Microbiome diversity declines while distinct expansions of Th17, iNKT, and dendritic cell subpopulations emerge after...

Anastomotic failure causes morbidity and mortality even in technically correct anastomoses. Initial leaks must be prevented by mucosal reapproximation across the anastomosis. Healing is a concerted effort between intestinal epithelial cells (IECs), immune cells, and commensal bacteria. IEC TLR4 activation and signaling is required for mucosal healing, leading to inflammatory factor release that recruits immune cells to limit bacteria invasion. TLR4 absence leads to mucosal damage from loss in epithelial proliferation, attenuated inflammatory response, and bacteria translocation. We hypothesize after anastomosis, an imbalance in microbiota will occur due to a decrease in TLR4 expression and will lead to changes in the immune milieu.

We isolated fecal content and small intestinal leukocytes from murine, Roux-en-Y and end-to-end anastomoses, to identify microbiome changes and subsequent alterations in the regulatory and pro-inflammatory immune cells 3 days post-operative. TLR4⁺ IECs were impaired after anastomosis. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. A distinct TCRβ^hi CD4⁺ T cells subset after anastomosis was 10-20-fold greater than in control mice. 84% were Th17 IL-17A/F⁺ IL-22⁺ and/or TNFα⁺. iNKT cells were increased and TCRβ^hi. 75% were iNKT IL-10⁺ and 13% iNKTh17 IL-22⁺. Additionally, Treg IL-10⁺ and IL-22⁺ cells were increased. A novel dendritic cell subset was identified in anastomotic regions that was CD11b^hi CD103^mid and was 93% IL-10⁺.

This anastomotic study demonstrated a decrease in IEC TLR4 expression and microbiome diversity which then coincided with increased expansion of regulatory and pro-inflammatory immune cells and cytokines. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.