Leucine-rich repeat kinase 2 promotes the development of experimental severe acute pancreatitis.

Translocation of gut bacteria into the pancreas promotes the development of severe acute pancreatitis (SAP). Recent clinical studies have also highlighted the association between fungal infections and SAP. The sensing of gut bacteria by pattern recognition receptors promotes the development of SAP via the production of proinflammatory cytokines; however, the mechanism by which gut fungi mediate SAP remains largely unknown. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that regulates innate immunity against fungi via Dectin-1 activation. Here, we investigated the role of LRRK2 in SAP development and observed that administration of LRRK2 inhibitors attenuated SAP development. The degree of SAP was greater in Lrrk2 transgenic (Tg) mice than in control mice and was accompanied by an increased production of nuclear factor-kappaB-dependent proinflammatory cytokines. Ablation of the fungal mycobiome by anti-fungal drugs inhibited SAP development in Lrrk2 Tg mice, whereas the degree of SAP was comparable in Lrrk2 Tg mice with or without gut sterilization by a broad range of antibiotics. Pancreatic mononuclear cells from Lrrk2 Tg mice produced large amounts of IL-6 and TNF-α upon stimulation with Dectin-1 ligands, and inhibition of the Dectin-1 pathway by a spleen tyrosine kinase inhibitor protected Lrrk2 Tg mice from SAP. These data indicate that LRRK2 activation is involved in the development of SAP through proinflammatory cytokine responses upon fungal exposure.

Activation of the aryl hydrocarbon receptor inhibits the development of experimental autoimmune pancreatitis through IL-22-mediated signaling pathways.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor expressed in hematopoietic and non-hematopoietic cells. Activation of the AhR by xenobiotics, microbial metabolites, and natural substances induces immunoregulatory responses. Autoimmune pancreatitis (AIP) is a chronic fibroinflammatory disorder of the pancreas driven by autoimmunity. Although AhR activation generally suppresses pathogenic autoimmune responses, the roles played by the AhR in AIP have been poorly defined. In this study, we examined how AhR activation affected the development of experimental AIP caused by the activation of plasmacytoid dendritic cells producing IFN-α and IL-33. Experimental AIP was induced in MRL/MpJ mice by repeated injections of polyinosinic-polycytidylic acid. Activation of the AhR by indole-3-pyruvic acid and indigo naturalis, which were supplemented in the diet, inhibited the development of experimental AIP, and these effects were independent of the activation of plasmacytoid dendritic cells producing IFN-α and IL-33. Interaction of indole-3-pyruvic acid and indigo naturalis with AhRs robustly augmented the production of IL-22 by pancreatic islet α cells. The blockade of IL-22 signaling pathways completely canceled the beneficial effects of AhR ligands on experimental AIP. Serum IL-22 concentrations were elevated in patients with type 1 AIP after the induction of remission with prednisolone. These data suggest that AhR activation suppresses chronic fibroinflammatory reactions that characterize AIP via IL-22 produced by pancreatic islet α cells.

Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression.

Mutations in nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease (CD). Although NOD2 activation contributes to the maintenance of intestinal homeostasis through the negative regulation of pro-inflammatory cytokine responses mediated by Toll-like receptors (TLRs), the effects of NOD2 activation on interferon (IFN)-α responses induced by TLR9 have been poorly defined. To explore the cross-talk between NOD2 and TLR9, human monocytes or dendritic cells (DCs) were stimulated with NOD2 and/or TLR9 ligands to measure IFN-α production. The severity of dextran sodium sulfate (DSS)-induced colitis was compared in mice treated with NOD2 and/or TLR9 ligands. Expression of IFN-α and IFN-stimulated genes (ISGs) was examined in the colonic mucosa of patients with inflammatory bowel disease (IBD). NOD2 activation reduced TLR9-induced IFN-α production by monocytes and DCs in a deubiquitinating enzyme A (DUBA)-dependent manner. Activation of DUBA induced by the co-stimulation of TLR9 and NOD2 inhibited Lys63-linked polyubiquitination of TRAF3 and suppressed TLR9-mediated IFN-α production. NOD2 activation in hematopoietic cells protected mice from TLR9-induced exacerbation of DSS-induced colitis by down-regulating IFN-α responses and up-regulating DUBA expression. Colonic mucosa of patients with active and remitted IBD phases was characterized by the enhanced and reduced expression of ISGs, respectively. Expression levels of IFN-α and IL-6 positively correlated in the active colonic mucosa of patients with ulcerative colitis and CD, whereas DUBA expression inversely correlated with that of IFN-α in patients with CD. Collectively, these data suggest that DUBA-dependent negative effect of NOD2 on TLR9-mediated IFN-α responses contributes to the maintenance of intestinal homeostasis.

