Lower respiratory tract infections caused by the paramyxoviruses human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are characterized by short-lasting virus-specific immunity and often long-term airway morbidity, both of which may be the result of alterations in the Ag-presenting function of the lung which follow these infections. In this study, we investigated whether hMPV and RSV experimental infections alter the phenotype and function of dendritic cell (DC) subsets that are recruited to the lung. Characterization of lung DC trafficking demonstrated a differential recruitment of plasmacytoid DC (pDC), conventional DC (cDC), and IFN-producing killer DC to the lung and draining lymph nodes after hMPV and RSV infection. In vitro infection of lung DC indicated that in pDC, production of IFN-α, TNF-α, and CCL5 was induced only by hMPV, whereas CCL3 and CCL4 were induced by both viruses. In cDC, a similar repertoire of cytokines was induced by hMPV and RSV, except for IFN-β, which was not induced by RSV. The function of lung pDC was altered following hMPV or RSV infection in vivo, as we demonstrated a reduced capacity of lung pDC to produce IFN-α as well as other cytokines including IL-6, TNF-α, CCL2, CCL3, and CCL4 in response to TLR9 stimulation. Moreover, we observed an impaired capacity of cDC from infected mice to present Ag to CD4+ T cells, an effect that lasted beyond the acute phase of infection. Our findings suggest that acute paramyxovirus infections can alter the long-term immune function of pulmonary DC.

Human metapneumovirus (hMPV)3 is a recently discovered pathogen first identified in respiratory specimens from young children suffering from clinical respiratory syndromes, ranging from a mild to severe lower respiratory tract illness (1). Seroprevalence studies have shown that by the age of 5 years, ∼70% of all children have developed Abs against hMPV (2). hMPV has a seasonal distribution, being usually isolated during the winter time, and is associated with both upper and lower respiratory tract infections in children and adults (3, 4, 5). hMPV is responsible for 10% of all hospitalizations of elderly patients with respiratory tract infections and those immunocompromised are also more susceptible to hMPV respiratory tract infections (5, 6). hMPV is a RNA virus of the Paramyxoviridae family and part of the Pneumovirinae subfamily along with human respiratory syncytial virus (RSV) (7). In young children, clinical symptoms associated with hMPV infections are virtually indistinguishable from those caused by RSV (8, 9), although some but not all studies have reported a lower severity of disease compared with RSV (5, 10). Infections caused by both hMPV and RSV are characterized by short-lasting immunity and as consequence reinfections occur throughout life (11). Moreover, both infections have been associated with long-term airway morbidity, including the development of wheezing and asthma (12, 13).

Dendritic cells (DC) play a central role as immunological sentinels (14, 15). They can efficiently sense invading pathogens by a set of pattern recognition receptors and because of their strategic localization at mucosal sites they are involved in response to viral infections (15, 16). After detection, uptake and degradative processing of invading pathogens, DC undergo maturation and migration to lymphoid tissue where they present processed viral Ag to lymphocytes (15, 17). Respiratory tract DC are present at high frequency within airway epithelium, submucosa, and associated lung parenchyma tissue under resting conditions (18). At least three subsets of pulmonary DC have been described in mice: 1) the CD11cintB220+Ly6C+ plasmacytoid DC (pDC), which are major producers of type I IFN in response to stimulation with enveloped viruses and hence are key effectors in the innate immune system (19, 20), 2) the CD11chighMHCIIhigh myeloid DC (cDC), which are the primary APCs, and 3) the CD11cintB220+CD49b+ NK IFN-producing killer DC (IKDC) that express cell surface markers of DC as well as NK cell markers (21, 22). IKDC could be considered as NK-like DC or DC-like NK cells playing a major role as a distinct population of innate effectors against viral pathogens (21, 23, 24). All of these cell types participate in the innate immune response and also are involved in either the generation or modulation of the adaptive immune response.

Despite the critical role of DC in the antiviral immune response, no data are available regarding the response of lung DC upon hMPV infection, whether this infection results in a distinct response compared with RSV and whether DC function may be altered beyond the period of acute infection, thus possibly affecting immune response in the convalescence period or even for longer period of times. Therefore, in this study, we investigated the effect of hMPV infection on trafficking and activation of lung DC in a mouse model of infection and compared it with RSV. We show that the recruitment and activation of lung DC were different following infection with RSV or hMPV. Moreover, we show that hMPV and RSV infections resulted in impaired ability of lung pDC to produce IFN-α and other cytokines in response to TLR9 stimulation and cDC to present Ag to CD4+ T cells. These data suggest that such subversion of pulmonary DC function may play an important role in the pathogenesis of acute infections caused by RSV and hMPV and possibly their long-term consequences, such as failure to develop antiviral immunologic memory, increased susceptibility to other infections, and altered response to bystander Ags.

