NLRP12 interacts with NLRP3 to block the activation of the human NLRP3 inflammasome.

Inflammasomes are multiprotein complexes that drive inflammation and contribute to protective immunity against pathogens and immune pathology in autoinflammatory diseases. Inflammasomes assemble when an inflammasome scaffold protein senses an activating signal and forms a signaling platform with the inflammasome adaptor protein ASC. The NLRP subfamily of NOD-like receptors (NLRs) includes inflammasome nucleators (such as NLRP3) and also NLRP12, which is genetically linked to familial autoinflammatory disorders that resemble diseases caused by gain-of-function NLRP3 mutants that generate a hyperactive NLRP3 inflammasome. We performed a screen to identify ASC inflammasome-nucleating proteins among NLRs that have the canonical pyrin-NACHT-LRR domain structure. Only NLRP3 and NLRP6 could initiate ASC polymerization to form "specks," and NLRP12 failed to nucleate ASC polymerization. However, wild-type NLRP12 inhibited ASC inflammasome assembly induced by wild-type and gain-of-function mutant NLRP3, an effect not seen with disease-associated NLRP12 mutants. The capacity of NLRP12 to suppress NLRP3 inflammasome assembly was limited to human NLRP3 and was not observed for wild-type murine NLRP3. Furthermore, peripheral blood mononuclear cells from patients with an NLRP12 mutant-associated inflammatory disorder produced increased amounts of the inflammatory cytokine IL-1ß in response to NLRP3 stimulation. Thus, our findings provide insights into NLRP12 biology and suggest that NLRP3 inhibitors in clinical trials for NLRP3-driven diseases may also be effective in treating NLRP12-associated autoinflammatory diseases.

Loss of NOD2 in macrophages improves colitis and tumorigenesis in a lysozyme-dependent manner.

Background: Crohn's disease (CD) is a complex and poorly understood myeloid-mediated disorder. Genetic variants with loss of function in the NOD2 gene confer an increased susceptibility to ileal CD. While Nod2 in myeloid cells may confer protection against T-cell mediated ileopathy, it remains unclear whether it may promote resolution of the inflamed colon. In this study, we evaluated the function of Nod2 in myeloid cells in a model of acute colitis and colitis-associated colon cancer (CAC). Methods: To ablate Nod2 specifically within the myeloid compartment, we generated LysMCre/+;Nod2fl/fl mice. The role of NOD2 was studied in a setting of Dextran Sodium Sulfate (DSS)-induced colitis and in azoxymethane (AOM)/DSS model. Clinical parameters were quantified by colonoscopy, histological, flow cytometry, and qRT-PCR analysis. Results: Upon DSS colitis model, LysMCre/+;Nod2fl/fl mice lost less weight than control littermates and had less severe damage to the colonic epithelium. In the AOM/DSS model, endoscopic monitoring of tumor progression revealed a lowered number of adenomas within the colon of LysMCre/+;Nod2fl/fl mice, associated with less expression of Tgfb. Mechanistically, lysozyme M was required for the improved disease severity in mice with a defect of NOD2 in myeloid cells. Conclusion: Our results indicate that loss of Nod2 signaling in myeloid cells aids in the tissue repair of the inflamed large intestine through lysozyme secretion by myeloid cells. These results may pave the way to design new therapeutics to limit the inflammatory and tumorigenic functions of NOD2.

NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

The regenerating family member 3 ß instigates IL-17A-mediated neutrophil recruitment downstream of NOD1/2 signalling for controlling colonisation resistance independently of microbiota community structure.

OBJECTIVE: Loss of the Crohn's disease predisposing NOD2 gene results in an intestinal microenvironment conducive for colonisation by attaching-and-effacing enteropathogens. However, it remains elusive whether it relies on the intracellular recruitment of the serine-threonine kinase RIPK2 by NOD2, a step that is required for its activation of the transcription factor NF-κB. DESIGN: Colonisation resistance was evaluated in wild type and mutant mice, as well as in ex-germ-free (ex-GF) mice which were colonised either with faeces from Ripk2-deficient mice or with bacteria with similar preferences for carbohydrates to those acquired by the pathogen. The severity of the mucosal pathology was quantified at several time points postinfection by using a previously established scoring. The community resilience in response to infection was evaluated by 16S ribosomal RNA gene sequence analysis. The control of pathogen virulence was evaluated by monitoring the secretion of Citrobacter-specific antibody response in the faeces. RESULTS: Primary infection was similarly outcompeted in ex-GF Ripk2-deficient and control mice, demonstrating that the susceptibility to infection resulting from RIPK2 deficiency cannot be solely attributed to specific microbiota community structures. In contrast, delayed clearance of Citrobacter rodentium and exacerbated histopathology were preceded by a weakened propensity of intestinal macrophages to afford innate lymphoid cell activation. This tissue protection unexpectedly required the regenerating family member 3ß by instigating interleukin (IL) 17A-mediated neutrophil recruitment to the intestine and subsequent phosphorylation of signal transducer and activator of transcription 3. CONCLUSIONS: These results unveil a previously unrecognised mechanism that efficiently protects from colonisation by diarrhoeagenic bacteria early in infection.

Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens.

Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium. Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen.

The battlefield in the war against attaching-and-effacing bacterial pathogens: Monocytes, macrophages and dendritic cells in action.

