Murine gut microbiota dysbiosis via enteric infection modulates the foreign body response to a distal biomaterial implant

Abstract

The gut microbiota influences systemic immunity and the function of distal tissues, including the brain, liver, skin, lung, and muscle. However, the role of the gut microbiota in the foreign body response and fibrosis is largely unexplored. To investigate this connection, we perturbed the homeostasis of the murine gut microbiota via infection with the pathogenic bacterial species enterotoxigenic Bacteroides fragilis (ETBF) and implanted particulate material (mean particle size <600 μm) of the synthetic polymer polycaprolactone (PCL) into a distal muscle injury. ETBF infection in mice led to increased neutrophil and γδ T cell infiltration into the PCL implant site. ETBF infection alone promoted systemic inflammation, increased levels of neutrophils in lymphoid tissues, and altered skeletal muscle gene expression. At the PCL implant site, we found significant changes in the transcriptome of sorted stromal cells between infected and control mice, including differences related to ECM components such as proteoglycans and glycosaminoglycans. However, we did not observe ETBF-induced differences in fibrosis levels. These results demonstrate the ability of the gut microbiota to mediate long-distance effects such as immune and stromal responses to a distal biomaterial implant.

Keywords: biomaterials; foreign body response; gut microbiota; immune response; infection.

Fig. 1.

Colonic ETBF infection increases inflammation and immune cell infiltration into PCL implant. (A) Experimental timeline. Abx = antibiotics (clindamycin and streptomycin) (see Materials and Methods for details). (B) H&E image of colon at the Day 0 time point (1-wk post gavage of DPBS or ETBF). (Scale bar, 100 μm.) (C) Stool detection of B. fragilis toxin (bft) and B. fragilis 16S at the 6-wk harvest time point. Copy number calculated through standard curve. n.d. = not detected. (D) Quadricep muscle with PCL implant expression of inflammatory genes at 6-wk post VML+PCL in mice gavaged with ETBF, normalized to the control condition. Rer1 used as reference housekeeper gene. (E) Quadricep muscle with PCL implant expression of Ly6g at 6-wk post VML+PCL in mice gavaged with ETBF, normalized to the control condition. Rer1 used as reference housekeeper gene. (F) Calculated neutrophil (CD11b⁺F4/80⁻Ly6G⁺Ly6C⁺) counts in quadricep muscle with PCL implant at 6-wk post VML+PCL in mice gavaged with DPBS or ETBF. (G) Immunofluorescence staining of DAPI (blue, nuclei), citrullinated histone H3 (red, NETs), and Ly6G (green, neutrophils) in PCL implant site of ETBF-infected mouse at 6-wk post VML+PCL. White arrows point to Ly6G⁺CitH3⁺ cells. (Scale bar, 10 μm.) (H) Calculated γδ T cell counts in quadricep muscle with PCL implant at 6-wk post VML+PCL in mice gavaged with DPBS or ETBF. Graphs show geometric mean ± geometric SD (C) or mean ± SD (D–F and H). N = 4 to 5 (B, D–F, and H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons (D) or two-tailed unpaired t test (E, F, and H).

Fig. 2.

ETBF infection impacts systemic immune and nonimmune tissues. (A) Experimental timeline. (B) Spleen expression of inflammatory genes at 1-wk post ETBF, normalized to the control condition. B2m used as reference housekeeper gene. (C) Neutrophil (CD11b⁺F4/80⁻Ly6G⁺Ly6C⁺) percentage of CD45⁺ cells in blood at 1-wk post gavage. (D) Neutrophil (CD11b⁺F4/80⁻Ly6G⁺Ly6C⁺) percentage of CD45⁺ cells in the spleen at 1-wk post gavage. (E) Neutrophil (CD11b⁺F4/80⁻Ly6G⁺Ly6C⁺) percentage of CD45⁺ cells in femur bone marrow at 1-wk post gavage. (F) Quadricep muscle expression of muscle and ECM-related genes at 1-wk post ETBF, normalized to the control condition. Rer1 used as reference housekeeping gene. Graphs show mean ± SD (B–F). N = 5 (B and F). N = 4 to 6 (C). N = 3 to 5 (D). N = 7 to 9 (E). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons (B and F) or two-tailed unpaired t test (C–E).

Fig. 3.

ETBF infection alters the transcriptional profile of fibroblasts at the PCL implant site. (A) Picrosirius Red images of PCL implant site at 6-wk post VML+PCL in mice gavaged with DPBS or ETBF. (Scale bar, 100 μm.) Quantification of mean red-green value on the CIELAB axis shown on Right. (B) Volcano plot of differential gene expression results of bulk RNA sequencing of sorted stromal cells (CD45⁻CD31⁻CD29⁺) from PCL implant site at 6-wk post VML+PCL. (C) Based on volcano plot in B, manual categorization of significant (P_adj < 0.05) differentially expressed genes of sorted stromal cells (CD45⁻CD31⁻CD29⁺) from PCL implant site at 6-wk post VML+PCL. (D) Normalized enrichment scores and adjusted P values of selected Reactome pathways from ranked gene set enrichment analysis. (E) Immunohistochemistry images of aggrecan in PCL implant site at 6-wk post VML+PCL in mice gavaged with DPBS or ETBF. Graphs show mean ± SD (A). N = 5 (A). Biological n = 4 (B–D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by the two-tailed unpaired t test (A). NES normalized to size of gene set (D).