The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes.

Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)-associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.

Pattern recognition receptor signaling in human dendritic cells is enhanced by ICOS ligand and modulated by the Crohn's disease ICOSLG risk allele.

Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune homeostasis and cytokine secretion. Multiple loci are associated with IBD, but a functional explanation is missing for most. Here we found that pattern-recognition receptor (PRR)-induced cytokine secretion was diminished in human monocyte-derived dendritic cells (MDDC) from rs7282490 ICOSLG GG risk carriers. Homotypic interactions between the costimulatory molecule ICOS and the ICOS ligand on MDDCs amplified nucleotide-binding oligomerization domain 2 (NOD2)-initiated cytokine secretion. This amplification required arginine residues in the ICOSL cytoplasmic tail that recruited the adaptor protein RACK1 and the kinases PKC and JNK leading to PKC, MAPK, and NF-κB activation. MDDC from rs7282490 GG risk-carriers had reduced ICOSL expression and PRR-initiated signaling and this loss-of-function ICOSLG risk allele associated with an ileal Crohn's disease phenotype, similar to polymorphisms in NOD2. Taken together, ICOSL amplifies PRR-initiated outcomes, which might contribute to immune homeostasis.

Disease Risk-Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor-Induced Outcomes in Human Macrophages.

STAT proteins can regulate both pro- and anti-inflammatory cytokine signaling. Therefore, identifying consequences of modulating expression of a given STAT is ultimately critical for determining its potential as a therapeutic target and for defining the mechanisms through which immune-mediated disease variants in STAT genes contribute to disease pathogenesis. Genetic variants in the STAT1/STAT4 region are associated with multiple immune-mediated diseases, including inflammatory bowel disease (IBD). These diseases are characterized by dysregulated cytokine secretion in response to pattern-recognition receptor (PRR) stimulation. We found that the common IBD-associated rs1517352 C risk allele increased both STAT1 and STAT4 expression in human monocyte-derived macrophages (MDMs). We therefore hypothesized that the STAT1/STAT4 variant might regulate PRR-initiated responses in a complementary and cooperative manner because of the important role of autocrine/paracrine cytokines in modulating PRR-initiated signaling. STAT1 and STAT4 were required for PRR- and live bacterial-induced secretion of multiple cytokines. These outcomes were particularly dependent on PRR-initiated autocrine/paracrine IL-12-induced STAT4 activation to generate IFN-γ, with autocrine IFN-γ then signaling through STAT1. STAT1 and STAT4 also promoted bacterial-induced cytokines in intestinal myeloid cells and PRR-enhanced antimicrobial pathways in MDMs. Importantly, MDMs from rs1517352 C IBD risk allele carriers demonstrated increased TLR4-, IFN-γ- and IL-12-induced STAT1 and STAT4 phosphorylation and cytokine secretion and increased TLR4-enhanced antimicrobial pathways. Taken together, STAT1 and STAT4 expression is coregulated by a shared genetic region, and STAT1 /STAT4-immune disease-associated variants modulate IFN-γ- and IL-12-associated outcomes, and in turn, PRR-induced outcomes, highlighting that these genes cooperate to regulate pathways relevant to disease pathogenesis.

Myeloid Cell-Intrinsic IRF5 Promotes T Cell Responses through Multiple Distinct Checkpoints In Vivo, and IRF5 Immune-Mediated Disease Risk Variants Modulate These Myeloid Cell Functions.

