Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infection

Abstract

Lymphocytes and neutrophils are rapidly depleted by systemic infection. Progenitor cells of the haematopoietic system, such as common myeloid progenitors and common lymphoid progenitors, increase the production of immune cells to restore and maintain homeostasis during chronic infection, but the contribution of haematopoietic stem cells (HSCs) to this process is largely unknown. Here we show, using an in vivo mouse model of Mycobacterium avium infection, that an increased proportion of long-term repopulating HSCs proliferate during M. avium infection, and that this response requires interferon-gamma (IFN-gamma) but not interferon-alpha (IFN-alpha) signalling. Thus, the haematopoietic response to chronic bacterial infection involves the activation not only of intermediate blood progenitors but of long-term repopulating HSCs as well. IFN-gamma is sufficient to promote long-term repopulating HSC proliferation in vivo; furthermore, HSCs from IFN-gamma-deficient mice have a lower proliferative rate, indicating that baseline IFN-gamma tone regulates HSC activity. These findings implicate IFN-gamma both as a regulator of HSCs during homeostasis and under conditions of infectious stress. Our studies contribute to a deeper understanding of haematological responses in patients with chronic infections such as HIV/AIDS or tuberculosis.

Figure 1

Infection with Mycobacterium avium induces changes in hematopoietic stem cells. (A) Absolute numbers of short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and long-term HSCs (KSL, Flk2−, CD34−) were determined after infection with M. avium. n=3-7. (B) LT-HSCs (side population, lineage-negative, Sca-1+, c-Kit+ (SP^KLS)) were not significantly changed after infection with M. avium. Plot is representative of three independent experiments, each with n=3-5. (C) BrdU incorporation in SP^KLS cells was determined at baseline and after infection. Data represent two independent experiments, each with n=2-5. (D) Engraftment efficiency was determined after transplantation of 500 SP^KLS from WT or M. avium-infected WT mice into lethally irradiated WT recipients. Data represent two independent experiments, each with n=2 to 6. (E) The percentage of LT-HSCs (KSL CD150+) in the spleen was determined at baseline and 4 weeks after M. avium infection. n=4 or 5.

Figure 2

The HSC response to M. avium infection is dependent upon intact IFNγ signaling. (A) IFNγ levels in bone marrow supernatant were quantified by cytokine bead array at baseline and 4 weeks postinfection with M. avium. n=3-6. (B) BrdU incorporation by HSCs (KSL CD150+) of naïve and M. avium-infected WT, Ifngr1-deficient, Stat1-deficient, and Ifnar1-deficient mice was quantified. n=3-6. (C) Absolute number of HSCs (KSL CD150+) in the whole bone marrow of naïve and infected WT, Ifngr1-deficient, Stat1-deficient, and Ifnar1-deficient mice was determined. n=3-7.

Figure 3

IFNγ is sufficient to induce HSC proliferation in vitro. (A) Relative quantities of Ifngr1 mRNA were determined in pooled samples of nucleated erythrocyte progenitors (“Eryth”), B-cells, T-cells, granulocytes (“Gran”), and LT-HSCs (SP^KLS). n=2-3 independent samples per cell type. (B) Irgm1 mRNA was quantified by real-time PCR in LT-HSCs (SP^KLS) after IFNγ treatment. (C) IRGM1 protein levels on LT-HSCs (SP^KLS) were determined by immunofluorescence. (D) BrdU incorporation by primitive hematopoietic cells (KSL CD150+) was assessed after 12-hour in vitro treatment with PBS or IFNγ. Data represent two independent experiments each performed in triplicate.

Figure 4

IFNγ is sufficient to induce HSC proliferation in vivo. (A) BrdU incorporation was measured in LT-HSCs (SP^KLS) of WT mice 24 hours after injection with IFNγ or PBS and 12 hours after injection with BrdU. n=5. (B) Whole bone marrow was isolated from PBS or IFNγ-injected WT mice 24 hours postinjection, and transplanted into lethally irradiated WT recipients in competitive transplant assays. Engraftment efficiency was determined 4, 8, 12, and 16 weeks after transplantation. n=5-6. (C) The percentage of primitive hematopoietic cells (KSL CD150+) in the spleen was determined 24 hours after injection with either PBS or IFNγ. n=3.

Figure 5

Basal IFNγ tone affects HSC cycling and function. (A) BrdU incorporation by LT-HSCs (SP^KLS) of WT and Ifng-deficient mice over 3 days was determined. n=4 or 5. (B) Engraftment efficiency was determined 4, 8, 12, and 16 weeks after transplantation of whole bone marrow from WT or Ifng-deficient mice into lethally irradiated WT recipients in either noncompetitive or competitive transplant assays. n=5.