620 - The Weaning Reaction is Disrupted in Mice Prone to Spontaneous Ileocolitis

Poster Number: 620
Publication Number: 620.200

Grace M. Callan, Medical College of Wisconsin, Wauwatosa, WI, United States; Calvin B. Williams, Medical College of Wisconsin, Milwaukee, WI, United States; Ryan Besserud, Medical College of Wisconsin, Milwaukee, WI, United States

Background: Appropriate colonization of the newborn GI tract is essential for proper immune development and long-term health. Bacterial blooms drive a strong immune response termed the “weaning reaction,” characterized by spikes in the pro-inflammatory cytokines TNF-α and IFN-γ, and by the induction of Foxp3+ regulatory T (Treg) cells that express the anti-inflammatory cytokine TGF-β1. Proper development of mucosal Treg cells prevents susceptibility to disease, including allergies and autoimmune diseases. Previously, we found that adult mice with spontaneous ileocolitis had decreased intestinal Treg cell development, dysbiosis, and dysregulated immune responses.

Objective: Determine whether newborn mice prone to spontaneous ileocolitis have a disrupted weaning reaction marked by alterations in the expression of the genes encoding TNF-α, IFN-γ, and TGF-β1 relative to healthy controls.

Design/Methods: Tissue from the distal small intestine of newborn mice was harvested at weekly intervals and used to extract mRNA, which was then reverse transcribed to cDNA and quantified using qPCR. Expression of Tnfa, Ifng, and Tgfb1 was normalized to the expression of Gapdh, Hprt, and Hsp90. Fold changes in expression were calculated relative to the cytokine levels observed in one week old mice. Relative cytokine levels were then plotted and compared between mice with spontaneous ileocolitis and two control strains. To assess downstream developmental consequences, CD4+ T cells sorted from mesenteric lymph nodes were analyzed by flow cytometry and single cell RNAseq at 100 days.

Results: In our experimental model, Tnfa expression peaked at 5 weeks, one week later than either control strain, declined, and then rose progressively as spontaneous ileocolitis developed. Ifng levels followed a similar trend, while Tgfb1 expression decreased and was similar across all 3 strains. At 100 days, the CD4+ T cell compartment reflected a pro-inflammatory predisposition with increased expression of Rorc (Th17 cells) and reduced expression of Foxp3.Conclusion(s): Mice prone to develop spontaneous ileocolitis had a disrupted weaning reaction marked by a delay in peak Tnfa and Ifng expression and progressively increased expression of these cytokines as the mice aged. Tissue Tgfb1 levels did not correlate with disease susceptibility. These data link delayed increases in the expression of Tnfa and Ifng during weaning with disease susceptibility, while also suggesting that tissue Tgfb1 levels are not adequate to assess the privileged role of Treg cell Tgfb1 in tolerance established during the weaning reaction.