Common IRF5 genetic risk variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern-recognition receptor (PRR)-induced cytokines in myeloid cells. However, how myeloid cell-intrinsic IRF5 regulates the multiple distinct checkpoints mediating T cell outcomes in vivo and IRF5-dependent mechanisms contributing to these distinct checkpoints are not well defined. Using an in vivo Ag-specific adoptive T cell transfer approach into Irf5-/- mice, we found that T cell-extrinsic IRF5 regulated T cell outcomes at multiple critical checkpoints, including chemokine-mediated T cell trafficking into lymph nodes and PDK1-dependent soluble Ag uptake, costimulatory molecule upregulation, and secretion of Th1 (IL-12)- and Th17 (IL-23, IL-1ß, and IL-6)-conditioning cytokines by myeloid cells, which then cross-regulated Th2 and regulatory T cells. IRF5 was required for PRR-induced MAPK and NF-κB activation, which, in turn, regulated these key outcomes in myeloid cells. Importantly, mice with IRF5 deleted from myeloid cells demonstrated T cell outcomes similar to those observed in Irf5-/- mice. Complementation of IL-12 and IL-23 was able to restore T cell differentiation both in vitro and in vivo in the context of myeloid cell-deficient IRF5. Finally, human monocyte-derived dendritic cells from IRF5 disease-associated genetic risk carriers leading to increased IRF5 expression demonstrated increased Ag uptake and increased PRR-induced costimulatory molecule expression and chemokine and cytokine secretion compared with monocyte-derived dendritic cells from low-expressing IRF5 genetic variant carriers. These data establish that myeloid cell-intrinsic IRF5 regulates multiple distinct checkpoints in T cell activation and differentiation and that these are modulated by IRF5 disease risk variants.

STAT3 and STAT5 Signaling Thresholds Determine Distinct Regulation for Innate Receptor-Induced Inflammatory Cytokines, and STAT3/STAT5 Disease Variants Modulate These Outcomes.

Genetic variants in the STAT3/STAT5A/STAT5B region are associated with immune-mediated diseases, including inflammatory bowel disease (IBD). However, how STAT3 and STAT5 regulate the critical balance between pro- and anti-inflammatory cytokines and how common disease-associated genetic variants (e.g., rs12942547) in the region modulate this balance are incompletely understood. We found that upon pattern-recognition receptor (PRR) stimulation of human monocyte-derived macrophages (MDMs), decreasing STAT3, STAT5a, and STAT5b expression led to a progressive decrease in anti-inflammatory cytokines, whereas proinflammatory cytokines initially decreased but then increased when STAT3 or STAT5 expression fell below a critical threshold. Mechanisms regulating STAT3- and STAT5-dependent inflammatory cytokine outcomes included negative feedback from autocrine/paracrine IL-10, TGF-ß, IL-4, IL-13, IL-22, and TSLP secretion and SOCS1/SOCS2/SOCS3 induction. MDMs from rs12942547 AA disease-risk carriers demonstrated increased STAT3, STAT5a, and STAT5b expression and increased PRR-induced STAT3 and STAT5 phosphorylation relative to GG MDMs. Both pro- and anti-inflammatory cytokine secretion was decreased in MDMs from GG carriers, as STAT3, STAT5a, and STAT5b expression was above the threshold for reciprocal regulation of these cytokines. Taken together, we identify that the threshold of STAT3, STAT5a, and STAT5b expression determines if PRR-induced proinflammatory cytokines are increased or decreased, define mechanisms for this reciprocal regulation, and elucidate consequences for disease variants in the STAT3/STAT5A/STAT5B region, indicating that considering signaling thresholds and targeting specific cell types might be beneficial when evaluating therapeutic interventions in this pathway.

Twist1 and Twist2 Induce Human Macrophage Memory upon Chronic Innate Receptor Treatment by HDAC-Mediated Deacetylation of Cytokine Promoters.

