In this study, we demonstrate that an intracellular bacterial infection causes major changes in the differentiation of hematopoietic progenitor cells via direct IFN-γ signaling. We conclude that while homeostatic production of monocytes and granulocytes is not dependent upon IFN-γ signaling, infection elicits the production of both populations of myeloid cells, and that this infection-induced process is critically dependent on IFN-γ signaling. These findings reveal a proposed, but largely unrecognized, role for IFN-γ in the direct modulation of hematopoiesis during bacterial infection. Because many pathogens induce IFN-γ production, these findings have implications for understanding host defense against a wide range of microbial infections. This conclusion is supported by a report that IFN-γ induces the expansion and proliferation potential of Lin−
). Moreover, studies of hematopoiesis during malaria infection revealed the emergence of a unique population of Lin−
progenitor cells that was found to require IFN-γ signaling (20
). We propose that these observations together provide a paradigm whereby inflammatory cytokines, such as IFN-γ, act to modulate the early differentiation of hematopoietic progenitor cells, thereby enhancing the production of the mature blood cells required to combat infection.
Although our findings have demonstrated an important role for IFN-γ in vivo, they contrast with earlier studies that described a negative effect of IFN-γ on hematopoiesis. In those studies, IFN-γ inhibited in vitro colony formation of human granulocyte-macrophage progenitor cells (14
), and even low levels of IFN-γ inhibited hematopoiesis in long-term bone marrow cultures (13
). We observed that the frequency of in vitro bone marrow colony-forming cells decreased during infection in wild-type mice. Although this finding would appear to be consistent with the earlier studies that demonstrated hematopoietic suppression, we propose instead that in vitro colony formation potential decreases because IFN-γ promotes hematopoietic progenitor cell differentiation, thus depleting the bone marrow of progenitor cells. In our studies, the classically defined myeloid progenitor cells were diminished postinfection in wild-type mice but not in IFN-γR–deficient mice, and in the wild-type mice, the frequency of Lin−
progenitors increased. Thus, one possible explanation is that the Sca-1−
cells have a greater ability to form colonies in vitro, whereas the infection-induced Lin−
population requires additional signals not provided in the standard in vitro culture conditions. Furthermore, in our studies of hematopoiesis in mixed bone marrow chimeric mice, we observed that infection caused a loss of wild-type cells within the Lin−
populations; in these mice the progenitor cells were mostly derived from IFN-γR–deficient donor cells. We think that this finding is consistent with our interpretation that the wild-type cells differentiated in response to infection-induced inflammation. An alternative explanation is that these cells were mobilized and emigrated from the bone marrow. We also observed high frequencies of wild-type–derived granulocytes within the bone marrow and spleen of infected mice, suggesting that the wild-type cells that were capable of IFN-γ signaling proliferated and differentiated. Under these same conditions, differentiating promyelocytes that could not signal via IFN-γ accumulated in the bone marrow and in the spleen. Thus, we propose that IFN-γ promotes, not suppresses, hematopoietic differentiation in vivo.
Our studies reveal an important role for IFN-γ in directing the development of both granulocytes and monocytes during infection. Although our previous studies of the bone marrow granulocyte population prompted our investigation of these cells, we noted alterations to the monocyte population as well. The expansion of blood monocytes observed in C57BL/6 mice was reduced in IFN-γR–deficient mice, suggesting that IFN-γ is required for the production of infection-induced monocytes. IFN-γR–deficient mice also exhibited pronounced neutrophilia postinfection, as compared with C57BL/6 mice, suggesting that granulopoiesis does not require IFN-γ signaling. However, more careful analysis of the infection-induced granulocytes in the IFN-γR–deficient mice revealed that this population of cells was defective, as these cells were infected with E. muris. Additionally, wild-type cells that expressed the IFN-γR dominated the population of mature granulocytes in the chimeric mice. Thus, our data support a requirement for IFN-γ–dependent signaling in the terminal differentiation of both granulocytes and monocytes during ehrlichial infection.
