Successful proliferation and differentiation of hematopoietic progenitor cells in bone marrow (BM) is essential to generate all mature blood cell types, including those involved in the immune response. Although vaccinia virus (VV) is known to induce a strong immune response, the effect of VV infection on hematopoiesis remains largely unknown. Here, we showed that in vivo VV infection results in the expansion of c-KithiSca-1+Lin− (KSL) hematopoietic stem cells. The in vivo expansion of the KSL population requires MyD88 that is a critical adaptor for Toll-like receptor-mediated signaling. Moreover, in BM of VV-infected mice, common myeloid progenitors (CMP) was decreased because of the rapid differentiation of CMP to more mature cells. However, the CMP compartment was not affected by VV infection in the absence of MyD88. The common lymphoid progenitor (CLP) cell population was increased regardless of MyD88 status, suggesting the independent regulation of CMP and CLP compartments by VV infection. VV infection also enhanced the potential of progenitors that preferentially induce the programming of dendritic cell (DC) development toward plasmacytoid DC. Therefore, the host immune response is gearing toward antiviral responses as early as at the precursor level upon VV infection.
Disclosure of potential conflicts of interest is found at the end of this article.
All cells in blood originate from pluripotent hematopoietic stem cells (HSC) in bone marrow (BM). HSC undergo a series of cell divisions and develop into lymphoid- or myeloid-committed progenitors . Lymphoid-restricted progenitors called common lymphoid progenitors (CLPs) give rise to T, B, and natural killer (NK) cells . Myeloid-committed progenitors include common myeloid progenitors (CMPs), granulocyte-monocyte progenitors, and megakaryocyte-erythrocyte progenitors . HSC and progenitor cells can be defined phenotypically by combinations of cell surface markers [2, 4, 5]. Hematopoiesis is a dynamic process and is modulated by environmental factors, including virus infection. For example, cytomegalovirus causes immune complications by affecting the differentiation of myeloid-committed progenitors , and changes in these progenitors can be modulated by NKT cells . Also, lympholytic choriomeningitis virus (LCMV) impairs development of dendritic cells (DC) .
Vaccinia virus (VV) belongs to the Poxviridae family of DNA viruses and has been successfully used as an attenuated vaccine to eradicate human smallpox [9, 10]. However, VV infection also alters host immune response, and vaccination has been associated with significant complications, particularly in immunocompromised individuals . VV inhibits DC functions and blocks cytokine signals by expressing decoy receptors for interleukin (IL)-1β, tumor necrosis factor-α, and interferon (IFN) [12, 13]. VV-infected antigen-presenting cells (APC) are impaired in their antigen presentation function [14, 15]. However, the effect of VV infection on hematopoiesis remains unknown. Here, we show that VV infection results in the expansion of phenotypically defined hematopoietic stem cells in a MyD88-dependent manner. This was associated with a drastic decrease in CMP and increased numbers of more restricted myeloid precursors and differentiated myeloid cells. Lastly, VV infection preferentially directed DC differentiation toward the plasmacytoid, not myeloid, DC lineage.
Materials and Methods
Mice and Reagents
C57BL/6 and MyD88−/− mice were used at 6–10 weeks of age. C57BL/6 mice were purchased from Harlan (Indianapolis, http://www.harlan.com). MyD88−/− mice were maintained in the laboratory animal resource facility at the Indiana University School of Medicine.