Intestinal tissues are continuously exposed to microbial products that stimulate pattern-recognition receptors (PRRs). Ongoing PRR stimulation can confer epigenetic changes in macrophages, which can then regulate subsequent immune outcomes and adaptation to the local environment. Mechanisms leading to these changes are incompletely understood. We found that short-term stimulation of the PRR NOD2 in primary human monocyte-derived macrophages resulted in increased H3 and H4 acetylation of cytokine promoters, consistent with the increased cytokine secretion observed. However, with prolonged NOD2 stimulation, both the acetylation and cytokine secretion were dramatically decreased. Chronic NOD2 stimulation upregulated the transcription factors Twist1 and Twist2, which bound to the promoters of the histone deacetylases HDAC1 and HDAC3 and induced HDAC1 and HDAC3 expression. HDAC1 and HDAC3 then mediated histone deacetylation at cytokine promoters and, in turn, cytokine downregulation under these conditions. Similar regulation was observed upon chronic stimulation of multiple PRRs. Consistent with the chronic microbial exposure in the intestinal environment, TWIST1, TWIST2, HDAC1, and HDAC3 were upregulated in human intestinal relative to peripheral macrophages. Importantly, complementing HDAC1 and HDAC3 in Twist1/Twist2-deficient monocyte-derived macrophages restored the reduced histone acetylation on cytokine promoters and the decreased cytokine secretion with chronic NOD2 stimulation. Taken together, we identify mechanisms wherein Twist1 and Twist2 promote chromatin modifications, resulting in macrophage instruction and adaptation to conditions in the intestinal microenvironment.

IRF5 Is Required for Bacterial Clearance in Human M1-Polarized Macrophages, and IRF5 Immune-Mediated Disease Risk Variants Modulate This Outcome.

Common IFN regulatory factor 5 (IRF5) variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern recognition receptor (PRR)-induced cytokines in macrophages. PRR-initiated pathways also contribute to bacterial clearance, and dysregulation of bacterial clearance can contribute to immune-mediated diseases. However, the role of IRF5 in macrophage-mediated bacterial clearance is not well defined. Furthermore, it is unclear if macrophages from individuals who are carriers of low IRF5-expressing genetic variants associated with protection for immune-mediated diseases might be at a disadvantage in bacterial clearance. We found that IRF5 was required for optimal bacterial clearance in PRR-stimulated, M1-differentiated human macrophages. Mechanisms regulated by IRF5 included inducing reactive oxygen species through p40phox, p47phox and p67phox, NOS2, and autophagy through ATG5. Complementing these pathways in IRF5-deficient M1 macrophages restored bacterial clearance. Further, these antimicrobial pathways required the activation of IRF5-dependent MAPK, NF-κB, and Akt2 pathways. Importantly, relative to high IRF5-expressing rs2004640/rs2280714 TT/TT immune-mediated disease risk-carrier human macrophages, M1-differentiated GG/CC carrier macrophages demonstrated less reactive oxygen species, NOS2, and autophagy pathway induction and, consequently, reduced bacterial clearance. Increasing IRF5 expression to the rs2004640/rs2280714 TT/TT levels restored these antimicrobial pathways. We define mechanisms wherein common IRF5 genetic variants modulate bacterial clearance, thereby highlighting that immune-mediated disease risk IRF5 carriers might be relatively protected from microbial-associated diseases.

IL23 induces IL23R recycling and amplifies innate receptor-induced signalling and cytokines in human macrophages, and the IBD-protective IL23R R381Q variant modulates these outcomes.

OBJECTIVE: The interleukin (IL)23 pathway contributes to IBD pathogenesis and is being actively studied as a therapeutic target in patients with IBD. Unexpected outcomes in these therapeutic trials have highlighted the importance of understanding the cell types and mechanisms through which IL23 regulates immune outcomes. How IL23 regulates macrophage outcomes and the consequences of the IL23R R381Q IBD-protective variant on macrophages are not well defined; macrophages are key players in IBD pathogenesis and inflammation. DESIGN: We analysed protein and RNA expression, signalling and localisation in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR, flow cytometry, immunoprecipitation and microscopy. RESULTS: IL23R was critical for optimal levels of pattern-recognition receptor (PRR)-induced signalling and cytokines in human MDMs. In contrast to the coreceptor IL12Rß1, IL23 induced dynamic IL23R cell surface regulation and this required clathrin and dynamin-mediated endocytosis and endocytic recycling-dependent pathways; these pathways were essential for IL23R-mediated outcomes. The IBD-protective IL23R R381Q variant showed distinct outcomes. Relative to IL23R R381, HeLa cells expressing IL23R Q381 showed decreased IL23R recycling and reduced assembly of IL23R Q381 with Janus kinase/signal transducer and activator of transcription pathway members. In MDMs from IL23R Q381 carriers, IL23R accumulated in late endosomes and lysosomes on IL23 treatment and cells demonstrated decreased IL23R- and PRR-induced signalling and cytokines relative to IL23R R381 MDMs. CONCLUSION: Macrophage-mediated inflammatory pathways are key contributors to IBD pathogenesis, and we identify an autocrine/paracrine IL23 requirement in PRR-initiated human macrophage outcomes and in human intestinal myeloid cells, establish that IL23R undergoes ligand-induced recycling, define mechanisms regulating IL23R-induced signalling and determine how the IBD-protective IL23R R381Q variant modulates these processes.

