Colonization with Altered Schaedler Flora Impacts Leukocyte Adhesion in Mesenteric Ischemia-Reperfusion Injury

Abstract

The microbiota impacts mesenteric ischemia-reperfusion injury, aggravating the interaction of leukocytes with endothelial cells in mesenteric venules. The role of defined gut microbiomes in this life-threatening pathology is unknown. To investigate how a defined model microbiome affects the adhesion of leukocytes in mesenteric ischemia-reperfusion, we took advantage of gnotobiotic isolator technology and transferred altered Schaedler flora (ASF) from C3H/HeNTac to germ-free C57BL/6J mice. We were able to detect all eight bacterial taxa of ASF in fecal samples of colonized C57BL/6J mice by PCR. Applying qRT-PCR for quantification of species-specific 16S rDNA sequences of ASF bacteria, we found a major shift in the abundance of ASF 500, which was greater in C57BL/6J mice relative to the C3H/HeNTac founder breeding pair. Using high-speed epifluorescence intravital microscopy to visualize the venules of the small bowel mesentery, we found that gnotobiotic ASF-colonized mice showed reduced leukocyte adherence, both pre- and post-ischemia. Relative to germ-free mice, the counts of adhering leukocytes were increased pre-ischemia but did not significantly increase in ASF-colonized mice in the post-ischemic state. Collectively, our results suggest a protective role of the minimal microbial consortium ASF in mesenteric ischemia-reperfusion injury.

Keywords: C–C motif chemokine ligand 5; acute mesenteric infarction; altered Schaedler flora; germ-free; leukocytes; mesenteric ischemia-reperfusion injury; microbiota; thrombosis.

Figure 1

Transfer of ASF from C3H/HeNTac mice onto C57BL/6J mice. (A). PCR based detection of ASF bacteria, performed with published primer pairs [50]. All ASF-specific bacteria were detected. (B). qRT-PCR based detection of ASF bacteria, performed with published primer pairs [50]. The graph compares the founder breeding pair (C3H/HeNTac; aged 20 weeks; pink, N = 2) with the C3H/HeNTac ASF offspring (aged 4 weeks; grey, N = 4) and the newly colonized C57BL6J mice (aged 17–19 weeks; green, N = 2). (C). Three male and female GF C57BL/6J mice aged 13–16 weeks were colonized with fecal contents harvested from 10 week old male and female C3H/HeNTac ASF mice. Fecal samples were collected at various time points from each individual mouse (h=hour; d=day). qRT-PCR results for ASF 360, ASF 361, ASF 457, and ASF 492 at different time points during colonization are shown for male and female C57BL/6J mice. Data are expressed as means ± SEM.

Figure 2

Leukocyte tethering to ischemia-reperfusion-injured mesenteric venules of gnotobiotic ASF-colonized mice. (A). Comparative in vivo analysis of adherent leukocytes in the mesenteric venules of CONV-R C57BL/6J (N = 12 mice per group) vs. ASF-colonized C57BL/6J mice (N = 9 mice per group), pre- and post-ischemia-reperfusion injury. Representative images are shown. Adhering leukocytes were stained with acridine orange (green). Scale bar: 100 µm. (B). Comparative in vivo analysis in the mesenteric venules of GF C57BL/6J (N = 5 mice per group) vs. ASF-colonized C57BL/6J mice (N = 9 mice per group), pre- and post-ischemia-reperfusion injury. Adhering leukocytes were stained with acridine orange (green). Scale bar: 50 µm. All data are expressed as means ± SEM. Statistical comparisons were performed using Student’s t-test (* p < 0.05, ** p < 0.01) or Mann–Whitney test (# p < 0.05).

Figure 3

Expression of CCL5 (RANTES) in isolated small intestinal epithelial cells and in the mid small intestine of gnotobiotic ASF-colonized mice relative to GF controls. (A). ELISA quantification of CCL5 protein levels in primary intestinal epithelial cell lysates from GF, ASF-colonized, and CONV-R C57BL/6J mice (N = 8 mice per group). (B). qRT-PCR quantification of small intestinal CCL5 mRNA levels in GF relative to ASF-colonized C57BL/6J mice (N = 7 mice per group). All data are expressed as means ± SEM. Statistical comparisons were performed using one-way ANOVA (* p < 0.05, ** p < 0.01) or Student’s t-test (** p < 0.01).