RSV A2 was grown in HEp-2 cells (American Type Culture Collection) and purified by polyethylene glycol precipitation, followed by centrifugation on 35–65% discontinuous sucrose gradients as described elsewhere (25). The hMPV strain CAN97–83 was obtained from the Centers for Disease Control (Atlanta, GA), with permission from Dr. G. Boivin (Research Center in Infectious Diseases, Regional Virology Laboratory, Laval University, Quebec City, Canada). Virus was propagated and titrated in LLC-MK2 cells (American Type Culture Collection) in MEM (without serum) containing 1.0 μg trypsin/ml (Worthington) as described elsewhere (26).

Female 8- to 10-wk-old BALB/c mice were purchased from Harlan and were housed under pathogen-free conditions in the animal research facility of the University of Texas Medical Branch (UTMB; Galveston, TX) in accordance with the National Institutes of Health and UTMB institutional guidelines for animal care. Under light anesthesia, mice were infected intranasally with 50 μl of RSV or hMPV diluted in PBS (final administered dose: 107 PFU) (27). Control mice were inoculated with the same volume of PBS (referred to hereinafter as mock infection). To monitor the progression of disease, daily determination of body weight was assessed.

Lungs and lung draining LN were harvested at different time points up to 14 days after hMPV, RSV, or mock infection. At the indicated time points (see Results), mice were anesthetized with an i.p. injection of ketamine and xylazine before the thoracic cavity was opened. Mice were exsanguinated and the trachea opened by incision of the cricothyroid membrane. Before harvest, lungs were gently perfused via the right ventricle with 5 ml of PBS containing 2 mM EDTA to remove blood cells from the pulmonary circulation. They were then cut into small pieces and incubated with collagenase A (0.5 mg/ml PBS; Sigma-Aldrich) and type IV bovine pancreatic DNase (20 μg/ml PBS; Sigma-Aldrich) for 1 h at 37°C. After digestion, lung cells were dispersed by shearing through a 20-gauge needle, followed by filtration through a nylon screen cell strainer (100 μm). Single-cell suspensions were washed and contaminating erythrocytes were lysed using ACK lysing buffer containing NH4Cl, KHCO3, and EDTA Na2 · 2H2O (BioSource International). Lung draining LN were consistently collected and digested to obtain single-cell suspensions as mentioned above for lung tissue.

Lung and LN cells were incubated with anti-FcγRII/FcγRIII mAb (24G2; BD Biosciences). After washing, cells were stained with the following anti-mouse Abs: anti-CD11c, anti-I-A/I-E (MHC-II), anti-CD11b, anti-CD49b/pan-NK (DX5), anti-Ly6C (Gr-1), anti-CD45R/B220, anti-CD103 (all from BD Pharmingen), and anti-mPDCA1 (Miltenyi Biotec). In a separate set of experiments, lung cells were stained with anti-CD11c, anti-MHC-II in combination with anti-CD40, anti-CD80, anti-CD86, anti-programmed death ligand 1 (PD-L1), and anti-PD-L2 (BD Pharmingen). Samples were stained at 4°C in PBS with 1% FBS and analyzed with a FACSCanto flow cytometer equipped with BD FACSDiva software (both from BD Biosciences Immunocytometry Systems). Analysis was performed using FlowJo software (version 7.2.2; Tree Star).

Lung cells were first enriched by MACS for specific CD11c+ and mPDCA-1+ populations (AutoMACS; Miltenyi Biotec), followed by FACS sorting isolation for cDC and pDC, respectively. CD11c+ cells were stained with Cy7-conjugated CD11c and FITC-conjugated MHC-II Abs and the mPDCA1+ cells were stained with FITC-conjugated CD11c, PerCP-conjugated B220, and PE-conjugated Gr-1 (RB6-8C5) Abs (BD Pharmingen). CD11chighMHC-IIhigh (cDC) and CD11cintB220+Gr1+ cells (pDC) were sorted using a FACSAria instrument (BD Biosciences Immunocytometry Systems). Routine postsorting analysis was performed to ensure that that the purity was >97%. DC were cultured in RPMI 1640 supplemented with 10% FCS, 2 mmol/L l-glutamine, 1 mM sodium pyruvate, and HEPES (complete medium).

