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. Author manuscript; available in PMC: 2010 May 4.
Published in final edited form as: Immunity. 2008 Mar 13;28(4):509–520. doi: 10.1016/j.immuni.2008.02.013

The Signal Transducer STAT5 Inhibits Plasmacytoid Dendritic Cell Development by Suppressing Transcription Factor IRF8

Eiji Esashi 1, Yui-Hsi Wang 1, Olivia Perng 1, Xiao-Feng Qin 1,2, Yong-Jun Liu 1,2,*, Stephanie S Watowich 1,2,*
PMCID: PMC2864148  NIHMSID: NIHMS46952  PMID: 18342552

SUMMARY

The development of distinct dendritic cell (DC) subsets is regulated by cytokines. Flt3-ligand- (Flt3L) is necessary for plasmacytoid (pDC) and conventional DC (cDC) maturation. GM-CSF inhibits Flt3L-driven pDC production while promoting cDC growth. We show that GM-CSF selectively utilizes STAT5 to block Flt3L-dependent pDC development from the lineage-negative, Flt3+ (lin−/Flt3+) bone marrow subset. STAT3, by contrast, is necessary for expansion of DC progenitors but not pDC maturation. In vivo, STAT5 suppresses pDC formation during repopulation of the DC compartment following bone marrow ablation. GM-CSF/STAT5 signaling rapidly extinguishes pDC-related gene expression in lin−/Flt3+ progenitors. Inspection of the Irf8 promoter revealed that STAT5 is recruited during GM-CSF-mediated suppression, indicating STAT5 directly inhibits transcription of this critical pDC gene. Our results therefore show that GM-CSF controls the production of pDCs by employing STAT5 to suppress IRF8 and the pDC transcriptional network in lin−/Flt3+ progenitors.

Keywords: plasmacytoid dendritic cells, GM-CSF, STAT5, development, FLT3

INTRODUCTION

DCs provide a critical link between the innate and adaptive immune systems by sensing foreign- and host-derived antigens and inducing lymphocyte activation or tolerance. DC subsets are distinguished phenotypically by cell surface markers and functionally by antigen presentation capabilities and cytokine secretion patterns (Banchereau and Steinman, 1998; Shortman and Liu, 2002). pDCs are a specialized lineage that serves as the professional interferon (IFN)-producing cells of the immune system (Asselin-Paturel et al., 2001; Cella et al., 1999; Nakano et al., 2001; Siegal et al., 1999). In response to infection, pDCs produce massive quantities of type I IFN and then undergo maturation to an antigen-presenting form (Kadowaki et al., 2000; Liu, 2005). pDCs are therefore considered to have an important role in establishing anti-viral immunity and shaping the outcome of immune responses.

DCs arise from hematopoietic stem cells in the bone marrow, with DC developmental potential restricted at the progenitor stage to the lin−/Flt3+ subset (D'Amico and Wu, 2003; Karsunky et al., 2003). pDCs and cDCs share a common precursor within the lin−/Flt3+ compartment (Naik et al., 2007; Onai et al., 2007), which expresses GM-CSFRα and transcription factors associated with DC development (Onai et al., 2007). Flt3L is necessary for steady state DC production (McKenna et al., 2000). Administration of exogenous Flt3L enhances DC production and stimulates DC mobilization (Karsunky et al., 2003; Maraskovsky et al., 1996; Pulendran et al., 2000). Flt3L/Flt3 signaling has instructive activity, modifying the developmental potential of Flt3− hematopoietic progenitors and Flt3− megakaryocyte/erythroid-restricted progenitors (MEPs) to produce pDCs and cDCs (Onai et al., 2006). The STAT3 transcription factor is crucial for delivering Flt3 signals to developing DCs in vivo. Hematopoietic-specific deletion of STAT3 results in acute loss of cDCs and attenuated responses to Flt3L in vivo (Laouar et al., 2003). Conversely, STAT3 overexpression promotes DC maturation from hematopoietic progenitors (Onai et al., 2006).

DC development is critically regulated by additional transcription factors beyond STAT3. IRF8 is required for maturation of pDCs and CD8α+ DCs (Schiavoni et al., 2002; Tsujimura et al., 2003), SpiB is necessary for pDCs (Schotte et al., 2004), and PU.1 is important for cDCs (Anderson et al., 2000; Guerriero et al., 2000). Flt3 signaling stimulates the expression of PU.1 and STAT3 (Onai et al., 2006), thus reinforcing pathways necessary for DC formation. The mechanisms that dictate specification of pDCs and cDCs from the common DC precursor are unclear, yet are likely to involve regulation of transcription factor expression or function.

GM-CSF is an important growth factor for cDCs (Daro et al., 2000; Inaba et al., 1992; Mach et al., 2000; Vremec et al., 1997) however it potently inhibits pDC production in Flt3L bone marrow cultures (Gilliet et al., 2002). GM-CSF activates several signaling pathways, including STATs 1, 3 and 5. Here, we show that GM-CSF utilizes STAT5 to block pDC-related gene expression and pDC development from lin−/Flt3+ progenitor cells. STAT5 directly inhibits transcription of IRF8, which is necessary for pDC development, thereby revealing a mechanism by which GM-CSF and STAT5 appear to control pDC lineage fate decisions within the DC progenitor compartment.

