Family Of Innate Immune Receptors Research Articles

Role and molecular mechanism of NOD2 in chronic non-communicable diseases.

Nucleotide-binding oligomerization domain containing 2 (NOD2), located in the cell cytoplasm, is a pattern recognition receptor belonging to the innate immune receptor family. It mediates the innate immune response by identifying conserved sequences in bacterial peptide glycans and plays an essential role in maintaining immune system homeostasis. Gene mutations of NOD2 lead to the development of autoimmune diseases such as Crohn's disease and Blau syndrome. Recently, NOD2 has been shown to be associated with the pathogenesis of diabetes, cardiac-cerebral diseases, and cancers. However, the function of NOD2 in these non-communicable diseases (CNCDs) is not well summarized in reviews. Our report mainly discusses the primary function and molecular mechanism of NOD2 as well as its potential clinical significance in CNCDs.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Ancient fish lineages illuminate toll-like receptor diversification in early vertebrate evolution.

Since its initial discovery over 50years ago, understanding the evolution of the vertebrate RAG- mediated adaptive immune response has been a major area of research focus for comparative geneticists. However, how the evolutionary novelty of an adaptive immune response impacted the diversity of receptors associated with the innate immune response has received considerably less attention until recently. Here, we investigate the diversification of vertebrate toll-like receptors (TLRs), one of the most ancient and well conserved innate immune receptor families found across the Tree of Life, integrating genomic data that represent all major vertebrate lineages with new transcriptomic data from Polypteriformes, the earliest diverging ray-finned fish lineage. Our analyses reveal TLR sequences that reflect the 6 major TLR subfamilies, TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11, and also currently unnamed, yet phylogenetically distinct TLR clades. We additionally recover evidence for a pulse of gene gain coincident with the rise of the RAG-mediated adaptive immune response in jawed vertebrates, followed by a period of rapid gene loss during the Cretaceous. These gene losses are primarily concentrated in marine teleost fish and synchronous with the mid Cretaceous anoxic event, a period of rapid extinction for marine species. Finally, we reveal a mismatch between phylogenetic placement and gene nomenclature for up to 50% of TLRs found in clades such as ray-finned fishes, cyclostomes, amphibians, and elasmobranchs. Collectively, these results provide an unparalleled perspective of TLR diversity and offer a ready framework for testing gene annotations in non-model species.

A cell reporter system to examine toll-like receptor ligands in channel catfish

Abstract Toll-like receptors (TLR) are a family of innate immune receptors that recognize microbe- or danger-associated molecular patterns (MAMPs or DAMPs). TLR ligand binding results in the recruitment of adaptor proteins, which initiate signaling cascades that lead to the activation of NF-κB and interferon regulatory factors (IRFs). Importantly, this signaling initiates both innate and adaptive immune responses. Channel catfish is an important aquacultural species, and the catfish genome encodes 20 TLRs. In contrast, mammalian genomes encode from 10 to 13 TLRs. Our hypothesis is that fish, due to their aquatic environment, express additional TLRs that provide protection from pathogens and pollutants. Our goal is to gain a functional understanding of catfish TLRs by identifying their ligands. To screen potential ligands, we use the HEK Dual-null cell line, which is engineered to not express endogenous TLRs. This cell line is stably transfected with a SEAP reporter gene under the control of NF-κB. Using this model, it was confirmed that TLR7 is activated by imiquimod, a synthetic ssRNA analog. In addition, we have identified the full repertoire of IFN genes in the catfish genome. This allows us to assess products downstream of TLR activation by qPCR. Taken together, this system permits us to study the TLR signaling pathways that drive catfish immune responses. In the future, once TLR signaling is better understood, specific ligands can be incorporated into vaccines as adjuvants to direct favorable immune responses and protect farmed and domesticated animal species. Supported by pre-doctoral fellowship USDA-NIFA-AFRI 2022-67011-36580 and grant NSF-IOS 1656419

MAVS Expression in Alveolar Macrophages Is Essential for Host Resistance against Aspergillus fumigatus.

