Immune Responses to Broad-Spectrum Antibiotic Treatment and Fecal Microbiota Transplantation in Mice

Abstract

Compelling evidence demonstrates the pivotal role of the commensal intestinal microbiota in host physiology and the detrimental effects of its perturbations following antibiotic treatment. Aim of this study was to investigate the impact of antibiotics induced depletion and subsequent restoration of the intestinal microbiota composition on the murine mucosal and systemic immunity. To address this, conventional C57BL/6j mice were subjected to broad-spectrum antibiotic treatment for 8 weeks. Restoration of the intestinal microbiota by peroral fecal microbiota transplantation (FMT) led to reestablishment of small intestinal CD4⁺, CD8⁺, and B220⁺ as well as of colonic CD4⁺ cell numbers as early as 7 days post-FMT. However, at d28 following FMT, colonic CD4⁺ and B220⁺ cell numbers were comparable to those in secondary abiotic (ABx) mice. Remarkably, CD8⁺ cell numbers were reduced in the colon upon antibiotic treatment, and FMT was not sufficient to restore this immune cell subset. Furthermore, absence of gut microbial stimuli resulted in decreased percentages of memory/effector T cells, regulatory T cells, and activated dendritic cells in the small intestine, colon, mesenteric lymph nodes (MLN), and spleen. Concurrent antibiotic treatment caused decreased cytokine production (IFN-γ, IL-17, IL-22, and IL-10) of CD4⁺ cells in respective compartments. These effects were, however, completely restored upon FMT. In summary, broad-spectrum antibiotic treatment resulted in profound local (i.e., small and large intestinal), peripheral (i.e., MLN), and systemic (i.e., splenic) changes in the immune cell repertoire that could, at least in part, be restored upon FMT. Further studies need to unravel the distinct molecular mechanisms underlying microbiota-driven changes in immune homeostasis subsequently providing novel therapeutic or even preventive approaches in human immunopathologies.

Keywords: antibiotics; bacterial recolonization; fecal microbiota transplantation; innate and adaptive immunity; microbiota; mucosal and systemic immune responses; secondary abiotic (gnotobiotic) mice.

Figure 1

Intestinal microbiota composition of conventional and secondary abiotic mice as compared to autoclaved food pellets. The intestinal microbiota composition was analyzed in fecal samples derived from conventionally colonized (SPF) mice and mice subjected to an 8 weeks course of broad-spectrum antibiotic treatment [thereby generating secondary abiotic (ABx) mice] by quantitative real-time PCR amplifying variable regions of the bacterial 16S rRNA gene and compared to the bacterial composition detected in sterilized (autoclaved) food pellets. The following main intestinal bacterial groups were determined (expressed as 16S rRNA gene numbers per nanogram DNA): enterobacteria (EB), enterococci (EC), lactic acid bacteria (LB), bifidobacteria (BIF), Bacteroides/Prevotella spp. (BP), Clostridium coccoides group (CLOCC), and Clostridium leptum group (CLEP). Numbers of samples harboring the respective bacterial group out of the total number of analyzed samples are given in parentheses.

Figure 2

Apoptotic and proliferating cells in small and large intestinal epithelia of secondary abiotic and microbiota-reconstituted mice. The average numbers of (A) apoptotic (positive for caspase-3, Casp3) and (B) proliferating cells (positive for Ki67) in at least six representative high power fields (HPFs, 400× magnification) per animal were determined in immunohistochemically stained small intestinal and colonic ex vivo biopsies derived from naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and abiotic mice reconstituted with murine microbiota at day (d) 7 (bars with vertical lines) and d28 (bars with horizontal lines) following fecal microbiota transplantation (FMT).

Figure 3

Ileal and colonic immune cell populations in secondary abiotic and microbiota-reconstituted mice. The average numbers of T lymphocytes [positive for CD3 (A,E)], B lymphocytes [positive for B220 (B,F)], regulatory T cells [Treg, positive for Foxp3 (C,G)], and macrophages/monocytes [positive for F4/80 (D,H)] from at least six representative high power fields (HPFs, 400× magnification) per animal were determined in ex vivo biopsies taken from the small intestine [upper panel (A–D)] and colon [lower panel (E–H)] of conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and with microbiota-reconstituted abiotic mice at day (d) 7 (bars with vertical lines) or d28 (bars with horizontal lines) following fecal microbiota transplantation (FMT).

Figure 4

CD4⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages [left panel (A,C,E,G)] and cell numbers [right panel (B,D,F,H)] of the CD4⁺ lymphocyte population within the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice on day (d) 7 (bars with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 4

CD4⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages [left panel (A,C,E,G)] and cell numbers [right panel (B,D,F,H)] of the CD4⁺ lymphocyte population within the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice on day (d) 7 (bars with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 5

CD8⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages [left panel (A,C,E,G)] and cell numbers [right panel (B,D,F,H)] of the CD8⁺ lymphocyte population within the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 5

CD8⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages [left panel (A,C,E,G)] and cell numbers [right panel (B,D,F,H)] of the CD8⁺ lymphocyte population within the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 6

Memory/effector T cell compartment in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The proportions of CD4⁺ memory/effector cells [CD4⁺CD44^hi, gated on CD4⁺ cells, left panel (A,C,E,G)] and CD8⁺ memory/effector cells [CD8⁺CD44^hi, gated on CD8⁺ cells, right panel (B,D,F,H)] within the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 6

Memory/effector T cell compartment in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The proportions of CD4⁺ memory/effector cells [CD4⁺CD44^hi, gated on CD4⁺ cells, left panel (A,C,E,G)] and CD8⁺ memory/effector cells [CD8⁺CD44^hi, gated on CD8⁺ cells, right panel (B,D,F,H)] within the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 7

IFN-γ-producing CD4⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages of IFN-γ-producing CD4⁺ cells in the (A) small intestine, (B) colon, (C) mesenteric lymph nodes (MLN), and (D) spleen of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 8

IL-17- and IL-22-producing CD4⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages of IL-17- [left panel (A,C,E,G)] and IL-22- [right panel (B,D,F,H)] producing CD4⁺ cells in the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 8

IL-17- and IL-22-producing CD4⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages of IL-17- [left panel (A,C,E,G)] and IL-22- [right panel (B,D,F,H)] producing CD4⁺ cells in the small intestine (A,B), colon (C,D), mesenteric lymph nodes (MLN) (E,F), and spleen (G,H) of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.

Figure 9

IL-10-producing CD4⁺ cells in intestinal and systemic compartments of secondary abiotic and microbiota-reconstituted mice. The percentages of IL-10-producing CD4⁺ cells in the (A) small intestine, (B) colon, (C) mesenteric lymph nodes (MLN), and (D) spleen of naive conventional mice (SPF, gray bars), secondary abiotic mice (ABx, white bars), and recolonized mice at day (d) 7 (boxes with vertical lines) and d28 (bars with horizontal lines) post-fecal microbiota transplantation (FMT) are depicted.