Microbe-associated molecular patterns derived from fungi and bacteria promote IgG4 antibody production in patients with type 1 autoimmune pancreatitis.

Enhanced IgG4 antibody (Ab) response is a prominent feature of type 1 autoimmune pancreatitis (AIP). Innate immune responses associated with IgG4 Ab production are poorly defined. We have previously reported that peripheral blood mononuclear cells (PBMCs) isolated from patients with type 1 AIP produce large amounts of IgG4 Abs upon stimulation with bacterial cell wall components. In addition, we showed that activation of plasmacytoid dendritic cells producing interferon (IFN)-α, interleukin (IL)-33, and B cell-activating factor (BAFF) upon sensing intestinal bacteria mediates the development of experimental AIP. In this study, we attempted to clarify the role of innate immunity against fungi in inducing enhanced IgG4 Ab responses in type 1 AIP. PBMCs isolated from healthy controls and patients with type 1 AIP were stimulated with a broad range of bacterial and fungal cell wall components. The concentrations of IgG1, IgG4, and cytokines were measured using enzyme-linked immunosorbent assays. Cell wall components derived from bacteria and fungi induced IgG1 and IgG4 Ab production in patients with type 1 AIP. Various types of microbe-associated molecular pattern motifs enhanced IgG4 Ab production in patients with type 1 AIP compared with the limited motifs in healthy controls. The enhanced IgG1 and IgG4 Ab production that followed in response to bacterial and fungal cell wall components was parallel to that of IFN-α, IFN-γ, IL-10, IL-33, and BAFF. In conclusion, cell wall components derived from fungi as well as bacteria promote IgG4 Ab responses in patients with type 1 AIP.

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.

Reciprocal regulation of protein arginine deiminase 2 and 4 expression in the colonic mucosa of ulcerative colitis.

Neutrophils express protein arginine deiminase 2 and PAD4, both of which mediate the citrullination of target proteins to induce production of neutrophil extracellular traps. Although PAD-dependent NETs trigger inflammatory bowel disease, the mechanisms governing the expression of PAD2 and PAD4 are poorly understood. In this study, we tried to clarify expression mechanisms of PAD2 and PAD4 in the colonic mucosa of patients with ulcerative colitis and Crohn's disease. Administration of Cl-amidine, a pan PAD-inhibitor, attenuated the development of dextran sodium sulfate-induced colitis, the effects of which were accompanied by reduced IL-6 and TNF-α production by colonic lamina propria mononuclear cells upon exposure to Toll-like receptor ligands. The mRNA expression of colonic PAD2 and PAD4 was negatively and positively correlated with disease activity and pro-inflammatory cytokine responses in patients with UC, respectively. Reciprocal regulation of PAD2 and PAD4 mRNA expression was observed in the colonic mucosa of UC patients, but not in those of CD patients. PAD4 mRNA expression was correlated with disease activity and pro-inflammatory cytokine responses in patients with CD. Collectively, these data suggest that reciprocal regulation of PAD2 and PAD4 expression is associated with disease activity in UC patients.

Crosstalk between NOD2 and TLR2 suppresses the development of TLR2-mediated experimental colitis.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular sensor for muramyl dipeptide (MDP), a degradation product of bacterial cell wall peptidoglycan (PGN). PGN stimulates cell-surface Toll-like receptor 2 (TLR2) independently of NOD2, indicating the presence of crosstalk between extracellular TLR2 and intracellular NOD2 upon exposure to PGN. NOD2-deficient mice were sensitive, while TLR2-deficient mice were resistant to experimental colitis induced by intrarectal administration of PGN. Severe colitis in NOD2-deficient mice was accompanied by increased expression of nuclear factor-kappa B-dependent cytokines and decreased expression of autophagy-related 16-like 1 (ATG16L1). MDP activation of NOD2 enhanced autophagy mediated by TLR2 in human dendritic cells. mRNA expression of TLR2 tended to be higher in the colonic mucosa of patients with active ulcerative colitis compared to that of those in remission. Induction of remission was associated with increased mRNA expression of ATG16L1 in both ulcerative colitis and Crohn's disease patients. Conversely, mRNA expression of receptor-interacting serine/threonine-protein kinase 2 was higher in the inflammatory colonic mucosa of patients with active disease than in the non-inflamed mucosa of patients in remission, in both ulcerative colitis and Crohn's disease. These findings highlight the role of NOD2-TLR2 crosstalk in the immunopathogenesis of colitis.

