Background: Loss of immune tolerance to commensal gut bacteria plays a critical role in the pathogenesis of inflammatory bowel disease (IBD). Prior studies have demonstrated that immunoglobulin (Ig) binding to the gut microbiota is altered in IBD and associated with disease activity. Since vitamin D has been linked to IBD clinical outcomes, gut microbiota, and B cell immunity, we investigated the effects of vitamin D on mucosal Ig-targeting of the gut microbiota and B cell immunophenotypes in patients with IBD.
Methods: We performed an open-label clinical trial (NCT04828031) of 48 patients with IBD with low vitamin D (≤ 25 ng/mL) treated with oral 50,000 IU vitamin D once weekly for 12 weeks. We obtained blood and stool samples and quantified changes in calprotectin and disease activity scores before and after vitamin D. We performed single-cell RNA-seq (scRNA-seq) and immune repertoire sequencing of peripheral blood mononuclear cells (PBMCs). We assessed Ig-binding of gut bacteria and microbiome composition with flow cytometry and 16S sequencing (Ig-Seq).
Results: Vitamin D supplementation increased serum vitamin D and decreased calprotectin and disease activity scores. Vitamin D increased IgA-only (IgA⁺IgG^-) (+17.9%, p<0.0001) and decreased all IgG ( IgA^-IgG⁺ & IgA⁺IgG⁺) binding of gut bacteria (-9.3%, p<0.05). In subgroup analysis, both patients with ulcerative colitis (UC) and Crohn’s disease (CD) had increases in IgA-only binding of gut bacteria, but only patients with UC had decreases in all IgG-binding of gut bacteria. Vitamin D increased IgA-bound Lachnospiraceae and decreased IgA-bound Proteobacteria and Pseudomonadaceae. IgG-bound Clostridiales was increased whereas IgG binding of probiotic, lactic acid-producing Lactobacillaceae and Pediococcus was decreased after vitamin D. scRNA-seq analyses revealed increased abundance of peripheral B cell neighborhoods after vitamin D which were skewed towards more immature B cell subtypes that expressed α4β7. There was a trend towards increased B cell receptor repertoire clonal diversity (p=0.10) after vitamin D. Systems pathway analyses revealed increased B-cell activating factor (BAFF) signaling between dendritic cell subsets (plasmacytoid dendritic cells (pDC), AXL+SIGLEC6+ dendritic cells (ASDC), conventional dendritic cell type 2 (cDC2) and all B cell subsets after vitamin D. Vitamin D activated BAFF signaling between pDC and B memory cells (via TNFSF13B:TNFRSF13C) and pDC and plasmablasts (via TNFSF13B: TNFRSF17).
Conclusions: Vitamin D treatment of patients with IBD alters host immune-microbe interactions by increasing anti-inflammatory mucosal IgA- and decreasing colitogenic IgG- binding of gut bacteria with differential targeting of specific bacterial taxa. Vitamin D may regulate tolerogenic pathways in IBD by inducing mucosal IgA through activation of dendritic cell-B cell BAFF signaling.

<b>Figure 1</b>.<b> Effects of Vitamin D on Mucosal Immunoglobulin Binding to Gut Microbiota and Peripheral B Immunophenotypes by scRNA-seq. </b>A) Flow cytometry of bacteria from stool before and after 12 weeks of vitamin D reveals differential shifts in IgA- and IgG-binding of gut bacteria in patients with IBD B) 16S sequencing differential abundance analyses using LEfSe reveals changes to specific bacterial taxa bound by immunoglobulins (IgA-Seq and IgG-Seq) with vitamin D C) UMAP demonstrating PBMC scRNA-seq atlas cell clusters by vitamin D status D) Differential abundance analyses of scRNA-seq cellular neighborhoods with miloR reveals alterations in peripheral B cell neighborhoods according to B cell subtype and α4β7 expression. E) Chord diagram demonstrating BAFF signaling pathway network according to vitamin D status. Nodes represent cell types. Arcs/flow connections represent significant weighted signaling interactions.

Figure 1. Effects of Vitamin D on Mucosal Immunoglobulin Binding to Gut Microbiota and Peripheral B Immunophenotypes by scRNA-seq. A) Flow cytometry of bacteria from stool before and after 12 weeks of vitamin D reveals differential shifts in IgA- and IgG-binding of gut bacteria in patients with IBD B) 16S sequencing differential abundance analyses using LEfSe reveals changes to specific bacterial taxa bound by immunoglobulins (IgA-Seq and IgG-Seq) with vitamin D C) UMAP demonstrating PBMC scRNA-seq atlas cell clusters by vitamin D status D) Differential abundance analyses of scRNA-seq cellular neighborhoods with miloR reveals alterations in peripheral B cell neighborhoods according to B cell subtype and α4β7 expression. E) Chord diagram demonstrating BAFF signaling pathway network according to vitamin D status. Nodes represent cell types. Arcs/flow connections represent significant weighted signaling interactions.

Justin Sonnenburg

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