High-fat diet aggravates experimental autoimmune pancreatitis through the activation of type I interferon signaling pathways.

Autoimmune pancreatitis (AIP) is an autoimmune disorder of the pancreas characterized by enhanced IgG4 antibody responses and multiple organ involvement. AIP is a pancreatic manifestation of the systemic IgG4-related disease (IgG4-RD). Although AIP and IgG4-RD predominantly occur in middle-aged and elderly men, the roles of eating habits and lifestyle in the pathogenesis of these conditions are poorly understood. In this study, we examined whether a high-fat diet (HFD), preferred by middle-aged and elderly men, increases sensitivity to experimental AIP. We modeled AIP in MRL/MpJ mice by repeated injections of polyinosinic:polycytidylic acid. HFD exacerbated AIP development and promoted pancreatic accumulation of interferon (IFN)-α-producing plasmacytoid dendritic cells (pDCs). However, HFD did not increase the severity of autoimmune sialadenitis, another disorder associated with AIP and IgG4-RD. Neutralization of type I IFN signaling pathways prevented the development of severe AIP induced by HFD. In contrast, leaky gut was less likely to be associated with the HFD-induced exacerbation of AIP, as was evidenced by the lack of significant alterations in the jejunal or ileal expression of tight junction proteins. These data suggest that HFD exacerbates experimental AIP through the activation of pDCs producing IFN-α.

Disruption of the intestinal barrier exacerbates experimental autoimmune pancreatitis by promoting the translocation of Staphylococcus sciuri into the pancreas.

Autoimmune pancreatitis (AIP) and IgG4-related disease (IgG4-RD) are new disease entities characterized by enhanced IgG4 antibody responses and involvement of multiple organs, including the pancreas and salivary glands. Although the immunopathogenesis of AIP and IgG4-RD is poorly understood, we previously reported that intestinal dysbiosis mediates experimental AIP through the activation of IFN-α- and IL-33-producing plasmacytoid dendritic cells (pDCs). Because intestinal dysbiosis is linked to intestinal barrier dysfunction, we explored whether the latter affects the development of AIP and autoimmune sialadenitis in MRL/MpJ mice treated with repeated injections of polyinosinic-polycytidylic acid [poly (I:C)]. Epithelial barrier disruption was induced by the administration of dextran sodium sulfate (DSS) in the drinking water. Mice co-treated with poly (I:C) and DSS, but not those treated with either agent alone, developed severe AIP, but not autoimmune sialadenitis, which was accompanied by the increased accumulation of IFN-α- and IL-33-producing pDCs. Sequencing of 16S ribosomal RNA revealed that Staphylococcus sciuri translocation from the gut to the pancreas was preferentially observed in mice with severe AIP co-treated with DSS and poly (I:C). The degree of experimental AIP, but not of autoimmune sialadenitis, was greater in germ-free mice mono-colonized with S. sciuri and treated with poly (I:C) than in germ-free mice treated with poly (I:C) alone, which was accompanied by the increased accumulation of IFN-α- and IL-33-producing pDCs. Taken together, these data suggest that intestinal barrier dysfunction exacerbates AIP through the activation of pDCs and translocation of S. sciuri into the pancreas.

Expression levels of cellular inhibitor of apoptosis proteins and colitogenic cytokines are inversely correlated with the activation of interferon regulatory factor 4.

Cellular inhibitors of apoptosis proteins 1 (cIAP1) and 2 (cIAP2) are involved in signaling pathways mediated by Toll-like receptors (TLRs) and tumor necrosis factor (TNF)-α. Excessive activation of TLRs and TNF-α underlies the immunopathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). However, the roles played by cIAP1 and cIAP2 in the development of CD and UC remain poorly understood. In this study, we attempted to clarify the molecular link between cIAP1/cIAP2 and colonic inflammation. Human monocyte-derived dendritic cells (DCs) treated with siRNAs specific for cIAP1 or cIAP2 exhibited reduced pro-inflammatory cytokine responses upon stimulation with TLR ligands. Expression of cIAP1 and cIAP2 in human DCs was suppressed in the presence of interferon regulatory factor 4 (IRF4). This effect was associated with inhibition of cIAP1 and cIAP2 polyubiquitination. To verify these in vitro findings, we created mice overexpressing IRF4 in DCs and showed that these mice were resistant to trinitrobenzene sulfonic acid-induced colitis as compared with wild-type mice; these effects were accompanied by reduced expression levels of cIAP1 and cIAP2. Pro-inflammatory cytokine production by mesenteric lymph node cells upon stimulation with TLR ligands was reduced in mice with DC-specific IRF4 overexpression as compared with that in wild-type mice. Finally, in clinical samples of the colonic mucosa from patients with CD, there was a negative relationship between the percentage of IRF4+ DCs and percentages of cIAP1+ or cIAP2+ lamina propria mononuclear cells. These data suggest that the colitogenic roles of cIAP1 and cIAP2 are negatively regulated by IRF4.