RESULTS

GM-CSF suppresses Flt3L-dependent development of pDCs from lin−/Flt3+progenitors

To determine if GM-CSF inhibited pDC development, we investigated its activity on lin−/Flt3+ cells and pDCs. Lin−/Flt3+ cells and pDCs were isolated from murine bone marrow by magnetic bead separation followed by FACS (Fig. 1A; data not shown). Post-sorting analysis confirmed ≥98% purity in the lin−/Flt3+ or CD11c+/CD11b−/B220+ pDC populations, respectively (Fig. 1B). Lin−/Flt3+ cells were cultured in Flt3L, GM-CSF or Flt3L plus GM-CSF for 7 d; subsequently, pDC and cDC production was measured. Flt3L stimulated pDC development while GM-CSF enhanced cDCs but not pDC maturation (Fig. 1C). The addition of GM-CSF to Flt3L cultures inhibited pDC development (Fig. 1C). Furthermore, GM-CSF plus Flt3L stimulated increased maturation of cDCs relative to Flt3L alone, approaching levels observed in GM-CSF alone (Fig. 1C). CFSE labeling showed similar levels of cellular proliferation in Flt3L or Flt3L plus GM-CSF conditions, indicating that GM-CSF does not affect Flt3L-dependent growth of lin−/Flt3+ cells (Fig. 1D). Moreover, the majority of the lin−/Flt3+ population was responsive to GM-CSF as the sole exogenous growth factor (Fig. 1D).

Figure 1. Cytokine-driven DC differentiation from lin−/Flt3+ bone marrow progenitors.

Figure 1

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(A) FACS purification of lin−/Flt3+ cells (lin−/PDCA1−/B220−/Flt3+/CD11c−) and pDCs (lin−/PDCA1+/B220+/CD11c+). (B) Post-sorting analysis of purified lin−/Flt3+ cells and pDCs. (C) Lin−/Flt3+ cells were cultured in 100ng/ml Flt3L, 50ng/ml GM-CSF or 100ng/ml Flt3L plus 50ng/ml GM-CSF for 7 d and analyzed by flow cytometry for pDCs (CD11c+/CD11b−/B220+ or CD11c+/CD11b−/PDCA1+ cells) and cDCs (CD11c+/CD11b+ cells). (D) Lin−/Flt3+ cells were labeled with CFSE, cultured for 3 d and analyzed by flow cytometry. (E) pDCs were cultured for 7 d (antibody stain) or 3 d (CFSE stain) prior to analysis.

Neither Flt3L, nor Flt3L plus GM-CSF, induced cellular proliferation of pDCs (Fig. 1E). Flt3L sustained pDC levels and addition of GM-CSF was incapable of promoting the formation of cDCs from pDCs (Fig. 1E). These results rule out the possibility that GM-CSF operates by stimulating further maturation of pDCs into cDCs. Collectively, our results show that GM-CSF acts upon the early lin−/Flt3+ DC progenitor stage to inhibit Flt3L-driven pDC development.

Signal transduction by Flt3L and GM-CSF in lin−/Flt3+ progenitors and pDCs

To examine the mechanisms involved in GM-CSF-mediated suppression of pDCs, we tested Flt3L- and GM-CSF-responsive STAT signal transduction. Lin−/Flt3+ cells were cultured in cytokine-free conditions for 4 h to reduce endogenous STAT activity, then stimulated with Flt3L, GM-CSF, or Flt3L plus GM-CSF for 20 min to study immediate signaling responses. Flt3L activated STAT3 yet did not activate STAT5 at detectable levels. GM-CSF alone, or in combination with Flt3L, stimulated both STAT3 and STAT5 (Fig. 2A).

Figure 2. Flt3L- and GM-CSF-responsive STAT activation and gene expression in Lin−/Flt3+ cells.

Figure 2

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(A) Lin−/Flt3+ cells were incubated without exogenous cytokine for 4 h, then stimulated with Flt3L (100ng/ml), GM-CSF (50ng/ml) or Flt3L (100ng/ml) and GM-CSF (50ng/ml) for 20 min, as indicated. Activated and total STAT protein levels were determined by immunoblotting. (B) Lin−/Flt3+ cells and pDCs were stimulated with Flt3L −/+ GM-CSF for 30 min. Expression of CIS and GAPDH was determined by RT-PCR. (C) Lin−/Flt3+ cells and pDCs were incubated −/+ GM-CSF for 4 h, then stimulated with Flt3L or maintained in GM-CSF alone for an additional 20 min. Activated and total STAT protein levels were assessed by immunoblotting. (D) Lin−/Flt3+ progenitors were stimulated with Flt3L (open circles) or Flt3L and GM-CSF (black circles) for 0, 16, 36 or 72 h. Gene expression was determined by quantitative PCR.

Selective activation of STAT5 by GM-CSF suggested STAT5 might induce a negative feedback pathway to suppress immediate Flt3-dependent signaling, and thus pDC development. Therefore, we measured expression of the STAT5 target gene cis, which is involved in cytokine negative regulation (Matsumoto et al., 1997). GM-CSF enhanced cis expression in lin−/Flt3+ cells and pDCs (Fig. 2B). Preincubation with GM-CSF, however, did not affect STAT3 activation by Flt3L in lin−/Flt3+ cells or pDCs (Fig. 2C). By contrast, activated STAT5 was reduced significantly after pre-treatment with GM-CSF (Fig. 2C). This suggests that GM-CSF-responsive negative feedback pathways, such as cis, repress GM-CSF-dependent STAT5 signaling but do not contribute substantially to inhibition of pDC development by blocking immediate Flt3L-dependent STAT3 activation.

To assess if GM-CSF regulated the expression of receptors with important roles in DCs, we examined Flt3, GM-CSFRα and TLR9 mRNA levels in lin−/Flt3+ cells stimulated with Flt3L or Flt3L plus GM-CSF. Flt3 expression declined during culture in Flt3L alone, and the addition of GM-CSF significantly accelerated Flt3 repression (Fig. 2D). GM-CSFRα expression was modestly induced by Flt3L, and the combination of Flt3L plus GM-CSF, to similar degrees (Fig. 2D). TLR9 expression was upregulated in Flt3L-stimulated progenitors, however this induction was suppressed in the presence of GM-CSF (Fig. 2D). Thus, GM-CSF inhibits the expression of Flt3 and TLR9, which are important for pDC development and function, in the developing lin−/Flt3+ progenitor population.