Our recent data demonstrate a critical role of the RIG-I-like receptor family in regulating antifungal immunity against Aspergillus fumigatus in a murine model. However, the importance of this pathway in humans and the cell types that use this innate immune receptor family to detect A. fumigatus remain unresolved. In this study, using patients who underwent hematopoietic stem cell transplantation, we demonstrate that a polymorphism in human MAVS present in the donor genome was associated with the incidence of invasive pulmonary aspergillosis. Moreover, in a separate cohort of confirmed invasive pulmonary aspergillosis patients, polymorphisms in the IFIH1 gene alter the inflammatory response, including IFN-responsive chemokines. Returning to our murine model, we now demonstrate that CD11c+ Siglec F+ alveolar macrophages require Mavs expression to maintain host resistance against A. fumigatus. Our data support the role of MAVS signaling in mediating antifungal immunity in both mice and humans at least in part through the role of MAVS-dependent signaling in alveolar macrophages.

Toll-Like Receptor-4 Antagonist Enhances the Repair of Ultraviolet Radiation-Induced DNA Damage and Augments Anti-Tumor Immune Responses in Mice.

Simple SummaryUltraviolet B (UVB) radiation is largely responsible for the development of skin cancer. When UVB-induced DNA damage in cells is not repaired, it can lead to the initiation of non-melanoma skin cancers. Xeroderma pigmentosum (XP) disease is caused by a defect in the repair of damaged DNA. Toll-like receptor-4 (TLR4) and NLR family pyrin domain containing 3 (NLRP3) belong to the family of innate immune receptors and are highly expressed in skin tumors. In this study, we determined the mechanism through which TLR4 inhibitor TAK-242 regulates inflammation and prevents skin cancer.Ultraviolet (UV) irradiation of the skin is related to the development of skin cancer. UVB also causes DNA damage in the form of cyclobutane pyrimidine dimers (CPDs), which can result in stable mutations. Toll-like receptor 4 (TLR4), a component of innate immunity, plays a key role in cancer. Previous studies from our laboratory have observed that TLR4 deficiency resulted in the repair of UVB-induced DNA damage, inhibition of UVB-induced immune suppression, and carcinogenesis. In this study, we determined the efficacy of TLR4 antagonist TAK-242 in regulation of UVB-induced DNA damage, inflammation, and tumor development. Our results indicate that TAK-242 treatment increased the expression of xeroderma pigmentosum group A (XPA) mRNA, resulting in the repair of UVB-induced CPDs in skin of SKH-1 mice. Treatment with TAK-242 also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) in UVB-exposed skin of SKH-1 mice. Cutaneous carcinogenesis was significantly reduced in mice treated with TAK-242 in comparison to vehicle-treated mice. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were also found to be significantly greater in vehicle-treated mice than TAK-242-treated mice. Finally, treatment with TAK-242 augmented anti-tumor immune responses in mice. Our data provide further evidence that activation of the TLR4 pathway promotes the development of UV-induced non-melanoma skin cancer mediated at least in part on its negative effects on DNA damage. Moreover, treatment with the TLR4 inhibitor TAK-242 may be effective for prevention of skin cancer.

Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system.

Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

Molecular characterization and cellular localization of a transmembrane C-type lectin receptor in hemocytes from the scallop Argopecten purpuratus

Recognition of potential pathogens is a crucial function of the immune system of every organism. In this context, great efforts have been made in the last years to characterize the diversity of pattern recognition receptors (PRRs) present in cultured aquatic species, such as bivalve mollusks. Among the great diversity of PRRs described so far, C-type lectin receptors (CLRs) comprise a large family of innate immune receptors that can recognize different carbohydrates present on the cell surface of microorganisms. CLRs can be found extracellularly in soluble form, or transmembrane, located on immune cell surfaces. To date, almost only soluble CLRs have been described in bivalves and so far, no transmembrane CLRs are identified in pectinids. In the present study, a sequence showing structural homologies to transmembrane CLRs (tmCLR) was characterized from scallop Argopecten purpuratus hemocytes, named ApCLR. The cDNA sequence displays 948 nucleotides, coding for a deduced protein of 316 amino acids with a molecular weight of 35.24 kDa. The protein sequence showed characteristic domains of a tmCLR, such as a signal peptide, an extracellular N-terminal carbohydrate recognition domain (CRD), one transmembrane domain and a cytoplasmic tail. Phylogenetic analysis revealed that ApCLR groups with soluble CLRs from scallops but also with transmembrane CLRs from vertebrates, such as chondrolectins and layilins. Gene expression analysis showed that ApCLR was constitutively present in gill, mantle, digestive gland, gonad and hemocyte tissues from non-immune challenged scallops and it was most strongly expressed in the gill and mantle tissues. Furthermore, ApCLR was significantly upregulated in scallop hemocytes at 6 h after scallop injection with heat-killed Vibrio splendidus. At the protein level, ApCLR is located at the plasma membrane of hemocytes, observed as punctate signals by immunofluorescence and confocal analysis. Results obtained in this study reveal the first transmembrane CLR in scallops. Also, results showed that ApCLR is present at the surface of hemocytes and increase its transcript availability during the immune challenge, suggesting a potential role in the recognition of microorganisms.