The recent adoption of a unified nomenclature for the mononuclear phagocyte system has already led to the generation of novel strategies for specifically depleting a single subset of phagocytes in the presence of intact lymphoid structures. Herein, we provide a detailed description of how the various types of tissue phagocyte orchestrate the host's defense against enteric bacterial infections. From a bench-to-bedside perspective, we expect that this paradigm will accelerate the development of novel adjuvants and vaccines in human and veterinary microbiology.

CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis.

Dendritic cells (DCs) and monocyte-derived macrophages (MΦs) are key components of intestinal immunity. However, the lack of surface markers differentiating MΦs from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, MΦs can be distinguished from DCs in the intestine of both mice and humans. On that basis, we revisit the phenotype of intestinal DCs in the absence of contaminating MΦs and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly-6C(hi) monocytes to intestinal MΦs. We determine how inflammation impacts this pathway and show that T cell-mediated colitis is associated with massive recruitment of monocytes to the intestine and the mesenteric lymph node (MLN). There, these monocytes differentiate into inflammatory MΦs endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. In the MLNs, inflammatory MΦs are located in the T-cell zone and trigger the induction of proinflammatory T cells. Finally, T cell-mediated colitis develops irrespective of intestinal DC migration, an unexpected finding supporting an important role for MLN-resident inflammatory MΦs in the etiology of T cell-mediated colitis.

DNGR-1 is a specific and universal marker of mouse and human Batf3-dependent dendritic cells in lymphoid and nonlymphoid tissues.

Mouse CD8α(+) dendritic cells (DCs) in lymphoid organs and CD103(+) CD11b(-) DCs in nonlymphoid tissues share phenotypic and functional similarities, as well as a unique shared developmental dependence on the transcription factor Batf3. Human DCs resembling mouse CD8α(+) DCs in phenotype and function have been identified in human blood, spleen, and tonsil. However, it is not clear whether such cells are also present in human nonlymphoid organs, and their equivalence to mouse CD8α(+) DC has recently been questioned. Furthermore, the identification of "CD8α(+) DC-like" cells across different tissues and species remains problematic because of the lack of a unique marker that can be used to unambiguously define lineage members. Here we show that mouse CD8α(+) DCs and CD103(+) CD11b(-) DCs can be defined by shared high expression of DNGR-1 (CLEC9A). We further show that DNGR-1 uniquely marks a CD11b(-) human DC population present in both lymphoid and nonlymphoid tissues of humans and humanized mice. Finally, we demonstrate that knockdown of Batf3 selectively prevents the development of DNGR-1(+) human DCs in vitro. Thus, high expression of DNGR-1 specifically and universally identifies a unique DC subset in mouse and humans. Evolutionarily conserved Batf3 dependence justifies classification of DNGR-1(hi) DCs as a distinct DC lineage.

Interleukin-9 stimulates the production of interleukin-5 in CD4+ T cells.

We recently showed that interleukin-9 (IL-9), a Th2 cytokine, promotes IL-5-mediated rejection of allografts in mice. This observation led us to investigate the functional link between IL-9 and IL-5 production during alloreactive T cell responses in vitro and in vivo. Firstly, we found that IL-9 was produced by alloreactive Th2 cells, and IL-9 mRNA was detected in skin allograft during Th2-type rejection. We then established that IL-5 production was impaired in alloreactive Th2 cells isolated from IL-9-deficient mice and that optimal IL-5 production after allogeneic stimulation requires a functional IL-9 receptor (IL-9R) on the responding cells. Finally, the production of IL-5 by anti-CD3-stimulated CD4+ T cells was abolished by neutralization of IL-9. Despite the fact that IL-9 promotes IL-5 production by alloreactive T cells, IL-9-deficient recipients of skin allografts still developed eosinophilic graft infiltrates and neither IL-9 nor IL-9R deficiency modified Th2-type allograft rejection.

Interleukin-9 promotes eosinophilic rejection of mouse heart allografts.

BACKGROUND: Eosinophils participate in allograft rejection when donor-reactive helper T lymphocytes are T-helper type 2 (Th2)-biased. Whereas the involvement of interleukin (IL)-4 and IL-5 in these forms of rejection is well established, the role of IL-9, another Th2-type cytokine promoting eosinophilia, has not been determined. METHODS: We first used real-time polymerase chain reaction to quantify IL-9 mRNA in rejected allografts in a mouse model of fully mismatched heart transplantation in which recipients were devoid of CD8 T cells and developed a Th2 alloimmune response. We then compared allograft survival in wild-type versus IL-9-deficient mice depleted of CD8 T cells. Finally, we compared the fate of major histocompatibility complex class II-mismatched cardiac transplants from wild-type versus IL-9 transgenic donors to determine the influence of IL-9 overexpression within the graft. RESULTS: The Th2 alloimmune response in CD8-deficient mice was associated with the accumulation of IL-9 mRNA in the rejected graft. In IL-9-deficient recipients depleted of CD8 T cells, eosinophil infiltration of heart allografts did not develop, but rejection still occurred. In the major histocompatibility complex class II disparate model, heart allografts from IL-9 transgenic donors were acutely rejected, whereas grafts from wild-type donors did not develop rejection. Acute rejection of IL-9 transgenic hearts was associated with massive eosinophil infiltration and prevented by neutralization of either IL-4 or IL-5. CONCLUSION: IL-9 is critically involved in heart transplant eosinophilia in conjunction with IL-4 and IL-5.

Cytokine mRNA quantification by real-time PCR.

Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.