Cytokine and chemokine profiles in ulcerative colitis relapse after coronavirus disease 2019 vaccination.

Coronavirus disease 2019 (COVID-19) vaccines are highly effective; however, vaccine-related adverse events, including autoimmunity, have been reported. Case reports describing relapse or new-onset of ulcerative colitis (UC) after COVID-19 mRNA vaccination are available. However, the molecular mechanisms underlying the development of colonic inflammation associated with COVID-19 mRNA vaccination are poorly understood. Furthermore, it is unclear whether the relapse of UC after COVID-19 vaccination is driven by unique cytokine responses that differ from those of UC not associated with vaccination. mRNAs derived from COVID-19 vaccines are potent inducers of type I IFN response. We encountered three cases of UC relapse after COVID-19 vaccination. mRNA expressions of IFN-α, IFN-ß, IL-1ß, and IL-12/23p40 showed higher tendency in the colonic mucosa of patients with UC associated with vaccination compared with those not associated with vaccination. In contrast, the expressions of C-X-C motif chemokine ligand 9 (CXCL9) and CXCL10 were comparable. Immunofluorescence analyses also showed higher expression of IFN-α in the colonic mucosa of patients with UC associated with COVID-19 vaccination than in those not associated with vaccination. Taken together, these data suggest that the colonic mucosa of patients with UC who relapsed after COVID-19 vaccination was characterized by enhanced type I IFN responses.

Oral administration of ovalbumin protects mice from concanavalin A-induced hepatitis through suppression of interferon-gamma responses.

The liver is a tolerogenic organ that exhibits hypo-responsiveness to antigens circulating in the portal vein. Antigens that are orally administered at high doses reach the liver. In our previous study, we demonstrated that administering ovalbumin (OVA) orally at high doses generates unique CD4+ T cells and tolerogenic dendritic cells, both of which can suppress T helper type 1 (Th1) responses, in the livers of two groups of mice: DO11.10 mice with transgenic CD4+ T cell receptors for OVA and BALB/c mice that received OVA-specific CD4+ T cells through adoptive transfer. This study aimed to investigate whether oral administration of OVA at high doses inhibits the development of hepatitis in the presence of OVA-specific CD4+ T cells. Oral administration of OVA at high doses inhibited the development of OVA-specific and concanavalin A (Con A)-induced hepatitis in DO11.10 mice, and these effects were associated with the downregulation of Th1 responses. Furthermore, the adoptive transfer of CD4+ T cells from the liver of OVA-fed DO11.10 mice inhibited the development of Con A-induced hepatitis in recipient BALB/c mice through the downregulation of Th1 responses. Finally, oral administration of OVA at high doses inhibited the development of Con A-induced hepatitis in BALB/c mice bearing naïve OVA-specific CD4+ T cells. These results suggest that the oral administration of antigens at high doses suppresses Th1-mediated hepatitis in an antigen-non-specific manner in the presence of antigen-specific CD4+ T cells.

Regulation of type I IFN responses by deubiquitinating enzyme A in inflammatory bowel diseases.

The development of Inflammatory bowel disease (IBD) is driven by excessive production of pro-inflammatory cytokines including TNF-α, IL-12, and IL-23. This notion is supported by the remarkable clinical success of biologics targeting these cytokines. Recognition of cell wall components derived from intestinal bacteria by Toll-like receptors (TLRs) induces the production of these pro-inflammatory cytokines by macrophages and dendritic cells in human IBD and experimental colitis model. Although sensing of bacterial nucleic acids by endosomal TLRs, specifically TLR3, TLR7, and TLR9 leads to robust production of type I IFNs, it remains debatable whether TLR-mediated type I IFN responses are pathogenic or protective in IBD patients. Additionally, recent studies identified deubiquitinating enzyme A (DUBA) as a novel negative regulator of TLR-mediated type I IFN responses. In light of these observations and their potential applications, in this review, we summarize recent findings on the roles of type I IFN responses and DUBA-mediated negative regulation of these responses in human IBD and experimental colitis model.

ATG16L1 negatively regulates RICK/RIP2-mediated innate immune responses.