Granulocyte macrophage-colony-stimulating factor (GM-CSF) was purchased from R&D Systems (Minneapolis, http://www.rndsystems.com), and Flt3-L was purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com). Methocult media and lineage depletion biotin selection kit were purchased from StemCell Technologies (Vancouver, BC, Canada, http://www.stemcell.com). To exclude dead cells during flow cytometry analysis, LIVE/DEAD Fixable Violet Dead Cell Stain Kit from Invitrogen (Carlsbad, CA, http://www.invitrogen.com) was used. The following antibodies for flow cytometry were purchased from eBioscience Inc. (San Diego, http://www.ebioscience.com): fluorescein isothiocyanate-conjugated anti-CD34 (RAM34), phycoerythrin (PE)-conjugated anti-IL7Rα (A7R34), PE-conjugated anti-Flt3 (A2F10.1), PE-Cy7-conjugated anti-c-kit (2B8), allophycocyanin-conjugated anti-Sca-1(D7), biotinylated anti-CD11b (M1/70), biotinylated anti-TER 119 (TER-119), biotinylated anti-Gr-1 (RB6–8C5), biotinylated anti-CD11c (p150/90), and biotinylated anti-B220 (RA3–6B2). APC-Cy7-conjugated streptavidin was purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml). Flow cytometry data were acquired by LSR II (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com), and data were analyzed using the CellQuest program (Becton Dickinson).
Vaccinia Virus Preparation and Infection
The Western Reserve strain of VV was propagated and titrated by using the TK cell line. Virus was harvested from infected cells by three cycles of freezing/thawing, sonication, and clarification by centrifugation. This VV preparation was further purified on a 36% sucrose gradient by ultracentrifugation. C57BL/6 and MyD88−/− mice were infected with 1 × 106 plaque-forming unit virus particles by intraperitoneal injection. Animals were sacrificed at the indicated times. BM cells were harvested from femur and tibia by repeated flushing with Hanks' balanced saline solution containing 2% bovine serum albumin.
Lineage Depletion and Analysis
Bone marrow cells were treated with FcR blocker (StemCell Technologies) and enriched for lineage-negative cells by incubation with anti-CD11b, anti-Ly6G, anti-TCR, anti-CD45R, and anti-TER119, followed by negative selection with a biotin selection system (StemCell Technologies). Lineage-negative cells were stained with Sca-1, c-kit, CD34, IL-7Rα, and Flt3 for detection of phenotypically defined hematopoietic stem and progenitor cells. Cells were then fixed with 1% paraformaldehyde and analyzed.
Lineage-depleted BM cells were harvested at indicated time points; stained with a cocktail of antibodies of c-Kit, Sca-1, and CD34 (all from eBioscience); fixed with paraformaldehyde; and then stained by the TW2.3 antibody recognizing the VV E3L protein . To detect the viral genome, the same cells were used to prepare RNA to do polymerase chain reaction (PCR). Primer sequences for E3L have been published .
Ex Vivo Culture
Lineage-negative BM cells were harvested from infected mice at 24 hours after infection and cultured with Stem Cells Pro medium supplemented with nutrients (Invitrogen) for 2 days. At the end of culture, cells were washed twice with phosphate-buffered saline (PBS); stained with Sca-1, c-Kit, and CD34 antibodies; and analyzed by flow cytometry. Dead cells were excluded by LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen).
In Vitro c-kithiSca-1+Lin− Culture
Feeder layers were prepared from total BM of C57BL/6 mice. BM cells were plated at 2× 106 cells per milliliter and expanded for 2 weeks. Cells were fed weekly with α-minimal essential medium supplemented with 2 mM l-glutamine, 0.16 mM i-Inositol, 16 μM folic acid, and 10−6 M hydrocortisone (StemCell Technologies). At the end of culture, feeder cells were inactivated by irradiating with 1,500 cGy from a gamma radiation source. Fluorescence-activated cell sorting-sorted c-kithiSca-1+Lin− (KSL) cells were plated on irradiated feeder layers in StemPro serum-free medium (Invitrogen), supplemented with 100 ng/ml lipopolysaccharide (LPS) or 10 μg/ml peptidoglycan, and incubated under hypoxic 5% O2/5% CO2 humidified conditions.