Lung cDC or pDC isolated from uninfected mice were infected in vitro with RSV or hMPV at a multiplicity of infection (MOI) of 3 for 24 h in 96-well plates (105 cells/well) in a total volume of 200 μl. Cell-free supernatant was collected and tested for multiple cytokines using the Bio-Plex Mouse Cytokine 23-Plex panel (Bio-Rad) according to the manufacturer’s instructions. The panel included the following cytokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, TNF-α, GM-CSF, IFN-γ, G-CSF, KC, CCL3 (MIP-1α), CCL4 (MIP-1β), CCL11 (eotaxin), CCL2 (MCP-1), and CCL5 (RANTES). IFN-α and IFN-β were measured by ELISA (PBL Biomedical Laboratories). The IFN-α ELISA recognizes the αA, α1, α4, α5, α6, and α9 isoforms. In a separate set of experiments, pDC were isolated from mock-, hMPV-, and RSV-infected mice after 3 days of infection. In brief, 105 cells/well were stimulated with 1 μg/ml stimulatory oligodeoxynucleotide (ODN) with mouse-specific CpGs. We used ODN 1826 sequence (5′-tccatgacgttcctgacgtt-3′; InvivoGen).

For T cell proliferation assays, FACS-sorted cDC from virus-infected or uninfected mice (104 cells/well) were cultured for 3 days with CD4+ T cells from DO11.10 mice (105 cells/well) in 96-well round-bottom microtiter plates in complete medium. cDC were loaded with 1 μM OVA peptide (323–339) 1 h before coculture with T cells. Cells were pulsed with 1 μCi/well [3H]thymidine for the last 18 h. Cells were harvested onto filters using a cell harvester (Brandel) and incorporation of [3H]thymidine to DNA was determined using a Wallac 1450 Microbeta/Trilux liquid scintillation counter (PerkinElmer).

Statistical analysis was performed using the InStat 3.05 biostatistics package (GraphPad) using a one-way ANOVA to ascertain differences between groups, followed by a Tukey-Kramer test to correct for multiple comparisons. Unless otherwise indicated, results are expressed as mean ± SEM.

Lung DC are the primary immunological sentinels that can efficiently sense invading pathogens by a set of pattern recognition receptors (14, 15). For an effective immune response to occur, DC must be able to sample the peripheral microenvironment and migrate through the afferent lymph to the LN where they prime naive T cells (28). Herein, we determined the recruitment of pDC, cDC, and IKDC to the lung and LN after hMPV infection and compared it with that induced by RSV. Mice were infected intranasally with hMPV, RSV, or mock inoculum and assessed daily for body weight loss to evaluate the progression of the disease. At different time points, mice were sacrificed to analyze the number of DC in lung tissue and LN. As shown in Fig. 1,A, hMPV-infected mice showed a biphasic body weight loss at days 1 (modest) and 7 and total recovery by day 11. On the other hand, maximum body weight loss in RSV-infected mice occurred at day 2 after infection with gradual recovery by day 7. Recruitment of pDC was evaluated by identifying cells expressing CD11cintB220+Gr-1+ (Fig. 1,B). Following hMPV infection, the number of pDC rapidly increased from 1 ± 0.1 × 105 cells/lung (in mock-infected mice) to 4.7 ± 0.8 × 105 cells/lung by day 1 and reaching a maximum of 8.4 ± 1.6 × 105 cells/lung on day 8 (Fig. 1,C). By day 14, number of lung pDC returned to levels similar to those of mock-infected mice. On the other hand, in RSV-infected mice, pDC peaked in the lung at day 3 after infection (8.2 ± 1.2 × 105 cells/lung) and returned to basal levels by day 8. In regard to the trafficking of pDC to LN, we generally observed an increase in size and total number of cells in LN from infected mice as early as day 1 after infection. Number of pDC in LN was similar for both hMPV and RSV infections, although pDC were recruited in slightly higher numbers in hMPV-infected mice (Fig. 1 D). pDC were rapidly recruited to LN by day 1 and peaked by day 6 (3.9 ± 0.3 × 104) after hMPV infection and started to decline by day 8. Similar results were observed when pDC were identified by their expression of mPDCA1 and B220 (data not shown). Overall, the data show that in this murine model hMPV and RSV induced a distinct kinetics of pDC recruitment to the lung that appeared overall to parallel the body weight curve. Pattern of pDC recruitment to the draining LN was similar for both viruses.