These studies also highlight a critical role for IFN-γ in driving the transcriptional regulation of hematopoiesis during infection. IFN-γ mediates a range of signaling events within target cells. As our knowledge of IFN-γ signaling has come primarily from studies of macrophages and other differentiated cell types in vitro, how such signals are regulated in hematopoietic progenitor cells is unknown. CDllblo
cells from infected IFN-γR–deficient mice expressed reduced amounts of irf-1
mRNA, relative to cells from wild-type mice. IRF-1 and IRF-8 (also known as IFN consensus sequence binding protein) are both known to play critical roles in hematopoiesis (33
). IRF-1 deficiency results in increased numbers of immature granulocytic precursors (33
), an observation that is similar to what we observed in infected IFN-γR–deficient mice. IRF-8 is required for normal monocyte and dendritic cell development (34
), which is consistent with our observation that fewer blood monocytes, but increased neutrophils, were detected postinfection in the IFN-γR–deficient mice. IRF-8 deficiency also results in myeloid hyperplasia (36
) and in increased numbers of myeloid-derived suppressor cells (37
); additionally, human myeloid malignancies show much lower than normal IRF-8 expression (38
). IRF-8 also regulates the proliferation-inducing effects of GM-CSF by inducing the expression of neurofibromin 1
, a tumor suppressor (39
). IRF-8 is also required for host defense: IRF-8–deficient mice are highly sensitive to Mycobacterium tuberculosis
and Plasmodium chabaudi
). IRF-8 acts to promote phagocyte development and maturation by inducing the expression of the phagocyte oxidase proteins gp91phox
). Our observation that a high frequency of granulocytes contained morulae in the spleens of IFN-γR–deficient mice is consistent with the role of IRF-8 in promoting phagocyte development during infection. Thus, the induction of irf-8
expression during ehrlichia infection likely plays an important role in regulating myelopoiesis by controlling proliferation, differentiation, and oxidative activity of phagocytes, and it is likely to be directly responsible for the defects in myelopoiesis in our infection model. Additionally, our studies show that IFN-γ–mediated signaling is critical for regulating irf-8
gene expression during infection.
IFN-γ–driven hematopoiesis can be viewed as a favorable response to infection, due to the production of pathogen-killing leukocytes. However, several studies have suggested that IFN signaling in HSCs may also have a negative effect. In this study, we have reported direct action of IFN-γ on myeloid progenitor cells, but it is likely that IFN-γ also acts on HSCs during ehrlichial infection, as several IFN-γ–inducible genes are vital to HSC maintenance (17
). The immunity-related GTPase Irgm1 (also known as Lrg-47) was induced in HSCs during infection and was required for the maintenance of the HSC pool (17
). The IFN-γ–inducible gene Adar1
was also shown to be critical for HSC maintenance: loss of ADAR1 in hematopoietic cells led to rapid apoptosis and the induction of IFN-responsive genes (18
). These studies provide strong evidence that IFN-γ acts directly on HSCs, and it was recently shown that IFN-γ signaling leads to HSC exhaustion during chronic mycobacterial infection (31
). This conclusion is consistent with the observation that HSCs lacking IRF-2, a transcriptional repressor of type I IFN signaling, undergo exhaustion when chronically stimulated with IFN-α (44
). Thus, although IFN-γ signaling may be an important driving force in responding to infection, this factor may also deplete stores of stem and progenitor cells within the hematopoietic compartment.
