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.

James Krueger

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