STAT3 controls Flt3L-dependent proliferation but not pDC differentiation or GM-CSF-mediated pDC suppression

To test STAT3 function, bone marrow cells from mice with hematopoietic conditional STAT3 deletion (e.g., TIE2cre/STAT3f/Δ) (Panopoulos et al., 2006) were cultured in Flt3L, GM-CSF or Flt3L plus GM-CSF and analyzed for proliferation and DC development. STAT3 was important for Flt3L-dependent growth of total bone marrow, yet was dispensable for GM-CSF- or Flt3L plus GM-CSF-responsive growth (Fig. 3A). CFSE labeling confirmed that STAT3 was crucial for Flt3L-dependent proliferation (Fig. 3B).

Figure 3. Flt3L- and GM-CSF-dependent DC development from STAT3-deficient bone marrow and lin−/Flt3+ progenitors.

Figure 3

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(A) 2 × 106 bone marrow cells from STAT3-deficient (STAT3KO) and wild type (WT) mice were cultured in Flt3L, GM-CSF or Flt3L plus GM-CSF for 7 d and cell numbers were determined by enumeration. The data represent mean values ± SD from three independent experiments. Error bars indicate SD. (B) Bone marrow cells were stained with CFSE, cultured as indicated for 3 d and analyzed by flow cytometry. (C, D) Bone marrow cells were cultured as indicated for 7 d; pDC (CD11c+/CD11b−/PDCA1+) and cDC (CD11c+/CD11b+) frequencies and absolute numbers were determined. Error bars indicate SD. (E) Lin−/Flt3+ cells were cultured for 7 d; pDC and cDC frequencies were analyzed. (F) WT and STAT3-deficient pDCs were isolated from fresh bone marrow samples by FACS, or following ex vivo differentiation in Flt3L (7 d). pDCs (4 × 104/100µl) were stimulated with 5µM CpG-A or left untreated for 24 h. IFNα production was determined by ELISA.

Flt3L stimulated pDC and cDC development from WT and STAT3-deficient total bone marrow samples (Fig. 3C), however, absolute DC numbers were severely reduced in STAT3-deficient cultures (Fig. 3D). Moreover, purified WT and STAT3-deficient lin−/Flt3+ progenitors generate pDCs and cDCs in response to Flt3L (Fig. 3E). These data collectively indicate an important role for STAT3 in Flt3L-mediated DC progenitor proliferation but not DC lineage commitment.

GM-CSF blocked Flt3L-driven pDC development from WT and STAT3-deficient bone marrow, as indicated by the reduced proportion and absolute number of pDCs in Flt3L plus GM-CSF cultures relative to Flt3L alone (Figs. 3C, 3D). Flt3L-driven pDC maturation from lin−/Flt3+ progenitors was also inhibited by GM-CSF (Fig. 3E). When used as the sole growth factor, GM-CSF stimulated cDC development yet failed to promote pDC maturation from WT and STAT3-deficient cells (Figs. 3C, 3D). Moreover, addition of GM-CSF to Flt3L cultures rescued the development of STAT3-deficient cDCs, restoring cDC numbers to a level similar to WT Flt3L cultures (Fig. 3D). Thus, STAT3 is dispensable for GM-CSF-dependent pDC suppression and cDC development.

To determine if STAT3 was important for terminal differentiation of pDCs, we examined whether STAT3-deficient pDCs could respond to CpG stimulation by producing IFN-α. Freshly isolated bone marrow pDCs or pDCs derived from Flt3L-stimulated bone marrow cultures produced similar levels of IFNα, regardless of STAT3 status (Fig. 3F). Taken together, therefore, our data suggest that STAT3 controls an essential Flt3L-driven proliferative phase in the DC developmental pathway, yet STAT3 does not dictate critical specification and maturation steps in the pDC lineage, or regulation of GM-CSF-dependent pDC suppression.

STAT5 is essential for GM-CSF-dependent suppression of pDC development from fetal liver progenitor cells

To test the function of STAT5 in GM-CSF-mediated suppression of pDC development, we utilized fetal liver (FL) hematopoietic progenitors from STAT5+/+ and STAT5−/− embryos since complete STAT5 deletion causes neonatal lethality (Cui et al., 2004). We found that Flt3L or Flt3L plus GM-CSF stimulated growth of STAT5+/+ and STAT5−/− FL cells (Fig. 4A). In addition, Flt3L supported pDC maturation from STAT5+/+ and STAT5−/− FL cells (Fig. 4B). Thus, STAT5 is dispensable for Flt3L-driven FL cell proliferation and pDC development.

Figure 4. Cytokine-driven DC development from STAT5−/− fetal liver cells.

Figure 4

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1 × 106 FL (E14.5) cells were cultured in 100ng/ml Flt3L −/+ 50ng/ml GM-CSF for 7 d. (A) Total cell number in each culture was determined by enumeration. Error bars indicate SD. (B) pDC (CD11c+/CD11b−/PDCA1+) and cDC (CD11c+/CD11b+) frequencies are shown. (C) Absolute numbers of pDCs, cDCs and CD11c+/CD11b− DCs (CD11b− DCs) were determined. The data represent mean values ± SD from three independent experiments.

GM-CSF inhibited Flt3L-driven pDC maturation from STAT5+/+ FL cells (Fig. 4B, 4C). Deletion of STAT5, however, enabled pDC development in Flt3L plus GM-CSF, as judged by the increased frequency and absolute number of pDCs in STAT5−/− FL cultures relative to STAT5+/+ (Fig. 4B, 4C). In addition, the absolute number of STAT5−/− pDCs was similar in Flt3L plus GM-CSF compared to Flt3L alone (Fig. 4C). STAT5 was dispensable for cDC and CD11b− DC development in response to Flt3L or Flt3L plus GM-CSF (Fig. 4B, 4C). Culture of STAT5+/+ FL progenitors in GM-CSF alone failed to promote pDC development, however a low level of pDCs matured from STAT5−/− progenitors grown in GM-CSF (data not shown). Thus, STAT5 has a fundamental role in mediating GM-CSF-dependent inhibition of pDC development but not GM-CSF-driven cDC maturation from FL progenitors ex vivo. Furthermore, the results suggest GM-CSF-dependent pDC inhibition is independent of global effects on cellular proliferation.

