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.

Obesity negatively impacts maintenance of antibody response to COVID-19 vaccines.

Severe obesity accelerates the decline of neutralizing antibodies to COVID-19 vaccines contributing to increased risk of hospitalization from breakthrough SARS-CoV-2 infections.1 These findings have repercussion on the vaccination policy for SARS-CoV-2 variants and other infectious diseases like influenza in obese population.

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 implication of anti-PD-1 therapy in cancer patients for the vaccination against viral and other infectious diseases.

The phenomenon of 'T cell exhaustion', a state of T cell dysfunction observed during chronic infections and cancers, has been a major obstacle in mounting appropriate immune responses against infectious agents or tumor antigens. The exhausted T cells are characterized by poor effector functions mainly due to the overexpression of inhibitory receptors such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin-domain containing 3 (TIM3), lymphocyte activation gene 3 (LAG3), and T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), commonly referred to as immune checkpoint (ICP) molecules. ICP blockade, especially of PD-1 that can potentially reverse T cell exhaustion and thereby re-stimulate the impaired immune system, is widely used in clinics as a promising therapeutic strategy for various cancers and is more recently being investigated in infectious diseases as well. In fact, cancer patients represent a population of immunocompromised individuals who are more susceptible to infections and associated complications, and thus the need for protective vaccinations against these diseases is of prime importance in this category. When it comes to vaccinating anti-PD-1-treated cancer patients against infectious diseases including COVID-19 and influenza, a special focus should be brought on the revived immune cells, which could be dynamically affected by the antigenic stimulation. However, since cancer patients are not generally included in clinical trials for designing vaccines against infectious diseases, the possible interaction between vaccine immune responses and ICP therapy is largely unexplored. Mechanistically, the reversal of T cell exhaustion by ICP in an otherwise immunocompromised population could be beneficial for the vaccine's efficacy, helping the immune system to mount a robust immune response. Nevertheless, patients with cancer undergoing anti-PD-1 blockade are known to experience immune-related adverse effects (irAEs). The risk of increasing the irAEs due to the overstimulation of the immune system during vaccination is a major concern. Therefore, while routine vaccination is indispensable for the protection of cancer patients, the impact of PD-1 blockade on vaccine responses against infectious agents requires careful consideration to avoid undesirable adverse effects that could impair the efficacy of anti-cancer treatment.

Tocilizumab-treated convalescent COVID-19 patients retain the cross-neutralization potential against SARS-CoV-2 variants.

Although tocilizumab treatment in severe and critical coronavirus disease 2019 (COVID-19) patients has proven its efficacy at the clinical level, there is little evidence supporting the effect of short-term use of interleukin-6 receptor blocking therapy on the B cell sub-populations and the cross-neutralization of SARS-CoV-2 variants in convalescent COVID-19 patients. We performed immunological profiling of 69 tocilizumab-treated and non-treated convalescent COVID-19 patients in total. We observed that SARS-CoV-2-specific IgG1 titers depended on disease severity but not on tocilizumab treatment. The plasma of both treated and non-treated patients infected with the ancestral variant exhibit strong neutralizing activity against the ancestral virus and the Alpha, Beta, and Delta variants of SARS-CoV-2, whereas the Gamma and Omicron viruses were less sensitive to seroneutralization. Overall, we observed that, despite the clinical benefits of short-term tocilizumab therapy in modifying the cytokine storm associated with COVID-19 infections, there were no modifications in the robustness of B cell and IgG responses to Spike antigens.

IFN-γ Induces PD-L1 Expression in Primed Human Basophils.

Programmed death-ligand 1 (PD-L1) plays a key role in maintaining immune tolerance and also in immune evasion of cancers and pathogens. Though the identity of stimuli that induce PD-L1 in various human innate cells and their function are relatively well studied, data on the basophils remain scarce. In this study, we have identified one of the factors, such as IFN-γ, that induces PD-L1 expression in human basophils. Interestingly, we found that basophil priming by IL-3 is indispensable for IFN-γ-induced PD-L1 expression in human basophils. However, priming by other cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF) and thymic stromal lymphopoietin (TSLP) was dispensable. Analyses of a published microarray data set on IL-3-treated basophils indicated that IL-3 enhances IFNGR2, one of the chains of the IFNGR heterodimer complex, and CD274, thus providing a mechanistic insight into the role of IL-3 priming in IFN-γ-induced PD-L1 expression in human basophils.

SARS-CoV-2 Induces Cytokine Responses in Human Basophils.

