Distinct Commensals Induce Interleukin-1ß via NLRP3 Inflammasome in Inflammatory Monocytes to Promote Intestinal Inflammation in Response to Injury.

The microbiota stimulates inflammation, but the signaling pathways and the members of the microbiota involved remain poorly understood. We found that the microbiota induces interleukin-1ß (IL-1ß) release upon intestinal injury and that this is mediated via the NLRP3 inflammasome. Enterobacteriaceae and in particular the pathobiont Proteus mirabilis, induced robust IL-1ß release that was comparable to that induced by the pathogen Salmonella. Upon epithelial injury, production of IL-1ß in the intestine was largely mediated by intestinal Ly6C(high) monocytes, required chemokine receptor CCR2 and was abolished by deletion of IL-1ß in CCR2(+) blood monocytes. Furthermore, colonization with P. mirabilis promoted intestinal inflammation upon intestinal injury via the production of hemolysin, which required NLRP3 and IL-1 receptor signaling in vivo. Thus, upon intestinal injury, selective members of the microbiota stimulate newly recruited monocytes to induce NLRP3-dependent IL-1ß release, which promotes inflammation in the intestine.

Interleukin-22 regulates the complement system to promote resistance against pathobionts after pathogen-induced intestinal damage.

Pathobionts play a critical role in disease development, but the immune mechanisms against pathobionts remain poorly understood. Here, we report a critical role for interleukin-22 (IL-22) in systemic protection against bacterial pathobionts that translocate into the circulation after infection with the pathogen Clostridium difficile. Infection with C. difficile induced IL-22, and infected Il22(-/-) mice harbored high numbers of pathobionts in extraintestinal organs despite comparable pathogen load and intestinal damage in mutant and wild-type mice. Pathobionts exhibited increased resistant against complement-mediated phagocytosis, and their intravenous administration resulted in high animal mortality. Selective removal of translocated commensals rescued Il22(-/-) mice, and IL-22 administration enhanced the elimination of pathobionts. Mechanistically, IL-22 augmented bacterial phagocytosis by increasing the expression and bacterial binding of complement C3. Our study demonstrates an unexpected role for IL-22 in controlling the elimination of pathobionts that enter the systemic circulation through the regulation of the complement system.

Lipopolysaccharide O structure of adherent and invasive Escherichia coli regulates intestinal inflammation via complement C3.

Gut dysbiosis associated with intestinal inflammation is characterized by the blooming of particular bacteria such as adherent-invasive E. coli (AIEC). However, the precise mechanisms by which AIEC impact on colitis remain largely unknown. Here we show that antibiotic-induced dysbiosis worsened chemically-induced colitis in IL-22-deficient mice, but not in wild-type mice. The increase in intestinal inflammation was associated with the expansion of E. coli strains with genetic and functional features of AIEC. These E. coli isolates exhibited high ability to out compete related bacteria via colicins and resistance to the host complement system in vitro. Mutation of wzy, the lipopolysaccharide O polymerase gene, rendered AIEC more sensitive to the complement system and more susceptible to engulfment and killing by phagocytes while retaining its ability to outcompete related bacteria in vitro. The wzy AIEC mutant showed impaired fitness to colonize the intestine under colitic conditions, but protected mice from chemically-induced colitis. Importantly, the ability of the wzy mutant to protect from colitis was blocked by depletion of complement C3 which was associated with impaired intestinal eradication of AIEC in colitic mice. These studies link surface lipopolysaccharide O-antigen structure to the regulation of colitic activity in commensal AIEC via interactions with the complement system.

WD40 Repeat Protein 26 Negatively Regulates Formyl Peptide Receptor-1 Mediated Wound Healing in Intestinal Epithelial Cells.