Cell Cycle Analysis
Cell cycle status of KSL cells was determined as previously described , with a minor modification. Briefly, lineage-negative BM cells were stained with antibodies recognizing surface molecules Sca-1 and c-Kit and then fixed with 4% paraformaldehyde on ice for 30 minutes. After being washed with PBS twice, cells were treated with 500 U/ml RNase A (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 37°C for 30 minutes. Cells were washed with PBS and stained with 40 μg/ml propidium iodide (Sigma). Data were acquired on a LSR II flow cytometer and analyzed with Cell Quest software (Becton Dickinson).
Dendritic Cell Generation from Bone Marrow Progenitor Cells
For generation of DC from progenitor cells, bone marrow cells were harvested, and red blood cells were depleted by lysis. Single-cell suspensions were seeded at 1 × 106 cells per milliliter in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin and streptomycin, and either GM-CSF (10 ng/ml) or Flt3-L (100 ng/ml). GM-CSF cultured cells were harvested at day 5, and Flt3-L cultured cells were harvested at day 12. Every 3 days, medium was replenished with fresh medium. At the end of each experiment, viable cell recovery was determined by trypan blue staining. To determine DC differentiation from BM cells, cells were stained with a mixture of antibodies recognizing CD11c, NK1.1, CD3, CD11b, and B220. CD11c+NK1.1−CD3− cells were considered DC.
The two-tailed Student's t test was used to calculate statistical significance. p values less than .05 were considered statistically significant.
VV Infection Induces the Expansion of the Lin−Sca-1+c-Kit+ HSC Population in the Bone Marrow
Both in vivo infection and in vitro infection by VV are known to modulate the host immune response. Because BM is the organ that generates precursors for all hematopoietic cells, we investigated the effect of VV infection on the hematopoietic precursor population in BM. We infected C57/BL6 mice with VV and examined BM cells at different days postinfection. To enrich HSC and multipotent progenitor cells (MPP), we first eliminated lineage+ cells that were differentiated cells, as described in Materials and Methods. Lineage-depleted cells were then used for further analysis. Cells from mock-infected mice showed a typical profile of KSL cells that comprise a minor population of HSC and MPP (Fig. 1A). However, upon VV infection, both the proportion and the number of KSL cells were dramatically increased as early as 1 day after infection. The KSL population returned to close to normal at day 7 postinfection (Fig. 1A). A similar increase was observed in Sca-1+c-Kit− cells in VV-infected mice, whereas the Sca-1−c-Kit+ cell population was decreased dramatically during the early period of infection (Fig. 1A).
Lack of Flt3 expression is used to distinguish the HSC population (Flt3− KSL), which possesses extensive and sustained long- and short-term self-renewal potential, from the MPP population (Flt3+ KSL), which has the ability to efficiently reconstitute lymphoid but not myeloid compartment . When we examined KSL cells using Flt3 and CD34 markers to compare HSC and MPP, Flt3− but not Flt3+ KSL cells were primarily expanded in VV-infected BM (Fig. 1B, 1C).
The alteration in the KSL population can be explained by at least two possibilities: cell intrinsic or extrinsic factors. It is possible that VV infects hematopoietic cells and that infected cells proliferate. Alternatively, the expansion of KSL could be caused by an indirect effect, such as environmental changes. To distinguish these two possibilities, we first investigated whether Lin− cells are infected by VV or not. We prepared RNA from Lin− BM cells from mock- and VV-infected mice and performed PCR to detect E3L, which is the early gene product of VV. As shown in Figure 1D, VV-infected BM cells expressed E3L, suggesting that VV infected Lin− BM cells in vivo.
We next asked whether VV-infected cells indeed expand or not. If the observed changes in KSL and Lin−Sca-1−Kit+ populations upon VV infection were due to intrinsic changes in these cells, similar increase and decrease of the two populations could emerge during the 2 days of culture. To get this answer, Lin− BM cells were prepared 24 hours postinfection and cultured for 2 days. Cells were then subjected to a cell viability analysis using LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen). We found that lineage-negative cells did not proliferate in vitro and that Lin+ cells were not present (Fig. 1E). These data suggest that the KSL expansion in vivo in VV-infected mice is likely not due to the direct effect of VV infection of precursor cells.
