Tissue-specific Positive Feedback Requirements for Production of Type I Interferon following Virus Infection^*

Mice

Mice deficient for STAT1 (stat1−/−) or for Rag1 (rag1−/−) and wild type controls on the same inbred background (BALB/c and C57BL6/J) were bred under specific pathogen-free conditions, in accordance with approved protocols at New York University School of Medicine. All mice were 8–12 weeks of age. In some experiments, IFN was induced in vivo by intraperitoneal injection of 100 μg of poly(I-C) per mouse 1 day before collecting organs for protein extraction. Individual whole organ protein extracts were obtained by crushing tissue between frosted glass slides and lysing the resulting released cells in immunoprecipitation lysis buffer (1% Triton X-100, 300 mm NaCl, 50 mm HEPES pH 7.6, 1 mm Na₃VO₄, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml aprotinin and leupeptin).

Enrichment of Splenic Subsets Using Magnetic Beads

Isolated spleens were disrupted by digestion with Liberase CI (1 ml/spleen of 1.67 Wunsch units/ml; Roche Applied Science) and DNase I (0.2 mg/ml; Roche Applied Science) for 30 min at 37 °C. Single cell suspensions were passed through a cell strainer (70 μm), and red blood cells were lysed using red blood cell lysis buffer (155 mm ammonium chloride, 0.1 mm EDTA, 12 mm sodium bicarbonate). Cell subtype enrichment involved anti-CD11c- and anti-CD11b-coupled magnetic beads and positive selection column MS+, according to the manufacturer’s instructions (MACS, Miltenyi Biotec). Positive MACS bead selections typically yielded cells that were greater than 90% pure after one round of selection, as judged by flow cytometry.

Flow Cytometric Analyses

Cell surface staining was carried out in phosphate-buffered saline containing 0.5 mm EDTA unless otherwise stated. Cells were incubated with the following fluorochrome-conjugated monoclonal antibodies: CD11b-fluorescein isothiocyanate, CD11c-phycoerythrin, and IgM-fluorescein isothiocyanate (BD Biosciences). Cells were washed and fixed in 1% paraformaldehyde before analysis.

Hematology

Splenic cell preparations or in vitro differentiated bone marrow (BM) were prepared by Cytospin centrifugation and stained with Wright-Giemsa stain.

Cell Isolation and Culture

BM cells were flushed from femurs and tibiae with RPMI 1640 medium with 25 mm Hepes. Red blood cells were lysed, and nucleated cells were plated in the appropriate medium. FLT3L-differentiated cells were plated at 10⁶/ml (day 0) and expanded in RPMI 1640 containing 1% syngeneic normal mouse serum, sodium pyruvate (1 mm), HEPES (25 mm), β-mercaptoethanol (2 mm), and antibiotics (penicillin-streptomycin; Cellgro), and were grown for 8 days in 24-well plates in the presence of 25 ng/ml FLT3L (Peprotech) and 10 ng/ml stem cell factor (Peprotech) with 80% of the medium replenished on day 5. GM-CSF-differentiated DC cultures were generated essentially as described by Inaba et al. (23). In brief, BM cells were plated at 700,000 cells/ml (day 0) in RPMI 1640 containing 10% fetal calf serum, HEPES (25 mm), sodium pyruvate (1 mm), and antibiotics (penicillin-streptomycin; Cellgro) and supplemented with 40 ng/ml GM-CSF (Peprotech). On day 2, 70% of the medium was replenished. On day 3, non-adherent cells were removed, retaining the loosely adherent DC progenitor cells. The resulting differentiated and expanded cells were collected on day 5. Other cytokines used in some cultures were interleukin-3 (10 ng/ml; Peptrotech) and IFNα/β (500 units/ml; Access Biomedical).

Virus Infections

Mice were infected with influenza A virus (WSN strain) by inhalation, as described previously (12). In brief, 10⁵ plaque-forming units in 50 μl of phosphate-buffered saline were instilled intranasally into each anesthetized wild type or STAT1 mutant mouse, and lungs of infected mice were harvested 1–3 d later. RNA was isolated from disrupted lungs by the TRIzol method, according to the manufacturer’s instructions (Invitrogen).

