Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling.

High-fat diet (HFD) and inflammation are key contributors to insulin resistance and type 2 diabetes (T2D). Interleukin (IL)-1ß plays a role in insulin resistance, yet how IL-1ß is induced by the fatty acids in an HFD, and how this alters insulin signaling, is unclear. We show that the saturated fatty acid palmitate, but not unsaturated oleate, induces the activation of the NLRP3-ASC inflammasome, causing caspase-1, IL-1ß and IL-18 production. This pathway involves mitochondrial reactive oxygen species and the AMP-activated protein kinase and unc-51-like kinase-1 (ULK1) autophagy signaling cascade. Inflammasome activation in hematopoietic cells impairs insulin signaling in several target tissues to reduce glucose tolerance and insulin sensitivity. Furthermore, IL-1ß affects insulin sensitivity through tumor necrosis factor-independent and dependent pathways. These findings provide insights into the association of inflammation, diet and T2D.

NLRX1 inhibits the early stages of CNS inflammation and prevents the onset of spontaneous autoimmunity.

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1-/- mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity.

Exome sequencing in multiple sclerosis families identifies 12 candidate genes and nominates biological pathways for the genesis of disease.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.

Dendritic Cells and Their Immunotherapeutic Potential for Treating Type 1 Diabetes.

Type 1 diabetes (T1D) results from the destruction of pancreatic beta cells through a process that is primarily mediated by T cells. Emerging evidence suggests that dendritic cells (DCs) play a crucial role in initiating and developing this debilitating disease. DCs are professional antigen-presenting cells with the ability to integrate signals arising from tissue infection or injury that present processed antigens from these sites to naïve T cells in secondary lymphoid organs, thereby triggering naïve T cells to differentiate and modulate adaptive immune responses. Recent advancements in our knowledge of the various subsets of DCs and their cellular structures and methods of orchestration over time have resulted in a better understanding of how the T cell response is shaped. DCs employ various arsenal to maintain their tolerance, including the induction of effector T cell deletion or unresponsiveness and the generation and expansion of regulatory T cell populations. Therapies that suppress the immunogenic effects of dendritic cells by blocking T cell costimulatory pathways and proinflammatory cytokine production are currently being sought. Moreover, new strategies are being developed that can regulate DC differentiation and development and harness the tolerogenic capacity of these cells. Here, in this report, we focus on recent advances in the field of DC immunology and evaluate the prospects of DC-based therapeutic strategies to treat T1D.

NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-κB signaling pathways.

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-ß, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

Elucidating the Role of Ezh2 in Tolerogenic Function of NOD Bone Marrow-Derived Dendritic Cells Expressing Constitutively Active Stat5b.

Tolerogenic dendritic cells (toDCs) are crucial to controlling the development of autoreactive T cell responses and the prevention of autoimmunity. We have reported that NOD.CD11cStat5b-CA transgenic mice expressing a constitutively active (CA) form of Stat5b under the control of a CD11c promoter are protected from diabetes and that Stat5b-CA-expressing DCs are tolerogenic and halt ongoing diabetes in NOD mice. However, the molecular mechanisms by which Stat5b-CA modulates DC tolerogenic function are not fully understood. Here, we used bone marrow-derived DCs (BMDCs) from NOD.CD11cStat5b-CA transgenic mice (Stat5b-CA.BMDCs) and found that Stat5b-CA.BMDCs displayed high levels of MHC class II, CD80, CD86, PD-L1, and PD-L2 and produced elevated amounts of TGFß but low amounts of TNFα and IL-23. Stat5b-CA.BMDCs upregulated Irf4 and downregulated Irf8 genes and protein expression and promoted CD11c+CD11b+ DC2 subset differentiation. Interestingly, we found that the histone methyltransferase Ezh2 and Stat5b-CA bound gamma-interferon activated site (GAS) sequences in the Irf8 enhancer IRF8 transcription, whereas Stat5b but not Ezh2 bound GAS sequences in the Irf4 promoter to enhance IRF4 transcription. Injection of Stat5b-CA.BMDCs into prediabetic NOD mice halted progression of islet inflammation and protected against diabetes. Importantly, inhibition of Ezh2 in tolerogenic Stat5b-CA.BMDCs reduced their ability to prevent diabetes development in NOD recipient mice. Taken together, our data suggest that the active form of Stat5b induces tolerogenic DC function by modulating IRF4 and IRF8 expression through recruitment of Ezh2 and highlight the fundamental role of Ezh2 in Stat5b-mediated induction of tolerogenic DC function.

Experimental Autoimmune Encephalomyelitis Potentiates Mouse Mast Cell Protease 4-Dependent Pressor Responses to Centrally or Systemically Administered Big Endothelin-1.

Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice. Chymase-specific enzymatic activity was also measured in the lung, brain, and mast cell extracts in vitro. Finally, the impact of EAE immunization was studied on the pulmonary and brain mRNA expression of different genes of the endothelin pathway, interleukin-33 (IL-33), and monitoring of immunoreactive tumor necrosis factor-α (TNF-α). Systemically or intrathecally administered big-ET-1 triggered increases in blood pressure in conscious mice. One week post-EAE, the pressor responses to big-ET-1 were potentiated in wild-type (WT) mice but not in mMCP-4 knockout (KO) mice. EAE triggered mMCP-4-specific activity in cerebral homogenates and peritoneal mast cells. Enhanced pulmonary, but not cerebral preproendothelin-1 and IL-33 mRNA were found in KO mice and further increased 1 week post-EAE immunization, but not in WT animals. Finally, TNF-α levels were also increased in serum from mMCP-4 KO mice, but not WT, 1 week post-EAE. Our study suggests that mMCP-4 activity is enhanced both centrally and systemically in a mouse model of EAE.

Loss of NLRX1 Exacerbates Neural Tissue Damage and NF-κB Signaling following Brain Injury.

Traumatic and nontraumatic brain injury results from severe disruptions in the cellular microenvironment leading to massive loss of neuronal populations and increased neuroinflammation. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity, and free-radical release, contribute to neural tissue damage. NLRX1 is a member of the NLR family of pattern recognition receptors and is a potent negative regulator of several pathways that significantly modulate many of these events. Thus, we hypothesized that NLRX1 limits immune system signaling in the brain following trauma. To evaluate this hypothesis, we used Nlrx1-/- mice in a controlled cortical impact (CCI) injury murine model of traumatic brain injury (TBI). In this article, we show that Nlrx1-/- mice exhibited significantly larger brain lesions and increased motor deficits following CCI injury. Mechanistically, our data indicate that the NF-κB signaling cascade is significantly upregulated in Nlrx1-/- animals. This upregulation is associated with increased microglia and macrophage populations in the cortical lesion. Using a mouse neuroblastoma cell line (N2A), we also found that NLRX1 significantly reduced apoptosis under hypoxic conditions. In human patients, we identify 15 NLRs that are significantly dysregulated, including significant downregulation of NLRX1 in brain injury following aneurysm. We further demonstrate a concurrent increase in NF-κB signaling that is correlated with aneurysm severity in these human subjects. Together, our data extend the function of NLRX1 beyond its currently characterized role in host-pathogen defense and identify this highly novel NLR as a significant modulator of brain injury progression.

The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to ß-amyloid peptide.

Recent studies have reported that many proteases, besides the canonical α-, ß-, and γ-secretases, cleave the amyloid precursor protein (APP) and modulate ß-amyloid (Aß) peptide production. Moreover, specific APP isoforms contain Kunitz protease-inhibitory domains, which regulate the proteolytic activity of serine proteases. This prompted us to investigate the role of matriptase, a member of the type II transmembrane serine protease family, in APP processing. Using quantitative RT-PCR, we detected matriptase mRNA in several regions of the human brain with an enrichment in neurons. RNA sequencing data of human dorsolateral prefrontal cortex revealed relatively high levels of matriptase RNA in young individuals, whereas lower levels were detected in older individuals. We further demonstrate that matriptase and APP directly interact with each other and that matriptase cleaves APP at a specific arginine residue (Arg-102) both in vitro and in cells. Site-directed (Arg-to-Ala) mutagenesis of this cleavage site abolished matriptase-mediated APP processing. Moreover, we observed that a soluble, shed matriptase form cleaves endogenous APP in SH-SY5Y cells and that this cleavage significantly reduces APP processing to Aß40. In summary, this study identifies matriptase as an APP-cleaving enzyme, an activity that could have important consequences for the abundance of Aß and in Alzheimer's disease pathology.

Constitutively active Stat5b signaling confers tolerogenic functions to dendritic cells of NOD mice and halts diabetes progression.

Defects in dendritic cells (DCs) development and function lead to autoimmune disorders. Autoimmune diabetes in humans and NOD mice results from a breakdown of self-tolerance, ending in T cell-mediated ß-cell destruction. DCs dysfunction in NOD mice results in part from a defect in the JAK-STAT5 signaling pathway associated with the idd4 susceptibility locus. The involvement of Stat5b in DCs tolerogenic functions remains unknown. We have generated transgenic mice (NOD.CD11cStat5b-CA) expressing a constitutively active form of the Stat5b gene (Stat5b-CA) under control of CD11c promoter. All NOD.CD11cStat5b-CA mice were protected against diabetes. Protection was associated with an increased in the pool and suppressive function of Tregs, a promotion of Th2 and Tc2 immune response and a decreased percentage of CD8+ T cells. Splenic DCs of NOD.CD11cStat5b-CA mice acquired a mature phenotype, promoted and induced better conversion of CD4+CD25-Foxp3- T cells into Tregs (CD4+CD25+Foxp3+ T cells) than DCs of NOD mice. Stat5b-CA.DC-educated CD4+CD25- T cells delayed diabetes onset whereas Stat5b-CA.DC-educated Tregs blocked ongoing diabetes in 8-10 weeks old NOD recipient mice. Importantly, injection of Stat5b.CA.DC to 8-10-week old NOD mice halted diabetes progression and educated their splenocytes to loose their diabetogenic potential when transferred to NOD.SCID mice. Our work is the first to report that an active form of Stat5b restored DCs tolerogenic functions that re-educated Tregs to re-establish and to sustain long-term protective immune response against diabetes in NOD mice.

Significant Contribution of Mouse Mast Cell Protease 4 in Early Phases of Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that reproduces cardinal signs of clinical, histopathological, and immunological features found in Multiple Sclerosis (MS). Mast cells are suggested to be involved in the main inflammatory phases occurring during EAE development, possibly by secreting several autacoids and proteases. Among the latter, the chymase mouse mast cell protease 4 (mMCP-4) can contribute to the inflammatory response by producing endothelin-1 (ET-1). The aim of this study was to determine the impact of mMCP-4 on acute inflammatory stages in EAE. C57BL/6 wild type (WT) or mMCP-4 knockout (KO) mice were immunized with MOG35-55 plus complete Freund's adjuvant followed by pertussis toxin. Immunized WT mice presented an initial acute phase characterized by progressive increases in clinical score, which were significantly reduced in mMCP-4 KO mice. In addition, higher levels of spinal myelin were found in mMCP-4 KO as compared with WT mice. Finally, whereas EAE triggered significant increases in brain levels of mMCP-4 mRNA and immunoreactive ET-1 in WT mice, the latter peptide was reduced to basal levels in mMCP-4 KO congeners. Together, the present study supports a role for mMCP-4 in the early inflammatory phases of the disease in a mouse model of MS.

The nucleotide-binding leucine-rich repeat (NLR) family member NLRX1 mediates protection against experimental autoimmune encephalomyelitis and represses macrophage/microglia-induced inflammation.

The nucleotide binding domain and leucine-rich repeat-containing (NLR) family of proteins is known to activate innate immunity, and the inflammasome-associated NLRs are prime examples. In contrast, the concept that NLRs can inhibit innate immunity is still debated, and the impact of such inhibitory NLRs in diseases shaped by adaptive immune responses is entirely unexplored. This study demonstrates that, in contrast to other NLRs that activate immunity, NLRX1 plays a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. When compared with wild-type controls, Nlrx1(-/-) mice have significantly worsened clinical scores and heightened CNS tissue damage during EAE. NLRX1 does not alter the production of encephalitogenic T cells in the peripheral lymphatic tissue, but Nlrx1(-/-) mice are more susceptible to adoptively transferred myelin-reactive T cells. Analysis of the macrophage and microglial populations indicates that NLRX1 reduces activation during both active and passive EAE models. This work represents the first case of an NLR that attenuates microglia inflammatory activities and protects against a neurodegenerative disease model caused by autoreactive T cells.

The nod-like receptor, Nlrp12, plays an anti-inflammatory role in experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is an organ-specific autoimmune disease resulting in demyelinating plaques throughout the central nervous system. In MS, the exact role of microglia remains unknown. On one hand, they can present antigens, skew T cell responses, and upregulate the expression of pro-inflammatory molecules. On the other hand, microglia may express anti-inflammatory molecules and inhibit inflammation. Microglia express a wide variety of immune receptors such as nod-like receptors (NLRs). NLRs are intracellular receptors capable of regulating both innate and adaptive immune responses. Among NLRs, Nlrp12 is largely expressed in cells of myeloid origins. It plays a role in immune inflammatory responses by negatively regulating the nuclear factor-kappa B (NF-κB) pathway. Thus, we hypothesize that Nlrp12 suppresses inflammation and ameliorates the course of MS. METHODS: We used experimental autoimmune encephalomyelitis (EAE), a well-characterized mouse model of MS. EAE was induced in wild-type (WT) and Nlrp12 (-/-) mice with myelin oligodendrocyte glycoprotein (MOG):complete Freud's adjuvant (CFA). The spinal cords of healthy and immunized mice were extracted for immunofluorescence and pro-inflammatory gene analysis. Primary murine cortical microglia cell cultures of WT and Nlrp12 (-/-) were prepared with cortices of 1-day-old pups. The cells were stimulated with lipopolysaccharide (LPS) and analyzed for the expression of pro-inflammatory genes as well as pro-inflammatory molecule secretions. RESULTS: Over the course of 9 weeks, the Nlrp12 (-/-) mice demonstrated increased severity in the disease state, where they developed the disease earlier and reached significantly higher clinical scores compared to the WT mice. The spinal cords of immunized WT mice relative to healthy WT mice revealed a significant increase in Nlrp12 messenger ribonucleic acid (mRNA) expression at 1, 3, and 5 weeks post injection. A significant increase in the expression of pro-inflammatory genes Ccr5, Cox2, and IL-1ß was found in the spinal cords of the Nlrp12 (-/-) mice relative to the WT mice (P < 0.05). A significant increase in the level of gliosis was observed in the spinal cords of the Nlrp12 (-/-) mice compared to the WT mice after 9 weeks of disease (P < 0.05). Primary Nlrp12 (-/-) microglia cells demonstrated a significant increase in inducible nitric oxide synthase (iNOS) expression (P < 0.05) and secreted significantly (P < 0.05) more tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and nitric oxide (NO). CONCLUSION: Nlrp12 plays a protective role by suppressing inflammation during the development of EAE. The absence of Nlrp12 results in an increased inflammatory response.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes.

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Involvement of neuronal IL-1ß in acquired brain lesions in a rat model of neonatal encephalopathy.

BACKGROUND: Infection-inflammation combined with hypoxia-ischemia (HI) is the most prevalent pathological scenario involved in perinatal brain damage leading to life-long neurological disabilities. Following lipopolysaccharide (LPS) and/or HI aggression, different patterns of inflammatory responses have been uncovered according to the brain differentiation stage. In fact, LPS pre-exposure has been reported to aggravate HI brain lesions in post-natal day 1 (P1) and P7 rat models that are respectively equivalent - in terms of brain development - to early and late human preterm newborns. However, little is known about the innate immune response in LPS plus HI-induced lesions of the full-term newborn forebrain and the associated neuropathological and neurobehavioral outcomes. METHODS: An original preclinical rat model has been previously documented for the innate neuroimmune response at different post-natal ages. It was used in the present study to investigate the neuroinflammatory mechanisms that underline neurological impairments after pathogen-induced inflammation and HI in term newborns. RESULTS: LPS and HI exerted a synergistic detrimental effect on rat brain. Their effect led to a peculiar pattern of parasagittal cortical-subcortical infarcts mimicking those in the human full-term newborn with subsequent severe neurodevelopmental impairments. An increased IL-1ß response in neocortical and basal gray neurons was demonstrated at 4 h after LPS + HI-exposure and preceded other neuroinflammatory responses such as microglial and astroglial cell activation. Neurological deficits were observed during the acute phase of injury followed by a recovery, then by a delayed onset of profound motor behavior impairment, reminiscent of the delayed clinical onset of motor system impairments observed in humans. Interleukin-1 receptor antagonist (IL-1ra) reduced the extent of brain lesions confirming the involvement of IL-1ß response in their pathophysiology. CONCLUSION: In rat pups at a neurodevelopmental age corresponding to full-term human newborns, a systemic pre-exposure to a pathogen component amplified HI-induced mortality and morbidities that are relevant to human pathology. Neuronal cells were the first cells to produce IL-1ß in LPS + HI-exposed full-term brains. Such IL-1ß production might be responsible for neuronal self-injuries via well-described neurotoxic mechanisms such as IL-1ß-induced nitric oxide production, or IL-1ß-dependent exacerbation of excitotoxic damage.

Inhibition of PI3K/C/EBPß axis in tolerogenic bone marrow-derived dendritic cells of NOD mice promotes Th17 differentiation and diabetes development.

Dendritic cells (DCs) are key regulators of the adaptive immune response. Tolerogenic dendritic cells play a crucial role in inducing and maintaining immune tolerance in autoimmune diseases such as type 1 diabetes in humans as well as in the NOD mouse model. We previously reported that bone marrow-derived DCs (BM.DCs) from NOD mice, generated with a low dose of GM-CSF (GM/DCs), induce Treg differentiation and are able to protect NOD mice from diabetes. We had also found that the p38 MAPK/C/EBPß axis is involved in regulating the phenotype, as well as the production of IL-10 and IL-12p70, by tolerogenic GM/DCs. Here, we report that the inhibition of the PI3K signaling switched the cytokine profile of GM/DCs toward Th17-promoting cytokines without affecting their phenotype. PI3K inhibition abrogated the production of IL-10 by GM/DCs, whereas it enhanced their production of IL-23 and TGFß. Inhibition of PI3K signaling in tolerogenic GM/DCs also induced naive CD4+ T cells differentiation toward Th17 cells. Mechanistically, PI3K inhibition increased the DNA-binding activity of C/EBPß through a GSK3-dependent pathway, which is important to maintain the semimature phenotype of tolerogenic GM/DCs. Furthermore, analysis of C/EBPß-/- GM/DCs demonstrated that C/EBPß is required for IL-23 production. Of physiological relevance, the level of protection from diabetes following transfusion of GM/DCs into young NOD mice was significantly reduced when NOD mice were transfused with GM/DCs pretreated with a PI3K inhibitor. Our data suggest that PI3K/C/EBPß signaling is important in controlling tolerogenic function of GM/DCs by limiting their Th17-promoting cytokines.

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome.

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1ß. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.

NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses.

The interplay between innate and adaptive immunity is important in multiple sclerosis (MS). The inflammasome complex, which activates caspase-1 to process pro-IL-1beta and pro-IL-18, is rapidly emerging as a pivotal regulator of innate immunity, with nucleotide-binding domain, leucine-rich repeat containing protein family, pyrin domain containing 3 (NLRP3) (cryopyrin or NALP3) as a prominent player. Although the role of NLRP3 in host response to pathogen associated molecular patterns and danger associated molecular patterns is well documented, its role in autoimmune diseases is less well studied. To investigate the role of NLRP3 protein in MS, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp3 expression was elevated in the spinal cords during EAE, and Nlrp3(-/-) mice had a dramatically delayed course and reduced severity of disease. This was accompanied by a significant reduction of the inflammatory infiltrate including macrophages, dendritic cells, CD4, and CD8(+) T cells in the spinal cords of the Nlrp3(-/-) mice, whereas microglial accumulation remained the same. Nlrp3(-/-) mice also displayed improved histology in the spinal cords with reduced destruction of myelin and astrogliosis. Nlrp3(-/-) mice with EAE produced less IL-18, and the disease course was similar to Il18(-/-) mice. Furthermore, Nlrp3(-/-) and Il18(-/-) mice had similarly reduced IFN-gamma and IL-17 production. Thus, NLRP3 plays a critical role in the induction of the EAE, likely through effects on capase-1-dependent cytokines which then influence Th1 and Th17.

Cutting edge: NLRP12 controls dendritic and myeloid cell migration to affect contact hypersensitivity.

Nucleotide-binding domain leucine-rich repeat (NLR) proteins are regulators of inflammation and immunity. Although first described 8 y ago, a physiologic role for NLRP12 has remained elusive until now. We find that murine Nlrp12, an NLR linked to atopic dermatitis and hereditary periodic fever in humans, is prominently expressed in dendritic cells (DCs) and neutrophils. Nlrp12-deficient mice exhibit attenuated inflammatory responses in two models of contact hypersensitivity that exhibit features of allergic dermatitis. This cannot be attributed to defective Ag processing/presentation, inflammasome activation, or measurable changes in other inflammatory cytokines. Rather, Nlrp12(-/-) DCs display a significantly reduced capacity to migrate to draining lymph nodes. Both DCs and neutrophils fail to respond to chemokines in vitro. These findings indicate that NLRP12 is important in maintaining neutrophils and peripheral DCs in a migration-competent state.

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1ß, IL-18, and TNF-α by resting macrophages. IL-1ß and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1ß and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.