IL-33 as a Critical Cytokine for Inflammation and Fibrosis in Inflammatory Bowel Diseases and Pancreatitis.

IL-33 is a pleiotropic cytokine that promotes inflammation and fibrosis. IL-33 is produced by a broad range of cells, including antigen-presenting cells (APCs), epithelial cells, and fibroblasts. IL-33 produced by the innate immune cells has been shown to activate pro-inflammatory T helper type 1 (Th1) and T helper type 2 (Th2) responses. The intestinal barrier and tolerogenic immune responses against commensal microbiota contribute to the maintenance of gut immune homeostasis. Breakdown of tolerogenic responses against commensal microbiota as a result of intestinal barrier dysfunction underlies the immunopathogenesis of inflammatory bowel diseases (IBD) and pancreatitis. Recent studies have provided evidence that IL-33 is an innate immune cytokine that bridges adaptive Th1 and Th2 responses associated with IBD and pancreatitis. In this Mini Review, we discuss the pathogenic roles played by IL-33 in the development of IBD and pancreatitis and consider the potential of this cytokine to be a new therapeutic target.

Case Report: Regulatory T Cell-Independent Induction of Remission in a Patient With Collagenous Colitis.

Collagenous colitis (CC), a prototypical microscopic colitis, is a chronic inflammatory disorder of the colon. The diagnosis of CC depends on the pathological examination. The colonic mucosa of patients with CC is characterized by the presence of a substantially thickened collagen band (>10µm) under the surface epithelium. In addition, intraepithelial and lamina propria lymphocytes are markedly increased in patients with CC. However, the roles played by the lymphocytes accumulating in the colonic mucosa of patients with CC are poorly defined. Recent studies indicate that T cells infiltrating the colonic mucosa of patients with CC are mainly represented by CD4+ T cells, CD8+ T cells, and forkhead box P3 (FOXP3)+ regulatory T cells (Tregs). Given that activation of CD4+/CD8+ T cells and FOXP3+ Tregs usually mediates pro-inflammatory and anti-inflammatory responses, respectively, alterations in the colonic numbers of these adaptive T cells might be related to the resolution of colitis in patients with CC. We determined alterations in the composition of colonic T cells by extensive immunohistochemical (IHC) analyses in a case of CC successfully treated with budesonide and metronidazole. Colonic lamina propria immune cells mainly comprised CD3+ T cells, CD4+ T cells, CD8+ T cells, CD68+ macrophages, and FOXP3+ Tregs, but not CD20+ B cells or myeloperoxidase (MPO)+ granulocytes in the active phase. During remission, the numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, and CD68+ macrophages did not change significantly in the colonic lamina propria, whereas FOXP3+ Tregs were markedly decreased, suggesting that induction of remission was achieved in a Treg-independent manner. Thus, our study indicates that accumulation of FOXP3+ Tregs in the colonic mucosa of patients with CC might be a counter-regulatory mechanism reflecting persistent inflammation and that induction of remission might be achieved without activation of Tregs.

Immunohistochemical characterization of granulomatosis with polyangiitis exhibiting spontaneous regression.

BACKGROUND: Granulomatosis with polyangiitis (GPA) is characterized by granulomatous inflammation, vasculitis, and elevated levels of serum proteinase 3 (PR3)-anti-neutrophil cytoplasmic antibody (PR3-ANCA). OBJECTIVE: We tried to characterize immune cells accumulated into the lung lesions of a GPA patient exhibiting spontaneous regression. METHODS: Transbronchial lung biopsy (TBLB) samples were subjected to immunohistochemical analyses. RESULTS: Multiple lung nodules were detected by CT. TBLB showed granulomatous inflammation and small vessel vasculitis. This case was diagnosed as GPA based on pathological findings and elevation of PR-3 ANCA levels. Spontaneous disappearance of multiple lung nodules was observed in CT. CD3+ T cells and CD20+ B cells accumulated in the inflammatory lesions surrounding the vessels whereas granulomatous inflammation was mainly comprised of CD3+ T cells and CD68+ macrophages, but not B cells or myeloperoxidase+ neutrophils. CONCLUSIONS: We characterized immune cell compositions of the lung lesions of a patient with GPA exhibiting spontaneous regression.

NOD2 deficiency protects mice from the development of adoptive transfer colitis through the induction of regulatory T cells expressing forkhead box P3.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular receptor for muramyl dipeptide derived from the intestinal microbiota. Loss-of-function mutations in Nod2 are associated with the development of Crohn's disease, suggesting that NOD2 signaling plays critical roles in the maintenance of intestinal immune homeostasis. Although NOD2 activation prevents the development of short-term experimental colitis, it remains unknown whether the sensitivity to long-term experimental colitis is influenced by NOD2. In this study, we explored the roles played by NOD2 in the development of long-term adoptive transfer colitis. Unexpectedly, we found that Rag1-/-Nod2-/- mice were more resistant to adoptive transfer colitis than Rag1-/- mice and had reduced proinflammatory cytokine responses and enhanced accumulation of regulatory T cells (Tregs) expressing forkhead box P3 in the colonic mucosa. Prevention of colitis in Rag1-/-Nod2-/- mice was mediated by TGF-ß1 because neutralization of TGF-ß1 resulted in the development of more severe colitis due to reduced accumulation of Tregs. Such paradoxical Treg responses in the absence of NOD2 could explain why Nod2 mutations in humans are not sufficient to cause Crohn's disease.

Case Report: A Case of Intestinal Behçet's Disease Exhibiting Enhanced Expression of IL-6 and Forkhead Box P3 mRNA After Treatment With Infliximab.

Behçet's disease (BD) is a rare inflammatory condition characterized by oral and genital ulcers, skin lesions, as well as ophthalmological, neurological, and gastrointestinal manifestations. BD involving the gastrointestinal tract is known as intestinal BD. The mucosa of the gastrointestinal tract of patients with intestinal BD exhibits enhanced levels of proinflammatory cytokines, such as IL-1ß, IL-6, and TNF-α. These proinflammatory cytokines play pathogenic roles in the development of BD, as evidenced by the fact that biologics targeting these cytokines effectively induce BD remission. It should be noted, however, that the molecular mechanisms by which the blockade of these cytokines suppresses chronic inflammatory responses in BD are poorly understood. Herein, we report a case of intestinal BD resistant to prednisolone that was successfully treated with infliximab (IFX). The induction of remission by IFX was accompanied by a marked elevation of IL-6 and forkhead box P3 (FOXP3) at mRNA level. This case suggests that induction of remission by IFX is mediated not only by the suppression of TNF-α-mediated signaling pathways, but also by the promotion of IL-6 expression and accumulation of regulatory T cells expressing FOXP3.

Development of a New Threshold of Toxicological Concern Database of Non-cancer Toxicity Endpoints for Industrial Chemicals.

In cases where chemical-specific toxicity data are absent or limited, the threshold of toxicological concern (TTC) offers an alternative to assess human exposure below which "there would be no appreciable risk to human health." The application of TTC to non-cancer systemic endpoints has been pursued for decades using a chemical classification and Point of Departure (POD). This study presents a new POD dataset of oral subacute/subchronic toxicity studies in rats for 656 industrial chemicals retrieved from the Hazard Evaluation Support System (HESS) Integrated Platform, which contains hundreds of reliable repeated-dose toxicity test data of industrial chemicals under the Chemical Substances of Control Law in Japan. The HESS TTC dataset was found to have less duplication with substances in other reported TTC datasets. Each chemical was classified into a Cramer Class, with 68, 3, and 29% of these 656 chemicals distributed in Classes III, II, and I, respectively. For each Cramer Class, a provisional Tolerable Daily Intake (TDI) was derived from the 5th percentile of the lognormal distribution of PODs. The TDIs were 1.9 and 30 µg/kg bw/day for Classes III and I, respectively. The TDI for Cramer Class II could not be determined due to insufficient sample size. This work complements previous studies of the TTC approach and increases the confidence of the thresholds for non-cancer endpoints by including unique chemical structures. This new TTC dataset is publicly available and can be merged with existing databases to improve the TTC approach.