Reactive granulopoiesis depends on T-cell production of IL-17A and neutropenia-associated alteration of gut microbiota

Abstract

Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1^-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.

Keywords: Ruminococcaceae; granulopoiesis; hematopoietic stem cell transplantation; microbiota; neutropenia.

Fig. 1.

IL-17A plays a critical role in reactive granulopoiesis in prolonged neutropenia. (A–C) Lethally irradiated B6 mice (CD45.2⁺) were transplanted with 7.5 × 10³ LSKs plus 2 × 10⁴ GMPs from B6-CD45.1⁺ donors. Absolute numbers of neutrophils in the peripheral blood (A) and plasma concentrations of IL-17A (B) and G-CSF (C) on day +18 after SCT (n = 15) and those in naive B6 mice (n = 6). (D) Naive or wild-type (WT) recipients were injected with BrdU (1 mg) on day + 18 after SCT and BM cells were harvested 2 h later (n: naive = 8, WT = 13). (E–L) Lethally irradiated IL-17A-deficient B6 mice (IL17A^−/−) and control B6-WT mice were transplanted from WT B6-Ly5a donors as Fig. 1A. Plasma concentrations of IL-17A (E) and G-CSF (F) on day +18 (n = 18/group). Absolute numbers of neutrophils (G) and T cells (H), hemoglobin concentrations (I), and absolute numbers of platelets (J) in the peripheral blood on day +18 after SCT (n = 10/group). (K) Phagocytic capacity of neutrophils was assessed by incubating peripheral blood neutrophils with pHrodo red-labeled Escherichia coli for 15 min at 37°C or on ice (n = 9–10/group). (L) ROS production was evaluated after DHR123 labeling followed by stimulation with PMA (n = 9–10/group). B6-WT (M-O) or B6-IL17A^−/− (P) mice were i.p. injected with 5-FU (200 mg/kg) or PBS on day 0. Absolute numbers of neutrophils in the peripheral blood (M, P) and plasma concentrations of IL-17A (N) and G-CSF (O) on day 14 (n = 12/group). Data from two (G–P), three (A–D), or four (E, F) similar experiments were combined and shown as mean ± SE. (G, I, J, and P) Gray bars on the left side of the figures represent reference ranges determined using data from naive mice (mean ± 2SD, n = 5) *P < 0.05, **P < 0.01, ***P < 0.005.

Fig. 2.

IL-17A plays a critical role in BM granulopoiesis and BM egress of neutrophils after SCT. Lethally irradiated B6-IL17A^−/− and B6-WT mice were transplanted as Fig. 1A. NCC (A), LSK (B), CMP (C), GMP (D), and MEP (E) in the BM of the bilateral femurs were enumerated on day +18 after SCT. Data from five similar experiments were combined and shown as mean ± SE (n = 22/group). (F) WT or IL17A^−/− recipients were injected with BrdU (1 mg) on day +18 after SCT and BM cells were harvested 2 h later. Data from three similar experiments were combined and shown as mean ± SE (n = 13–14/group). (G–I) Fractions 1–5 within granulopoietic precursors gated as SI Appendix, Fig. S1E were enumerated in the femur 18 d after SCT. Representative dot plots (G), proportion (H), and absolute numbers (I) of each fraction. Data from four similar experiments were combined and shown as mean ± SE (n = 17/group). (J) WT (n = 5) or IL17A^−/− (n = 3) recipient mice were i.p. injected with 1-mg BrdU on day +18 after SCT. Proportion of BrdU⁺ neutrophils in the peripheral blood is shown as mean ± SE. Data from one of two experiments are shown. *P < 0.05, *** P < 0.005.

Fig. 3.

T cell production of IL-17A plays a critical role in reactive granulopoiesis after SCT. SCT was performed as in Fig. 1A. (A) Splenic T cells were purified from SCT recipients on day +18 after SCT (n = 7–9/group). mRNA extracted from T cells was subjected to Q-PCR targeting IL-17A. (B–D) B6-WT (n = 9) or B6-RAG1^−/− (n = 6) were transplanted as in Fig. 1A. Plasma concentration of IL-17A and G-CSF, and numbers of neutrophils in the peripheral blood on day +18. (E–I) RAG1^−/− recipients were injected with 6 × 10⁶ T cells from WT (n = 10–17) or IL17A^−/− (n = 9–12) mice on day 0 of SCT or left untreated (n = 3–11). Numbers of T cells in the blood (E), plasma concentrations of IL-17A (F) and G-CSF (G), numbers of and GMPs in the bilateral femoral BM (H), proportion of fraction #1 to #4 (I) and fraction #5 (J) in the BM, numbers of neutrophils in the blood (K) on day +18. (L, M) IL17A^−/− recipients were injected with 6 × 10⁶ T cells from WT B6 mice on day 0 of SCT or left untreated (n = 7/group). Numbers of and GMPs in the bilateral femoral BM (J), and neutrophils in the blood (K) on day +18. Data from two (A–D,J, and K) and three (E–I) experiments were combined and shown as mean ± SE. *P < 0.05, **P < 0.01, and ***P < 0.005.