Polymorphisms in the autophagy-related protein 16 like 1 (ATG16L1) and nucleotide-binding oligomerization domain 2 (NOD2) genes are associated with Crohn's disease (CD). Impaired interaction between ATG16L1 and NOD2 underlies CD immunopathogenesis. Although activation of the receptor-interacting serine-threonine kinase (RICK, also known as RIP2), a downstream signaling molecule for NOD2 and multiple toll-like receptors (TLRs), plays a pathogenic role in the development of inflammatory bowel disease, the molecular interaction between ATG16L1 and RICK/RIP2 remains poorly understood. In this study, we examined the physical interaction between ATG16L1 and RICK/RIP2 in human embryonic kidney 293 cells and human monocyte-derived dendritic cells (DCs) expressing excessive and endogenous levels of these proteins, respectively. We established that ATG16L1 binds to RICK/RIP2 kinase domain and negatively regulates TLR2-mediated nuclear factor-kappa B (NF-κB) activation and pro-inflammatory cytokine responses by inhibiting the interaction between TLR2 and RICK/RIP2. Binding of ATG16L1 to RICK/RIP2 suppressed NF-κB activation by down-regulating RICK/RIP2 polyubiquitination. Notably, the percentage of colonic DCs expressing ATG16L1 inversely correlated with IL-6 and TNF-α expression levels in the colon of CD patients. These data suggest that the interaction between ATG16L1 and RICK/RIP2 maintains intestinal homeostasis via the down-regulation of TLR-mediated pro-inflammatory cytokine responses.

A case with Crohn's disease-associated spondyloarthritis exhibiting enhanced pro-inflammatory cytokine responses to Toll-like receptor ligands.

BACKGROUND: Despite the high incidence of spondyloarthritis (SpA) as an extra-intestinal manifestation of Crohn's disease (CD), the immunopathogenesis of CD-associated SpA remains largely unknown. OBJECTIVE: We tried to explore molecular mechanisms accounting for the development of CD-associated SpA in a patient successfully treated with infliximab. METHODS: Peripheral blood mononuclear cells (PBMCs) before infliximab treatment were stimulated with Toll-like receptor (TLR) ligands to measure pro-inflammatory cytokine responses. Endoscopic biopsy samples before and after infliximab treatment were subjected to quantitative polymerase chain reaction. RESULTS: PBMCs from this CD-associated SpA patient exhibited higher production of pro-inflammatory cytokines upon stimulation with TLR ligands than PBMCs from healthy controls. Induction of remission by infliximab was associated with the downregulation of pro-inflammatory cytokine responses in the small intestinal mucosa, which is continually exposed to TLR ligands. CONCLUSIONS: Excessive pro-inflammatory cytokine responses to TLR ligands might underlie the immunopathogenesis of CD-associated SpA.

Tumor invasion to the arteries feeding the gallbladder as a novel risk factor for cholecystitis after metallic stent placement in distal malignant biliary obstruction.

BACKGROUND AND AIM: Cholecystitis is a major complication after self-expandable metallic stent (SEMS) placement for malignant biliary obstruction. Ischemia is one of the risk factors for cholecystitis, but little is known about the influence of tumor invasion to the feeding artery of the gallbladder on the onset of cholecystitis after SEMS placement. The aim of the present study was to identify risk factors for cholecystitis after SEMS placement. METHODS: Incidence and nine predictive factors of cholecystitis were retrospectively evaluated in 107 patients who underwent SEMS placement for unresectable distal malignant biliary obstruction at Kyoto University Hospital and Otsu Red Cross Hospital between January 2012 and June 2016. RESULTS: Cholecystitis occurred in 13 of 107 patients (12.1%) after SEMS placement during the median follow-up period of 262 days. Univariate analyses showed that tumor invasion to the feeding artery of the gallbladder and tumor involvement to the orifice of the cystic duct were significant predictors of cholecystitis (P = 0.001 and P < 0.001). Multivariate analysis confirmed that these two factors were significant and independent risks for cholecystitis with odds ratios of 22.13 (95% CI, 3.57-137.18; P = 0.001) and 25.26 (95% CI, 4.12-154.98; P < 0.001), respectively. CONCLUSIONS: This study showed for the first time that tumor invasion to the feeding artery of the gallbladder as well as tumor involvement to the orifice of the cystic duct are independent risk factors for cholecystitis after SEMS placement.

NOD2-mediated dual negative regulation of inflammatory responses triggered by TLRs in the gastrointestinal tract.

