Broad-Spectrum Antibiotics Deplete Bone Marrow Regulatory T Cells

Abstract

Bone marrow suppression, including neutropenia, is a major adverse effect of prolonged antibiotic use that impairs the clinical care and outcomes of patients with serious infections. The mechanisms underlying antibiotic-mediated bone marrow suppression remain poorly understood, with initial evidence indicating that depletion of the intestinal microbiota is an important factor. Based on our earlier studies of blood and bone marrow changes in a mouse model of prolonged antibiotic administration, we studied whether changes in megakaryocytes or regulatory T cells (Tregs), two cell types that are critical in the maintenance of hematopoietic stem cells, contribute to antibiotic-mediated bone marrow suppression. Despite increased platelet numbers, megakaryocytes were unchanged in the bone marrow of antibiotic-treated mice; however, Tregs were found to be significantly depleted. Exogenous addition of Tregs was insufficient to rescue the function of bone marrow from antibiotic-treated mice in both colony formation and transplantation assays. These findings indicate that the intestinal microbiota support normal Treg development to protect healthy hematopoiesis, but that the restoration of Tregs alone is insufficient to restore normal bone marrow function.

Keywords: T regulatory cells; antibiotics; bone marrow suppression; hematopoietic stem cell.

Figure 1

Antibiotic administration alters peripheral blood and bone marrow cell composition. (A) Antibiotic treatment experiment design. (B) White blood cell counts (WBC), (C) hemoglobin, (D) platelet counts from peripheral blood using automated hematologic cell counter and compared between control vs. antibiotic-treated (antibiotics). (E) Whole bone marrow (WBM) cells comparing control vs. antibiotics were counted using automated cell counter. (F,G) Neutrophil population from WBM for control vs. antibiotics are shown using flow cytometry. (H) CD4:CD8 ratio in WBM for control vs. antibiotics are shown using flow cytometry. Results are compiled from 3 independent experiments (n = 5–8 per experiment). Graphs show mean + SEM. Statistical significance was determined by Mann–Whitney U test. ns, not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 2

Antibiotic administration does not change megakaryocyte progenitors (MkP) but leads to decrease in regulatory T cells (Treg) in bone marrow. (A) MkP population from whole bone marrow (WBM) for control vs antibiotic-treated (antibiotics) are shown using flow cytometry. MkP (solid arrows) size, shape and number from bone marrow are compared between (B) control and (C) antibiotics (hematoxylin and eosin staining of bone marrow from femur, 10×). (D) Flow cytometry plot of Treg population comparing control and antibiotics-treated group. (E) Treg population from WBM of female mice for control vs antibiotics. (F) Number of Treg per tibia for male and female control vs antibiotics-treated mice are shown. Results are compiled from 2–3 independent experiments (n = 5–8 per experiment). Graphs show mean + SEM. Graphs show mean + SEM. Statistical significance was determined by Mann-Whitney U test. ns, not significant, * p < 0.05, ** p < 0.01.

Figure 3

Addition of Treg (CD25+ cells) to antibiotic-treated mice whole bone marrow (WBM) improve cell count in vitro and engraftment in vivo. (A) Methocult culture experiment design (B) Colonies from WBM cells were counted in six replicates on day 5 for each group. Results are compiled from 1 independent experiment, n = 2–3. (C) Bone marrow transplant (BMT) experiment design using Treg-depleted recipient mice (D) Engraftment at 12 weeks after transplant analyzed from WBC from donor cells using flow cytometry. (E) Treg population from WBM at 16 weeks after transplant using flow cytometry. (F) CD4:CD8 ratio from WBM at 16 weeks after transplant. (G) B cell contribution from donor-derived WBC at 12 weeks after transplant. N = 3–5. Graph show mean + SEM. No statistical differences by Kruskal-Wallace.