Fig. 4.

The microbiota is critical in reactive granulopoiesis after SCT and chemotherapy. (A–I) Recipient mice were orally treated with three antibiotics (3ABx) including ABPC, SM, and VCM, or control diluent (Ctrl) from day −7 and transplanted as in Fig. 1A. (A) Fecal bacterial load on day 0 was measured using Q-PCR targeting 16S rRNA gene (n = 4/group). Numbers of neutrophils (B, C) and T cells (D) in the blood, and CMP (E) and GMP (F) in the right femoral BM on day +18 after SCT (n = 13/group). (G) Il17a expression in splenic T cells purified from naive B6, or Ctrl- or 3Abx-treated SCT recipients on day +18 (n = 5–7/group). Plasma concentrations of IL-17 (H) and G-CSF (I) from naive B6 (n = 6), or Ctrl- (n = 25) or 3Abx-treated (n = 19) recipients on day +18. (J–L) B6 mice treated with 3ABx or Ctrl for 7 d were i.p. injected with 5-FU (200 mg/kg) on day 0. Plasma concentrations of IL-17A (J) and G-CSF (K), and numbers of neutrophils in the blood (L) on days +7, +10, and +14 after 5-FU treatment (n = 12/group). Data from two (B–G, J–L) or three (H, I) similar experiments were combined and shown as mean ± SE. *P < 0.05, **P < 0.01, and ***P < 0.005.

Fig. 5.

Prolonged neutropenia after SCT and 5-FU alters composition of intestinal microbiota. (A–C) SCT was performed as in Fig. 1A. 16S rRNA sequencing of fecal samples was performed on day +10. (A) Principal coordinate analysis of genus compositions of intestinal microbiota from one of three similar experiments (n = 4–6/group). Bacterial composition at phylum level (B, n = 15/group), and the genera significantly increased or decreased in SCT recipients compared with naive controls with relative abundance ≥ 0.1% in any groups (C, n = 15/group). (D,E) Lethally irradiated B6 mice were transplanted with 5 × 10⁶ BM cells from syngeneic donors. Numbers of neutrophils in the blood on day +7 (n = 3–5/group) (D) and principal coordinate analysis of genus compositions of intestinal microbiota (E) on day +10 after BMT. (F, G) B6 mice were i.p. injected with 5-FU (200 mg/kg) on day 0, and 16S rRNA sequencing were performed on day +10 (n = 4–6/group). Venn diagrams of bacterial genus, the relative abundance of which was significantly increased (F) or decreased (G) in SCT recipients (blue), 5-FU-treated mice (yellow), or BMT recipients (green) compared with naive mice. Fold change of relative abundance of Ruminococcaceae UCG-014 in 5-FU-treated mice (H) and BMT recipients (I) compared with naive controls. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.

Fig. 6.

Intestinal microbiota induced by prolonged neutropenia promotes reactive granulopoiesis after SCT. Lethally irradiated B6 mice were transplanted as in Fig. 1A. Fecal suspension was prepared using feces obtained from SCT recipients on day +10 after SCT. Another group of B6 mice were treated with 3ABx and transplanted as Fig. 4A. 3ABx was lifted on day +13 and FMT was performed on day +14 using fecal suspension from SCT recipients or naive mice. (A) Experimental scheme of FMT. (B, C) 16S rRNA sequencing of fecal samples was performed on day +20 after SCT (n = 3–5/group). Principal coordinate analysis of genus compositions of intestinal microbiota (B) and fold change of relative abundance of Ruminococcaceae (C). (D) Plasma concentrations of IL-17A measured on 4 d after FMT from naive mice or SCT recipients (n = 8/group). (E) GMPs in the femur enumerated 4 d after FMT (n = 15/group). (F) Absolute numbers of neutrophils in the peripheral blood enumerated 1–6 d after FMT (n = 8–9/group). Data from two (D, F) or three (E) experiments were combined and shown as mean ± SE *P < 0.05, **P < 0.01, and ***P < 0.005.