JAK2 Disease-Risk Variants Are Gain of Function and JAK Signaling Threshold Determines Innate Receptor-Induced Proinflammatory Cytokine Secretion in Macrophages.

JAK2 genetic variants are associated with inflammatory bowel disease (IBD) and JAK inhibitors are being evaluated for therapy targeting immune-mediated diseases, including IBD. As JAK pathway-mediated cytokine regulation varies across cell types and stimulation conditions, we examined how JAK signaling and IBD-associated JAK2 variants regulate distinct acute and chronic microbial product exposure outcomes in human myeloid cells, consistent with the conditions of initial entry and ongoing intestinal tissue residence, respectively. Macrophages from controls and ulcerative colitis patients carrying the IBD-risk rs10758669 CC genotype showed increased JAK2 expression and nucleotide-binding oligomerization domain 2-induced JAK2 phosphorylation relative to AA carriers. Interestingly, the threshold of JAK2 expression and signaling determined pattern-recognition receptor (PRR)-induced outcomes; whereas anti-inflammatory cytokines progressively decreased with lower JAK2 expression, proinflammatory cytokines switched from decreased to increased secretion below a certain JAK2 expression threshold. Low JAK2-expressing rs10758669 AA macrophages were above this threshold; consequently, both PRR-induced pro- and anti-inflammatory cytokines were decreased. However, relative to rs10758669 CC risk carriers, AA carrier macrophages switched to increased nucleotide-binding oligomerization domain 2-induced proinflammatory cytokines at lower therapeutically used JAK inhibitor doses. Importantly, JAK inhibitors increased proinflammatory cytokines secreted by peripheral macrophages following chronic PRR stimulation and by human intestinal myeloid cells following exposure to intestinal pathogens. Mechanistically, the decreased response to and secretion of autocrine/paracrine IL-10, IL-4, IL-22 and thymic stromal lymphopoietin regulated these JAK-dependent outcomes in myeloid cells. Taken together, the JAK signaling threshold determines whether PRR-induced pro- and anti-inflammatory cytokines are reciprocally regulated in myeloid cells; consideration of JAK2 genotype and targeting of specific cell types might improve JAK-targeted therapy in immune-mediated diseases.

IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine.

Chronic mucosal inflammation and tissue damage predisposes patients to the development of colorectal cancer. This association could be explained by the hypothesis that the same factors and pathways important for wound healing also promote tumorigenesis. A sensor of tissue damage should induce these factors to promote tissue repair and regulate their action to prevent development of cancer. Interleukin 22 (IL-22), a cytokine of the IL-10 superfamily, has an important role in colonic epithelial cell repair, and its levels are increased in the blood and intestine of inflammatory bowel disease patients. This cytokine can be neutralized by the soluble IL-22 receptor, known as the IL-22 binding protein (IL-22BP, also known as IL22RA2); however, the significance of endogenous IL-22BP in vivo and the pathways that regulate this receptor are unknown. Here we describe that IL-22BP has a crucial role in controlling tumorigenesis and epithelial cell proliferation in the colon. IL-22BP is highly expressed by dendritic cells in the colon in steady-state conditions. Sensing of intestinal tissue damage via the NLRP3 or NLRP6 inflammasomes led to an IL-18-dependent downregulation of IL-22BP, thereby increasing the ratio of IL-22/IL-22BP. IL-22, which is induced during intestinal tissue damage, exerted protective properties during the peak of damage, but promoted tumour development if uncontrolled during the recovery phase. Thus, the IL-22-IL-22BP axis critically regulates intestinal tissue repair and tumorigenesis in the colon.

