a strong candidate gene. We then sequenced DNA in and around the gene to identify additional SNPs. We carried out a second round of tests of association using some of these additional SNPs, thus elucidating the association with disease in the gene and its extended promoter region in fine detail.
Association analyses resulted in our identifying as a candidate gene NALP1, which encodes NACHT leucine-rich-repeat protein 1, a regulator of the innate immune system. Fine-scale association mapping with the use of DNA from affected families and additional SNPs in and around NALP1 showed an association of specific variants with vitiligo alone, with an extended autoimmune and autoinflammatory disease phenotype, or with both. Conditional logistic-regression analysis of NALP1 SNPs indicated that at least two variants contribute independently to the risk of disease.
DNA sequence variants in the NALP1 region are associated with the risk of several epidemiologically associated autoimmune and autoinflammatory diseases, implicating the innate immune system in the pathogenesis of these disorders.
Excerpt
It is a great privilege to introduce this Cold Spring Harbor Symposium on quantitative biology on the subject of immune recognition. My predecessors in this role, Macfarlane Burnet (1967) and Niels Jerne (1976), had a profound effect on the development of ideas in our field, and many of the participants have made great contributions to our understanding of the subject of this symposium and are clearly more qualified than I to introduce it.
The first Cold Spring Harbor Symposium to consider the immune system was held in 1967 on the subject of antibodies. It marked the acceptance of the clonal selection theory as the central paradigm of immunology (Jerne 1967). That meeting also brought together sufficient data on antibody structure to promote the idea that immunoglobulins are encoded in two distinct types of gene segments, variable segments and constant segments, and that a genetic mechanism must exist to direct the...
Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1beta. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.
Generation of Interleukin (IL)-1beta via cleavage of its proform requires the activity of caspase-1 (and caspase-11 in mice), but the mechanism involved in the activation of the proinflammatory caspases remains elusive. Here we report the identification of a caspase-activating complex that we call the inflammasome. The inflammasome comprises caspase-1, caspase-5, Pycard/Asc, and NALP1, a Pyrin domain-containing protein sharing structural homology with NODs. Using a cell-free system, we show that proinflammatory caspase activation and proIL-1beta processing is lost upon prior immunodepletion of Pycard. Moreover, expression of a dominant-negative form of Pycard in differentiated THP-1 cells blocks proIL-1beta maturation and activation of inflammatory caspases induced by LPS in vivo. Thus, the inflammasome constitutes an important arm of the innate immunity.
Large mammalian proteins containing a nucleotide-binding domain (NBD) and C-terminal leucine-rich repeats (LRR) similar in structure to plant disease resistance proteins have been suggested as critical in innate immunity. Our interest in CIITA, a NBD/LRR protein, and recent reports linking mutations in two other NBD/LRR proteins to inflammatory disorders have prompted us to perform a search for other members. Twenty-two known and novel NBD/LRR genes are spread across eight human chromosomes, with multigene clusters occurring on 11, 16, and 19. Most of these are telomeric. Their N termini vary, but most have a pyrin domain. The genomic organization demonstrates a high degree of conservation of the NBD- and LRR-encoding exons. Except for CIITA, all the predicted NBD/LRR proteins are likely ATP-binding proteins. Some have broad tissue expression, whereas others are restricted to myeloid cells. The implications of these data on origins, expression, and function of these genes are discussed.
Legionella pneumophila is a gram-negative bacterial pathogen that is the cause of Legionnaires' Disease. Legionella produces disease because it can replicate inside a specialized compartment of host macrophages. Macrophages isolated from various inbred mice exhibit large differences in permissiveness for intracellular replication of Legionella. A locus affecting this host-resistance phenotype, Lgn1, has been mapped to chromosome 13, but the responsible gene has not been identified.
Here, we report that Naip5 (also known as Birc1e) influences susceptibility to Legionella. Naip5 encodes a protein that is homologous to plant innate immunity (so-called "resistance") proteins and has been implicated in signaling pathways related to apoptosis regulation. Detailed recombination mapping and analysis of expression implicates Naip5 in the Legionella permissiveness differences among mouse strains. A bacterial artificial chromosome (BAC) transgenic line expressing a nonpermissive allele of Naip5 exhibits a reduction in macrophage Legionella permissiveness. In addition, morpholino-based antisense inhibition of Naip5 causes an increase in the Legionella permissiveness of macrophages.