Basophils play a key role in the orientation of immune responses. Though the interaction of SARS-CoV-2 with various immune cells has been relatively well studied, the response of basophils to this pandemic virus is not characterized yet. In this study, we report that SARS-CoV-2 induces cytokine responses and in particular IL-13, in both resting and IL-3 primed basophils. The response was prominent under IL-3 primed condition. However, either SARS-CoV-2 or SARS-CoV-2-infected epithelial cells did not alter the expression of surface markers associated with the activation of basophils, such as CD69, CD13 and/or degranulation marker CD107a. We also validate that human basophils are not permissive to SARS-CoV-2 replication. Though increased expression of immune checkpoint molecule PD-L1 has been reported on the basophils from COVID-19 patients, we observed that SARS-CoV-2 does not induce PD-L1 on the basophils. Our data suggest that basophil cytokine responses to SARS-CoV-2 might help in reducing the inflammation and also to promote antibody responses to the virus.

Interplay between SMAD2 and STAT5A is a critical determinant of IL-17A/IL-17F differential expression.

Interleukins (IL)-17A and F are critical cytokines in anti-microbial immunity but also contribute to auto-immune pathologies. Recent evidence suggests that they may be differentially produced by T-helper (Th) cells, but the underlying mechanisms remain unknown. To address this question, we built a regulatory graph integrating all reported upstream regulators of IL-17A and F, completed by ChIP-seq data analyses. The resulting regulatory graph encompasses 82 components and 136 regulatory links. The graph was then supplemented by logical rules calibrated with original flow cytometry data using naive CD4+ T cells, in conditions inducing IL-17A or IL-17F. The model displays specific stable states corresponding to virtual phenotypes explaining IL-17A and IL-17F differential regulation across eight cytokine stimulatory conditions. Our model analysis points to the transcription factors NFAT2A, STAT5A and SMAD2 as key regulators of the differential expression of IL-17A and IL-17F, with STAT5A controlling IL-17F expression, and an interplay of NFAT2A, STAT5A and SMAD2 controlling IL-17A expression. We experimentally observed that the production of IL-17A was correlated with an increase of SMAD2 transcription, and the expression of IL-17F correlated with an increase of BLIMP-1 transcription, together with an increase of STAT5A expression (mRNA), as predicted by our model. Interestingly, RORγt presumably plays a more determinant role in IL-17A expression as compared to IL-17F expression. In conclusion, we propose the first mechanistic model accounting for the differential expression of IL-17A and F in Th cells, providing a basis to design novel therapeutic interventions in auto-immune and inflammatory diseases.

A Quantitative Multivariate Model of Human Dendritic Cell-T Helper Cell Communication.

Cell-cell communication involves a large number of molecular signals that function as words of a complex language whose grammar remains mostly unknown. Here, we describe an integrative approach involving (1) protein-level measurement of multiple communication signals coupled to output responses in receiving cells and (2) mathematical modeling to uncover input-output relationships and interactions between signals. Using human dendritic cell (DC)-T helper (Th) cell communication as a model, we measured 36 DC-derived signals and 17 Th cytokines broadly covering Th diversity in 428 observations. We developed a data-driven, computationally validated model capturing 56 already described and 290 potentially novel mechanisms of Th cell specification. By predicting context-dependent behaviors, we demonstrate a new function for IL-12p70 as an inducer of Th17 in an IL-1 signaling context. This work provides a unique resource to decipher the complex combinatorial rules governing DC-Th cell communication and guide their manipulation for vaccine design and immunotherapies.

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.

Constitutive expression of TNF-related activation-induced cytokine (TRANCE)/receptor activating NF-κB ligand (RANK)-L by rat plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are a subset of DCs whose major function relies on their capacity to produce large amount of type I IFN upon stimulation via TLR 7 and 9. This function is evolutionary conserved and place pDC in critical position in the innate immune response to virus. Here we show that rat pDC constitutively express TNF-related activation-induced cytokine (TRANCE) also known as Receptor-activating NF-κB ligand (RANKL). TRANCE/RANKL is a member of the TNF superfamily which plays a central role in osteoclastogenesis through its interaction with its receptor RANK. TRANCE/RANK interaction are also involved in lymphoid organogenesis as well as T cell/DC cross talk. Unlike conventional DC, rat CD4(high) pDC were shown to constitutively express TRANCE/RANKL both at the mRNA and the surface protein level. TRANCE/RANKL was also induced on the CD4(low) subsets of pDC following activation by CpG. The secreted form of TRANCE/RANKL was also produced by rat pDC. Of note, levels of mRNA, surface and secreted TRANCE/RANKL expression were similar to that observed for activated T cells. TRANCE/RANKL expression was found on pDC in all lymphoid organs as well blood and BM with a maximum expression in mesenteric lymph nodes. Despite this TRANCE/RANKL expression, we were unable to demonstrate in vitro osteoclastogenesis activity for rat pDC. Taken together, these data identifies pDC as novel source of TRANCE/RANKL in the immune system.