In vitro differentiated BM-derived cells (1 × 10⁶) were cultured in 50 μl of RPMI 1640 containing 25 mm Hepes and 0.1% bovine serum albumin, either in the presence or in the absence of 50 μl (128 hemaglutination units/ml) of Newcastle disease virus (NDV). After 1 h at 37 °C, cells were plated in medium supplemented with 10% fetal calf serum for 4–12 h, as indicated, and cells were lysed in immunoprecipitation lysis buffer. For RNA analysis, 2 × 10⁶ BM-derived cells were used for each infection, and medium, virus, and reagents were scaled up appropriately.

Antibodies

Immunoblotting was performed using the following polyclonal antibodies: anti-IRF7, anti-IRF3, anti-IRF4, and anti-IRF8 (Zymed Laboratories Inc.) and monoclonal α-tubulin (Sigma).

Quantitative Real-time RT-PCR Analysis

RNA was extracted from at least 2 × 10⁶ BM-derived cells using the RNeasy Minikit (Qiagen). Reverse transcription was performed using 3 μg of total RNA, and the resulting cDNA was used for real-time PCR analysis, using gene-specific primer pairs in the presence of Sybr® Green dye (Molecular Probes, Eugene, OR). Each target transcript was quantified by comparison with a standard curve generated by serial dilution of an abundant sample following normalization to the amount of glyceraldehyde-3-phosphate dehydrogenase or ribosomal protein L32 mRNA. Sequences of real-time PCR primers are available upon request.

Gene Transduction

STAT1-deficient fibroblasts were reconstituted with IRF7 by recombinant retroviral infection, which was performed using Phoenix cells, following the Nolan laboratory protocol. In brief, Phoenix cells, which are 293T cells stably expressing ecotropic packaging proteins, were transfected with retroviral constructs (green fluorescent protein or empty vector retroviruses as controls) in the presence of 25 μm chloroquine using a standard calcium phosphate protocol. 48 h later, supernatants were collected, spun, and used directly for infection of 3T3 cells preplated a day earlier at 1 × 10⁵/6 wells in the presence of 40 μg/ml polybrene.

Effective IFN Expression in Lungs following Infection Requires Intact IFN Signaling

An essential role for type I IFN in resolving respiratory infections and coordinating an appropriate T cell response has been documented previously (12, 13). Importantly, those studies revealed a role for IFN locally at the site of infection to orchestrate the inflammatory response as well as systemically to limit the spread of virus to peripheral tissues. In contrast to wild type animals, IFN-unresponsive mice (STAT1- or IFNAR-deficient) failed to contain influenza virus within the respiratory tract or establish an effective Th1-biased immune response. We investigated whether impaired IFN production at the site of infection due to the absence of positive feedback contributed to the phenotype of STAT1-deficient mice.

Induction of IFN was compared in wild type and STAT1 mutant mice following intranasal influenza viral infection in vivo. Although IFN was readily detected in infected lungs from wild type mice, IFN production was markedly impaired in STAT1-null lungs when compared with the wild type lungs (Fig. 1A, black squares). Additionally, the mutant lungs failed to induce the accumulation of IRF7 message to levels equivalent to wild type controls (Fig. 1A, open triangles). Thus, the inability to support positive feedback in vivo via induction of IRF7 within the respiratory epithelium likely contributed to the failure of STAT1 mutant mice to mount an effective IFN response in response to respiratory infection.

IRF7 Protein Is Inducible in Most Tissues but Constitutively Expressed in Spleen

IRF7 was initially identified as an IFN-inducible transcription factor in hematopoietic cells (24). Given its induction in response to virus infection in lung, we examined its expression in multiple organs. To mimic systemic viral infection, mice were intraperitoneally injected with poly(I-C), a well characterized type I IFN inducer (25), and 1 day after poly(I-C) injection, selected organs were examined for IRF protein expression. Abundance of IRF7 protein increased in response to poly(I-C) injection in most tissues (Fig. 1B).