We conclude that polymorphisms in Naip5 are involved in the permissiveness differences of mouse macrophages for intracellular Legionella replication. We speculate that Naip5 is a functional mammalian homolog of plant "resistance" proteins that monitor for, and initiate host response to, the presence of secreted bacterial virulence proteins.
Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.
Mutations within the NALP3/cryopyrin/CIAS1 gene are responsible for three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA. The NALP3 protein is homologous to NALP1, which is a component of the inflammasome, a molecular platform that activates the proinflammatory caspases-1 and -5. NALP3 (and other members of the NALP family) lacks the C-terminal, CARD-containing sequence of NALP1, and its role in caspase activation is unclear. Here, we report that NALP2 and NALP3 associate with ASC, the CARD-containing protein Cardinal, and caspase-1 (but not caspase-5), thereby forming an inflammasome with high proIL-1beta-processing activity. Macrophages from Muckle-Wells patients spontaneously secrete active IL-1beta. Increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with NALP3-dependent autoinflammatory disorders.
Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively. The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the 'inflammasome', a complex comprising several adaptors. Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function. ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages. Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1beta and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock. Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages. Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S. typhimurium. Caspase-1 activation was not compromised by loss of RIP2. These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens. Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.
The newly discovered CATERPILLER (CLR) gene family encodes proteins with a variable but limited number of N-terminal domains, followed by a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). The N-terminal domain consists of transactivation, CARD, Pyrin, or BIR domains, with a minority containing undefined domains. These proteins are remarkably similar in structure to the TIR-NBD-LRR and CC-NBD-LRR disease resistance (R) proteins that mediate immune responses in plants. The NBD-LRR architecture is conserved in plants and vertebrates, but only remnants are found in worms and flies. The CLRs regulate inflammatory and apoptotic responses, and some act as sensors that detect pathogen products. Several CLR genes have been genetically linked to susceptibility to immunologic disorders. We describe prominent family members, including CIITA, CARD4/NOD1, NOD2/CARD15, CIAS1, CARD7/NALP1, and NAIP, in more detail. We also discuss implied roles of these proteins in diversifying immune detection and in providing a check-and-balance during inflammation.
CARD12 (Ipaf/Clan) is an important regulator of caspase-1 activation. It belongs to the family of the nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins. The NBS domain of the NBS-LRR proteins contains putative ATP/GTPase-specific P-loop and Mg2+-binding site motifs. However, the nucleotide-binding properties and the function of the NBS domain are unknown. We developed a nucleotide-binding assay and investigated nucleotide binding to CARD12. We find that the NBS domain of CARD12 contains a nucleotide-binding pocket with specificity for ATP/dATP. A point mutation in the P-loop (K175R) of the NBS domain abolishes ATP/dATP binding. We further demonstrate that the nucleotide-binding site is required for CARD12-mediated caspase-1 activation. CARD12 self-association and association with procaspase-1 in transfected cells were markedly decreased by the P-loop mutation K175R. Furthermore, the P-loop mutation greatly reduced caspase-1 activation-dependent proIL-1beta processing. Thus, CARD12 function is dependent on the nucleotide-binding site. Our data provide insights into the molecular mechanisms of CARD12-mediated caspase-1 activation.
Eukaryotes have evolved systems to detect bacterial intrusion. Recognition of bacteria relies on the sensing of pathogen associated molecular patterns (PAMPs) by host pattern recognition molecules (PRMs), which include various families of leucine-rich repeat (LRR) bearing proteins in plants and animals. Detection of microbes often occurs outside the cell. Recent findings now indicate that mammals have also evolved strategies to recognize bacteria inside the cell via members of the NACHT-LRR protein family (NLRs). Here, we review the biology of these mammalian NLRs and the emerging view of their important, role not solely as PRMs but as signalling platforms and regulators of immunity.
Cytokines of the interleukin-1 (IL-1) family, such as IL-1 alpha/beta and IL-18, have important functions in host defense, immune regulation, and inflammation. Insight into their biological functions has led to novel therapeutic approaches to treat human inflammatory diseases. Within the IL-1 family, IL-1 alpha/beta, IL-1Ra, and IL-18 have been matched to their respective receptor complexes and have been shown to have distinct biological functions. The most prominent orphan IL-1 receptor is ST 2. This receptor has been described as a negative regulator of Toll-like receptor-IL-1 receptor signaling, but it also functions as an important effector molecule of T helper type 2 responses. We report a member of the IL-1 family, IL-33, which mediates its biological effects via IL-1 receptor ST 2, activates NF-kappaB and MAP kinases, and drives production of T(H)2-associated cytokines from in vitro polarized T(H)2 cells. In vivo, IL-33 induces the expression of IL-4, IL-5, and IL-13 and leads to severe pathological changes in mucosal organs.
