Altered macrophage population and phenotype in mesenteric artery adventitia during inflammatory bowel disease pathogenesis

Abstract

Objective: Vascular dysfunction associated with Inflammatory Bowel Disease (IBD) is poorly understood. Preliminary studies show increased macrophage marker expression and accumulation in mesenteric artery (MA) adventitia at a single timepoint with established IBD. Depletion of these macrophages results in improved vasodilation via adventitial sensory nerves. We hypothesize that increased macrophage infiltration and polarization to an M1-like phenotype occurs early in IBD pathogenesis before the development of severe colitis. Thus, we aimed to quantify macrophage accumulation and polarization in MAs during IBD pathogenesis. Methods: Male and female IL10-/- mice were gavaged with H. hepaticus at weaning and developed IBD for 1, 5, 10, 20, 40, 80, 160, and 240 days. Age-matched C57BL/6 mice served as controls, and non-gavaged Day 0 mice were included in each group. The mesenteric arteries were dissected, fixed, and immunolabeled for total macrophages (MT, F4/80), M1-like macrophages (M1, CD68), and M2-like macrophages (M2, CD206) and then confocally imaged. Fluorescence area and M2:M1 ratios were quantified in Image J from max z-projections through one wall of each MA. Differences within timepoints were analyzed by nested one-way ANOVA, and differences across timepoints were compared with two-way ANOVA. Results: There was a main effect of both disease and time for MA MTs (F4/80+, P<0.05). In Controls, MTs varied by day without a clear trend. In contrast, MTs from IBD mice steadily increased from Days 0-20, then decreased to a plateau from Days 40-160, followed by an increase of MT at day 240. By Day 240, MTs of IBD surpassed MTs of Controls (P=0.005). MA M1s (CD68+) showed opposite trends. In Controls, there was a steady increase in M1s from Days 0-240. In contrast, IBD M1s varied by day with no clear trend but surpassed M1s of Controls by Day 240 (P=0.015), which could account for the increased MTs seen in IBD by Day 240. Like MA MTs, there was a main effect of both disease and time for MA M1s (CD68+, P<0.05). In contrast, there was a main effect of timepoint, but not disease for M2s (CD206+). In Controls, M2s increased until Day 40, followed by plateau through Day 240. IBD M2s increased until Day 20 and then decreased steadily to a plateau by Day 160. The M2:M1 ratio in Controls showed a biphasic response, decreasing until Day 40, then increasing until Day 240. The M2:M1 ratio in IBD started high at Day 0, then steadily decreased to a plateau by Day 240. Conclusions: Overall, preliminary and current data suggest that increased macrophage content, particularly M1s, in the MA adventitia may contribute to vascular dysfunction in IBD. Additional studies are needed, as the current quantitation method does not consider changes in inflammatory mediator release by the macrophages or the responses of vascular cells to those mediators. However, perivascular macrophages represent a promising target to improve mesenteric blood flow. Funding: T34 GM 136493 to KRR, R01HL157038, R00HL129196 to EMB. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.