Toll-like receptors form different complexes with UNC93B1.

The multipass transmembrane protein UNC93B1 is critical for the proper trafficking and function of many members of the Toll-like receptor (TLR) family of innate immune receptors. A new study reports two structures of UNC93B1 in complex with full-length TLR3 or TLR7 and sheds light on how this single chaperone may differentially interact with and regulate the function of individual TLRs.

Expressional correlation of Toll-like Receptor 9 (TLR9) with angiogenic factors and anti-apoptotic markers in cervical cancer cells

Cervical cancer is one of the most frequently occurring oncological malignancies which in turn is attributed with the chronic persistence of human papillomavirus (HPV). Recent explorations into cancer immunobiology have elucidated that innate immune response are important for proliferation of cancerous cells. Toll-like receptors (TLRs), belonging to the pattern recognition receptors (PRRs) family of innate immune receptors have been reported for their expression in various cancers, where they aid cancer cell proliferation along with tumor development. The present study focuses on investigating TLR9 mRNA within HPV18⁺ HeLa (cervical cancer cells) and its correlation with angiogenic factors and anti-apoptotic markers. The expression of TLR9 mRNA was substantially higher in HeLa cancer cells in comparison with normal kidney cells. MTT assay demonstrated that treatment with CpG ODN significantly increased the proliferation in dose-dependent manner. Augmented levels of cell percentage within G2/M phase was also recorded post CpG ODN treatment within HeLa cells. qRT-PCR analysis also elucidated elevated levels of Bcl-2, Bcl-X_L, COX-2, VEGF, and TLR9 within HeLa cells. Thus, to conclude, our study established that HeLa cells have increased TLR9 mRNA expression concomitant with elevated COX-2, Bcl-2, Bcl-X_L and VEGF. These results indicate toward potential applicability of targeting TLR9 as a therapeutical intervention for the treatment and management of this pernicious malignancy.

Equilibrium Binding Model for CpG DNA-Dependent Dimerization of Toll-like Receptor 9 Ectodomain.

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

Effects of NLRP6 in Cerebral Ischemia/Reperfusion (I/R) Injury in Rats.

The NOD-like receptor protein 6 (NLRP6), an intracytoplasmic pattern recognition receptor in the nucleotide-binding domain, leucine-rich repeat-containing (NLR) innate immune receptor family, influences the inflammation reaction. The role of NLRP6 in cerebral ischemia-reperfusion (I/R) injury in rats is unclear. We explore the function of NLRP6 in cerebral I/R injury. The investigators used a middle cerebral artery occlusion/reperfusion model (MCAO) to imitate ischemic injury. We found the peak expression of NLRP6 is in 48-h post-cerebral I/R injury. The expression of NLRP6 siRNA, as well as the expression of protein and mRNA, was detected by Western blot and qRT-PCR. The degree of IL-1β and IL-18 was assessed by ELISA. After downregulating NLRP6, the expression of IL-1β, IL-18, cleaved Caspase-1, and myeloperoxidase (MPO) were reduced. In HE and Nissl staining, pathological injury of brain tissue after downregulating NLRP6 was improved. NLRP6 siRNA decreased the NLRP6-ASC binding states by CO-IP. NRP6 has a pro-inflammatory effect in cerebral I/R injury, which may provide a new target for the treatment of cerebral I/R injury.