MTMR3 risk allele enhances innate receptor-induced signaling and cytokines by decreasing autophagy and increasing caspase-1 activation.

Inflammatory bowel disease (IBD) is characterized by dysregulated host:microbial interactions and cytokine production. Host pattern recognition receptors (PRRs) are critical in regulating these interactions. Multiple genetic loci are associated with IBD, but altered functions for most, including in the rs713875 MTMR3/HORMAD2/LIF/OSM region, are unknown. We identified a previously undefined role for myotubularin-related protein 3 (MTMR3) in amplifying PRR-induced cytokine secretion in human macrophages and defined MTMR3-initiated mechanisms contributing to this amplification. MTMR3 decreased PRR-induced phosphatidylinositol 3-phosphate (PtdIns3P) and autophagy levels, thereby increasing PRR-induced caspase-1 activation, autocrine IL-1ß secretion, NFκB signaling, and, ultimately, overall cytokine secretion. This MTMR3-mediated regulation required the N-terminal pleckstrin homology-GRAM domain and Cys413 within the phosphatase domain of MTMR3. In MTMR3-deficient macrophages, reducing the enhanced autophagy or restoring NFκB signaling rescued PRR-induced cytokines. Macrophages from rs713875 CC IBD risk carriers demonstrated increased MTMR3 expression and, in turn, decreased PRR-induced PtdIns3P and autophagy and increased PRR-induced caspase-1 activation, signaling, and cytokine secretion. Thus, the rs713875 IBD risk polymorphism increases MTMR3 expression, which modulates PRR-induced outcomes, ultimately leading to enhanced PRR-induced cytokines.

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators.

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-ß dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to proinflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages.

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl, and Mer (TAM) receptors in regulating chronic pattern recognition receptor stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and proinflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGF-ß-dependent TAM upregulation in human macrophages, which, in turn, upregulated suppressor of cytokine signaling 3 expression. Restoring suppressor of cytokine signaling 3 expression under TAM knockdown conditions restored chronic NOD2-mediated proinflammatory cytokine downregulation. In contrast to the upregulated proinflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K, and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating proinflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment.

A TNFSF15 disease-risk polymorphism increases pattern-recognition receptor-induced signaling through caspase-8-induced IL-1.

Inflammatory diseases are characterized by dysregulated cytokine production. Altered functions for most risk loci, including the inflammatory bowel disease and leprosy-associated tumor necrosis factor ligand superfamily member 15 (TNFSF15) region, are unclear. Regulation of pattern-recognition-receptor (PRR)-induced signaling and cytokines is crucial for immune homeostasis; TNFSF15:death receptor 3 (DR3) contributions to PRR responses have not been described. We found that human macrophages expressed DR3 and that TNFSF15:DR3 interactions were critical for amplifying PRR-initiated MAPK/NF-κB/PI3K signaling and cytokine secretion in macrophages. Mechanisms mediating TNFSF15:DR3 contributions to PRR outcomes included TACE-induced TNFSF15 cleavage to soluble TNFSF15; soluble TNFSF15 then led to TRADD/FADD/MALT-1- and caspase-8-mediated autocrine IL-1 secretion. Notably, TNFSF15 treatment also induced cytokine secretion through a caspase-8-dependent pathway in intestinal myeloid cells. Importantly, rs6478108 A disease risk-carrier macrophages demonstrated increased TNFSF15 expression and PRR-induced signaling and cytokines. Taken together, TNFSF15:DR3 interactions amplify PRR-induced signaling and cytokines, and the rs6478108 TNFSF15 disease-risk polymorphism results in a gain of function.

A TPL2 (MAP3K8) disease-risk polymorphism increases TPL2 expression thereby leading to increased pattern recognition receptor-initiated caspase-1 and caspase-8 activation, signalling and cytokine secretion.