STAT5 regulates the production of pDCs during bone marrow reconstitution following ablative irradiation

To examine STAT5 function in vivo, FL progenitors from STAT5+/+ or STAT5−/− d14.5 embryos (CD45.2+) were transplanted into lethally irradiated CD45.1+ congenic recipients. Chimerism and DC levels in the bone marrow and spleen were analyzed at 8 weeks post transplant. STAT5−/− FL progenitors repopulated the bone marrow compartment with modestly reduced ability relative to STAT5+/+ progenitors yet showed decreased ability to repopulate the spleen (Fig. 5), consistent with their lymphoid deficiency (Yao et al., 2006).

Figure 5. Development of pDCs and cDCs from STAT5−/− fetal liver progenitors in vivo.

Figure 5

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(A, D) Left panels: The frequency of donor-derived CD45.2+ cells in the bone marrow or spleen of WT and STAT5KO transplant recipients was determined. Second column: The percentage of CD11c+/CD11b−, CD11c+/CD11b+ and CD11c−/CD11b+ cells within the bone marrow or spleen CD45.2+ subset from WT and STAT5KO recipients are shown. The CD45.2+/CD11c+/CD11b− (R1, boxed) and CD45.2+/CD11c+/CD11b+ (R2, boxed) subsets were analyzed for PDCA1 expression. Levels of donor-derived pDCs (CD45.2+/CD11c+/CD11b−/PDCA1+ cells), cDCs (CD45.2+/CD11c+/CD11b+ cells, which are negative for PDCA1) and CD11b− DCs (CD45.2+/CD11c+/CD11b−/PDCA1−) from a representative experiment are shown. (B, E) Relative levels of pDCs, CD11b− DCs and cDCs within the donor-derived CD45.2+/CD11c+ DC population in the bone marrow or spleen of WT and STAT5KO recipient mice were determined. The data represent individual (open circles) and mean (bar) values for STAT5+/+ or STAT5−/− chimeric mice (n=8/genotype) from 2 independent experiments. P values determined by unpaired two-tailed Student’s t test are indicated. (C) pDCs were recovered from STAT5KO (black bars) and WT (white bars) recipient mice by FACS; cells were stimulated with CpG-A (5µM) and IFNα production was measured. The data represent mean values ± SD from two independent experiments. (F) The donor-derived CD45.2+ population from the spleen of WT and STAT5KO transplant recipient mice was analyzed for CD11chi/CD4+/CD11b+, CD11chi/CD8α+ and CD11chi/CD8α− DC subsets by flow cytometry.

STAT5−/− FL progenitors contributed to the pDC lineage more efficiently than STAT5+/+ progenitors, as judged by comparison of relative pDC levels within the donor-derived (i.e., CD45.2+/CD11c+) DC subset (Fig. 5A, 5B). STAT5−/− pDCs were also present at higher levels in the total donor-derived bone marrow compartment, relative to STAT5+/+ pDCs (Table I). While similar absolute pDC numbers were found in STAT5+/+ and STAT5−/− chimeras, the total donor-derived bone marrow cell number was reduced in STAT5−/− recipients, indicating an enhanced propensity for STAT5−/− progenitors to generate pDCs (Table I). Furthermore, we found a decreased proportion and absolute number of STAT5−/− CD11c+/CD11b−/PDCA1− DCs (CD11b− DCs) within the donor-derived DC bone marrow subset, relative to STAT5+/+ recipients (Fig. 5A, 5B). This reduction corresponded to lower bone marrow cell numbers in STAT5−/− chimeras (Table I). Similar proportions and absolute numbers of cDCs were observed in the donor-derived DC subset and total bone marrow compartment of STAT5+/+ and STAT5−/− chimeras (Fig. 5A, 5B, Table I). Overall, STAT5−/− progenitors repopulated the total bone marrow DC compartment better than STAT5+/+ progenitors, as judged by an increased proportion of CD45.2+/CD11c+ cells in STAT5−/− recipients (Fig. 5A). Significantly, STAT5 was not essential for pDC functional maturation since equivalent levels of IFN-α were produced by pDCs isolated from the bone marrow of STAT5+/+ and STAT5−/− chimeric mice in response to CpG stimulation (Fig. 5C).

Table I.

Absolute number of donor-derived cells. Absolute numbers of total donor-derived CD45.2+ cells, donor-derived CD45.2+ pDCs, CD45.2+ CD11b− DCs and CD45.2+ cDCs were determined within the bone marrow and spleen of WT and STAT5KO transplant recipients. Values reflect the mean ± SD from 8 bone marrow chimeric mice (two independent experiments).

BM
Total (×106) pDCs (×105) CD11b− DCs (×105) cDCs (×105)
WT 63.93 ± 7.35 8.80 ± 1.36 2.65 ± 0.56 4.83 ± 0.88
STAT5KO 45.88 ± 20.55 9.05 ± 2.26 1.91 ± 0.76 4.18 ± 0.90
Spleen
Total (×106) pDCs (×105) CD11b− DCs (×105) cDCs (×105)
WT 203.04 ± 31.40 17.28 ± 8.51 20.43 ± 3.45 43.86 ± 15.81
STAT5KO 17.05 ± 8.58 7.03 ± 2.18 6.14 ± 3.15 15.79 ± 5.30
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The relative proportions of DC subsets in the spleen of transplant recipient animals showed a similar trend to that observed in the bone marrow. We found increased pDC levels, reduced CD11b− DCs, and similar cDC levels in the donor-derived splenic CD45.2+/CD11c+ DC compartment of STAT5−/− recipients relative to STAT5+/+ recipients (Fig. 5D, 5E). Examination of the splenic CD11chi population revealed elevated levels of CD8α+ DCs and reduced CD4+/CD11b+ DCs in STAT5−/− chimeric mice compared to STAT5+/+ chimeras (Fig. 5F). Splenic cellularity was dramatically decreased in STAT5−/− recipients, leading to a significant reduction in the absolute numbers of splenic pDCs, CD11b− DCs and cDCs, relative to STAT5+/+ recipients (Table I). The overall proportion of donor-derived DCs was higher in the spleen of STAT5−/− recipients compared to STAT5+/+ recipients (Fig. 5D, middle panels), similar to observations in the bone marrow, and thus supporting the notion that lymphoid repopulation but not DC repopulation is affected in STAT5−/− recipients. Hence, STAT5 has an important role in controlling the proportion of pDC and CD11b− DC subsets within the bone marrow and spleen during hematopoietic reconstitution following ablative irradiation. In these hematopoietic tissues, STAT5 represses pDC development and concomitantly enhances CD11b− DC formation.

