Microbiome profile between non-surgical, Roux-en-Y, and end-to-end anastomosis intestinal regions

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 impede TLR4 expression and impair crosstalk with the immune milieu. We isolated small intestinal leukocytes and fecal content from murine, Roux-en-Y and end-to-end anastomoses, to identify regulatory and pro-inflammatory immune cells and microbiome changes 3 days post-operative. TLR4+ IECs were impaired after anastomosis. A distinct TCRbhi 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 TNFa+. iNKT cells were increased and TCRbhi. 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 CD11bhi CD103mid and was 93% IL-10+. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. This anastomotic study demonstrated an increased expansion of regulatory and pro-inflammatory immune cells and cytokines that coincided with decreased IEC TLR4+ expression and microbiome diversity. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.