*Purpose: We previously demonstrated that gut microbiome alterations, through fecal microbiota transfer (FMT) or single bacteria transfer, had specific immune effects for cardiac allograft outcome. The present study aimed to characterize immune stimulatory or suppressive effects of gut bacteria on cells and structures of the host immune system.

*Methods: Antibiotic-pretreated mice received vascularized cardiac grafts and tacrolimus. Mice were gavaged with Bifidobacterium pseudolongum (Bifido), a dominant gut member in immuno-suppressed hosts, or Desulfovibrio desulfuricans (Desulfo), abundant in colitis. Longitudinal characterization of the mouse gut microbiota was performed using 16S rRNA gene sequencing. The effect of shifts in microbiota on immune responses was assessed with immunohistochemistry and flow cytometry of intestinal segments and lymph nodes (LN). Bone marrow derived dendritic cells (BMDC), peritoneal macrophages (MΦ), and B cells were stimulated with bacteria or isolated Bifido exopolysaccharides (EPS), and cytokine responses measured by ELISA and activation markers by flow cytometry.

*Results: Bifido decreased graft inflammation and fibrosis and prolonged allograft survival. Desulfo resulted in poor histology and reduced graft survival. Bifido significantly (p=0.016) increased LN Foxp3⁺ CD4 regulatory T cells (Treg) and decreased (p=0.002) LN and spleen activated CD44^hiCD69⁺ CD4 effector T cells (Teff) compared to Desulfo. Intestinal segments and mesenteric LN, but not peripheral LN, showed increases in Foxp3⁺ Treg cells, F4/80⁺ MΦ and CD11c⁺ DC early after Bifido treatment but not Desulfo treatment. These bacteria also profoundly altered the structure of the LN stromal laminin fibers to create suppressive or inflammatory niches, with increased laminin α4:α5 ratios in the Bifido group and decreased ratios in the Desulfo group. Stimulation of myeloid cells with EPS did not affect expression of CD40, CD80, CD86, or MHC II, but prevented lipopolysaccharide (LPS) induced TNFα and IL-6 expression by BMDC. Stimulation of BMDC with Bifido cells or EPS induced the expression of the anti-inflammatory cytokines IL-10 and CCL19, but induced lesser amounts of the pro-inflammatory cytokines TNFα and IL-6. Microbiota characterization using 16S RNA gene sequencing and Principal Coordinate Analysis of Jensen-Shannon divergence showed gut microbiome profiles clustered by study group over time.

*Conclusions: These results demonstrate immunomodulatory properties of gut microbiota, selected bacterial strains, and specific bacterial surface structural component. They have specific effects on myeloid cells and LN stromal fibers in different immune compartments. Characterization of the microbiota in organ transplantation provides novel insights into functional pathways involved in modulating inflammation and immunity.

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