STAT5 controls GM-CSF-mediated suppression of pDCs from purified adult lin−/Flt3+ progenitors

Hematopoietic progenitors from fetal liver and adult bone marrow have differences in developmental potential. To determine if STAT5 was necessary for GM-CSF-mediated suppression of pDC development from adult bone marrow progenitor cells, we recovered donor-derived lin−/Flt3+ progenitors (i.e., CD45.2+/lin−/Flt3+ cells) from the bone marrow of STAT5+/+ and STAT5−/− recipient mice. Lin−/Flt3+ cells were cultured in the presence of Flt3L, GM-CSF or Flt3L plus GM-CSF for 7 d, and subsequently analyzed for pDC and cDC levels. STAT5 was dispensable for Flt3L-driven pDC development, as anticipated (Fig. 6A). STAT5 function was required, however, for efficient suppression of Flt3L-driven pDC development by GM-CSF. An increased proportion of pDCs developed from STAT5−/− lin−/Flt3+ progenitors in Flt3L plus GM-CSF, relative to STAT5+/+ progenitors in corresponding culture conditions, approaching pDC levels found in Flt3L alone (Fig. 6A). In addition, GM-CSF induced pDCs from STAT5−/− lin−/Flt3+ progenitors but not STAT5+/+ progenitors (Fig. 6A), suggesting GM-CSF signals are sufficient for pDC maturation in cells lacking functional STAT5. Thus, STAT5 is necessary for GM-CSF-mediated pDC suppression from the purified lin−/Flt3+ progenitor subset.

Figure 6. STAT5 function in DC development and gene expression.

Figure 6

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Donor-derived CD45.2+/lin−/Flt3+ progenitor cells were recovered from the bone marrow of STAT5+/+ (WT) and STAT5−/− (STAT5KO) transplant recipient mice by FACS. (A) CD45.2+/lin−/Flt3+ cells were cultured as indicated for 7 d; pDC (CD11c+/CD11b−/PDCA1+) and cDC (CD11c+/CD11b+) levels were determined. (B) STAT5+/+ CD45.2+/lin−/Flt3+ cells were cultured for 0, 16, 36 or 72 hours in Flt3L (solid line, open circles) or Flt3L plus GM-CSF (hatched line, black circles). Real-time PCR was used to determine transcription factor expression levels. (C) CD45.2+/lin−/Flt3+ cells from WT and STAT5KO recipient mice were cultured in Flt3L (white bars) or Flt3L plus GM-CSF (black bars) for 36 h. Transcription factor expression levels were determined by real time PCR. Error bars represent SD. (D) D2SC/1 cells were stimulated with GM-CSF or left untreated for 120 min; expression of IRF4 and IRF8 mRNA was measured by real time PCR. Error bars represent SD. (E) D2SC/1 cells stably expressing STAT5a were stimulated with GM-CSF or left untreated for 30 min. Nuclear extracts were recovered and used in EMSAs with or without STAT5 supershift antibody or competitor oligonucleotides (D, IRF8 STAT site; mD, mutated IRF8 STAT site; S5, β-casein STAT site; P, IRF8 STAT-like site). GM-CSF-responsive STAT5 DNA binding and STAT5 antibody supershift complexes are indicated (open and closed stars, respectively). (F) Luciferase assays were performed as described in the Experimental Procedures. Error bars represent SD. (G) D2SC/1 cells were stimulated with GM-CSF for 60 min or left untreated. Chromatin immunoprecipitations were performed with anti-STAT5 antibodies (STAT5) or an irrelevant IgG (IgG). PCR reactions were performed, using primers specific for the murine IRF4 and IRF8 promoters, on total cell lysates (input) or immunoprecipitation samples, as indicated. PCR reactions were visualized by ethidium bromide staining of agarose gels.

STAT5 controls the expression of critical DC lineage transcription factors

To assess the mechanism of GM-CSF/STAT5-mediated suppression of pDCs, donor-derived lin−/Flt3+ cells from STAT5+/+ and STAT5−/− chimeras were stimulated with Flt3L or Flt3L plus GM-CSF and examined for the expression of transcription factors that are important in DCs, including PU.1, IRF2, IRF4, IRF7, IRF8 and SpiB (Wu and Liu, 2007; Zenke and Hieronymus, 2006). PU.1 and IRF2 were regulated similarly in STAT5+/+ lin−/Flt3+ cells in Flt3L or Flt3L plus GM-CSF (Fig. 6B,), suggesting they are not involved in pDC suppression by GM-CSF/STAT5. By contrast, IRF4 expression was strongly induced in STAT5+/+ lin−/Flt3+ cells in Flt3L plus GM-CSF, relative to Flt3L alone (Fig. 6B). Furthermore, expression of IRF7 and IRF8 was inhibited by GM-CSF plus Flt3L versus Flt3L alone, while SpiB induction was delayed by GM-CSF (Fig. 6B). Since IRF4 is important for cDC development, and IRF8, SpiB and IRF7 have critical roles in pDCs, our results suggest GM-CSF extinguishes a transcriptional network necessary for pDC development while enhancing pathways that promote cDC maturation.