Interestingly, IRF7 protein was expressed at relatively high levels in thymus and spleen, even in the absence of poly(I-C) treatment. In contrast, IRF3 was expressed in all tissues examined, and its expression was not affected by IFN induction (Fig. 1B, lower panel). Similar results were obtained after infection of mice with negative-strand RNA viruses (data not shown). Thus, like lung, most peripheral tissues required induction to accumulate significant levels of IRF7 protein expression and would likely exhibit positive feedback-dependent production of IFN following viral infection. However, constitutive IRF7 accumulation in lymphoid organs indicated the possibility of a distinct type of response to infection in these tissues.

IFN Feedback Does Not Account for IRF7 Protein Levels in the Spleen

A possible explanation for elevated constitutive levels of IRF7 in spleen and thymus could be the low levels of type I IFN observed in lymphoid organs even in the absence of viral infection (26–28). To examine this possibility, IRF7 protein expression was quantified in multiple organs, including spleens and thymi of stat1−/− mice that are incapable of responding to IFN. Stat1−/− mice expressed levels of IRF7 in spleen and thymus comparable with wild type mice, whereas in other tissues, such as lung, heart, kidney, and skeletal muscle, IRF7 accumulation strictly required STAT1-dependent induction (Fig. 1C and data not shown). Therefore, IFN feedback does not appear to be required for abundant constitutive IRF7 protein expression in lymphoid organs.

Mature T Cells and B Cells Are Not Required for Splenic IRF7 Expression

The mouse spleen is composed largely of B and T cells, together constituting ~90% of splenic cellularity. To determine whether B and T cells were the origin of the abundant resting IRF7 levels observed in spleen, mice deficient for Rag1 protein and therefore lacking mature B and T cells were examined (Fig. 1D). Rag1−/− mouse spleen expressed equivalent levels of IRF7 protein when compared with wild type spleen, indicating that mature B and T cells were not the exclusive source for splenic IRF7 expression.

IRF7 protein expression was also directly measured in lymphocytes. T and B cells from wild type spleens were purified by magnetic bead selection using anti-CD3 and anti-CD19 antibodies, respectively. Little IRF7 protein was detected in lymphocytes not treated with IFN (data not shown), suggesting that splenic IRF7 expression was localized to a non-T non-B splenocyte population.

Kinetics of IFN Production and IRF7 Induction in Fibroblasts and Splenocytes

We examined how constitutive expression of IRF7 in lymphoid tissue impacted the kinetics of IFN production following virus infection. Comparing fibroblasts (inducible IRF7) with splenocytes (constitutive IRF7), we measured the rate of IFN induction and the role of positive feedback in response to infection. As shown in Fig. 2A, IRF7 mRNA was expressed at least 3 orders of magnitude higher in uninfected splenocytes (triangles) when compared with uninfected fibroblasts (squares). The presence or absence of STAT1 did not significantly affect constitutive IRF7 expression in spleen (compare open and closed symbols), confirming the lack of a role for constitutive IFN in maintaining high basal IRF7 expression in spleen. IRF7 levels were further induced following viral infection, but to a significant extent only in wild type cells, suggesting that positive feedback can further augment IRF7 production in response to autocrine IFN. As shown previously, IRF7 was also highly inducible in wild type fibroblasts, reaching levels equivalent to induced splenocytes but only at late times post-infection (p.i.). However, IRF7 induction in STAT1-deficient cells was significantly impaired.

Expression of high levels of constitutive IRF7 protein in wild type splenocytes correlated with accelerated and enhanced induction of type I IFN following virus infection (Fig. 2B and C). IFN levels produced by infected splenocytes at 4 h p.i. were comparable with those induced in infected wild type fibroblasts at 8–12 h p.i., subsequent to IRF7 induction. At 4 h p.i., IFN production by STAT1 mutant splenocytes was equivalent to that produced by wild type cells, which was consistent with their equivalent IRF7 levels. Greater IFN production was observed in wild type splenocytes at later time points, reflecting the induction of IRF7 (Fig. 2A), presumably in response to autocrine IFN. Little IFN was produced by STAT1-deficient fibroblasts, the IRF7 levels of which started out and remained low throughout the course of the infection.