Although it is not clear how Sca-1 functions in progenitor cells, it is thought that Sca-1 can regulate different signaling pathways by modulating lipid raft composition, and it has been shown that overexpression of Sca-1 can inhibit myeloid differentiation (45
). This observation is inconsistent with our hypothesis that IFN-γ drives myelopoiesis during infection. We propose that other genes, expressed during infection, modify the effects of Sca-1 signaling in vivo, as it is well known that IFN-γ signaling is context-dependent. Indeed, the observation that Lin−
cells can be induced to proliferate in response to IFN-γ (30
) suggests that the outcome of IFN-γ signaling may differ in less differentiated progenitor cells. In support of this idea, it has been shown that Sca-1 is required for IFN-α–driven HSC proliferation (19
Several reports have also revealed that TLR signaling is critical for HSC and progenitor cell differentiation (1
), indicating that some pathogens interact directly with stem and progenitor cells. For example, monopoiesis was impaired in Listeria monocytogenes
-infected MyD88-deficient mice (48
). We observed that MyD88-deficient mice exhibited a phenotype similar to that of E. muris
-infected IFN-γR–deficient mice with respect to the reduced Sca-1 upregulation on bone marrow progenitor cells (K. C. MacNamara and G. M. Winslow, unpublished observations). However, E. muris
is not known to encode canonical TLR ligands (i.e., LPS and peptidoglycan), and we failed to detect any differences in hematopoiesis in infected mice deficient in TLR2, TLR4, or TLR9 (K. C. MacNamara and G. M. Winslow, unpublished observations). The TLR-associated signaling molecule MyD88 forms a physical association with the IFN-γR, and MyD88 can stabilize IFN-γ–induced gene transcripts (49
). Thus, we propose that an alternative TLR-independent role for MyD88 in infection-induced hematopoiesis is via the IFN-γ pathway or in IL-1β or IL-18 signaling.
The role for IFN-γ specifically during periods of stress and infection is highlighted by the fact that under steady-state conditions the bone marrow, blood, and spleen have nearly identical frequencies of leukocytes in both IFN-γR–deficient mice and C57BL/6 mice. However, infection of IFN-γR–deficient mice resulted in enhanced frequencies and numbers of myeloid progenitor cells, as compared with C57BL/6 mice. In one model of why myelopoiesis is enhanced in the absence of IFN-γ, it was proposed that IFN-γ acts to suppress the innate immune response by acting on myeloid progenitor cells (16
). This model is in conflict with our finding that in mice containing both wild-type and IFN-γR–deficient cells, most mature neutrophils were found to be of wild-type origin. Our data support the notion that IFN-γ induced during infection is required for the production of mature neutrophils. However, an alternative possibility is that IFN-γ acts to increase the lifespan of mature neutrophils, thus favoring the accumulation of wild-type cells in the spleen. We propose a model whereby IFN-γ acts directly on progenitor cells and immature cells, thereby accelerating their movement out of the pool of earlier progenitor cells. This would explain why the pool of Lin−
cells is composed of IFN-γR–deficient cells in the infected chimeric mice: in the absence of IFN-γ signaling they fail to differentiate and, thus, dominate this pool.
Our data suggest that the hematological alterations observed during ehrlichiosis are due to the direct effects of inflammation on hematopoietic stem and progenitor cells. We propose that anemia and thrombocytopenia, which are hallmarks of ehrlichiosis, occur at the expense of promoting the production of granulocytes. The observation that the onset of these hematological abnormalities is delayed in IFN-γR–deficient mice, where we also see a reduced loss of classically defined myeloid progenitor cells, supports our conclusion that anemia and thrombocytopenia are a direct result of altered functional capacity of myeloid progenitor cells. The cost of producing more granulocytes may therefore be the transient loss of hemoglobin and platelet production; this outcome may be favorable to the host, as wild-type mice survive infection and IFN-γR–deficient mice are unable to control the infection.
Most infectious diseases cause inflammation and induce the expression of IFN-γ, so similar alterations in hematopoiesis likely occur in many infections. A better understanding of how infections alter hematopoiesis and progenitor cell function and capacity is critical not only for our overall understanding of immunity, but for our general knowledge of how hematopoiesis is modulated in the face of inflammation and immunologic stress.