STAT5 was dispensable for the expression of each factor in Flt3L alone (Fig. 6C), consistent with Flt3L-driven pDC and cDC development from STAT5−/− progenitors ex vivo and repopulation of the pDC compartment in STAT5−/− transplant recipient mice (Fig. 4, Fig. 5, Fig. 6A). By contrast, GM-CSF inhibited the expression of IRF7, IRF8 and SpiB in lin−/Flt3+ cells via a STAT5-dependent mechanism, while GM-CSF-dependent induction of IRF4 was attenuated in STAT5−/− lin−/Flt3+ cells (Fig. 6C). These results point to an essential role for STAT5 in mediating the effects of GM-CSF upon the DC transcriptional network in developing lin−/Flt3+ bone marrow progenitor cells.

STAT5 directly suppresses IRF8 expression by a mechanism involving STAT5 recruitment at the IRF8 promoter

The IRF4 and IRF8 promoters contain STAT consensus sites (Kanno et al., 1993; Matsuyama et al., 1995), however the SpiB promoter (−2000bp) lacks STAT consensus sites as judged by nucleotide sequence analysis (not shown). We therefore focused on STAT5 regulation of IRF4 and IRF8. We found that GM-CSF-mediated downregulation of IRF8 was recapitulated in the murine splenic DC cell line D2SC/1 (Granucci et al., 1994), while induction of IRF4 by GM-CSF was not observed by 2 h stimulation (Fig. 6D). These results suggested that IRF8 but not IRF4 was a direct target of the GM-CSF/STAT5 pathway, and indicated that D2SC/1 cells provide a model system for investigation of IRF8 regulation by STAT5. We found that GM-CSF suppressed reporter activity of an IRF8 promoter construct, consistent with its activity on endogenous IRF8 expression (Fig. 6F). EMSAs showed that GM-CSF stimulated DNA binding to a STAT consensus site in the IRF8 promoter, previously shown to be IFN-responsive (Kanno et al., 1993). STAT5 interacted specifically with this STAT site, as judged by STAT5 antibody supershifts and competition with a STAT5 consensus oligonucleotide (Fig. 6E). Chromatin immunoprecipitation experiments showed that STAT5 was recruited to the proximal IRF8 promoter upon GM-CSF stimulation but was not detectable at the IRF4 promoter (Fig. 6G). Thus, GM-CSF stimulates STAT5 interaction at the endogenous IRF8 promoter to inhibit IRF8 expression, indicating a direct mechanism of IRF8 regulation by GM-CSF/STAT5.

DISCUSSION

STAT proteins have a central role in blood cell production in steady state conditions and physiological stress situations such as infection. Distinct DC subsets reside throughout the body, yet the basic mechanisms that control DC development by cytokines and STATs remain relatively elusive. The Flt3L/STAT3 pathway has emerged as a major regulator of DC production in vivo (Karsunky et al., 2003; Laouar et al., 2003; Maraskovsky et al., 1996; Pulendran et al., 2000). In agreement, we found that STAT3 was necessary for proliferation of bone marrow progenitors to Flt3L, and absolute pDC and cDC numbers were reduced in STAT3-deficient bone marrow cultures as a consequence. STAT3 was dispensable for pDC specification and maturation, however, as STAT3-deficient pDCs responded to CpG stimulation by producing IFN-α, a hallmark of differentiated pDCs. Our data support the idea that STAT3 regulates the proliferation and/or maintenance of the Flt3+ progenitor population and thereby controls absolute production of DCs (Laouar et al., 2003), yet STAT3 is not essential for the commitment or development of the pDC lineage.

GM-CSF and STAT5 override Flt3L-dependent signals in early stages of the pDC developmental pathway to block pDC commitment and/or terminal maturation. This was shown by GM-CSF/STAT5-mediated inhibition of absolute pDC production in ex vivo FL cultures and selective blockade of pDC maturation from purified lin−/Flt3+ bone marrow progenitors. GM-CSF was unable to induce the conversion or maturation of pDCs to cDCs, underscoring the idea that it operates early in pDC development. GM-CSF may act on the common DC progenitor within the adult lin−/Flt3+ bone marrow subset (Onai et al., 2007) to suppress pDC lineage commitment and/or maturation, while concomitantly supporting or enhancing cDC specification. The DC progenitor population is less characterized during fetal development, yet we show that development of FL pDCs are inhibited by GM-CSF and STAT5, indicating the possibility of an equivalent to the adult common DC progenitor in FL. Moreover, since both FL progenitors and adult lin−/Flt3+ bone marrow cells responded to the inhibitory actions of GM-CSF and STAT5, this pathway may be important in regulating pDC levels during ontogeny.

pDC repopulation following bone marrow ablation was negatively regulated by STAT5, suggesting that the DC progenitor compartment may respond to STAT5-activating cytokines to control balanced production of the DC lineages in vivo. By contrast, STAT5 was unnecessary for repopulation of cDCs, consistent with the fact that GM-CSF appears to be largely dispensable for cDC maturation in vivo (Vremec et al., 1997). In ex vivo conditions, however, STAT5 was important for GM-CSF-driven cDC development from lin−/Flt3+ progenitors. It remains to be determined whether cDC development in vivo is regulated by other cytokines beyond Flt3L; our results suggest that STAT5-acting cytokines may have some ability to control cDC production from bone marrow yet this pathway would be redundant or secondary to Flt3L/STAT3. For pDCs, additional studies should assess the function of other STAT5-activating cytokines, or endogenous and pharmacological levels of GM-CSF in steady state and ‘emergency’ conditions such as infection, to determine if they repress pDC maturation in vivo. Modulation of pDCs by STAT5-acting cytokines may influence the outcome of immune responses in certain settings, and may provide a therapeutic approach to regulate the level of this important cell lineage.

