Society: AGA

Background A defective gastrointestinal epithelial tight junction (TJ) barrier has been implicated as a key pathogenic factor in inflammatory bowel diseases (IBD) and other gut inflammatory conditions. An important therapeutic target to treat IBD is to enhance or restore the intestinal TJ barrier function. We recently showed that Bifidobacterium bifidum (BB) causes a marked enhancement of the intestinal epithelial TJ barrier in a strain-specific manner. However, the role of BB (and the possible mechanism involved) in protecting against cytokine-induced increase in intestinal permeability remains unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the proinflammatory cytokines (TNF-α and IL-1β) induced increase in intestinal TJ permeability and the mechanism involved. Methods: Filter-grown Caco-2 monolayers (in vitro) and recycling intestinal perfusion of live mice (in vivo) were used to assess intestinal TJ permeability. Results: TNF-α and IL-1β caused rapid activation of NF-kB and NF-kB-dependent activation of myosin light chain kinase (MLCK) gene expression and activity in Caco-2 monolayers and mouse enterocytes in live mice. BB inhibited the TNF-α and IL-1β increase in intestinal TJ permeability in a strain-specific manner. BB preservation of the TJ barrier was preceded by increased peroxisome proliferator-activated receptor-gamma (PPAR-γ) activity, phosphorylation, and inhibition of NF-kB activation. BB also inhibited the TNF-α and IL-1β increase in MLCK expression and activity in Caco-2 monolayers and in mouse enterocytes. BB inhibition of TNF-α and IL-1β induced an increase in NF-kB activation, MLCK expression and activity, and TJ permeability was abolished by siRNA-induced knockdown of PPAR-γ in Caco-2 monolayers. The Villin-cre intestinal epithelial-specific PPAR-γ knock-out mice were generated to study the role of mouse enterocytes PPAR-γ in BB protective effect. BB did not inhibit the TNF-α and IL-1β induced NF-kB activation, increase MLCK expression and activity, or increased mouse intestinal permeability in PPAR-γ intestinal epithelial cell-deficient mice (Villin Cre). Conclusion: These studies provide a novel insight into the mechanism of BB preservation of the intestinal TJ barrier and protection against cytokine-induced increase in intestinal TJ permeability. These data show for the first time, BB (or probiotic bacteria) preserves and protects the intestinal barrier function by PPAR-γ-dependent inhibition of NF-kB activation and the subsequent MLCK gene activation and phosphorylation.

Background: Monoclonal antibodies targeting the interleukin (IL)-23p19 subunit are effective in the treatment of inflammatory bowel diseases (IBD), but have different molecular attributes that may translate to differences in clinical efficacy. Within this class, guselkumab (GUS) is a fully human IgG1 monoclonal antibody with a native Fc region, and risankizumab (RIS) is a humanized IgG1 antibody with a mutated Fc region. Binding of these therapeutic antibodies to Fcγ receptor (FcγR) I, or CD64, is of particular interest, as CD64⁺ IL-23-producing myeloid cells are increased in the inflamed colon in IBD and correlated with endoscopic disease severity (Chapuy L, et al. Mucosal Immunol. 2019; Chapuy et al. J Crohns Colitis. 2020). Here, we compared functional characteristics of the antigen-binding and Fc regions of GUS and RIS.
Methods: IL-23 binding affinity of GUS and RIS was compared in vitro using KinExA and surface plasmon resonance. In vitro cellular potency of GUS and RIS was measured by impact on IL-23-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human peripheral blood mononuclear cells. Binding of GUS and RIS to FcγRs was assessed in cells transfected with individual FcγRs. In primary human “inflammatory” monocytes differentiated with granulocyte-macrophage colony-stimulating factor and interferon-γ (IFN-γ) followed by toll-like receptor stimulation to induce IL-23 production, binding of GUS and RIS to CD64 and capture of endogenously secreted IL-23 was assessed using flow cytometry. The potential impact of GUS binding to CD64 was assessed in IFN-γ primed monocytes using a human 41-plex cytokine bead assay.
Results: GUS and RIS displayed comparable picomolar binding affinity for IL-23 and equivalent high potency for inhibition of IL-23-induced STAT3 phosphorylation. GUS showed strongest binding to CD64 compared with other FcγRs, whereas RIS had negligible binding to any FcγR. GUS, but not RIS, showed dose-dependent Fc-mediated binding to CD64 in primary human “inflammatory” monocytes. Moreover, CD64-bound GUS was able to simultaneously capture IL-23 endogenously secreted from the same cells (Figure). GUS binding to CD64 on monocytes did not induce cytokine production.
Conclusion: GUS, but not RIS, can simultaneously bind CD64⁺myeloid cells via its Fc region and neutralize IL-23 with high affinity and potency. Our in vitro data suggest a mechanistic benefit through enhanced localization of GUS within the inflamed colon, where CD64⁺ IL-23-producing myeloid cells are increased, and GUS can potently neutralize IL-23 at its source of production. These findings may contribute to differences in therapeutic profiles between antibodies.