N-formyl peptide receptors (FPRs) serve as phagocyte pattern-recognition receptors that play a crucial role in the regulation of host defense against infection. Epithelial cells also express FPRs, and their activation during inflammation or injury results in enhanced epithelial migration and proliferation and improved mucosal wound repair. However, signaling mechanisms that govern epithelial FPR1 activity are not well understood. This study identified a novel FPR1-interacting protein, WD40 repeat protein (WDR)-26, which negatively regulates FPR1-mediated wound healing in intestinal epithelial cells. We show that WDR26-mediated inhibition of wound repair is mediated through the inhibition of Rac family small GTPase 1 and cell division cycle 42 activation, as well as downstream intracellular reactive oxygen species production. Furthermore, on FPR1 activation with N-formyl-methionyl-leucyl phenylalanine, WDR26 dissociates from FPR1, resulting in the activation of downstream cell division cycle 42/Rac family small GTPase 1 signaling, increased epithelial cell migration, and mucosal wound repair. These findings elucidate a novel regulatory function of WDR26 in FPR1-mediated wound healing in intestinal epithelial cells.

Staphylococcus δ-toxin induces allergic skin disease by activating mast cells.

Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Regulation of the gut microbiota by the mucosal immune system in mice.

The benefits of commensal bacteria to the health of the host have been well documented, such as providing stimulation to potentiate host immune responses, generation of useful metabolites, and direct competition with pathogens. However, the ability of the host immune system to control the microbiota remains less well understood. Recent microbiota analyses in mouse models have revealed detailed structures and diversities of microbiota at different sites of the digestive tract in mouse populations. The contradictory findings of previous studies on the role of host immune responses in overall microbiota composition are likely attributable to the high ß-diversity in mouse populations as well as technical limitations of the methods to analyze microbiota. The host employs multiple systems to strictly regulate their interactions with the microbiota. A spatial segregation between the host and microbiota is achieved with the mucosal epithelium, which is further fortified with a mucus layer on the luminal side and Paneth cells that produce antimicrobial peptides. When commensal bacteria or pathogens breach the epithelial barrier and translocate to peripheral tissues, the host immune system is activated to eliminate them. Defective segregation and tissue elimination of commensals result in exaggerated inflammatory responses and possibly death of the host. In this review, we discuss the current understanding of mouse microbiota, its common features with human microbiota, the technologies utilized to analyze microbiota, and finally the challenges faced to delineate the role of host immune responses in the composition of the luminal microbiota.

Protective role of commensals against Clostridium difficile infection via an IL-1ß-mediated positive-feedback loop.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotic-treated individuals. Commensal bacteria are known to have a significant role in the intestinal accumulation of C. difficile after antibiotic treatment, but little is known about how they affect host immunity during C. difficile infection. In this article, we report that C. difficile infection results in translocation of commensals across the intestinal epithelial barrier that is critical for neutrophil recruitment through the induction of an IL-1ß-mediated positive-feedback loop. Mice lacking ASC, an essential mediator of IL-1ß and IL-18 processing and secretion, were highly susceptible to C. difficile infection. ASC(-/-) mice exhibited enhanced translocation of commensals to multiple organs after C. difficile infection. Notably, ASC(-/-) mice exhibited impaired CXCL1 production and neutrophil influx into intestinal tissues in response to C. difficile infection. The impairment in neutrophil recruitment resulted in reduced production of IL-1ß and CXCL1 but not IL-18. Importantly, translocated commensals were required for ASC/Nlrp3-dependent IL-1ß secretion by neutrophils. Mice lacking IL-1ß were deficient in inducing CXCL1 secretion, suggesting that IL-1ß is the dominant inducer of ASC-mediated CXCL1 production during C. difficile infection. These results indicate that translocated commensals play a crucial role in CXCL1-dependent recruitment of neutrophils to the intestine through an IL-1ß/NLRP3/ASC-mediated positive-feedback mechanism that is important for host survival and clearance of translocated commensals during C. difficile infection.

Glycan sequence-dependent Nod2 activation investigated by using a chemically synthesized bacterial peptidoglycan fragment library.