At the molecular level, signaling via Flt3L rapidly induced the expression of key pDC genes in lin−/Flt3+ progenitors, including PU.1, IRF7, IRF8, SpiB and TLR9. It is notable that this group includes three transcription factors with clear roles in pDC lineage specification or maturation (i.e., PU.1, IRF8, SpiB) (Anderson et al., 2000; Guerriero et al., 2000; Schiavoni et al., 2002; Schotte et al., 2004; Tsujimura et al., 2003), suggesting that Flt3L may initiate and/or sustain the transcriptional network required for pDC development. Others have shown a limited instructive function for STAT3 in redirecting megakaryocyte-erythroid progenitors (MEPs) to a pDC and cDC cell fate (Onai et al., 2006), although it is not yet known if STAT3 directly or indirectly controls Flt3L-mediated DC gene expression. Furthermore, STAT3-independent commitment and maturation pathways also exist for the pDC lineage since functional pDCs mature from STAT3-deficient bone marrow progenitors. The collective data suggest that STAT3 supports and/or reinforces Flt3L-mediated execution of the pDC developmental program, presumably in a permissive environment, in which pDC-related genes are accessible to the transcriptional machinery.

STAT5 inhibited the propensity of bone marrow progenitors to generate pDCs in vivo, indicating it could affect DC cell fate decisions. These results are reminiscent of the lymphoid to myeloid lineage conversion function for GM-CSF in CLPs and early T cell progenitors (Iwasaki-Arai et al., 2003; Kondo et al., 2000), and of IL-2/STAT5-mediated inhibition of Th17 development (Laurence et al., 2007), a process which is dependent upon STAT3 and STAT3-activating cytokines (Nurieva et al., 2007; Yang et al., 2007). Molecularly, GM-CSF and STAT5 appeared to exert a broad effect on the DC transcriptional network in lin−/Flt3+ progenitors by overriding Flt3L-mediated induction and/or reinforcement of pDC-related gene expression, to suppress Flt3, TLR9, IRF7, IRF8 and SpiB, and to induce IRF4, a key factor in cDC development (Suzuki et al., 2004; Tamura et al., 2005). Promoter sequence analysis initially suggested IRF8 and IRF4 as the most likely direct targets of GM-CSF/STAT5, and between these two genes only IRF8 appears to be regulated directly by GM-CSF-activated STAT5. Since IRF8 is crucial for pDC maturation (Schiavoni et al., 2002; Tsujimura et al., 2003), inhibition of its expression by GM-CSF/STAT5 indicates this is an essential step in suppressing pDC production. Blockade of IRF8 expression may negatively affect the expression of regulatory transcription factors (e.g., SpiB, IRF7) and receptors (e.g., Flt3, TLR9) that are necessary for pDC development and function. Furthermore, STAT5-dependent inhibition of IRF8 may lead to the induction of IRF4 and a concomitant activation or reinforcement of the pathways that control cDC development. Interestingly, our analysis of splenic DC subsets showed that STAT5−/− chimeric mice have elevated levels of CD8α+ DCs and reduced CD4+/CD11b+ DCs within the CD11chi population, compared to STAT5+/+ chimeras. IRF8 is essential for the development of CD8α+ DCs, while IRF4 is important for formation of the splenic CD4+/CD11b+ DC subset (Schiavoni et al., 2002; Suzuki et al., 2004). Thus, the control of IRF4/IRF8 expression levels by GM-CSF/STAT5 might factor in the regulation of these DC subsets in vivo. It is not yet known if GM-CSF/STAT5-mediated suppression of IRF8 operates in other hematopoietic lineages to regulate their development and/or function, although our preliminary studies with the D2SC/1 (DC) and J774 (macrophage) cell lines suggested this pathway may be restricted to DCs (data not shown). To understand the molecular pathways by which GM-CSF regulates DC production from bone marrow precursors, the direct target genes of STAT5 and IRF8 must be characterized in lin−/Fl3+ cells.

In summary, we show an essential function for GM-CSF and STAT5 in controlling the transcriptional program in lin−/Flt3+ progenitors required for development of the pDC lineage. These results have important implications: GM-CSF is naturally induced during infection and this property is exploited clinically to treat myelosuppression. Are pDCs adversely affected in these situations, and how does this impact the immune response? Equally significant, can the function of GM-CSF be harnessed to treat conditions such as psoriasis that are associated with pDC overproduction, or provide an effective approach to modulate DCs in cancer therapy? The answers to these questions lie in developing a further understanding of the DC developmental pathway and the molecular machinery that controls DC cell fate decisions and functional maturation. Ultimately this knowledge may assist in redirecting the immune response to treat immunological disease and cancer.

EXPERIMENTAL PROCEDURES

STAT-deficient mice; isolation of bone marrow and spleen

Bone marrow STAT3-deficient mice were obtained by breeding Tie2cre transgenic and floxed Stat3 mice (Panopoulos et al., 2006). STAT5+/− mice with targeted disruption of Stat5a and Stat5b (Cui et al., 2004) were intercrossed; E14.5 fetuses were obtained for fetal liver (FL) cell isolation. Animals were housed in specific pathogen-free barrier facilities. All experiments were performed according to institutional guidelines at UT MD Anderson Cancer Center.

Antibody staining and flow cytometry; CFSE staining; isolation of lin−/Flt3+ progenitors and pDCs

Single cell suspensions of bone marrow and spleen were labeled with antibodies (see figure legends) and analyzed on a FACS-Calibur machine. CFSE labeling was performed according to the manufacturer’s protocol (Invitrogen). Lin−/Flt3+ progenitors and pDCs were isolated from total bone marrow by magnetic bead separation followed by FACS. Negative immunoselection of lin− bone marrow progenitors was performed with biotin-CD3, biotin-CD11b, biotin-CD19 and biotin-Ter119 Abs, followed by SA-coated micro beads (Miltenyi Biotec). Lin−/PDCA1−/B220−/Flt3+/CD11c− cells (lin−/Flt3+ progenitors) and lin−/B220+/CD11c+ cells (pDCs) were isolated on a FACS Aria machine (BD Biosciences) using FITC-PDCA1, FITC-B220, PE-Flt3, APC-CD11c and a mixture of PerCP-Cy5.5-labeled mAbs against lineage markers CD3, CD11b and CD19.