Killer dendritic cells link innate and adaptive immunity against established osteosarcoma in rats.

We have previously reported that a distinct subset of splenic CD4(-) rat dendritic cells (DC) induces a rapid and caspase-independent apoptosis-like cell death in a large number of tumor cells in vitro. The killing activity of these killer DC (KDC) was restricted to their immature state and was immediately followed by their engulfment of the apoptotic target cells, suggesting that these KDC could directly link innate and adaptive immunity to tumors. Here, we addressed this question using a transplantable model of rat osteosarcoma. First, we showed that rat KDC have an MHC II(+)CD103(+)CD11b(+)NKp46(-) phenotype and are therefore distinct from natural killer cells, which are MHC II(-)CD103(-)CD11b(-)NKp46(+). KDC numbers could be specifically and strongly (up to 10-fold) enhanced by Flt3L in vivo. The OSRGa cell line derived from the osteosarcoma tumor was killed and phagocytosed in vitro by both normal and Flt3L-induced splenic KDC. Such tumor antigen-loaded KDC were used to s.c. vaccinate progressive tumor-bearing rats. Vaccination with OSRGa-loaded KDC but not KDC loaded with irrelevant tumor cells (Jurkat) delayed tumor progression or even induced tumor regression. This vaccine effect was not observed in CD8 T cell-depleted animals and protective against tumor rechallenge. These results suggest that KDC possess the intrinsic capability not only to kill and then engulf tumor cells but also to efficiently cross-present tumor cell-derived antigen in vivo and subsequently induce an adaptive antitumor immune response.

Dendritic cells as killers: mechanistic aspects and potential roles.

Dendritic cells (DC) are professional APC endowed with the unique capacity to activate naive T cells. DC also have important effector functions during the innate immune response, such as pathogen recognition and cytokine production. In fact, DC represent the crucial link between innate and adaptive immune responses. However, DC are quite heterogeneous and various subsets endowed with specific pathogen recognition mechanisms, locations, phenotypes, and functions have been described both in rodents and in humans. A series of studies indicated that rodent as well as human DC could also mediate another important innate function, i.e., cell-mediated cytotoxicity, mostly toward tumor cells. In this article, we will review the phenotypes of these so-called killer DC, their killing mechanism, and putative implication in the immune response.

Immature CD4- CD103+ rat dendritic cells induce rapid caspase-independent apoptosis-like cell death in various tumor and nontumor cells and phagocytose their victims.

We previously reported the characterization of a MHC class II(low) CD4- CD103+ (CD4-) subset of dendritic cells (DC) in rat spleen that exhibit a Ca2+-, Fas ligand-, TRAIL- and TNF-alpha-independent cytotoxic activity against specific targets in vitro. In this study, we demonstrate that this DC subset was also found in lymph nodes. Freshly extracted and, therefore, immature CD4- DC exhibited a potent cytotoxic activity against a large panel of tumor cell lines as well as primary endothelial cells. The cytotoxic activity of immature CD4- DC required cell-to-cell contact and de novo protein expression. CD4- DC-mediated cell death resembled apoptosis, as evidenced by outer membrane phosphatidylserine exposure and nuclear fragmentation in target cells, but was caspase as well as Fas-associated death domain and receptor-interacting protein independent. Bcl-2 overexpression in target cells did not protect them against DC-mediated cell death. Immature CD4- DC phagocytosed efficiently apoptotic cells in vitro and, therefore, rapidly and specifically engulfed their victims following death induction. Maturation induced a dramatic down-regulation of the killing and phagocytic activities of CD4- DC. In contrast, CD4+ DC were both unable to kill target cells and to phagocytose apoptotic cells in vitro. Taken together, these data indicate that rat immature CD4- CD103+ DC mediate an unusual cytotoxic activity and can use this function to efficiently acquire Ag from live cells.

A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases.

In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3' end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3-Jun/Fos-Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.