The newly described CATERPILLER family (also known as NOD-LRR or NACHT-LRR) is comprised of proteins with a nucleotide-binding domain and a leucine-rich region. This family has gained rapid prominence because of its demonstrated and anticipated roles in immunity, cell death and growth, and diseases. CATERPILLER proteins are structurally similar to a subgroup of plant-disease-resistance (R) proteins and to the apoptotic protease activating factor 1 (APAF1). They provide positive and negative signals for the control of immune and inflammatory responses, and might represent intracellular sensors of pathogen products. Most importantly, they are genetically linked to several human immunological disorders.
Mutations in the NALP3/CIAS1/cryopyrin gene are linked to three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and chronic infantile neurologic cutaneous and articular syndrome. NALP3, with the adaptor molecule ASC, has been proposed to form a caspase-1-activating "inflammasome," a complex with pro-IL1beta-processing activity. Here, we demonstrate the effect of NALP3 deficiency on caspase-1 function. NALP3 was essential for the ATP-driven activation of caspase-1 in lipopolysaccharide-stimulated macrophages and for the efficient secretion of the caspase-1-dependent cytokines IL-1alpha, IL-1beta, and IL-18. IL-1beta has been shown to play a key role in contact hypersensitivity; we show that ASC- and NALP3-deficient mice also demonstrate an impaired contact hypersensitivity response to the hapten trinitrophenylchloride. NALP3, however, was not required for caspase-1 activation by Salmonella typhimurium, and NALP3 deficiency only partially protects mice from the lethal effects of endotoxin. These data suggest that NALP3 plays a specific role in the caspase-1 activation pathway.
Macrophages respond to Salmonella typhimurium infection via Ipaf, a NACHT-leucine-rich repeat family member that activates caspase-1 and secretion of interleukin 1beta. However, the specific microbial salmonella-derived agonist responsible for activating Ipaf is unknown. We show here that cytosolic bacterial flagellin activated caspase-1 through Ipaf but was independent of Toll-like receptor 5, a known flagellin sensor. Stimulation of the Ipaf pathway in macrophages after infection required a functional salmonella pathogenicity island 1 type III secretion system but not the flagellar type III secretion system; furthermore, Ipaf activation could be recapitulated by the introduction of purified flagellin directly into the cytoplasm. These observations raise the possibility that the salmonella pathogenicity island 1 type III secretion system cannot completely exclude 'promiscuous' secretion of flagellin and that the host capitalizes on this 'error' by activating a potent host-defense pathway.
The innate immune system relies on its capacity to rapidly detect invading pathogenic microbes as foreign and eliminate them. Indeed, Toll-like receptors are a class of membrane receptors that sense extracellular microbes and trigger anti-pathogen signalling cascades. Recently, intracellular microbial sensors have also been identified, including NOD-like receptors and the helicase-domain-containing antiviral proteins RIG-I and MDA5. Some of these cytoplasmic molecules sense microbial, as well as non-microbial, danger signals, but the mechanisms of recognition used by these sensors remain poorly understood. Nonetheless, it is apparent that these proteins are likely to have critical roles in health and disease.
Many pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.
NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions.
Nod-like receptors (NLRs), Nod1 and Nod2 are cytosolic detectors of pathogen-associated molecular patterns (PAMPs). Nod1 is a three-domain protein, consisting of a caspase activation and recruitment domain (CARD), a nucleotide-binding oligomerization domain (NOD), and a leucine-rich repeat domain (LRR). The binding of PAMPs to the LRR results in the activation of signaling through homophilic CARD-CARD interactions. Several CARD structures have been determined, including a recent NMR structure of Nod1 CARD. In contrast to the reported NMR structure, the crystal structure reported here is a dimer, where the sixth helix is swapped between two monomers. While the overall structure is very similar to the known CARD structures, this is the first report of a homodimeric CARD structure. The ability of the CARD to exist in monomeric and dimeric forms suggests another level of regulation in the activation of NLR proteins.
Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.