In vitro culture of lin−/Flt3+ progenitors and pDCs; immunoblot analysis

Lin−/Flt3+ progenitors and pDCs were cultured in RPMI 1640 medium containing 10% FCS, 1 mM sodium pyruvate, hepes, penicillin, streptomycin and β-mercaptoethanol (RPMI/FCS) supplemented with murine Flt3L (100 ng/ml) and/or GM-CSF (50 ng/ml) (R&D Systems), or lacking exogenous cytokine, as described in the figure legends. Cells were recovered at the indicated timepoints for RNA or whole cell lysate preparation. Immunoblots of whole cell lysates were performed with anti-phospho–STAT3 Ab (Cell Signaling), anti-STAT3 (C-20, Santa Cruz), anti-phospho–STAT5 Ab (Cell Signaling) and anti–STAT5 Ab (Santa Cruz). Enhanced chemiluminescence was used for detection.

In vivo reconstitution assays and isolation of donor-derived cells

Congenic CD45.1+ mice were lethally irradiated (950rad) and injected intravenously with single cell suspensions containing 2×106 fetal liver cells (CD45.2+) from E14.5 STAT5+/+ or STAT5−/− embryos and 2×105 host (CD45.1+) bone marrow cells; the latter were used to provide radioprotection. After 8 weeks, mice were sacrificed and donor-derived cells (CD45.2+) in bone marrow and spleen were analyzed by flow cytometry.

For isolation of donor-derived lin−/Flt3+ progenitors, lin− cells were obtained from bone marrow by negative immunoselection using a mixture of rat mAbs against the lineage markers CD3, CD11b, CD11c, CD19 and Ter119, followed by anti-rat IgG-coated micro beads (Miltenyi Biotec). Subsequently, lin−/PDCA1−/B220−/Flt3+/CD45.2+ cells were isolated on a FACS Aria machine (BD Biosciences) using FITC-PDCA1, FITC-B220, PE-Flt3, PerCP-Cy5.5-CD45.2 and a mixture of APC-labeled mAbs against lineage markers CD3, CD11b, CD11c and CD19.

RNA isolation; reverse-transcription and real-time PCR assays

Total RNA was extracted (Qiagen RNeasy mini protocol) and reverse-transcribed using oligo-dT, random hexamers and SuperScript RT II (Invitrogen). Semi-quantitative reverse-transcription PCR (RT-PCR) was performed with previously described primers for cis and GAPDH (Shigematsu et al., 2004). Real-time PCR for Flt3, GM-CSFRα, TLR9, PU.1, IRF2, IRF4, IRF7, IRF8, SpiB and RPL13A was performed with Power SYBR® Green PCR Master Mix reagent (Applied Biosystems) and a sequence detector (ABI PRISM 7500) (primer sequences are available upon request). Threshold cycle (CT) values for each gene were normalized to RPL13A using the equation 1.8 (RPL13A-GENE) (100,000), as recommended by the manufacturer, where RPL13A was the mean CT of duplicate RPL13A runs, GENE was the mean CT of duplicate runs of the gene of interest, and 100,000 was an arbitrarily chosen factor to bring all values above zero.

D2SC/1 cell culture; retroviral infection; chromatin immunoprecipitations (ChIPs); EMSAs; reporter assays

The murine splenic DC cell line D2SC/1 (Granucci et al., 1994) was maintained in Iscove’s modified Dulbecco’s medium containing 10% heat-inactivated fetal calf serum and 1% penicillin-streptomycin (Invitrogen-Gibco). D2SC/1 cells were infected with the bicistronic vector pMIG carrying STAT5 or were mock-infected; after 2–3 days growth, GFP+ cells were isolated by FACS. Cells were stimulated with recombinant murine GM-CSF (50ng/mL) as indicated in the figure legends. Chromatin immunoprecipitation (ChIP) experiments were performed using a mixture of antibodies for STAT5A and STAT5B (R&D Systems; Santa Cruz), following the recommendation of the manufacturer of the ChIP reagents (Upstate Biotechnology, Inc.). Nuclear extracts (10µg) were used in EMSAs with 32P-labeled oligonucleotides corresponding to the STAT consensus site in the murine IRF8 promoter (Kanno et al., 1993) or bovine β-casein promoter (Santa Cruz Biotechnology), in the absence or presence of competitor oligonucleotide or STAT5 antibody, as described (Onishi et al., 1998). For reporter assays, an IRF8 promoter fragment (−351 to +52) was cloned into pGL4.12 (Promega). D2SC/1 cells were transiently transfected with pGL4.12(−351 to +52), pME18S-STAT5a and phRL-TK (Promega) using Lipofectamine2000 (Invitrogen). After 32 hours, cells were stimulated for 2 h with GM-CSF or left untreated. Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System.

Statistical analysis

Data are presented as mean values +/− one standard deviation (SD); P values were calculated using an unpaired two-tailed Student’s t test.

ACKNOWLEDGEMENTS

We thank Dr. Lothar Hennighausen for the gift of STAT5+/− mice, Dr. Athanasia D. Panopoulos for STAT3-deficient mice and Dr. Michel Gilliet for his advice on various aspects of this project. We thank Karen Ramirez, Zhiwei He and Dr. Eric Wieder for assistance with cell sorting and Ling Zhang for technical support. We are grateful to Drs. Peter J. Murray and Kimberly S. Schluns for their critical review of the manuscript. This work was supported by grants from the NIH (to Y.J.L.); NIH, Gillson Longenbaugh Foundation and the MDACC Institutional Grants Program (to S.S.W.).

Footnotes

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