In Vivo Expansion of KSL Cells by VV Infection Is MyD88-Dependent
A recent study reported the presence of toll-like receptors (TLRs) on KSL and progenitor cells . In addition, the VV protein A52R disrupts the formation of TLR complexes that play an important role in delivering signals in many immune cells . Therefore, we evaluated whether expansion of the KSL population in VV-infected BM requires TLR-mediated signals. For this purpose, we analyzed mice deficient in MyD88 (MyD88−/−), a critical adaptor for TLR-mediated signal transduction . MyD88−/− mice and wild-type (WT) littermates were infected, and the KSL populations were examined 2 days after infection, at which time the maximum expansion of progenitor cells was observed in normal mice. In vivo VV infection of WT mice resulted in dramatic increases in both percentages and the cell numbers of the KSL population, similar to the pattern observed in C57BL/6 mice (Fig. 2A). In contrast, a marginal induction of KSL was observed in BM from MyD88−/− mice (Fig. 2A). Moreover, the KSL population in MyD88−/− BM was composed of comparable proportions of CD34+Flt3− and CD34+Flt3+ cells, whereas WT BM cells have a preferential expansion of CD34+Flt3− cells after infection (Fig. 2B).
We next asked whether the lack of KSL expansion in MyD88−/− mice was due to a defect in virus infection or replication. When we examined the expression of E3L in the lineage-depleted cell population, the percentage of E3L+ cells was comparable between WT and MyD88−/− cells (Fig. 2C). The data suggest that infection and replication of VV were not compromised in the absence of MyD88. Instead, MyD88−/− cells may not proliferate as much as wild-type cells. To test this, we performed cell cycle analysis. As reported previously , only a small fraction of KSL cells was found in the S-G2-M phase of cell cycle in both WT and MyD88−/− mice without VV infection (Fig. 2D). After infection, however, we observed that a higher proportion of WT cells than MyD88−/− cells were cycling (Fig. 2D). In agreement with this observation, the proliferative potential of WT KSL in response to TLR ligands such as LPS or peptidoglycan was greater than that of MyD88−/− KSL (Fig. 2E). Together, VV infection induces expansion of CD34+Flt− KSL cells through a mechanism involving MyD88-dependent signaling.
Changes in the Common Myeloid and Lymphoid Progenitor Cells by VV Infection
Having observed increases in the KSL population upon VV infection, we then investigated the myeloid versus lymphoid progenitor cell compartments. Using Sca-1, c-Kit, CD34, and IL-7Rα, we assessed effects on the CMP that are Lin− IL-7Rα− CD34+ c-kithi Sca-1− and the CLP with the phenotype Lin− IL-7Rα+c-Kitlo Sca-1lo . As shown in Figure 3A, the CMP and CLP populations were decreased and increased, respectively, during 1–2 days of infection (Fig. 3A). Similar to changes in KSL cells, the CMP and CLP compartments returned to normal 7 days postinfection.
Decreases in CMP could be due to either a decline in generation of progenitor cells or enhanced differentiation of more mature cells. Because KSL numbers were increased, we hypothesized that loss of CMP is likely due to the rapid differentiation to mature cells. In this context, we examined cells expressing CD11bintGr-1int because these makers are known to be associated with more differentiated myeloid precursors. As shown in Figure 3B, VV-infected BM showed the increase in CD11bintGr-1int cells. Consistent with this observation, more myeloid cells were present in blood after VV infection (Fig. 3C). Therefore, the reduction of CMP is likely due at least in part to enhanced differentiation.