Loss-of-function mutations in nucleotide-binding oligomerization domain 2 (NOD2) constitute the primary risk factors for Crohn's disease. NOD2 is an intracellular sensor for muramyl dipeptide (MDP), a small molecule derived from the peptidoglycan layer of bacterial cell wall. Although NOD2 is involved in host immune responses, much attention has been paid to the involvement of NOD2 in the maintenance of intestinal homeostasis. Despite the fact that the proinflammatory cytokine and chemokine responses induced by NOD2 activation alone are weaker than those induced by toll-like receptors (TLRs), NOD2 plays a crucial role in host defense against invading pathogens and in the regulation of immune responses. Recent studies have highlighted the importance of negative regulatory functions of NOD2 in TLRs-mediated proinflammatory cytokine responses. MDP-mediated activation of NOD2 induces interferon regulatory factor 4 (IRF4) expression, thereby suppressing nuclear factor-κB-dependent colitogenic cytokine responses through the inhibition of Lys(K)63-linked polyubiquitination on receptor-interacting serine/threonine protein kinase 2. MDP-mediated activation of NOD2 also downregulates TLR9-induced type I IFN responses by inhibiting the K63-linked polyubiquitination of TNF receptor-associated factor 3 via deubiquitinating enzyme A (DUBA) expression. Thus, NOD2 exerts dual negative regulation of TLRs-mediated proinflammatory cytokine and type I IFN responses by inducing the expression of IRF4 and DUBA, respectively. In this review, we summarize the molecular mechanisms whereby NOD2 activation suppresses TLRs-mediated proinflammatory and type I IFN responses. In addition, we discuss the clinical relevance of the NOD2-mediated negative regulation of TLRs in inflammatory bowel disease.

Circumferential Stenosis of the Second Part of the Duodenum Caused by Eosinophilic Gastroenteritis.

Isolated eosinophilic gastroenteritis (EGE) of the second part of the duodenum is rare. We herein report a case of EGE limited to the second part of the duodenum that caused circumferential stenosis due to massive wall thickening. A boring biopsy was useful to verify the accumulation of eosinophils. Induction of remission by prednisolone was accompanied by a marked reduction in the mRNA expression of interleukin-6, C-C motif chemokine ligand 17 (CCL17), and CCL26 without any reduction in prototypical EGE-associated T helper type 2 cytokines (IL-5, IL-13). Thus, the enhanced expression of IL-6, CCL17, and CCL26 might be involved in the development of EGE in this case.

Concurrent occurrence of ulcerative duodenitis and ulcerative colitis displaying unique responses to golimumab and ustekinumab.

Recent studies have reported the occurrence of upper gastrointestinal (UGI) inflammation in patients with ulcerative colitis (UC). However, whether UC-associated UGI and colorectal lesions share pathogenic cytokine profiles and responses to biologics remains unknown. Herein, we report a case of concurrent UC and ulcerative duodenitis (UD) that displayed unique responses to biologic treatment. Although treatment with prednisolone (PSL) failed to induce remission in both disorders, golimumab (GLM) and ustekinumab (UST) were effective against UD and UC, respectively, and remission of both disorders was achieved using UST. Immunofluorescence analyses revealed that numbers of immune cells expressing TNF-α were comparable in both duodenal and rectal mucosa before the treatment. GLM or UST treatment markedly decreased numbers of TNF-α-expressing duodenal immune cells, suggesting the presence of correlation between TNF-α expression and disease activity of UD. In contrast, TNF-α expression was not parallel to disease activity of UC because GLM or PSL failed to induce remission despite a marked reduction in TNF-α expression. Responsiveness to GLM or UST together with immunofluorescence studies suggests that TNF-α and IL-12/23p40 are pathogenic cytokines causing UD and UC, respectively, in the present case.

Visualization of Gastrointestinal Bezoar Movement Causing and Releasing Small Bowel Obstruction on Computed Tomography in a Patient With Diabetes Mellitus.

Although delayed gastric emptying promotes gastrointestinal bezoar formation in patients with diabetes mellitus (DM), the association between movement of gastrointestinal bezoars and glycemic status remains unclear. We report a case of small bowel obstruction (SBO) caused by impaction of the migrated gastric bezoar into the small bowel in a patient with DM. Correction of hyperglycemia and lactic acidosis led to normalization of gastrointestinal motility, followed by expulsion of the impacted bezoar and resolution of SBO. This case suggests a link between hyperglycemia, metabolic acidosis, and gastrointestinal motility based on visualization of gastrointestinal bezoar movement in the gastrointestinal tract using computed tomography.

Ulcerative Colitis-associated Spondyloarthritis Successfully Treated with Infliximab in the Absence of Enhanced TNF-α Responses.