OBJECTIVE: IBD is characterised by dysregulated intestinal immune homeostasis and cytokine secretion. In the intestine, properly regulating pattern recognition receptor (PRR)-mediated signalling and cytokines is crucial given the ongoing host-microbial interactions. TPL2 (MAP3K8, COT) contributes to PRR-initiated pathways, yet the mechanisms for TPL2 signalling contributions in primary human myeloid cells are incompletely understood and its role in intestinal myeloid cells is poorly defined. Furthermore, functional consequences for the IBD-risk locus rs1042058 in TPL2 are unknown. METHODS: We analysed protein, cytokine and RNA expression, and signalling in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR and flow cytometry. RESULTS: PRR-induced cytokine secretion was increased in MDMs from rs1042058 TPL2 GG risk individuals. TPL2 activation by the Crohn's disease-associated PRR nucleotide-oligomerisation domain (NOD)2 required PKC, and IKKß, IKKα and IKKγ signalling. TPL2, in turn, significantly enhanced NOD2-induced ERK, JNK and NFκB signalling. We found that another major mechanism for the TPL2 contribution to NOD2 signalling was through ERK-dependent and JNK-dependent caspase-1 and caspase-8 activation, which in turn, led to early autocrine interleukin (IL)-1ß and IL-18 secretion and amplification of long-term cytokines. Importantly, Salmonella typhimurium-induced cytokines from human intestinal myeloid-derived cells required TPL2 as well as autocrine IL-1ß and IL-18. Finally, rs1042058 GG risk carrier MDMs from healthy individuals and patients with Crohn's disease had increased TPL2 expression and NOD2-initiated TPL2 phosphorylation, ERK, JNK and NFκB activation, and early autocrine IL-1ß and IL-18 secretion. CONCLUSIONS: Taken together, the rs1042058 GG IBD-risk polymorphism in TPL2 results in a gain-of-function by increasing TPL2 expression and signalling, thereby amplifying PRR-initiated outcomes.

The IL18RAP region disease polymorphism decreases IL-18RAP/IL-18R1/IL-1R1 expression and signaling through innate receptor-initiated pathways.

Fine-tuning of cytokine-inducing pathways is essential for immune homeostasis. Consistently, a dysregulated increase or decrease in pattern-recognition receptor (PRR)-induced signaling and cytokine secretion can lead to inflammatory bowel disease. Multiple gene loci are associated with inflammatory bowel disease, but their functional effects are largely unknown. One such region in chromosome 2q12 (rs917997), also associated with other immune-mediated diseases, encompasses IL18RAP. We found that human monocyte-derived macrophages (MDMs) from rs917997 AA risk carriers secrete significantly less cytokines than G carriers upon stimulation of multiple PRRs, including nucleotide-binding oligomerization domain 2 (NOD2). We identified that IL-18 signaling through IL-18RAP was critical in amplifying PRR-induced cytokine secretion in MDMs. IL-18RAP responded to NOD2-initiated early, caspase-1-dependent autocrine IL-18, which dramatically enhanced MAPK, NF-κB, PI3K, and calcium signaling. Reconstituting MAPK activation was sufficient to rescue decreased cytokines in NOD2-stimulated IL-18RAP-deficient MDMs. Relative to GG carriers, MDM from rs917997 AA carriers had decreased expression of cell-surface IL-18RAP protein, as well as of IL-18R1 and IL-1R1, genes also located in the IL18RAP region. Accordingly, these risk-carrier MDMs show diminished PRR-, IL-18-, and IL-1-induced MAPK and NF-κB signaling. Taken together, our results demonstrate clear functional consequences of the rs917997 risk polymorphism; this polymorphism leads to a loss-of-function through decreased IL-18RAP, IL-18R1, and IL-1R1 protein expression, which impairs autocrine IL-18 and IL-1 signaling, thereby leading to decreased cytokine secretion in MDMs upon stimulation of a broad range of PRRs.

IRF5 risk polymorphisms contribute to interindividual variance in pattern recognition receptor-mediated cytokine secretion in human monocyte-derived cells.

Monocyte-derived cells display highly variable cytokine secretion upon pattern recognition receptor (PRR) stimulation across individuals; such variability likely affects interindividual inflammatory/autoimmune disease susceptibility. To define mechanisms for this heterogeneity, we examined PRR-induced monocyte-derived cell cytokine secretion from a large cohort of healthy individuals. Although cytokine secretion ranged widely among individuals, the magnitude of cytokine induction after individual nucleotide-binding oligomerization domain 2 (Nod2) and TLR2 stimulation (a cohort of 86 individuals) or stimulation of multiple TLRs (a cohort of 77 individuals), either alone or in combination with Nod2, was consistent intraindividually across these stimuli. Nod2 and TLRs signal through IFN regulatory factor 5 (IRF5), and common IRF5 polymorphisms confer risk for autoimmunity. We find that cells from rs2004640 IRF5 risk-associated allele carriers secrete increased cytokines upon individual or synergistic PRR stimulation in a gene dose- and ligand dose-dependent manner in both monocyte-derived dendritic cells and monocyte-derived macrophages. IRF5 expression knockdown in IRF5 risk allele carrier cells significantly decreases PRR-induced cytokines. Moreover, we find that IRF5 knockdown profoundly decreases Nod2-mediated MAPK and NF-κB pathway activation, whereas the PI3K and mammalian target of rapamycin pathways are not impaired. Finally, the IRF5 rs2004640 polymorphism is a major determinant of the variance (r(2) = 0.53) in Nod2-induced cytokine secretion by monocyte-derived cells from different individuals. We therefore show a profound contribution of a single gene to the variance in interindividual PRR-induced cytokines. The hyperresponsiveness of IRF5 disease-associated polymorphisms to a wide spectrum of microbial triggers has broad implications on global immunological responses, host defenses against pathogens, and inflammatory/autoimmune disease susceptibility.

NLRP1 and NLRP3 inflammasomes are essential for distinct outcomes of decreased cytokines but enhanced bacterial killing upon chronic Nod2 stimulation.

Upon chronic microbial exposure and pattern-recognition receptor (PRR) stimulation, myeloid-derived cells undergo a distinct transcriptional program relative to acute PRR stimulation, with proinflammatory pathways being downregulated. However, other host-response pathways might be differentially regulated, and this concept has been relatively unexplored. Understanding mechanisms regulating chronic microbial exposure outcomes is important for conditions of ongoing infection or at mucosal surfaces, such as the intestine. The intracellular PRR nucleotide oligomerization domain 2 (Nod2) confers the highest genetic risk toward developing Crohn's disease (CD). We previously identified mechanisms mediating downregulation of proinflammatory pathways upon chronic Nod2 stimulation; here we sought to define how chronic Nod2 stimulation regulates bacterial killing. We find that, despite downregulating cytokine secretion upon restimulation through PRR and live bacteria, chronic Nod2 stimulation of human monocyte-derived macrophages enhances bacterial killing; this dual regulation is absent in CD Nod2-risk carriers. We show that chronic Nod2-mediated reprogramming of human monocyte-derived macrophages to a state of enhanced bacterial killing requires upregulated reactive oxygen/nitrogen species pathway function through increased p67phox/p47phox/nitric oxide synthase-2 expression; selectively knocking down each of these genes reverses the enhanced bacterial killing. Importantly, we find that, during chronic Nod2 stimulation, NLRP3/NLRP1 inflammasome-mediated caspase-1 activation with subsequent IL-1 secretion is essential for the subsequent bifurcation to downregulated proinflammatory cytokines and upregulated bacterial killing. Therefore, we identify mechanisms mediating the distinct inflammatory and microbicidal outcomes upon chronic stimulation of the CD-associated protein Nod2.

Nod2-induced autocrine interleukin-1 alters signaling by ERK and p38 to differentially regulate secretion of inflammatory cytokines.

BACKGROUND & AIMS: Stimulation of nucleotide-binding oligomerization domain-containing (Nod)2 and other pattern recognition receptors (PRR) in human monocyte-derived macrophages induces interleukin (IL)-1, which increases mitogen-activated protein kinase (MAPK) activation and cytokine secretion. Activation of MAPK by PRR has varied effects on inflammatory cytokine secretion. We investigated whether different levels of autocrine IL-1 mediate these varied effects. METHODS: Macrophage responses to PRR ligands were analyzed by enzyme-linked immunosorbent assay and flow cytometry. We overexpressed or reduced MAPK levels (using small inhibitory RNA). RESULTS: Nod2 and other PRR activated signaling via extracellular signal-related kinase (ERK) and p38 that inhibited inflammatory cytokine production by human monocyte-derived macrophages; autocrine IL-1 production prevented this inhibition. ERK and p38 inhibited inflammatory cytokine production by human macrophages that produce low levels of IL-1 (such as M2, endotoxin-tolerant, and intestinal macrophages); adding exogenous IL-1 caused ERK and p38 to stimulate production of inflammatory cytokines in these cells. In mouse macrophages, which do not produce IL-1 in response to PRR stimulation alone, addition of exogenous IL-1 reversed the ERK-mediated inhibition of IL-12p40. Increasing activation of c-Jun N-terminal kinase in Nod2-stimulated human monocyte-derived macrophages, in the absence of autocrine IL-1 signaling, caused ERK and p38 to stimulate inflammatory cytokines secretion. Importantly, infection of human intestinal macrophages with pathogens that induce IL-1 production reversed the inhibition of inflammatory cytokine production by ERK and p38. CONCLUSIONS: In response to PRR stimulation of macrophages, the level of MAPK signaling is regulated by autocrine IL-1 and determines whether production of inflammatory cytokines is inhibited or stimulated. This mechanism could account for reported differences in MAPK regulation of inflammatory cytokines and propagate the inflammatory response to pathogens.

Distinct roles for Nod2 protein and autocrine interleukin-1beta in muramyl dipeptide-induced mitogen-activated protein kinase activation and cytokine secretion in human macrophages.

Elucidating factors regulating Crohn's disease-associated nucleotide-binding oligomerization domain 2 (Nod2) responses is critical to understanding the mechanisms of intestinal immune homeostasis. Stimulation of primary monocyte-derived macrophages by muramyl dipeptide (MDP), a component of bacterial peptidoglycan and specific Nod2 ligand, produces cytokines, including IL-1ß. We found that IL-1ß blockade profoundly inhibits MDP-induced cytokine production in human monocyte-derived macrophages, demonstrating a key role for IL-1ß autocrine secretion in Nod2-mediated responses. Importantly, although MAPK activation has previously been attributed directly to Nod2 signaling, we determined that the IL-1ß autocrine loop is responsible for the majority of MDP-induced MAPK activation. Because the critical effects of IL-1ß autocrine secretion on MAPK activation are observed as early as 10 min after Nod2 stimulation, we hypothesized that secretion of IL-1ß from preexisting intracellular pro-IL-1ß stores is necessary for optimal MDP-mediated cytokine induction. Consistently, we detected IL-1ß secretion within 10 min of MDP treatment. Moreover, caspase-1 inhibition significantly attenuates MDP-mediated early MAPK activation. Importantly, selective JNK/p38 activation is sufficient to rescue the decreased cytokine secretion during Nod2 stimulation in the absence of autocrine IL-1ß. Finally, we found that the IL-1ß autocrine loop significantly enhances responses by a broad range of pattern recognition receptors. Taken together, MDP stimulation activates Nod2 to process and release preexisting pro-IL-1ß stores in a caspase-1-dependent fashion; this secreted IL-1ß, in turn, contributes to the majority of MDP-initiated MAPK activation and leads to subsequent cytokine secretion. Our findings clarify mechanisms of IL-1ß contributions to Nod2 responses and elucidate the dominant role of IL-1ß in MDP-initiated MAPK and cytokine secretion.