Matija Hedl

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.

Chronic stimulation of Nod2 mediates tolerance to bacterial products

The Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (Nod) families of proteins are critical for bacterial recognition, and, acutely, this frequently leads to proinflammatory responses. Polymorphisms in Nod2 (CARD 15) are associated with an increased likelihood of developing Crohns disease. However, it is not yet clear how Nod2 dysfunctions lead to defects in human intestinal immune homeostasis. Studies to date have focused on functions after acute, rather than chronic, Nod2 stimulation. However, the intestine is an environment of chronic bacterial product exposure with tolerance to luminal flora. We therefore hypothesized that long-term Nod2 stimulation contributes to down-regulation of inflammatory responses from innate immune receptors. We found that pretreatment with muramyl dipeptide (MDP), a ligand for Nod2, significantly decreased production of the proinflammatory cytokines TNF-α, IL-8, and IL-1β upon Nod2, TLR4, and TLR2 restimulation in primary human monocyte-derived macrophages from a large cohort of individuals. Importantly, TNF-α-induced production of proinflammatory cytokines remained intact in these same cells. MDP-stimulated macrophages from Crohns disease-relevant Leu1007insC Nod2 homozygote individuals were deficient in their ability to cross-tolerize to subsequent treatment with TLR2 and TLR4 ligands. We show that acute Nod2 stimulation induced IRAK-1 activation, and that chronic MDP treatment down-regulated IRAK-1 activation upon Nod2 or TLR4 restimulation. In a subset of individuals, chronic Nod2 stimulation induced expression of the IRAK-1 inhibitory protein IRAK-M. Significantly, intestinal macrophages exhibit tolerance to MDP per production of inflammatory cytokines. These results illustrate a role for chronic stimulation of Nod2 in mediating tolerance to bacterial products.

Distinct Roles for Nod2 Protein and Autocrine Interleukin-1β in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages

Elucidating factors regulating Crohns 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-1β. We found that IL-1β blockade profoundly inhibits MDP-induced cytokine production in human monocyte-derived macrophages, demonstrating a key role for IL-1β autocrine secretion in Nod2-mediated responses. Importantly, although MAPK activation has previously been attributed directly to Nod2 signaling, we determined that the IL-1β autocrine loop is responsible for the majority of MDP-induced MAPK activation. Because the critical effects of IL-1β autocrine secretion on MAPK activation are observed as early as 10 min after Nod2 stimulation, we hypothesized that secretion of IL-1β from preexisting intracellular pro-IL-1β stores is necessary for optimal MDP-mediated cytokine induction. Consistently, we detected IL-1β 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-1β. Finally, we found that the IL-1β 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-1β stores in a caspase-1-dependent fashion; this secreted IL-1β, in turn, contributes to the majority of MDP-initiated MAPK activation and leads to subsequent cytokine secretion. Our findings clarify mechanisms of IL-1β contributions to Nod2 responses and elucidate the dominant role of IL-1β in MDP-initiated MAPK and cytokine secretion.

Secretory Mediators Regulate Nod2-Induced Tolerance in Human Macrophages

BACKGROUND & AIMS Nucleotide oligomerization domain 2 (Nod2) polymorphisms increase the risk of developing Crohns disease, which is characterized by chronic intestinal inflammation. Bacterial peptidoglycan products chronically stimulate Nod2 in the intestine. Recent studies found that chronic Nod2 stimulation in human macrophages down-regulates proinflammatory cytokines on Nod2 or Toll-like receptor (TLR) restimulation. Therefore, an emerging hypothesis is that Nod2-mediated cytokine down-regulation is required for intestinal homeostasis, but the mechanisms mediating this down-regulation are incompletely understood. METHODS Utilizing primary human macrophages, we examined secretory mediators as a mechanism of Nod2-mediated tolerance by inhibiting their function and assessing tolerance reversal through cytokine secretion. Signaling pathways contributing to secretory mediator induction and Nod2-mediated tolerance were identified through pathway inhibition. RESULTS We found that chronic Nod2 stimulation cross-tolerizes not only to TLRs but also to the interleukin (IL)-1 receptor. Moreover, chronic IL-1β stimulation down-regulates Nod2 responses. Accordingly, IL-1β blockade partially reverses Nod2-mediated tolerance. We found that an additional essential mechanism for Nod2-mediated tolerance is the early secretion of the anti-inflammatory mediators IL-10, transforming growth factor β, and IL-1Ra. Importantly, the mammalian target of rapamycin (mTOR) pathway, involved in cell growth, differentiation, and activation, significantly contributes to Nod2-induced anti-inflammatory as opposed to proinflammatory cytokines and to Nod2-mediated tolerance. CONCLUSIONS Inflammatory responses through the IL-1R are down-regulated upon chronic Nod2 stimulation, secretory mediators are a critical mechanism for Nod2-mediated cytokine down-regulation, and the mTOR pathway is crucial for Nod2-mediated tolerance. These results further contribute to our understanding of the mechanisms through which Nod2, a protein critical to intestinal homeostasis, down-regulates cytokine responses.

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 (r2 = 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.

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.

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

Significance This study describes a role for tumor necrosis factor ligand superfamily member 15 (TNFSF15), a molecule being considered for therapy in inflammatory diseases. We identify that death receptor 3 (DR3) is clearly expressed on human macrophages and define a new role for TNFSF15:DR3 signaling in mediating innate immune outcomes in human myeloid-derived cells, as well as mechanisms mediating these outcomes. We further define the disease-relevant, functional consequences of polymorphisms in the TNFSF15 region, one of the 163 inflammatory bowel disease risk loci. To our knowledge, our studies highlight a previously unknown mechanism through which TNFSF15:DR3 can contribute to intestinal inflammation, namely by enhancing signaling and cytokine secretion upon stimulation of a broad range of pattern-recognition-receptors, mycobacterial components, and live bacteria. 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.

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 Crohns disease phenotype, similar to polymorphisms in NOD2. Taken together, ICOSL amplifies PRR-initiated outcomes, which might contribute to immune homeostasis.

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.

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 Crohns 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.