Expression of IFNα subtypes other than IFNα4 is completely dependent on the presence of IRF7 and therefore on IFN sensitivity, at least in fibroblasts (4). A similar correlation with the presence of IRF7 was observed in splenocytes (Fig. 2C). The non-α4 IFN subset was induced in both wild type and STAT1-deficient splenocytes that expressed IRF7. Equal levels of IFN induction were observed at 4 h, although wild type cells expressed higher levels at later time points, subsequent to further induction of IRF7. Thus, expression of IFNα was dependent on IRF7 in both fibroblasts and splenocytes; however, the higher initial set point of IRF7 abundance in splenocytes allowed robust IFN production prior to feedback induction of IRF7 following autocrine IFN stimulation.

A DC Subset Is the Source of Abundant IRF7 in Splenocytes

To identify the cell type that accounted for high levels of IRF7 expression in spleen, individual non-B and non-T splenic cell populations were isolated and assayed for IRF7 expression. Splenic CD11c⁺ DC expressed relatively high levels of IRF7 protein, as well as moderately elevated levels of IRF8 (Fig. 3A, left panel). In contrast, other IRF proteins, such as IRF3, were expressed equally by both CD11c⁺ and CD11c⁻ cell populations.

To further characterize the IRF7-expressing splenocyte population, subset enrichment was carried out by sequential positive selection. CD11b⁺ cells were first selected to isolate CD11b⁺CD11c⁺ DC along with CD11b⁺ splenic macrophages. Subsequently, CD11c⁺ cells were selected from the CD11b⁻ population, to isolate the CD11b⁻CD11c⁺ DC subset. The remaining double negative cells after CD11c isolation were also examined. FACS staining confirmed the effectiveness of the selections through the expression of CD11b and CD11c markers on the appropriate cell populations (data not shown). As shown in Fig. 3A (right panel), CD11b⁻CD11c⁺ DC expressed greater levels of IRF7 protein when compared with CD11b⁻ or double negative cell populations. Again, IRF8 was also enriched in CD11b⁻ CD11c⁺ cells, whereas other IRF proteins were expressed at similar levels in all subsets. Thus, IRF7 is expressed at highest levels in CD11b⁻CD11c⁺ cells, suggesting that it is highly expressed in a DC subset.

FLT3L-differentiated Bone Marrow Generates IRF7-expressing DC

Dendritic cells can be broadly divided into distinct subtypes, including myeloid DC (mDC) and plasmacytoid DC (pDC). Similar cells can be differentiated and expanded in vitro from BM progenitors. Conventional mDC expand in the presence of GM-CSF, whereas pDC progenitors respond to FLT3L (29). We used this system to examine IRF protein levels in BM-derived DC. FLT3L-derived pDC expressed high levels of IRF7, as well as IRF3 (Fig. 3B, upper panels). In contrast, GM-CSF-derived mDC expressed IRF3 but markedly less IRF7. Naive BM progenitors expressed undetectable levels of either IRF protein (Fig. 3B, upper panels, first lane).

In vitro differentiation of DC in response to FLT3L yielded heterogeneous populations, with typically 30–40% being CD11b⁻CD11c^int. Wright-Giemsa staining of cytospin preparations confirmed that the major cell population displayed plasmacytoid morphology (Fig. 3C) and were B220⁺ (data not shown). In contrast, GM-CSF treatment resulted in the generation of a DC population that was 75–90% CD11b⁺CD11c⁺B220⁻ and resembled DC with a myeloid morphology (Fig. 3C). The addition of interleukin-3 to differentiating bone marrow cultures, either alone or in the presence of FLT3L or GM-CSF, skewed cell differentiation away from DC and yielded cultures dominated by granulocyte/macrophage lineages that did not express IRF7 and expressed little IRF3 (Fig. 3B and C, and data not shown). IFN has been suggested to bias DC differentiation toward pDC morphology (30), but we observed no such effect when IFNα was added alone or in the presence of FLT3L (Fig. 3B and data not shown). However, we found that inclusion of stem cell factor increased the yield of FLT3-derived pDC, probably by enhancing progenitor proliferation during the first few days of culture (data not shown).