Elementary Steps in Plant‐Pathogen Recognition Revealed by Enhancing the Stability and Structure of Domains of Plant Innate Immune Receptors

Pathogens elicit an immune response in plants through the recognition of the avirulent effectors by multi‐domain intracellular disease resistance receptors. The nature and specificity of plant‐pathogen interactions and the molecular mechanism of eliciting an immune response is being explored using the N protein from Nicotianaglutinosa. The N protein is an archetype of the nucleotide binding (NB), leucine rich repeat (LRR) family of innate immune receptors (NLRs), and the interaction with its avirulent effector, the helicase domain (HEL) of the replicase protein of tobacco mosaic virus (TMV) triggers a classic hypersensitive cell death response in plants. An initial step in activating cell death pathways upon pathogen recognition is a structure change that enables NLR assembly through their N‐terminal Toll‐Interlukin1 Receptor‐like (TIR) domains. Stabilized domains with increased tendency to self‐associate have been constructed by focused mutagenesis guided by computational modeling of a 3.0 Å crystal structure of the N‐TIR domain. The energetics of TIR dimerization is being correlated with the severity of the hypersensitive response by measuring salicylic acid using LC‐MS to examine the hypothesis that receptor assembly is correlated with NLR signaling. Yeast surface display is being used to probe the manner whereby host NLRs recognize pathogenic determinants, and reveals that the interaction of N with its pathogen effector, the HEL domain of TMV, is mediated via its LRR domain. Binding analyses show that a stabilized LRR domain binds HEL about 250‐fold more strongly than a non‐virulent homologue that does not display a hypersensitive response. Variants of the full‐length N protein are being rebuilt from the individually stabilized TIR and LRR domains optimized for functional analyses to enable biochemical and structural investigations of plant NLR receptors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NLRP12 Regulates Anti-viral RIG-I Activation via Interaction with TRIM25

Establishing the balance between positive and negative innate immune mechanisms is crucial for maintaining homeostasis. Here we uncover the regulatory crosstalk between two previously unlinked innate immune receptor families: RIG-I, an anti-viral cytosolic receptor activated type I interferon production, and NLR (nucleotide-binding domain, leucine repeat domain-containing protein). We show that NLRP12 dampens RIG-I-mediated immune signaling against RNA viruses by controlling RIG-I's association with its adaptor MAVS. The nucleotide-binding domain of NLRP12 interacts with the ubiquitin ligase TRIM25 to prevent TRIM25-mediated, Lys63-linked ubiquitination and activation of RIG-I. NLRP12 also enhances RNF125-mediated, Lys48-linked degradative ubiquitination of RIG-I. Vesicular stomatitis virus (VSV) infection downregulates NLRP12 expression to allow RIG-I activation. Myeloid-cell-specific Nlrp12-deficient mice display a heightened interferon and TNF response and are more resistant to VSV infection. These results indicate that NLRP12 functions as a checkpoint for anti-viral RIG-I activation.

The protective roles of NLRP6 in intestinal epithelial cells.

The evolution of chronic inflammatory diseases is thought to be due to a combination of host genetic variations and environmental factors that include the alteration of intestinal flora, termed “dysbiosis.” The intestinal mucosal barrier includes a chemical barrier and physical barrier that have important roles in protecting the intestine against inflammatory injury. The chemical barrier includes antimicrobial peptides (AMPs), and the physical barrier includes a mucous layer, a monolayer of intestinal epithelial cells and cell junctions. The intestinal mucosal barrier is not a static barrier, but rather, it strongly interacts with the gut microbiome and cells of the immune system. Correct expression of AMPs, together with mucus and balanced epithelial cell proliferation, prevents the occurrence of disease. NLRP6, a member of the nucleotide‐binding domain, leucine‐rich repeat‐containing (NLR) innate immune receptor family, participates in the progression of intestinal inflammation and enteric pathogen infections. It has become apparent in recent years that NLRP6 is important in disease pathogenesis, as it responds to internal ligands that lead to the release of AMPs and mucus, thus regulating the regeneration of intestinal epithelial cells. This review summarizes the activation of NLRP6 and its protective role in the intestinal epithelial cell.

The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity

Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ Tcells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ Tcell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limitNF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ Tcell signaling and metabolism.

Evolutionary Regression and Species-Specific Codon Usage of TLR15.