We then compared the CLP and CMP compartments between WT and MyD88−/− mice 2 days after VV infection. Unlike WT mice, the CMP population was not decreased in MyD88−/− BM, whereas CLP cells were comparable between WT and MyD88−/− mice (Fig. 3D). Thus, differentiation of CMP requires a signal mediated by MyD88, and CLP and CMP compartments appear to be regulated independently by VV infection.
VV Infection Preferentially Increases Development of Plasmacytoid DC
DC are the most potent professional APC and play essential roles in both innate and adaptive immune responses. Among different DC subsets, plasmacytoid dendritic cells (pDC) are important for antiviral immunity by virtue of their ability to produce type I IFN . Moreover, LCMV infection alters DC subsets from pDC to the myeloid dendritic cell (mDC) subset . We questioned whether VV infection also influenced DC differentiation from its progenitors. To address this issue, we first compared the differentiation potential of BM cells with that of the mDC versus pDC subset ex vivo.
BM cells were harvested from mock or VV-infected WT and MyD88−/− mice and cultured in the presence of GM-CSF or Flt3-L. GM-CSF preferentially induces differentiation of mDC from CMP, whereas Flt3-L promotes the generation of DC from both CLP and CMP . In the presence of GM-CSF, DC were primarily CD11c+CD11b+B220− with or without VV infection (Fig. 4A, top left). However, the numbers of recovered DC at the end of the culture were reduced, if BM cells were from mice infected with VV for 2 days, regardless of the type of mice (Fig. 4A, top right). In contrast, a majority of CD11c+ DC in the Flt-3 culture expressed B220, a marker of pDC, and DC generation was more efficient when precursors were prepared from VV-infected mice (Fig. 4A, bottom group). Moreover, BM cells from VV-infected MyD88−/− mice did not expand as much as from WT mice, which were associated with decreased CD11b+B220+ cells. We next examined whether the similar expansion of pDC occurs during in vivo infection. When freshly isolated BM cells were examined, the pDC population in BM of WT mice was increased upon VV infection (Fig. 4B). However, this increase was not observed in the absence of MyD88 (Fig. 4B). The hallmark of pDC is the ability to produce type I IFN upon virus infection . Our data also showed that pDC generated from VV-infected BM cells in vitro produced IFN-β and that the amount of IFN-β was higher in the culture of WT than MyD88−/− pDC (Fig. 4C).
Microbial infection stimulates rapid emigration of peripheral leukocytes to the infected area, a necessity to initiate an innate immune response [27, 28]. Thus, large numbers of cells are generated to replenish the pool upon infection to maintain homeostasis and provide sufficient cells to fight against the microbial pathogens. Our current study supports this concept because VV infection induces expansion of hematopoietic stem cells and progenitor cells at early stages of infection. In addition, VV infection preferentially promotes rapid differentiation of CMP in vivo and efficient generation of plasmacytoid dendritic cells from BM precursors during the ex vivo culture. We also observed a dramatic increase in neutrophils and monocytes in peripheral blood shortly after VV infection (data not shown), which may reflect rapid differentiation and expansion of precursors upon infection. Increased blood neutrophils and monocytes may also reflect enhanced release of these cells to the blood from marrow.
Rapid increase and decrease in KSL cells during VV infection appear to control KSL generation to maintain the homeostasis of hematopoietic cells. Expansion of KSL cells immediately after VV infection would accommodate the demand of cells to clear the virus. However, this increase subsides, and the hematopoietic cell compartment returns to normal within 1 week. Our results suggest that this controlling mechanism is likely operated by an indirect effect. Evidence supporting this includes death of BM cells when they were infected in vitro, very low representation of VV-infected Lin− cells in BM of VV-infected mice, and the decrease in the viability and the number of KSL cells from VV-infected BM during ex vivo culture.