Background: Anti-tumor necrosis factor-α (TNF-α) antibodies have markedly improved the outcome of inflammatory bowel disease (IBD); however, half of patients do not respond to the treatment. Interleukin-18 (IL-18) gene polymorphism is associated with resistance to anti-TNF-α antibodies, but therapies targeting IL-18 have not been clinically applied. IL-18 is synthesized as an inactive precursor, cleaved by caspase-1 to yield a mature protein. Only the mature IL-18 is biologically active, and mature IL-18 levels are elevated in intestinal tissue and serum of patients who do not respond to anti-TNF-α antibodies. Here, we aimed to investigate whether specific inhibition of mature IL-18 using a monoclonal antibody (mAb) against a neoepitope of caspase-cleaved mature IL-18 could be a novel therapeutic approach for IBD.
Methods: The mature form of IL-18 was produced by cleaving pro IL-18 with caspase-1, and mAbs against the mature form of IL-18 were established. Structural analysis and functional inhibition assays were performed to select antibodies with inhibitory effects. DSS-induced acute and chronic colitis model and TNBS-induced colitis model were created, and anti-mature IL-18 mAbs were administered intraperitoneally, and the disease activity index and weight loss rate were evaluated. Pathological evaluation, expression of proinflammatory cytokines, measurement of FITC-Dextran permeability, and analysis of microbiota using a next-generation sequencer were performed. In the chronic colitis model, sirius red staining, Masson trichrome staining and immunostaining of α-SMA were perfomed to assess the effect of anti-mature Il-18 mAb on fibrosis.
Results: We succeeded in producing an anti-mature IL-18 mAb that binds to IL-18 with high affinity and strongly inhibits IL-18-induced IFN-γ production. In both DSS-induced colitis model and TNBS-induced colitis model, treatment with the anti-mature Il-18 mAb resulted in improvement of intestinal inflammation, suppression of IFN-γ and TNF-α expression, reduction of FITC-Dextran permeability, improvement of decreased diversity of intestinal microbiota, increase of Bacteroidetes phylum, and decrease of Firmicutes phylum. In addition, the combination with anti-TNF-α mAb inhibited the intestinal inflammation synergistically. In DSS-induced chronic colitis model, treatment with anti-mature IL-18 mAb resulted in suppression of collagen accumulation in intestinal tissue. Conclusions: The anti-mature IL-18 mAb improved the acute and chronic intestinal inflammation, and in combination with anti-TNF-α mAb inhibited the intestinal inflammation synergistically. In addition, treatment with anti-mature Il-18 mAb suppressed intestinal fibrosis. Monoclonal antibody against neoepitope of caspase-cleaved mature Il-18 is expected to be a novel therapeutic agent for IBD.

Background: The pathogenesis of inflammatory bowel disease (IBD) is still unclear. Immune abnormality is considered as the important element. CD8⁺T cells play critical roles in IBD immunodeficiencies through devastating intestinal mucosal homeostasis and inducing inflammation. Inerleukin 36 (IL-36), as a new cytokine, has the function of activating CD8⁺T cells, enhancing the tumor-specific CD8⁺T cell immune response. However, its regulatory mechanism in IBD remain understood and need further exploration.

Methods: Clinical samples collected from UC patients and dextran sulfate sodium (DSS) induced colitis mice were used to determine the mRNA and protein expressions of IL-36α, β, γ and CD8 with RT-PCR, immunohistochemical staining and western blot respectively. Intestinal inflammation was evaluated after IL-36 receptor antagonist (IL-36Ra) treatment. The expression and proportion of CD8⁺T cells as well as its related secretory factor in gut-associated lymphoid tissue were determined by flow cytometry. Subsequently, native CD8⁺T cells from mice splenocytes were purified, and cultured in vitro with IL-36α, β, γ and IL-36Ra for 72h, in order to observe the direct effect of IL-36 ligand on the proliferation, differentiation and cytokine secretion of CD8⁺T cells in vitro.

Result: The mRNA and protein expressions of IL-36α, β, γ and CD8 were significantly increased not only in UC patients but also in mice with colitis compared with the corresponding controls. And there was a positive correlation between IL-36 mRNA and CD8 mRNA expression. Meanwhile, the proportion of CD8⁺T cells in spleen, mesenteric lymph nodes and colonic lamina propria were obvious higher in DSS mice. At the same time, CD8⁺T cells that secreting granzyme B (GZMB) and perforin (PRF1) were increased significantly. Further detection of CD8⁺T subsets secreting different cytokines showed that the proportion of Tc1 cells which secreting interferon gama (IFNγ), Tc2 cells which secreting IL-4 and Tc17 cells which secreting IL-17 were increased in spleen, lymph nodes and colonic lamina propria. Then IL-36Ra were treated for DSS induced colitis mice and found that IL-36Ra not only effectively ameliorated the inflammation, but also decreased the proportion of CD8⁺T and its pro-inflammatory cytokines in spleen, lymph nodes and colon mucosa. In vitro, IL-36α, β, γ directly stimulated native CD8⁺T cells and promoted the cell secretion of GZMB, IFNγ, IL-17 and other cell cytokines, while this effect of native CD8⁺T cells can be effectively inhibited by IL-36Ra.

Conclusions: Our results suggest that IL-36 could aggravte intestinal inflammation through regulating CD8⁺T cells homeostasis, promoting its differentiation, and stimulating CD8⁺T cells to secrete GZMB, PRF1 and pro-inflammatory cytokine such as IFNγ and IL-17.

Keywords：Inflammatory bowel disease; interleukin 36; immunity; CD8⁺ T cells

Background: Inflammatory bowel diseases (IBD) are characterized by chronic inflammation and dysbiosis. Dysbiosis in IBD involves an expansion of Proteobacteria, which contain lipopolysaccharides (LPS) in their outer membrane. Dual oxidase 2 (DUOX2) is an epithelial NADPH oxidase that prevents bacterial colonization of the mucosa through the production of hydrogen peroxide (H₂O₂) and is consistently altered in IBD patients before the onset of disease and in association with dysbiosis. We and others have shown that the LPS sensing toll-like receptor 4 (TLR4) is overexpressed in IBD inflammation. We have also established a link between TLR4 activation and increased DUOX2 expression and activity. Additionally, we have demonstrated that the overactivation of DUOX2 promotes pathology. Here, we aim to define the dynamics of DUOX2 activity in IBD inflammation and explore the effects of the microbiota and TLR4-targeted small-molecule inhibitors on DUOX2.
Methods: C57BI/6J mice raised in specific-pathogen free (SPF) and germ free (GF) conditions underwent a model of dextran sulfate sodium (DSS)-induced colitis for 6 days. Separately, SPF IL-10-KO mice, which develop spontaneous colitis, underwent a piroxicam-accelerated colitis model for 14 days. A subset of these mice had TAK242 (Resatorvid, TLR4-inhibitor in clinical development) administered daily throughout the experiment (3 mg/kg). Following euthanasia, colons were collected for histology, qPCR and H₂O₂ determinations. Wild Type (WT) colonoids were pretreated with TAK242 and stimulated with the heat-killed IBD-relevant bacteria adherent invasive Escherichia coli (AIEC). DUOX2 activity (H₂O₂) and expression were determined.
Results: SPF mice undergoing DSS-induced colitis developed overt inflammation accompanied by upregulation of Duox2 and Duoxa2 and increased production of H₂O₂. DSS-treated GF mice developed a mild inflammation causing increased H₂O₂ production and Duoxa2 upregulation. SPF IL-10-KO mice undergoing piroxicam-accelerated colitis showed overt inflammation and decreased colon lengths, upregulation of Duox2 and Duoxa2, and increased production of H₂O₂ which was abrogated upon administration of TAK-242. In-vitro, AIEC led to the induction of DUOX2 activity and expression which was abrogated upon pharmacological inhibition of TLR4 by TAK-242.
Conclusions: Our results show that both acute and chronic colitis induce DUOX2 expression and activity even in the absence of the microbiota. In addition, we show that the TLR4 antagonist TAK-242 inhibits DUOX2 activity. Our results highlight the need to screen other FDA approved therapeutics for use against DUOX2.

Nivis Brito

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