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.

Hiroki Kawashima

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