Hematopoietic progenitor cells express several TLRs, which are responsible for rapid replenishment of the innate immune system during infection [19, 21, 29]. Our study suggests physiological significance of TLR expression in hematopoietic cells during in vivo VV infection. Absence of MyD88 prevented differentiation of CMP to more committed monocytes and granulocytes precursors, but not CLP expansion upon VV infection. This implicates a greater contribution of MyD88-mediated signaling to CMP than CLP cell differentiation and supports a study by others that showed that TLR ligands selectively stimulate differentiation of CMP . However, it is not clear whether MyD88 expression in HSC progenitors plays a direct role for VV-induced proliferation or whether expansion is mediated indirectly by other factors, such as cytokines, adhesion molecules, or signaling molecules, that are regulated by MyD88. Our results showed that VV-infected cells themselves do not survive and thus cannot contribute to the expansion and differentiation. Perhaps VV infection induces an environmental change caused by VV products or death of VV-infected cells. Uninfected neighboring cells then may sense the virus products released by dying cells via TLRs and activate the MyD88 signaling pathway. It is possible that CLP and CMP express different members of the TLR family, and thus the response of the two against the environmental change could be different. In addition, it is not clear how MyD88 delivers the signal to cell cycle machinery in KSL cells. Further investigation is necessary to delineate signaling molecules that control expansion of KSL cell upon VV infection. Whatever the mechanisms involved, our data clearly demonstrate the essential role of MyD88 in hematopoiesis during VV infection.
Viral infection has been reported to change the differentiation potential and the function of DC [8, 25]. In contrast to LCMV and measles virus, which cause immunosuppression by interfering with DC differentiation from BM precursor [25, 30], VV infection increased the generation of pDC precursors in a MyD88-dependent manner. Although the precise mechanisms responsible for the different potential of viruses are less clear, different viruses would produce a distinct set of gene products that likely modulate the host immune response differently. In VV-infected mice, we showed increases in CLP that correlate with a higher yield of pDC that can be generated from BM cells prepared from VV-infected mice compared with that generated from mock-infected mice. pDC are considered the first line of defense against virus infection because of their ability to produce type I IFN . Type I IFN plays a critical role in virus clearance, as evidenced by studies that showed high susceptibility of mice deficient in type I IFN receptors to VV infection  and that supplying IFN-β to the DC culture in vitro prevents the inhibitory effect mediated by VV . Thus, the production of IFN-β by VV-infected DC in the presence of Flt3-L may facilitate better cell survival.
Previously, we have shown that DC from VV-infected mice are impaired in their antigen presentation by MHC class II-mediated pathway . However, the same DC expressed increased levels of MHC I and costimulatory molecules, and the ability to produce IFN-β and inflammatory cytokines was not compromised. Therefore, VV specifically target MHC class II antigen presentation by DC, which in turn prevents the activation of CD4 T cells. These data together with the current study demonstrating VV-mediated alteration in hematopoietic precursors provide evidence that VV infection results in multiple changes in host immune responses. Healthy individuals can recover from VV infection, and they seem to able to mount an appropriate immune response against subsequent infection [9, 10, 32, 33]. However, immunocompromised individuals suffer from clinical complications associated with VV infection, suggesting that the ability to mount the initial immune response is critical to protecting the host.
The current study demonstrated that VV infection induces rapid and extensive expansion and differentiation followed by the constriction of hematopoietic stem and progenitor cells in BM. This change in BM that requires MyD88-mediated signaling is likely to facilitate the demand of immune cells to clear the viruses. Therefore, the immune response against VV infection seems to be controlled as early as the hematopoietic progenitor level to mount appropriate protective immunity.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
We are grateful to Dr Renukaradhya Gourapura, Kristin Gillett, and Beau Champ for conducting vaccinia infection and providing organs from infected mice and Drs. Janice Blum, Randy Brutkiewicz, and Mark Kaplan for helpful discussions. This study was supported by PO1 A1056097 (to C.-H.C. and S.-C.H.) and in part by NIH R01-DE-13988 (to S.-C.H.). C.-H.C. is currently affiliated with the Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI.