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Originally published online as doi:10.1189/jlb.0406246 on September 11, 2006

Published online before print September 11, 2006
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(Journal of Leukocyte Biology. 2006;80:1500-1511.)
© 2006 by Society for Leukocyte Biology

IRF-1 deficiency skews the differentiation of dendritic cells toward plasmacytoid and tolerogenic features

L. Gabriele*, A. Fragale{dagger}, P. Borghi*, P. Sestili*, E. Stellacci{dagger}, M. Venditti*, G. Schiavoni*, M. Sanchez*, F. Belardelli* and A. Battistini{dagger},1

* Departments of Cell Biology and Neurosciences and
{dagger} Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy

1 Correspondence: Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 299, Rome 00161, Italy. E-mail: battist{at}iss.it


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ABSTRACT
 
Members of the IFN regulatory factors (IRFs) family are transcriptional regulators that play essential roles in the homeostasis and function of the immune system. Recent studies indicate a direct involvement of some members of the family in the development of different subsets of dendritic cells (DC). Here, we report that IRF-1 is a potent modulator of the development and functional maturation of DC. IRF-1-deficient mice (IRF-1–/–) exhibited a predominance of plasmacytoid DC and a selective reduction of conventional DC, especially the CD8{alpha}+ subset. IRF-1–/– splenic DC were markedly impaired in their ability to produce proinflammatory cytokines such as IL-12. By contrast, they expressed high levels of IL-10, TGF-β, and the tolerogenic enzyme indoleamine 2,3 dioxygenase. As a consequence, IRF-1–/– DC were unable to undergo full maturation and retained plasmacytoid and tolerogenic characteristics following virus infection ex vivo and in vivo. Accordingly, DC from IRF-1–/– mice were less efficient in stimulating the proliferation of allogeneic T cells and instead, induced an IL-10-mediated, suppressive activity in allogeneic CD4+CD25+ regulatory T cells. Together, these results indicate that IRF-1 is a key regulator of DC differentiation and maturation, exerting a variety of effects on the functional activation and tolerogenic potential of these cells.

Key Words: cytokines • virus infections • tolerance • IFN regulatory factors


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INTRODUCTION
 
Dendritic cells (DC) play different key functions in immune regulation, including initiation and regulation of adaptive immune responses to pathogens such as viruses, as well as establishment and maintenance of central and peripheral tolerance [1 ]. The functional diversity of DC in evoking this range of immune responses can be attributed in part to the existence of distinct subsets that can be distinguished on the basis of phenotypic and functional features, localization, and differentiation status [2 ]. Based on the levels of CD11c expression, two major DC subsets have been identified in the mouse, CD11chigh conventional DC (cDC) and CD11clow plasmacytoid DC (pDC). Subsets of cDC can be distinguished by differing expression of the CD8{alpha} marker, as CD8{alpha}+ and CD8{alpha}– DC that display different anatomic distributions in lymphoid organs [3 ]. CD8{alpha}– DC in the spleen can be divided further into CD4+ and CD4– subsets [2 , 3 ]. After activation, cDC exhibit distinct cytokine profiles and biological functions, such as the ability to induce Th1 and Th2 responses [4 , 5 ]. pDC are the major IFN-producing cells [6 7 8 ]. In response to different pathogens, they produce large amounts of IFN-{alpha}/β, which contribute to initiation of the adaptive immune response by affecting the activation of many other cell types including monocytes, cDC, T cells, and B cells [9 ]. However, under certain circumstances, cDC can also act as specialized, IFN-producing cells [10 ].

Although DC have been considered mainly for their role in activating the immune system [11 , 12 ], it is now well known that DC can also induce tolerance [13 , 14 ], and immature or partially matured DC are most active [13 , 15 ]. Most DC found in lymphoid organs in the steady state exhibit an immature or partially mature phenotype in terms of the expression of MHC Class II and costimulatory molecules and can present antigens in such a manner that they induce tolerance [13 , 15 ]. In particular, B220+ pDC and CD11clowB220–CD45RB+ DC may induce the differentiation of distinct subsets of T regulatory cells (Treg) [16 17 18 ]. pDC, stimulated by virus, have also been shown to induce CD4+ Treg [19 ]. In addition, it was found that murine and human DC expressing the enzyme indoleamine 2,3-dioxygenase (IDO) have potent, suppressive functions [20 , 21 ].

Transcription factors of the IFN regulatory factor (IRF) family participate in the early host response, to pathogens in immunomodulation and hematopoietic differentiation. In addition, family members, including IRF-8, IRF-2, and IRF-4, have been shown to play critical roles in the development and maturation of different DC subsets, further emphasizing their importance to the homeostasis and functions of the immune system [22 ].

IRF-1 was first identified by its ability to induce the transcription of Type I IFN and IFN-inducible genes [23 ]. Extensive analyses of IRF-1 revealed remarkable functional diversity in regulating cellular responses by the differential modulation of distinct sets of genes. The specific effects were dependent on the cell type, its state of differentiation, and the nature of the stimulus. These studies showed that IRF-1 plays key regulatory roles in the development and function of specific cell populations of the immune system [24 ]. Others and we [25 26 27 28 29 ] have shown that IRF-1 affects the differentiation of the lymphoid and myeloid lineages. IRF-1-deficient (IRF-1–/–) mice have also been found to exhibit marked impairment of CD8+ T and NK cell maturation, impaired IL-12 production, and exclusive Th2 differentiation associated with defects in Th1 responses [25 26 27 ]. As a result, these mice are highly susceptible to a number of infectious agents including Leishmania major and Lymphocytic choriomeningitis virus, of which effective host control is associated with a Th1 response in normal mice [26 ]. Conversely, IRF-1–/– mice exhibit significantly reduced susceptibility to autoimmune diseases including Type II collagen-induced arthritis, experimental autoimmune encephalomyelitis, and diabetes [30 31 32 ]. Moreover, it has been shown that IRF-1–/– mice infected with Helicobacter pylori are resistant to gastritis as a result of the lack of an adaptive immune response [33 ]. These observations suggest that IRF-1 plays a role in regulating inflammation and autoimmunity, processes finely modulated by DC.

In the present study, we address the role of IRF-1 in the differentiation and functional maturation of DC and show that IRF-1–/– mice exhibit a prevalence of DC subsets with an immature and tolerogenic phenotype, which were unable to undergo full maturation even following treatment with strong activation stimuli. These findings define a critical role for IRF-1 in DC activity and highlight IRF-1 as a new target for the control of diseases associated with immune dysfunction.


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MATERIALS AND METHODS
 
Mice
The generation of IRF-1–/– mice has been already described [25 ]. IRF-1–/– mice (Jackson Laboratories, Bar Harbor, ME) were kindly provided by Dr. Yutaka Tagaya [National Cancer Institute, National Institutes of Health (NCI, NIH), Bethesda, MD]. The B6.129-IRF1–/– mice, which are on a hybrid C57BL/6 x 129SV background, have been crossed with C57BL/6, and homozygous mice were obtained by backcross. The knockout and wild-type (WT) generations were housed in a specific pathogen-free facility and used between 8 and 12 weeks of age. The Institutional Animal Care of the Istituto Superiore di Sanità (Rome, Italy) approved procedures. CD1 mice were obtained from Jackson Laboratories.

Isolation of DC, CD4+CD25+ Treg cells, and CD4+ and CD8+ T cells
DC were isolated from lymphoid organs using the method described by Schiavoni et al. [34 ]. Briefly, after enzymatic digestion of tissues, released cells were washed, counted, and subjected to Nycodenz gradient. The low-density fraction containing DC was collected, washed, and used directly for phenotypic analysis or further incubated with anti-CD11c MicroBeads (Miltenyi Biotec, Auburn, CA). The positive fraction was recovered using a MACS separation column and checked on a FACSsort® (Becton Dickinson, San Jose, CA) for purity. The cells obtained were routinely >95% CD11c+. For further cell sorting, CD11c+-selected cells were stained with rat antimouse, FITC-conjugated CD11c (HL3), PE-conjugated CD8{alpha} (53-6.7), and PE-Cy5-conjugated CD45R/B220 (RA3-6B2; all from BD PharMingen, San Diego, CA). Three different populations expressing CD11chighCD8{alpha}+, CD11chighCD8{alpha}–, and CD11clowCD45R/B220+ were purified simultaneously by sorting using the FACSAria (Becton Dickinson) equipped with three air-cooled and solid-state lasers (488 nm, 633 nm, and 407 nm). Cells labeled with fluorochrome-conjugated isotypic antibodies (BD PharMingen) were used to gate nonspecific fluorescence signals, and dead cells were excluded on the basis of propidium iodide (5 µg/mL, Sigma-Aldrich, St. Louis, MO) fluorescence intensity. The purity of DC preparations was always between 95% and 99%. Twenty percent to 30% were CD8{alpha}+, and 70–80% were CD8{alpha}–.

Lymphocyte pellets were collected, washed, and used for isolation of CD4+CD25+ T cells from spleens of CD1 mice. To this end, cells were incubated with a cocktail of lineage-specific, biotin-conjugated antibodies against CD8 (Ly-2), CD11b (membrane-activated complex 1), CD45R (B220), CD49b (DX5), and Ter-119 in combination with antibiotin MicroBeads. Subsequently, the pre-enriched, negatively selected CD4+ T cells were positively selected with CD25-PE and anti-PE MicroBeads (Miltenyi Biotec). Starting from 500 x 106 lymphocytes, 2–4 x 106 CD4+CD25+ cells were recovered with a purity ranging from 88% to 96%. CD4+CD25– T cells were also collected, and purity ranged from 95% to 99%. Unselected, splenic lymphocytes were used as a source of total T cells. CD4+ and CD8+ T cells were isolated by magnetic-activated sorting (Miltenyi Biotec) and were used as responder cells in MLR assays. The cells were checked on a FACSsort® for purity and were routinely >95% CD4+ and CD8+.

DC cultures from bone marrow (BM)
BM cells were isolated by flushing femurs with PBS as described previously by Brasel et al. [35 ]. Briefly, BM mononuclear cells were cultured at 1 x 106 /mL in IMDM supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, 10 mM Hepes, 1 mM Na pyruvate, 1 mM nonessential amino acids (NEA), and antibiotics (all from BioWhittaker, Walkersville, MD), along with 100 ng/mL recombinant human fetal liver tyrosine kinase 3 ligand (Flt3L) for 9 days (PeproTech, Rocky Hill, NJ). Fresh factors were added to the culture medium every 48 h of culture.

mAb and flow cytometry
For phenotypical analyses, the following mAb (all from BD PharMingen) were used: anti-CD8{alpha} (53-6.7), PE- or FITC-labeled; anti-CD45R/B220 (RA3-6B2), PE- or FITC-labeled; anti-plasmacytoid dentritic cell antigen (PDCA)-1-PE; anti-CD40-biotin (HM40-3); anti-CD80-biotin (16-10A1); anti-CD86-biotin (GL1); anti-MHC class II molecule (I-A)d/I-Ed-biotin (2G9); anti-Ly6C-biotin (AL-21); anti-CD45RB-biotin (16A); anti-CD45RA-biotin (14.8); anti-CD4-FITC (H129.19); anti-CD25-PE (PC61); and anti-CD11c (HL3), which was used in PE-, FITC-, or biotin-conjugated form. Biotinylated mAb were detected with streptavidin-Red 670 (Life Technologies, Gaithersburg, MD). Anti-PDCA-1-PE was from Miltenyi Biotec. Stained cells were analyzed on a FACSsort® flow cytometer (Becton Dickinson). Viable cells were selected for analysis based on forward- and side-scatter properties.

MLR
For MLR, increasing numbers of CD11c+ DC, freshly isolated from WT or IRF-1–/– mice (1.25x104–105/well), were seeded in triplicates in 96-well plates and incubated with 105 cells/well magnetically sorted CD4+, CD8+, or total T splenic lymphocytes isolated from allogeneic CD1 mice in 200 µl RPMI-1640 medium (BioWhittaker), supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, 10 mM Hepes, 1 mM Na pyruvate, 1 mM NEA, 100 U/ml penicillin, and 100 µg/ml streptomycin for 3 days and then pulsed with 1 µCi [3H]-thymidine (Amersham Pharmacia Biotech, UK) for the last 16 h of culture. For suppression assays, fixed numbers of CD11c+ DC, freshly isolated from WT or IRF-1–/– mice (105/well), were seeded in triplicates in 96-well plates and incubated with 2.5 x 104–105 cells/well magnetically sorted CD4+CD25+ with 105 cells/well CD4+CD25– T cells as targets in the presence of 0.5 µg/ml-purified, soluble antimouse CD3 (145-2C11; BD PharMingen) in 96-well, round-bottom, microtiter plates. Neutralizing purified antimouse IL-10 antibodies (JES5-16E3) or an isotype antibody (BD PharMingen) were added to the culture medium at 2.5 µg/ml concentration. Plates were incubated for 5 days and pulsed with 1 µCi [3H]-thymidine (Amersham Pharmacia Biotech.) for the last 16 h of culture.

In vitro stimulation of DC
Magnetically sorted, splenic DC (sDC; >97% CD11c+) were infected with Newcastle disease virus [NDV; 576 hemagglutinin (HA)/ml] for 1 h at 37°C, 5% CO2, then washed, and cultured at 106 cell/ml for different times. In parallel, CD11c+ DC were treated with 0.5 µg/ml LPS (Sigma-Aldrich), cultured for different times in IMDM medium, supplemented with 10% heat-inactivated FCS and antibiotics, or left untreated in the same culture medium. The cells were then double-stained for CD8{alpha} and I-A and analyzed by flow cytometry as indicated above or collected for RT-PCR analysis (see below).

IFN bioassay and cytokine assays
Culture supernatants of DC stimulated as described above were harvested and assayed for IFN Type I biological activity by measuring their ability to confer resistance to encephalomyelocarditis virus infection upon L929 cells as described previously [34 ]. ELISAs were used to assay for IL-12p40 and IL-10 in the same supernatants using the Quantikine ELISA kits (R&D Systems, Minneapolis, MN).

In vivo infection
Mice were anesthetized and inoculated i.v. with 128 HA NDV. Twenty-four hours after infection, mice were anesthetized and then were bled and killed by CO2 inhalation. Spleens were collected and treated for the DC enrichment by the Nycodenz gradient, and then cells were analyzed by FACS analysis. Serum was prepared from whole blood by centrifugation at 6000 rpm for 30 min at 4°C.

RT-PCR and analysis of amplified products
Total RNA was extracted from 0.5–2 x 106 freshly isolated or in vitro-treated, magnetically purified CD11c+ sDC by using the Miniprep total RNA purification kit (Qiagen, Valencia, CA). RNA was DNase I-digested (Roche, East Sussex, UK) and reverse-transcribed as described previously [34 ]. Nonsaturating PCR was performed on 2 µl each cDNA sample using specific primer pairs; β-actin RT-PCRs were run to normalize the levels of mRNA in the samples. The primers used and PCR conditions were: TLR3 5'-TCGGATTCTTGGTTTCAAGG-3', 5'-CTTGCTGAACTGCGTGATGT-3', 56°C, 35 cycles; TLR4 5'-AGTGGGTCAAGGAACAGAAGCA-3', 5'-CTTTACCAGCTCATTTCTCACC-3', 60°C, 35 cycles; IL-12p40 5'-AACTGGCGTTGGAAGCACGG-3', 5'-GAACACATGCCCACTTGCTG-3', 56°C, 30 cycles; IL-15 5'-CATATGGAATCCAACTGGATAGATGTAAGATA-3', 5'-CATATGCTCGAGGGACGTGTTGATGAACAT-3', 56°C, 30 cycles; β-actin 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', 5'-CTAGAAGCATTGCGGTGGAGCATGGAGGG-3', 67°C, 24 cycles [34 ]; IDO 5'-GAAGGATCCTTGAAGACCAC-3', 5'-GAAGTCGCGATTTCCACCAA-3', 57°C, 30 cycles [36 ]; IL-4 5'-ATGGGTCTCAACCCCCAGCTAGT-3', 5'-GCTCTTTAGGCTTTCCAGGAAGTC-3', 63°C, 30 cycles; IL-10 5'-TCAAACAAAGGACCAGCTGGACAACATACTG-3', 5'-CTGTCTAGGTCCTGGAGTCCAGCAGACTCA-3', 67°C, 30 cycles; IFN-{gamma} 5'-CATGAAAATCCTGCAGAGCCAG-3', 5'-TGCTGGCAGAATTATTCTTATTGG-3', 57°C, 30 cycles; TGF-β 5'-CTCCCACTCCCGTGGCTTCTAG-3', 5'-GTTCCACATGTTGCTCCACACTTG-3', 62°C, 30 cycles; TNF-{alpha} 5'-CCACGTCGTAGCAAACCACC-3', 5'-AAGTACTTGGGCAGATTGACCTC-3', 60°C, 30 cycles [37 ]; TLR9 5'-CCGCAAGACTCTATTTGTGCTGG-3', 5'-TGTCCCTAGTCAGGGCTGTACTCAG-3', 62°C, 30 cycles; TLR2 5'-TCTAAAGTCGATCCGCGACAT-3', 5'-TACCCAGCTCGCTCACTACGT-3', 58°C, 30 cycles; TLR7 5'-TTCCGATACGATGAATATGCACG-3', 5'-TGAGTTTGTCCAGAAGCCGTAAT-3', 56°C, 30 cycles [38 ]; IRF7 5'-AAACCATAGAGGCACCCAAG-3', 5'-TTGGGAGTTGGGATTCTGAGTCAAGGC-3', 60°C, 30 cycles [39 ]; IRF1 5'-ACAAAGCAGGAGAAAAAGAGCCAG-3', 5'- TTCCTGGTGAGGGGTGGCAGCATC-3', 68°C, 35 cycles.

Statistical analysis
Student’s t-tests were used to calculate differences between the groups. Differences in P values of 0.05 or less were considered significant.


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RESULTS
 
IRF-1–/– mice are characterized by a predominance of pDC with tolerogenic features
We characterized DC from spleen, thymus, skin-draining lymph nodes (LN), and mesenteric LN of IRF-1–/– and WT mice. The total numbers of DC-enriched fraction obtained from spleens, LN, and thymus were comparable for WT and IRF-1–/– mice. Staining for CD11c allowed us to gate on CD11chigh cDC and CD11clow pDC. As shown in Figure 1 , the frequency of the entire CD11c+ population was decreased in spleens and unaffected in other lymphoid organs of IRF1–/– mice as compared with control littermates. Quantitative analysis of the CD11chigh and CD11clow DC subsets revealed that the former population was decreased of twofolds, and the latter was markedly increased in IRF-1–/– mice (Fig. 1) , indicating that the development of DC was skewed significantly toward the pDC subset in these mice. As CD11clow pDC comprise different subsets characterized by CD11clowB220+CD45RAhigh and CD11clowB220–CD45RBhigh phenotype [16 , 17 ] and endowed with tolerogenic potential, we assessed the distribution of these subsets in IRF-1–/– mice. sDC-enriched cells were first double-stained for CD11c and B220 or PDCA-1. As shown in Figure 2A , the frequency of CD11clow cells in IRF-1–/– mice (gate R1) was increased significantly over the levels found in WT mice. In addition, PDCA-1-specific staining was higher in R1-gated pDC from IRF-1–/– (filled histogram) than WT mice (open histogram). It is notable that CD11clowB220+ and CD11clowB220– populations were highly represented in the knockouts. We then extended the characterization of these subsets using three-color flow cytometry to analyze the expression of CD11c, B220, CD45RB, CD45RA, Ly6C, and CD8{alpha}. It is interesting that cells with a CD11clowB220+ DC plasmacytoid phenotype expressing CD45RA, CD45RB, Ly6C, and CD8{alpha} were present at significantly higher levels in IRF-1–/– as compared with WT mice (Fig. 2B) . Furthermore, the frequency of CD11clowB220– pDC-expressing CD45RB was higher in IRF-1–/– than WT mice (Fig. 2B) . To determine whether the enhanced representation of the pDC subset was an indirect effect of the loss of IRF-1 in the periphery or was instead a cell-intrinsic feature at the progenitor level, we used a Flt3L-based culture system that supports the generation of pDC from BM cells. We found that under these experimental conditions, IRF-1–/– progenitors gave rise to significantly higher percentages of CD11clowB220+ pDC than WT progenitors (34.3% vs. 21.3%, respectively; Fig. 2C ). These data indicate that IRF-1 plays a role in governing the development of pDC at the level of BM progenitors.


Figure 1
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  		Figure 1. IRF-1–/– mice exhibit an altered DC compartment. DC were isolated from spleen, thymus, and skin-draining and mesenteric LN from IRF-1–/– and WT mice. Nycodenz-enriched DC were stained for a CD11c marker. The dot plot analysis shows the percentage of cDC (gate R1) and pDC (gate R2) analyzed for CD11 positivity and by forward/side-scatter (FSC) properties. Data are representative of one experiment of three performed.


Figure 2
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  		Figure 2. IRF-1–/– mice show increased numbers of pDC with tolerogenic phenotype. (A and B) Spleens from IRF-1–/– and WT mice were enriched for DC by Nycodenz density-gradient centrifugation, as described in Materials and Methods. The low-density cell fraction was double-stained for CD11c and PDCA-1 or triple-stained for CD11c, B220, and alternatively, CD45RB, CD45RA, Ly6C, or CD8{alpha} markers and then analyzed by flow cytometry for detection of pDC. A region (R1 gate) was drawn on pDC-expressing CD11c at low levels and analyzed further for the expression of the PDCA-1 marker or the B220 marker and forward/side-scatter properties. Histograms show PDCA-1-specific staining in R1-gated pDC in IRF-1–/– (filled) or WT (open) mice (A). Bars show specific staining for the indicated markers in the B220+CD11clow and B220–CD11clow-gated populations in IRF-1–/– (shaded bars) and WT (open bars) mice (B). (C) BM cells from IRF-1–/– or WT mice were cultured with Flt3L for 9 days and then were double-stained for CD11c and B220 expression and analyzed by flow cytometry. Results are representative of one from at least five independent experiments.

Decreased numbers and immature phenotype of cDC in IRF-1–/– mice
We next analyzed the distribution of the CD11chighCD8{alpha}– and CD11chighCD8{alpha}+ subsets of cDC in lymphoid organs of IRF-1–/– and control mice. Compared with WT mice, IRF-1–/– mice exhibited a marked reduction of CD11chighCD8{alpha}+ DC in all tissues tested (Fig. 3A and data not shown), whereas the percentage of the CD11chighCD8{alpha}– DC subset was slightly, but significantly, decreased. To further analyze the maturation and the activation phenotype of CD8{alpha}+ and CD8{alpha}– subsets of sDC from IRF-1–/– mice, we performed three-color flow cytometric analysis using antibodies to CD11c and CD8{alpha}, together with antibodies to the costimulatory antigens CD40, CD80, and CD86 and the surface MHC Class II molecule (IA). These analyses showed that CD11chighCD8{alpha}+ DC from IRF-1–/– mice had markedly reduced expression of IA and significantly lower levels of CD40, CD80, and CD86 (Fig. 3B) . In addition, the IRF-1–/– CD11chighCD8{alpha}– DC subset displayed a moderate reduction in the expression of all markers tested when compared with WT DC (Fig. 3B) .


Figure 3
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  		Figure 3. Defective development and maturation of CD11chigh sDC in IRF-1–/– mice. (A) The low-density cell fraction obtained from spleens was double-stained for CD11c and CD8{alpha} expression. The dot plots show the percentage of CD8{alpha}+ (R2 gate) and CD8{alpha}– (R3 gate) CD11c+ DC subsets. (B) DC were additionally stained for MHC Class II (I-A), CD40, CD80, or CD86 surface markers. Histograms show specific staining for the indicated antigens in CD8{alpha}+ CD11c+ (upper)- and CD8{alpha}– CD11c+ (lower)-gated populations in IRF-1–/– (filled histograms) and WT (open histograms) mice. Results are representative of at least five independent experiments. (C) Bars show the percentage of CD11chigh DC double-stained for CD4 and CD8{alpha} in IRF-1–/– (shaded bars) and WT (open bars) mice. The means are from four independent experiments ± SD (*, P<0.05; **, P<0.01). (D) Purification of CD8{alpha}+ CD11chigh, CD8{alpha}– CD11chigh, and B220+CD11clow. CD11c+ cells were selected from spleens of WT mice and CD8{alpha}+ CD11chigh, CD8{alpha}– CD11chigh, and B220+CD11clow were purified simultaneously by sorting using a FACSAria as described in Materials and Methods. Representative FACS diagrams indicate the percentages of the different cell populations obtained after each step of purification. (E) mRNA was extracted from sorted populations and RT-PCR performed using IRF-1 and β-actin-specific probes. Results are representative of two independent experiments.

CD11chighCD8{alpha}– DC can be subdivided further into CD4–CD8{alpha}– and CD4+CD8{alpha}– subsets, and it has been suggested that the CD4–CD8{alpha}– DC subset might constitute an activated or more differentiated population than the CD4+CD8{alpha}– cDC [2 , 40 ]. Analysis of the distribution of these two subsets in IRF-1–/– and WT mice showed that the CD4+CD8{alpha}– subset was present at higher levels in the knockout mice (Fig. 3C) . In contrast, the CD4–CD8{alpha}– subset was present at a higher frequency in WT mice. These results indicated that although the total CD11chighCD8{alpha}– DC population was only affected slightly by the expression of IRF-1, these cells displayed a less mature phenotype. Taken together, these results suggest that cDC in IRF-1–/– mice have an immature phenotype and a profound alteration in the distribution of their subsets characterized by a marked decrease in CD8{alpha}+ cDC. The analysis of the CD11chighCD8{alpha}+and CD11chighCD8{alpha}– subsets in GM-CSF-cultured BM resembled the results obtained by the analysis of the spleen (data not shown).

To study IRF-1 expression by different DC subsets, CD11chighCD8{alpha}+, CD11chighCD8{alpha}–, and CD11clowB220+ cells were isolated by FACS sorting; cDNA was prepared and subjected to RT-PCR. As shown in Figure 3D , IRF-1 message was clearly present in CD8{alpha}– and CD8{alpha}+ but was absent in CD11clowB220+. This analysis indicated a selective expression of IRF-1 in DC subsets and supports the described, altered distribution of DC subsets in knockout mice, in particular, the significant increase in CD11clowB220+ (Figs. 2 and 3) .

sDC from IRF-1–/– mice express significantly increased amounts of tolerogenic cytokines
The results described above indicated that a deficiency in IRF-1 results in a marked decrease in cDC characterized by an immature phenotype and in the dominance of pDC subsets endowed with tolerogenic potential. We therefore examined whether these features of IRF-1–/– DC were reflected in a specific cytokine-expression profile. As shown in Figure 4A , sDC from IRF-1–/– mice expressed significantly higher levels of transcripts for the anti-inflammatory and tolerogenic cytokines TGF-β and IL-10 and lower levels of transcripts for the proinflammatory cytokines TNF-{alpha}, IFN-{gamma}, IL-12 p40, and IL-15 than cells from WT mice. Moreover, sDC from IRF-1–/– mice expressed IL-4 at high levels, a finding consistent with the Th2-polarized phenotype of these mice described previously [41 ]. It is interesting that expression of the tryptophan-degrading enzyme, IDO, was increased significantly in IRF-1–/– sDC (Fig. 4B) . This enzyme has been shown to be immunosuppressive in certain settings and tolerogenic in others, and its expression is positively regulated by IL-10 [20 ].


Figure 4
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  		Figure 4. Expression of cytokines and TLR in sDC from IRF-1–/– mice. Total RNA was extracted from freshly sorted CD11c+ DC from pooled spleens of IRF-1–/– and WT mice and assayed by nonsaturating RT-PCR for the expression of the indicated cytokines (A), IDO (B), or TLRs (C) and β-actin as control. Representative data from one experiment out of three performed are shown.

TLRs trigger DC maturation and inflammatory responses through the recognition of a broad range of microbial compounds [42 , 43 ]. Studies of TLR expression showed that IRF-1–/– sDC exhibited extremely low levels of TLR3 and TLR4 as compared with WT cells (Fig. 4C) . It is interesting that IRF-1–/– DC expressed higher levels of TLR9, which is known to be associated primarily with pDC [44 ]. No alterations in the expression of TLR2 or TLR7 were observed.

sDC from IRF-1–/– mice retain tolerogenic features following infection with NDV
The altered distribution of DC subsets in IRF-1–/– mice and their bias toward immature and tolerogenic characteristics prompted us to test whether strong stimuli for DC maturation, such as NDV or LPS, might restore immunogenic features in these cells. As shown in Figure 5A (gate R1), infection of sDC from WT mice with NDV induced the generation of a substantial number of I-AhighCD8{alpha}+ cells, 35.6% of all cells. In contrast, only 12.1% I-AhighCD8{alpha}+ cells were detected following infection of sDC from IRF-1–/– mice. Similarly, a reduced percentage of CD86+ CD8{alpha}+ cells was expressed by infected sDC from IRF-1–/– mice as compared with infected WT DC (data not shown).


Figure 5
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  		Figure 5. sDC from IRF-1–/– mice show reduced activation after NDV and LPS stimulation in vitro. Magnetically purified CD11c+ DC from spleens of IRF-1–/– and WT mice were left untreated and infected with NDV or treated with LPS as described in Materials and Methods. (A) Control (untreated), NDV-infected, or LPS-treated cells were double-stained for CD8{alpha} and MHC Class II (I-A) and analyzed by flow cytometry. The R1 gate was drawn on highly activated CD8 {alpha}highI-Ahigh DC, whereas the R2 gate indicated poorly activated CD8{alpha}lowI-Alow DC. (B) Total RNA was extracted from sDC of IRF-1–/– and WT mice untreated [control (CTL)], infected with NDV, or treated with LPS. IL-10, IL-12 p40, IRF-7, and β-actin mRNA levels were detected by RT-PCR. Data are representative of three independent experiments. (C) sDC from IRF-1–/– and WT mice were infected with NDV or treated with LPS as described in Materials and Methods. At the indicated time-points, supernatants were harvested, and IL-12p40 and IL-10 production was measured with each specific ELISA kit. The same supernatants were also assayed for IFN-{alpha}/β bioactivity, as described in Materials and Methods. Data are expressed as means ± SD of culture triplicates and are representative of two experiments.

It is interesting that the frequency of I-AlowCD8{alpha}– DC was increased significantly in IRF-1–/– cultures as compared with those from WT mice (Fig. 5A , gate R2). The persistence of an immature phenotype of IRF-1–/– DC was also observed following LPS treatment. In fact, the increase of highly activated I-AhighCD8{alpha}+ cells in stimulated cultures from WT mice was not observed in cultures of IRF-1–/– DC (Fig. 5A , gate R1). In addition, expression of PDCA-1 and Ly6C was increased significantly on IRF-1–/– DC infected with NDV (data not shown).

To understand the basis for the failure of IRF-1–/– DC to undergo full maturation following infection with NDV or treatment with LPS, we evaluated stimulated sDC for expression of IL-12p40 and IL-10, representative of proinflammatory and anti-inflammatory responses, respectively. As shown in Figure 5B , IRF-1–/– DC, at variance with WT DC, infected with NDV or stimulated with LPS, did not up-modulate IL-12p40. In contrast, a clear-cut increase in IL-10 expression was present in stimulated IRF-1–/– DC as compared with WT cultures, which exhibited only mild increases in IL-10 expression. It is interesting that we found that IRF-7 was expressed at the same levels in untreated and treated DC from IRF-1–/– and WT mice (Fig. 5B) . We also measured by ELISA the IL-12p40 and IL-10 proteins secreted in the medium on a kinetic base. As shown in Figure 5C , high levels of IL-12p40 were induced in response to LPS in WT cells. Conversely, IL-12p40 secretion was decreased dramatically in sDC from IRF-1–/– mice stimulated with LPS at any time-point. Conversely, production of IL-10 was enhanced significantly in IRF-1–/– sDC, as compared with WT cultures infected with NDV or stimulated with LPS at any time-point. Biological assay of IFN Type I production in the same cultures indicated that the magnitude of the IFN response to infection with NDV was at any time-point increased in IRF-1–/– as compared with WT cultures, the increase being significant at early time-points and remaining constant over the time. To gain further insight into the immature and tolerogenic characteristics of IRF-1–/– DC, we investigated the effects of infecting mice with NDV. DC isolated from mice injected i.v. with NDV 24 h earlier were stained for CD11c and B220. As shown in Figure 6A (gate R1), infection with NDV induced a marked change in the features of IRF-1–/– CD11c+ DC when compared with their WT counterparts. Studies of CD11chigh DC subsets from knockout and WT mice revealed that cells from IRF-1–/– mice were greatly impaired in their ability to expand the population of CD8{alpha}+ DC following infection with NDV (Fig. 6B , gate R2). It is important that this lack of responsiveness was associated with a clear-cut increase in the percentages of PDCA-1+ CD11clowCD8{alpha}low DC (R4-gated) from IRF-1–/– mice, confirming the strong propensity of these cells to adopt pDC features (Fig. 6C) . Similarly, expression of the B220 marker was significantly higher on IRF-1–/– DC than WT counterparts (Fig. 6C) .


Figure 6
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  		Figure 6. NDV in vivo infection skews sDC differentiation toward a tolerogenic phenotype. IRF-1–/– and WT mice were infected with NDV, as described in Materials and Methods. After 24 h, Nycodenz-enriched sDC from pooled spleens were triple-stained for CD11c, B220, and alternatively, PDCA-1 or CD8{alpha} markers and then analyzed by flow cytometry. (A) sDC were analyzed for CD11c positivity and by forward/side-scatter properties from NDV-infected IRF-1–/– and WT mice. (B) The dot plots show the percentage of CD8{alpha}+ (R2 gate) and CD8{alpha}– (R3 gate) CD11c+ DC subsets in pooled spleens from uninfected and NDV-infected IRF-1–/– and WT mice. (C) R4-gated sDC were analyzed for PDCA-1 positivity and forward/side-scatter properties or for B220 marker. Filled and open histograms show specific staining for B220 of sDC from IRF-1–/– and WT mice, respectively. (D) IFN-{alpha}/β levels assayed in the sera of IRF-1–/– and WT mice after 8 h of NDV infection. Data are expressed as means ± SD of culture triplicates and are representative of two experiments.

Of note, infection of IRF-1–/– and WT mice with NDV resulted in comparable levels of serum IFN (Fig. 6D) , suggesting the involvement of distinct pathways in DC maturation and IFN production.

sDC from IRF-1–/– mice are poised to induce a suppressive response
To determine T-stimulatory function of DC from IRF-1–/– mice, we performed MLR and suppression assays. The proliferative response of allogeneic T cells cultured with different numbers of WT and IRF-1–/– CD11c+ DC (Fig. 7A ) indicated that DC from IRF-1–/– mice exhibited a decreased ability to stimulate the proliferation of allogeneic CD4+, CD8+, and total T cells as compared with WT cells. In suppression assays using naïve, allogeneic CD4+CD25– T cells as target T cells, DC isolated from WT or IRF-1–/– mice were cultured for 5 days with increasing numbers of allogeneic CD4+CD25+ Treg purified from CD1 mice. As shown in Figure 7B , Treg cocultured with DC from IRF-1–/– mice had greater suppressive activity than the same cells cocultured with DC from WT mice. To determine whether elevated expression of IL-10 might contribute to the enhanced, suppressive activity of IRF-1–/– DC, saturating concentrations of neutralizing antibodies to IL-10 were added to the culture medium. In these cultures, the suppressive capacity of Treg cocultured with IRF-1–/– DC was reduced dramatically, and that of Treg cocultured with WT DC was affected to a lesser extent. This indicated that IL-10 secreted by IRF-1–/– DC was mainly responsible for the increased suppressive activity of DC from the knockout mice.


Figure 7
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  		Figure 7. sDC from IRF-1–/– mice exhibit an impaired T cell allostimolatory activity and induce suppression activity in allogeneic CD4+CD25+ Treg cells. (A) Stimulatory activity of CD11c+ sDC from IRF-1–/– and WT mice was analyzed on CD4+, CD8+, and total lymphocytes by MLR assay. Data are representative of one experiment out of three performed in triplicates. (B) CD11c+ sDC from IRF-1–/– and WT mice were cultured with a fixed amount of responder T cells and different concentrations of CD4+CD25+ Treg cells. An isotypic antibody (IgG) or antibodies anti-IL-10 were added at saturating concentrations as described in Materials and Methods. Data are expressed as means ± SD (*, P<0.05; **, P<0.001), representative of one experiment out of three, performed in triplicates.


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DISCUSSION
 
DC are recognized for their ability to induce not only strong adaptive immunity but also immunologic unresponsiveness and tolerance [1 , 13 , 45 ]. Whether suppression or stimulation of the immune response predominates is determined by the conditions used to induce maturation, the maturation signals themselves, the state of DC activation, and their cellular origins [46 ].

In the present study, we identified IRF-1 as a factor critical for differentiation and full maturation of DC and as a key negative regulator of DC subsets characterized by tolerogenic potential. Several lines of evidence support these conclusions: the profound reduction in the number and maturation potential of cDC in all lymphoid organs of IRF-1–/– mice; the substantial increase in pDC and DC endowed with a tolerogenic phenotype in the spleen of knockout mice; the functional alteration in DC from IRF-1–/– mice, which prevent them from maturing in response to viral or bacterial stimuli; and the ability of DC from IRF-1–/– mice to express increased levels of immunosuppressive cytokines and to exhibit a heightened ability to elicit IL-10-mediated, suppressive activity from allogeneic Treg.

It has been shown that resting but fully differentiated DC can induce tolerance, whereas activated, mature DC induce T cell immunity [47 ]. Moreover, DC, which are in an immature state, exhibit tolerogenic features [48 49 50 51 ]. We found that the CD11chigh DC population and specifically, the CD8{alpha}+ subset were reduced significantly in central and peripheral lymphoid organs of IRF-1–/– mice. It is remarkable that limitations in the activation of CD8{alpha}+ DC were associated with the decreased expression of CD80, CD86, and MHC Class II. Although the absence of IRF-1 affected the number and activation status of CD8{alpha}– DC to a lesser extent than for CD8{alpha}+ DC, it significantly influenced the frequencies of CD4+ and CD4– cells in this subset. As it has been suggested that these subtypes may reflect different maturational stages [2 , 40 ], the prevalence of CD4+ CD8{alpha}– DC in IRF-1–/– mice is likely to be associated with a specific role of IRF-1 in controlling DC maturation. In contrast to cDC, the frequency of CD11clow pDC was increased greatly in all lymphoid organs of IRF-1–/– mice with a preponderance of immature CD11clowB220+CD45RAhigh cells in the spleen. It is interesting that we also observed a significant increase of the CD11clowB220–CD45RBhigh cells, a pDC subset that is endowed with a potent, tolerogenic potential [17 ]. Of note, the effects of IRF-1 deficiency on pDC development were determined at the level of BM progenitors, as reflected by the significant increase of pDC from in vitro BM cultures generated with Flt3L.

The absence of proinflammatory cytokines is considered one of the critical factors in determining the tolerogenic potential of DC [13 , 15 ]. It is striking that the cytokine profile of DC from IRF-1–/– mice is fully in keeping with their tolerogenic characteristics. Specifically, IRF-1–/– DC expressed lower levels of the proinflammatory cytokines IFN-{gamma}, IL-15, IL-12 p40, and TNF-{alpha} as compared with DC from WT mice. By contrast, DC from IRF-1–/– mice expressed high levels of the tolerogenic cytokines IL-10 and TGF-β. It is important that other studies have shown that autocrine production of IL-10 is a crucial factor in modulating the tolerogenic function of DC [20 ]. An additional feature likely to be associated with tolerogenic potential of IRF-1–/– DC is the high expression of the inducible enzyme IDO. This enzyme has been described recently as a crucial mediator of suppression of T cell proliferation by tolerogenic DC, and it is interesting that IL-10 sustains the IDO expression during DC maturation [51 ].

The potent, regulatory effects exerted by IRF-1 on the differentiation and maturation of DC were also evident in the responses of DC to stimulation with NDV and LPS, which failed to induce full phenotypic activation of DC from IRF-1–/– mice. It has been reported that under conditions of infection or inflammation, the induction of tolerance or immunity by DC depends on whether mature DC have been activated by "danger signals" that include viral or bacterial products [52 ]. We found that following infection with NDV, a potent inducer of DC maturation, IRF-1–/– sDC were driven to acquire features of pDC, including expression of B220 and a CD11clow phenotype. They also retained an immature phenotype characterized by a reduced capacity to generate mature CD11chighI-AhighCD8{alpha}+ cells. Moreover, following NDV infection and to a lesser extent, exposure to LPS, sDC from IRF-1–/– mice strongly up-regulated IL-10 production but were not able to up-modulate IL-12 secretion. It is intriguing that IRF-1–/– DC also exhibited almost undetectable levels of TLR3 and TLR4, receptors engaged by dsRNA and LPS, respectively, but they displayed high levels of TLR9, a receptor expressed prominently by pDC [43 , 44 ]. Recently, it has been suggested that TLR3 promotes cross-priming of viral antigens by ensuring the maturation of immature mouse CD8{alpha}+ DC [53 ]. It is, therefore, tempting to speculate that the impaired expression of TLR3 in IRF-1–/– DC may be related to the inability of IRF-1–/– CD8{alpha}+ DC to mature after exposure to viral stimuli. Conversely, IRF-1–/– DC responded to NDV infection by differentiating into DC with tolerogenic features but retained the ability to produce substantial levels of Type I IFN. In this respect, analysis of pDC, deficient in each TLR, revealed that TLR7 plays an important role primarily in NDV recognition, although TLR9 also participates partially in this process [54 ]. It is important that TLR7- and TLR9-mediated Type I IFN production in pDC is dependent on expression of IRF-7, which was up-modulated to WT levels in virus-stimulated IRF-1–/– DC. Moreover, it was found that retinoic acid inducible gene I, but not the TLR system, is essential for induction of Type I IFN after NDV infection in fibroblasts and cDC [54 ]. Therefore, we suggest that pathways alternative to those activated by TLR3 may play a crucial role in NDV-induced expression of Type I IFN in IRF-1–/– DC.

One of the most telling findings of this study is that the tolerogenic characteristics of IRF-1–/– DC reflect a profound alteration in their functional state. Indeed, IRF-1–/– DC were less effective in stimulating proliferation of allogeneic CD4+ and CD8+ T cells. In this respect, increased IDO expression, as seen in IRF-1–/– DC, has been related to the inability of DC to stimulate T cell proliferation in a MLR [20 ]. It is remarkable that the decreased ability of IRF-1–/– DC to induce proliferation of allogeneic T cells was mirrored by a greater ability to induce a suppressive response from Treg. This suppressive activity was largely mediated by IL-10, produced by DC themselves, or induced in Treg cells [55 ]. Although our experiments failed to clarify this point, we believe that the high production of IL-10 in IRF-1–/– DC plays a crucial role in determining the suppressive features of these cells directly and/or modifying the cytokine milieu or repressing the sustained IDO expression, especially in IRF-1–/– cultures. In this respect, it is noteworthy that a recent report showed that virus-stimulated pDC are capable of inducing anergic and regulatory properties from CD4+ T cells in a Type I IFN- and IL-10-dependent manner [19 ].

A wealth of information obtained with mice bearing knockouts of different IRF genes indicates that this gene family plays multiple roles in the generation and development of DC through diverse processes. It is interesting that IRFs have common and distinct activities that promote shared as well as DC subset-specific pathways of gene expression [22 ]. The role played by IRF-1 in governing DC differentiation and function appears to be distinct from that of other IRFs.

We suggest that the tolerogenic characteristics of IRF-1–/– DC account not only for the impaired responses of these mice to infectious agents and their reduced susceptibility to induction of autoimmune disorders but also for constitutive, suppressor mechanisms including the maintenance of an immature/anergic DC phenotype and the induction of activated Treg (manuscript in preparation).

Our results, although enhancing our understandings of the Th2 bias exhibited by IRF-1-deficient mice [41 ], unearth a new role for IRF-1 in regulating the tolerogenic features of DC, which are promising tools for immunotherapy of various diseases. The ability to modulate IRF-1 expression in DC may thus open new strategies for shaping immune responses.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Italian AIDS Project, the Italian Ministry of Health, and ISS-NIH Scientific Cooperation agreement to A. B. and F. B., and by a grant from the Italian Association for Cancer Research (Project AIRC n. F89) to F. B. We thank Dr. Yuzaka Tagaya (NCI) for providing IRF-1–/– mice and M. D’Urso and A. M. Pacca for excellent technical assistance with breeding. We thank Roberto Gilardi for artwork and Sabrina Tocchio and Cinzia Gasparrini for editorial assistance.

Received April 5, 2006; revised July 14, 2006; accepted August 8, 2006.


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