Nucleotide oligomerization domain-containing protein 2 (Nod2), an innate immune receptor, recognizes bacterial cell-wall peptidoglycan (PGN), the minimum ligand of which is muramyl dipeptide (MDP). Enzymatic digestion of PGN appears to be important for Nod2 recognition. PGN is degraded by muramidase or glucosamidase through a process that produces two types of glycan sequence; glycans containing GlcNAcß(1→4)MurNAc or MurNAcß(1→4)GlcNAc. In this report, a range of disaccharide or tetrasaccharide fragments of each sequence were chemically synthesized, and their activities in stimulating human Nod2 (hNod2) were investigated. The results reveal that hNod2 recognitions is dependent on the glycan sequence, as demonstrated by comparing the activities of glycans with the same peptide moieties. (MurNAcß(1→4)GlcNAc)(2) -containing structures exhibited stronger activity than those containing (GlcNAcß(1→4)MurNAc)(2) . The results suggest that differences in the enzymatic degradation process affect the host's immunomodulation process.

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1ß in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.

Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeostasis of the colon to prevent spontaneous development of murine inflammatory bowel disease.

Fine-tuning of host cell responses to commensal bacteria plays a crucial role in maintaining homeostasis of the gut. Here, we show that tumor necrosis factor receptor-associated factor (Traf)2(-/-) mice spontaneously developed severe colitis and succumbed within 3 weeks after birth. Histological analysis revealed that apoptosis of colonic epithelial cells was enhanced, and B cells diffusely infiltrated into the submucosal layer of the colon of Traf2(-/-) mice. Expression of proinflammatory cytokines, including Tnfa, Il17a, and Ifng, was up-regulated, whereas expression of antimicrobial peptides was down-regulated in the colon of Traf2(-/-) mice. Moreover, a number of IL-17-producing helper T cells were increased in the colonic lamina propria of Traf2(-/-) mice. These cellular alterations resulted in drastic changes in the colonic microbiota of Traf2(-/-) mice compared with Traf2(+/+) mice. Treatment of Traf2(-/-) mice with antibiotics ameliorated colitis along with down-regulation of proinflammatory cytokines and prolonged survival, suggesting that the altered colonic microbiota might contribute to exacerbation of colitis. Finally, deletion of Tnfr1, but not Il17a, dramatically ameliorated colitis in Traf2(-/-) mice by preventing apoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, and restoration of wild-type commensal bacteria. Together, TRAF2 plays a crucial role in controlling homeostasis of the colon.

Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo.

Nod1 is a member of family of intracellular proteins that mediate host recognition of bacterial peptidoglycan. To characterize immune responses mediated by Nod1, synthetic ligand compounds possessing enhanced ability to stimulate Nod1 were developed to study the function of Nod1. Stimulation of epithelial cells with Nod1 stimulatory molecules induced chemokines and other proinflammatory molecules that are important for innate immune responses and recruitment of acute inflammatory cells. Administration of Nod1 ligands into mice induced chemokines and recruitment of acute inflammatory cells, an activity that was abolished in Nod1-null mice. Microarray analysis revealed that Nod1 stimulation induces a restricted number of genes in intestinal epithelial cells compared with that induced by tumor necrosis factor (TNF) alpha. Nod1 stimulation did not induce TNFalpha, interleukin 12, and interferon gamma, suggesting that the primary role of Nod1 is to induce the recruitment of immune cells. These results indicate that Nod1 functions as a pathogen recognition molecule to induce expression of molecules involved in the early stages of the innate immune response.

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation.

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-kappaB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-kappaB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IkappaB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-kappaB activation.

Characterization of natural human nucleotide-binding oligomerization domain protein 1 (Nod1) ligands from bacterial culture supernatant for elucidation of immune modulators in the environment.

Nucleotide-binding oligomerization domain protein 1 (Nod1) is an intracellular protein involved in recognition of the bacterial component peptidoglycan. This recognition event induces a host defense response to eliminate invading pathogens. The genetic variation of Nod1 has been linked to several inflammatory diseases and allergies, which are strongly affected by environmental factors. We have found that many of the bacteria that contain DAP-type peptidoglycan release Nod1 ligands into the environment. However, the structures of natural Nod1 ligands in the environment are not well understood. Herein, we report the isolation and structural elucidation of natural human Nod1 (hNod1) ligands from the Escherichia coli K-12 culture supernatant. The supernatant was fractionated with reversed-phase high performance liquid chromatography (RP-HPLC), resulting in the isolation of several hNod1 stimulatory fractions. Structural characterization studies demonstrated that the molecular structure of the most active fraction was the native hNod1 ligand GlcNAc-(beta1-4)-(anhydro)MurNAc-l-Ala-gamma-d-Glu-meso-DAP. We also found other peptidoglycan fragments using the 7-(diethylamino)coumarin-3-carbonyl labeling method to enhance sensitivity in mass spectroscopy studies. These results suggested that DAP-containing bacteria release certain hNod1 ligands to the environment, and these ligands would accumulate in the environment and regulate the immune system through Nod1.

Mechanism and repertoire of ASC-mediated gene expression.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor molecule that mediates inflammatory and apoptotic signals. Although the role of ASC in caspase-1-mediated IL-1beta and IL-18 maturation is well known, ASC also induces NF-kappaB activation and cytokine gene expression in human cells. In this study, we investigated the molecular mechanism and repertoire of ASC-induced gene expression in human cells. We found that the specific activation of ASC induced AP-1 activity, which was required for optimal IL8 promoter activity. ASC activation also induced STAT3-, but not STAT1-, IFN-stimulated gene factor 3- or NF-AT-dependent reporter gene expression. The ASC-mediated AP-1 activation was NF-kappaB-independent and primarily cell-autonomous response, whereas the STAT3 activation required NF-kappaB activation and was mediated by a factor that can act in a paracrine manner. ASC-mediated AP-1 activation was inhibited by chemical or protein inhibitors for caspase-8, caspase-8-targeting small-interfering RNA, and p38 and JNK inhibitors, but not by a caspase-1 inhibitor, caspase-9 or Fas-associated death domain protein (FADD) dominant-negative mutants, FADD- or RICK-targeting small-interfering RNAs, or a MEK inhibitor, indicating that the ASC-induced AP-1 activation is mediated by caspase-8, p38, and JNK, but does not require caspase-1, caspase-9, FADD, RICK, or ERK. DNA microarray analyses identified 75 genes that were induced by ASC activation. A large proportion of them was related to transcription (23%), inflammation (21%), or cell death (16%), indicating that ASC is a potent inducer of inflammatory and cell death-related genes. This is the first report of ASC-mediated AP-1 activation and the repertoire of genes induced downstream of ASC activation.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Transitions in oral and intestinal microflora composition and innate immune receptor-dependent stimulation during mouse development.

Commensal bacteria possess immunostimulatory activities that can modulate host responses to affect development and homeostasis in the intestine. However, how different populations of resident bacteria stimulate the immune system remains largely unknown. We characterized here the ability of intestinal and oral microflora to stimulate individual pattern recognition receptors (PRRs) in bone marrow-derived macrophages and mesothelial cells. The intestinal but not oral microflora elicited age- and cell type-specific immunostimulation. The immunostimulatory activity of the intestinal microflora varied among individual mice but was largely mediated via Toll-like receptor 4 (TLR4) during breast-feeding, whereas it became TLR4 independent after weaning. This transition was associated with a change from a microflora rich in TLR4-stimulatory proteobacteria to one dominated by Bacteroidales and/or Clostridiales that poorly stimulate TLR4. The major stimulatory activity of the intestinal microflora was still intact in NOD1-, NOD2-, TLR2-, TLR4-, TLR5-, TLR9-, TLR11-, ASC-, or RICK-deficient cells but still relied on the adaptor MyD88. These studies demonstrate a transition in the intestinal microflora accompanied by a dynamic change of its ability to stimulate different PRRs which control intestinal homeostasis.

Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (Villin-CreERT2; Dsc2 fl/fl) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell-matrix traction forces, decreased levels of integrin ß1 and ß4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.

Synthesis of diaminopimelic acid containing peptidoglycan fragments and tracheal cytotoxin (TCT) and investigation of their biological functions.

Bacterial cell wall peptidoglycan (PGN) is a potent immunostimulator and immune adjuvant. The PGN of Gram-negative bacteria and some Gram-positive bacteria contain meso-diaminopimelic acid (meso-DAP), and we have recently shown that the intracellular protein Nod1 is a PGN receptor and recognizes DAP-containing peptides. In this study, we achieved the synthesis of DAP-containing PGN fragments, including the first chemical synthesis of tracheal cytotoxin (TCT), GlcNAc-(beta1-4)-(anhydro)MurNAc-L-Ala-gamma-D-Glu-meso-DAP-D-Ala, and a repeating-unit of DAP-type PGN, GlcNAc-(beta1-4)-MurNAc-L-Ala-gamma-D-Glu-meso-DAP-D-Ala. For the synthesis of PGN fragments, we first established a new synthetic method for an orthogonally protected meso-DAP derivative, and then we constructed the glycopeptide structures. The ability of these fragments to stimulate human Nod1, as well as differences in Nod1 recognition of the variety of synthesized ligand structures were examined. The results showed that the substitution of the N terminus of iE-DAP is necessary for stronger Nod1 recognition, but the structure of the substituent seems not to be strictly recognized. The importance of the carboxyl group at the 2-position of DAP for human Nod1 stimulation was also shown.

MEX is a testis-specific E3 ubiquitin ligase that promotes death receptor-induced apoptosis.

In the present study, we report the identification and characterization of MEX (MEKK1-related protein X), a protein with homology to MEKK1 that is expressed uniquely in the testis. MEX is comprises four putative zinc-binding domains including an N-terminal SWIM (SWI2/SNF2 and MuDR) domain of unknown function and two RING (really interesting new gene) fingers separated by a ZZ zinc finger domain. Biochemical analyses revealed that MEX is self-ubiquitinated and targeted for degradation through the proteasome pathway. MEX can act as an E3, Ub (ubiquitin) ligase, through the E2, Ub-conjugating enzymes UbcH5a, UbcH5c or UbcH6. A region of MEX that contains the RING fingers and the ZZ zinc finger was required for interaction with UbcH5a and MEX self-association, whereas the SWIM domain was critical for MEX ubiquitination. The expression of MEX promoted apoptosis that was induced through Fas, DR (death receptor) 3 and DR4 signalling, but not that mediated by the BH3 (Bcl-2 homology 3)-only protein BimEL or the chemotherapeutic drug adriamycin. The enhancement of apoptosis by MEX required a functional SWIM domain, suggesting that MEX ubiquitination is critical for the enhancement of apoptosis. These results indicate that MEX acts as an E3 Ub ligase, an activity that is dependent on the SWIM domain and suggest a role for MEX in the regulation of death receptor-induced apoptosis in the testes.

[The molecular mechanism of autoinflammatory disease--lessons from the function of NOD protein families].

The latest decade, our understanding of pattern-recognizing receptors involved in innate immune system has been accumulated. One class of the pattern recognizing receptors, the toll-like receptors (TLRs) are well known to detect extracellular pathogens on the cell surface membrane. On the other hand, recently discovered the nucleotide-binding oligomerization domain proteins (NODs) are involved in recognizing intracellular pathogens. Since Nod2, one of the NODs, mutations were found to associate with susceptibility of Crohn's disease, the NODs have been highlighted. For example, cryopyrin mutations have been reported to associate with Familial cold urticaria (FCU)/Familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), Neonatal onset multisystem inflammatory disease (NOMID)/Chronic infantile neurologic cutaneous and articular syndrome (CINCA). Here, we summarize the discovery of the NODs and related molecules, and also discuss the function of the NODs and molecular mechanisms of the autoinflammatory diseases.