Infection-induced myelopoiesis during intracellular bacterial infection is critically dependent upon IFN-γ signaling

Abstract

Although microbial infections can alter steady-state hematopoiesis, the mechanisms that drive such changes are not well understood. We addressed a role for IFN-γ signaling in infection-induced bone marrow suppression and anemia in a murine model of human monocytic ehrlichiosis, an emerging tick-borne disease. Within the bone marrow of Ehrlichia muris-infected C57BL/6 mice, we observed a reduction in myeloid progenitor cells, as defined both phenotypically and functionally. Infected mice exhibited a concomitant increase in developing myeloid cells within the bone marrow, an increase in the frequency of circulating monocytes, and an increase in splenic myeloid cells. The infection-induced changes in progenitor cell phenotype were critically dependent on IFN-γ, but not IFN-α, signaling. In mice deficient in the IFN-γ signaling pathway, we observed an increase in myeloid progenitor cells and CDllb(lo)Gr1(lo) promyelocytic cells within the bone marrow, as well as reduced frequencies of mature granulocytes and monocytes. Furthermore, E. muris-infected IFN-γR-deficient mice did not exhibit anemia or an increase in circulating monocytes, and they succumbed to infection. Gene transcription studies revealed that IFN-γR-deficient CDllb(lo)Gr1(lo) promyelocytes from E. muris-infected mice exhibited significantly reduced expression of irf-1 and irf-8, both key transcription factors that regulate the differentiation of granulocytes and monocytes. Finally, using mixed bone marrow chimeric mice, we show that IFN-γ-dependent infection-induced myelopoiesis occurs via the direct effect of the cytokine on developing myeloid cells. We propose that, in addition to its many other known roles, IFN-γ acts to control infection by directly promoting the differentiation of myeloid cells that contribute to host defense.

FIGURE 1

E. muris infection causes a loss of CMPs in the bone marrow. A, Lin⁻ IL-7Rα⁺ and IL-7Rα⁻ bone marrow cells from mock-infected or E. muris-infected mice (left panels) were analyzed for c-Kit and Sca-1 expression. The regions in the right panels demarcate the CLPs (Lin⁻IL-7Rα⁺c-Kit^lo Sca-1⁺), CMPs (Lin⁻IL-7Rα⁻c-Kit^hi Sca-1⁻), and the infection-induced Lin⁻IL-7Rα⁻c-Kit^hiSca-1⁺ progenitor cells. B, The Lin⁻IL-7Rα⁻ cells were analyzed for expression of c-Kit and Sca-1 in C57BL/6, IFN-αR–deficient, and IFN-γR–deficient mice. C, The frequencies of the CLPs, CMPs, and Lin⁻IL-7Rα⁻c-Kit^hiSca-1⁺ progenitor cells in the bone marrow were determined in C57BL/6, IFN-αR–deficient, and IFN-γR–deficient mice. The error bars represent the means and SD. D, The number of Sca-1⁻ and Sca-1⁺ cells within the Lin⁻IL-7Rα⁻c-Kit^hi population is indicated for each strain of mouse. The lineage markers used were the following: CD3, B220, CDllb, Gr-1, and Ter119. The error bars represent the averages and SEM. The bone marrow was harvested from both femurs. The data are representative of at least three separate experiments, where each experiment included at least three mice per group. A Student t test was used to evaluate statistically significant differences between the indicated groups, and significant differences are indicated as follows: *p < 0.05; **p < 0.001. dpi, day postinfection; n.s., not significant.

FIGURE 2

Classically defined myeloid progenitor cells are diminished and infection-induced myeloid progenitors dominate the bone marrow compartment during E. muris infection. A, Classically defined myeloid progenitor cells were identified in the Lin⁻IL7Rα⁻c-Kit^hiSca-1⁻ bone marrow cells by examining the expression of CD34 and FcγRII/III in C57BL/6 (top panels) and IFN-γR–deficient (bottom panels) mice. MEPs were identified as CD34⁻FcγRII/III⁻, CMPs were identified as CD34^loFcγRII/III⁻, and GMPs were CD34⁺FcγRII/III⁺, as indicated in the text. B, The total number of each population was determined in mock-infected and E. muris-infected C57BL/6 and IFN-γR-deficient mice. C, Infection-induced myeloid progenitor cell subsets (identified within the Sca-1–expressing population of Lin⁻IL-7Rα⁻c-Kit^hi cells) were identified based on the pattern of CD34 and FcγRII/III expression used above in C57BL/6 (top panels) and IFN-γR–deficient (bottom panels) mock and E. muris-infected mice. D, The numbers of cells within each population were determined in the bone marrow from the femurs. The error bars indicate the averages and SEM. *p < 0.05.

FIGURE 3

IFN-γ–dependent reduction of in vitro colony formation and alterations in peripheral blood cell subsets. A, Bone marrow cells were harvested from C57BL/6 and IFN-γR–deficient mice and were analyzed by in vitro colony-forming assay. Total colonies derived from granulocyte-macrophage (CFU granulocyte, macrophage) and multipotential (CFU granulocyte, erythrocyte, macrophage, megakaryocyte) progenitor cells were enumerated on day 7 of culture. B–D, Peripheral blood was analyzed at the indicated times postinfection for hemoglobin concentration (B), platelet count (C), and the frequencies of leukocyte subsets (D).

FIGURE 4

Infection-induced granulopoiesis is altered in mice incapable of IFN-γ signaling. A, Bone marrow harvested from C57BL/6 or IFN-γR–deficient mice was analyzed for expression of CDllb and Gr-1. B, The numbers of CDllb^loGr-1^lo, CDllb^loGr-1^hi, and CDllb^hiGr-1^hi cells identified in A are indicated. C and D, Splenocytes from the same mice were analyzed as in A, and the numbers of each population are indicated. E, Spleen cell preparations from mock and day 15 E. muris-infected mice were generated by touch impression and were analyzed by Giemsa staining. Neutrophils in the IFN-γR–deficient (bottom panels), but not in C57BL/6 mice (top panels), contained ehrlichia morulae (arrows). None of the neutrophils in the C57BL/6 mice were infected, but 24.7% of the IFN-γR–deficient neutrophils contained morulae (5 of 19, 3 of 17, and 10 of 33 cells enumerated contained morulae in the IFN-γ–deficient mice; three individual fields were counted from three separate mice; original magnification ×100).

FIGURE 5

IFN-γ acts on hematopoietic progenitor cells during infection. C57BL/6 mice (CD45.2) and IFN-γR–deficient mice (CD45.2) were lethally irradiated and were reconstituted with wild-type congenic (CD45.1) bone marrow. Eight weeks postreconstitution, mice were either mock-infected or were infected with E. muris. Bone marrow was analyzed on day 8 postinfection for expression of c-Kit and Sca-1 on Lin⁻ donor cells.

FIGURE 6

Infection-induced myelopoiesis requires IFN-γ signaling. A, A schematic of the experimental strategy and the gating and analysis approach used in the study are shown. Donor bone marrow-derived cells (all CD45.2) were identified, and Lin⁻IL-7Rα⁻ cells were further analyzed for GFP expression to identify the wild-type and IFN-γR–deficient donor cells. B, Bacterial infection was evaluated by quantitative PCR in spleen tissue from E. muris-infected C57BL/6, IFN-γR–deficient, and bone marrow chimeric mice. C, Lin⁻IL-7Rα⁻ cells were analyzed for expression of c-Kit and Sca-1 (left panels), and GFP expression was analyzed in the c-Kit^hiSca-1⁻ and c-Kit^hiSca-1⁺ cells (right panels). D, The frequency of C57BL/6 (filled bars) or IFN-γR–deficient (open bars) donor cells within each population is shown. E, Bone marrow was harvested from mock and infected chimeric mice, and the immature and mature granulocyte populations were identified on the basis of expression of CDllb and Gr-1. F, The frequencies of C57BL/6 and IFN-γR–deficient donor-derived cells in bone marrow were determined for each of the indicated granulocyte populations. G and H, Spleens were harvested from mock and infected chimeric mice, and the granulocyte populations and the frequencies of C57BL/6 and IFN-γR–deficient donor-derived cells were determined as described for the bone marrow. The data are from one experiment that is representative of two experiments of similar design.

FIGURE 7

IFN-γ induces the transcription of myeloid-specific genes during infection. RNA was isolated from bone marrow CD11b^loGr-1^lo cells purified from mock-infected or E. muris-infected C57BL/6 or IFN-γR–deficient mice on day 15 postinfection, and mRNA expression was analyzed. The data indicate gene expression in infected cells, relative to cells from mock-infected C57BL/6 mice (shaded histograms) or IFN-γR–deficient mice (open histogram). The relative expression of irf1–irf8 (A) and other genes known to be regulated by IFN-γ (B) are shown. *p < 0.05; **p < 0.001 (for C57BL/6 mice); ^#p < 0.05; ^##p < 0.001 (for IFN-γR–deficient mice).