Similarly, high levels of IRF7 were detected in splenic pDC populations isolated by FACS from spleens of uninfected mice (Fig. 3D). These CD11c^intLy6^hiB220⁺ cells expressed much higher levels of IRF7 than the moderate levels of IRF7 expressed in a mixed DC population isolated by sorting splenocytes only on the basis of CD11c or the minimal levels of IRF7 expressed by in vitro differentiated mDC. Taken together, these results indicate that the major if not exclusive cells expressing constitutive IRF7 are pDC.

Bone Marrow-derived pDC Produce IFNα in Response to Viral Infection

In vitro differentiated pDC and mDC populations were infected with NDV and assayed for levels of IRF7 protein and IFNα mRNA. FLT3L-differentiated pDC populations expressed IRF7 protein prior to infection, and the level of IRF7 increased modestly following infection (Fig. 3B, lower panels). IRF7 protein was nearly undetectable in naive mDC, but it was inducible, although to relatively lower levels, following viral infection. Consistent with the higher constitutive levels of IRF7, infected pDC produced ~10-fold more IFNα (all subtypes) than did mDC. These cells expressed ~8-fold more of the IRF7-dependent non-α4 subtypes of IFNα than did mDC, and expression peaked more rapidly, at 4 h p.i. (Fig. 4A and data not shown). These amounts may underestimate the potential of pDC to produce IFNα since these in vitro derived cultures were only ~30–40% pDC by flow cytometric and histologic criteria (data not shown). These results demonstrate that IRF7 is highly expressed in IFN-producing pDC in a largely IFN-independent manner, enabling rapid and robust IFN production in response to infection. Early IFN production included production of IRF7-dependent non-α4 subtypes, normally expressed only with delayed kinetics in other cell types.

Differential Expression of TLR Proteins by Mouse DC Subsets

The ability of DC to respond to pathogens is determined at least in part by TLR proteins, although the role of these receptors in viral infections is controversial (31). We examined the levels of TLR mRNA expression in pDC and mDC. Interestingly, TLR2, TLR3, TLR4, and TLR8 were expressed at significantly higher levels in mDC than in pDC. Only TLR9 was expressed at a higher level in pDC populations (Fig. 4B), consistent with other reports (reviewed in Refs. 32–34). Since TLR9 is a DNA receptor, these results suggest that the enhanced IFN production by pDC cannot be explained by the presence of TLR proteins traditionally associated with ligands that could be derived from RNA viruses. Instead, IFN production by pDC more closely correlates with IRF7 abundance (Fig. 3). IRF8 was also modestly enriched in pDC (Fig. 3 and data not shown) and may contribute to the IFN-producing ability of these cells. However, IRF8 is required for DC differentiation (35, 36) and likely contributes to DC lineage commitment rather than directly to function.

Ectopic Expression of IRF7 Augments the Kinetics, Magnitude, and Diversity of IFNα Production

We designed an experiment to test whether IRF7 constitutive expression was a major determinant for the robust IFN production observed in pDC. Fibroblasts lack IRF7 expression prior to IFN exposure and therefore produce IFN in a delayed-early manner following viral infection, especially the non-α4 subset (4). We ectopically expressed IRF7 (or green fluorescent protein, as control) in mouse fibroblasts and measured IFN production following infection with NDV. As shown in Fig. 4C, IRF7-expressing fibroblasts responded to infection by making 100-fold more IFNα than control cells and produced it more rapidly, with significant levels detectable at 4 h p.i. The IFN produced included multiple subtypes and was not limited to the IFNα4 subtype produced in the absence of positive feedback. Thus, although additional components of the IFN signaling pathway are also induced during positive feedback, IRF7 expression alone is sufficient to drive the rapid production of large quantities of multiple isoforms of type I IFN following virus infection.