Toll-like receptors (TLRs) form an ancient family of innate immune receptors that detect microbial structures and activate the host immune response. Most subfamilies of TLRs (including TLR3, TLR5, and TLR7) are highly conserved among vertebrate species. In contrast, TLR15, a member of the TLR1 subfamily, appears to be unique to birds and reptiles. We investigated the functional evolution of TLR15. Phylogenetic and synteny analyses revealed putative TLR15 orthologs in bird species, several reptilian species and also in a shark species, pointing to an unprecedented date of origin of TLR15 as well as large scale reciprocal loss of this TLR in most other vertebrates. Cloning and functional analysis of TLR15 of the green anole lizard (Anolis carolinensis), salt water crocodile (Crocodylus porosus), American alligator (Alligator mississippiensis), and chicken (Gallus gallus) showed for all species TLR15 specific protease-induced activation of NF-κB, despite highly variable TLR15 protein expression levels. The variable TLR15 expression was consistent in both human and reptilian cells and could be attributed to species-specific differences in TLR15 codon usage. The species-specific codon bias was not or barely noted for more evolutionarily conserved TLRs (e.g., TLR3). Overall, our results indicate that TLR15 originates before the divergence of chondrichthyes fish and tetrapods and that TLR15 of both avian and reptilian species has a conserved function as protease activated receptor. The species-specific codon usage and large scale loss of TLR15 in most vertebrates suggest evolutionary regression of this ancient TLR.

Role of triggering receptor expressed on myeloid cells 2 in neuroinflammation and neurodegeneration of the central nervous system

AbstractTriggering receptor expressed on myeloid cells 2 (TREM2) is a member of the TREM family of innate immune receptors. It is expressed on the cell membrane of myeloid lineage cells including osteoclasts and microglia. Since it was reported that rare TREM2 variants, such as R47H and R62H, increase the risk of late‐onset Alzheimer's disease, it has been pointed out that many TREM2 variants relate to other neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and frontotemporal dementia. Although there are still conflicting reports as to whether TREM2 promotes or suppresses neurodegenerative or neuroinflammatory diseases, TREM2 has recently been considered to play a key role in the concept of disease‐associated microglia. In disease‐associated microglia induction, TREM2 might have a role as a microglia‐specific checkpoint.

Reduced expression of the NLRP6 inflammasome in the colon of patients with Hirschsprung’s disease

PurposeHirschsprung's associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung's Disease (HSCR). The pathogenesis of HAEC remains unsatisfactorily understood. Mounting evidence of an altered microbiome in patients with HSCR adds a new angle to the pathogenesis of HAEC. NLRP6 is a member of the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) innate immune receptor family, a multiprotein complex that functions as a sensor of damage-associated molecular patterns. Known as inflammasomes they have been implicated in the regulation of colonic microbial ecology and by alteration, regulators of inflammation. We designed this study to test the hypothesis that NLRP6 expression is altered in the colon of patients with HSCR. MethodsWe investigated NLPR6 protein expression in both the aganglionic and ganglionic regions of HSCR patients (n = 10) versus healthy control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expressions were quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. Main resultsqPCR and Western blot analysis revealed that NLRP6 is expressed in the colon of patients with HSCR. NLRP6 expression was significantly decreased (p < 0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that NLRP6 expression in colonic epithelium was markedly decreased in HSCR specimens compared to controls. ConclusionWe demonstrate for the first time the expression and distribution of NLRP6 in patients with HSCR. The observed decreased expression of NLRP6 may contribute to an altered colonic microbiome in patients with HSCR and increases the susceptibility to develop HAEC.

NLRP6 Induces Pyroptosis by Activation of Caspase-1 in Gingival Fibroblasts

NLRP6, a member of the nucleotide-binding domain, leucine-rich repeat-containing (NLR) innate immune receptor family, has been reported to participate in inflammasome formation. Activation of inflammasome triggers a caspase-1–dependent programming cell death called pyroptosis. However, whether NLRP6 induces pyroptosis has not been investigated. In this study, we showed that NLRP6 overexpression activated caspase-1 and gasdermin-D and then induced pyroptosis of human gingival fibroblasts, resulting in release of proinflammatory mediators interleukin (IL)–1β and IL-18. Moreover, NLRP6 was highly expressed in gingival tissue of periodontitis compared with healthy controls. Porphyromonas gingivalis, which is a commensal bacterium and has periodontopathic potential, induced pyroptosis of gingival fibroblasts by activation of NLRP6. Together, we, for the first time, identified that NLRP6 could induce pyroptosis of gingival fibroblasts by activation of caspase-1 and may play a role in periodontitis.