Although concurrent occurrence of spondyloarthritis (SpA) and ulcerative colitis (UC) is sometimes seen, the profiles of cytokines have been poorly understood in UC-associated SpA. We herein report a case of UC-associated SpA successfully treated with infliximab (IFX). Profiles of cytokines in the serum and colonic mucosa were characterized by an enhanced expression of IL-6 but not tumor necrosis factor (TNF)-α. Successful induction of remission by IFX was associated with the downregulation of IL-6 expression but no significant alteration in TNF-α expression. These findings suggest that some cases of UC-associated SpA might be driven by IL-6, and IFX might be effective in cases lacking enhanced TNF-α responses.

Successful initial tofacitinib treatment for acute severe ulcerative colitis with steroid resistance: a case series.

Background: The standard therapy for acute severe ulcerative colitis (ASUC) is intravenous corticosteroids; however, 30% of ulcerative colitis (UC) patients do not recover with corticosteroids alone. Few studies have reported the efficacy and safety of tofacitinib for ASUC with steroid resistance. We report a case series of successful first-line treatment consisting of tofacitinib (20 mg/day) administered to ASUC patients with steroid resistance. Methods: Patients diagnosed with ASUC at our institution between October 2018 and February 2020 were retrospectively evaluated. They were administered a high dose of tofacitinib (20 mg) after showing no response to steroid therapy in a dose of 1-1.5 mg/kg/day. Results: Eight patients with ASUC, 4 (50%) men, median age 47.1 (range 19-65) years, were included. Four patients were newly diagnosed, and the median UC duration was 4 (range 0-20) years. Six of the 8 patients were able to avoid colectomy. One patient (patient 2) had no response; however, remission was achieved after switching from tofacitinib to infliximab. One patient (patient 6) with no response to tofacitinib underwent total colectomy. Only one patient (patient 4) experienced an adverse event, local herpes zoster, treated with acyclovir without tofacitinib discontinuation. Conclusions: Clinical remission without serious adverse events can be achieved with high probability and colectomy can be avoided by first administering high-dose tofacitinib to steroid-resistant ASUC patients. Tofacitinib may be one of the first-line treatment options for steroid-resistant ASUC.

Multicenter phase II randomized study evaluating dose-response of antiperistaltic effect of L-menthol sprayed onto the gastric mucosa for upper gastrointestinal endoscopy.

AIM: Peppermint oil solution was found to be effective for reducing gastric spasm during upper gastrointestinal endoscopy. The aim of the present study was to assess whether the gastric peristalsis-suppressing effect is dose-dependently induced by L-menthol, the major constituent of peppermint oil, and to determine the recommended dose of an L-menthol preparation. METHODS: In this phase II, multicenter, double-blind, dose-response study, 131 eligible patients were randomly assigned to receive 20 mL of 0.4% L-menthol (n = 32), 0.8% L-menthol (n = 35), 1.6% L-menthol (n = 30), or placebo (n = 34). The primary efficacy measure was the proportion of subjects with no peristalsis in two time periods, 75 to 105 s after treatment and immediately before the completion of endoscopy. RESULTS: The peristalsis-suppressing effect of L-menthol increased dose dependently (5.6%, 32.0%, 47.4% and 52.9% in the 0%, 0.4%, 0.8% and 1.6% groups, respectively: P < 0.001, one-tailed Cochran-Armitage trend test). As compared with the placebo group, the proportion of subjects with no peristalsis after administration was significantly higher in the 0.8% group (P = 0.015) and 1.6% group (P = 0.009). Adverse events in the L-menthol dose groups occurred with similar frequencies in the placebo group. CONCLUSION: L-menthol suppresses peristalsis in a dose-dependent manner, and the dose-response reaches a plateau at 0.8% L-menthol. Further Phase III studies are needed to establish the superiority of 0.8% L-menthol over placebo.

[A case of endocrine carcinoma of the cecum treated with CDDP/VP-16].

A 41-year-old man was admitted to our hospital because of multiple liver tumors. Colonoscopy showed a mass lesion in the cecum. He was given a diagnosis of endocrine cell carcinoma by immunostaining technique, and received chemotherapy of CAPOX regimen for 3 courses. After that, he underwent second-line chemotherapy of EP(CDDP/VP-16)regimen due to deterioration of his performance status(PS), and his tumor marker NSE. He then showed dramatically improved PS, and improvement in the size of liver mets and NSE(4. 3mg/mL).