FIGURE 1.

pDC recruitment to the lung in hMPV- and RSV-infected mice. BALB/c mice were infected with hMPV or RSV (107 PFU/mouse). Mock-infected mice were inoculated with 50 μl of PBS. Mice were monitored daily for body weight loss. Lungs and LN were removed at different time points and tissue was dispersed by collagenase digestion. Cells were stained with anti-B220-PerCP, anti-CD11c-PE-Cy7, and anti-Gr-1-PE. Cells were acquired in a FACSCanto and data were analyzed by FlowJo software. A, Body weight curve. B, Dot plot indicates gated population of B220+CD11cint cells. pDC were identified by the additional expression of Gr-1. pDC from mock (gray-shaded histogram)-, RSV (open histogram)-, and hMPV-infected mice (dotted line histogram) are shown. C, Total lung pDC. D, Total LN pDC. Graphs represent mean ± SEM (n = 9–16 mice/group/time point). ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001, compared with mock-infected mice. Data are representative of three independent experiments with similar results.

FIGURE 1.

pDC recruitment to the lung in hMPV- and RSV-infected mice. BALB/c mice were infected with hMPV or RSV (107 PFU/mouse). Mock-infected mice were inoculated with 50 μl of PBS. Mice were monitored daily for body weight loss. Lungs and LN were removed at different time points and tissue was dispersed by collagenase digestion. Cells were stained with anti-B220-PerCP, anti-CD11c-PE-Cy7, and anti-Gr-1-PE. Cells were acquired in a FACSCanto and data were analyzed by FlowJo software. A, Body weight curve. B, Dot plot indicates gated population of B220+CD11cint cells. pDC were identified by the additional expression of Gr-1. pDC from mock (gray-shaded histogram)-, RSV (open histogram)-, and hMPV-infected mice (dotted line histogram) are shown. C, Total lung pDC. D, Total LN pDC. Graphs represent mean ± SEM (n = 9–16 mice/group/time point). ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001, compared with mock-infected mice. Data are representative of three independent experiments with similar results.

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Recruitment of cDC to the lung and LN during hMPV or RSV infection was examined by the expression of CD11chigh and MHC-IIhigh (Fig. 2,A). As shown in Fig. 2,B, number of cDC increased from 1.6 ± 0.2 × 105 cells/lung on day 1 to a maximum of 32.8 ± 7.9 × 105 cells/lung on day 10 after infection with hMPV. Thereafter, the number of lung pDC declined slowly, yet remained significantly elevated until day 18 (12.2 ± 1.3 × 105 cells/lung) compared with mock-infected mice (2.2 ± 0.1 × 105 cells/lung). Similar recruitment pattern was observed when mice were infected with RSV but lung cDC peaked earlier since a maximum of 32.3 ± 1.7 × 105 cells/lung were observed at day 8 after infection. Trafficking of cDC to mediastinal LN was almost identical in hMPV or RSV infections, with an increasing time-dependent number of cells and a peak at day 6 after infection (Fig. 2 C).

FIGURE 2.

cDC recruitment to the lung in hMPV- and RSV-infected mice. Cells from lung and LN of hMPV-, RSV-, or mock-infected mice were stained with anti-MHC-II-FITC and anti-CD11c-PE-Cy7. Cells were acquired in a FACSCanto and data were analyzed by FlowJo software. A, Dot plots indicate gated cDC identified as CD11chighMHC-IIhigh. B, Total lung cDC. C, Total LN cDC. Graphs represent mean ± SEM (n = 8–16 mice/group/time point). ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001, compared with mock-infected mice. Data are representative of four independent experiments.

FIGURE 2.

cDC recruitment to the lung in hMPV- and RSV-infected mice. Cells from lung and LN of hMPV-, RSV-, or mock-infected mice were stained with anti-MHC-II-FITC and anti-CD11c-PE-Cy7. Cells were acquired in a FACSCanto and data were analyzed by FlowJo software. A, Dot plots indicate gated cDC identified as CD11chighMHC-IIhigh. B, Total lung cDC. C, Total LN cDC. Graphs represent mean ± SEM (n = 8–16 mice/group/time point). ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001, compared with mock-infected mice. Data are representative of four independent experiments.

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IKDC were identified by expression of CD11cintB220+DX5+. Fig. 3,A shows the morphology of sorted lung IKDC stained by Wright-Giemsa. As shown in Fig. 3,B, in hMPV-infected mice IKDC recruitment resembled that of pDC, i.e., as early as day 1 and with a first peak at day 2 (3.4 ± 0.4 × 105 cells/lung) and a second peak at day 8 after infection (4.0 ± 0.1 × 105 cells/lung). By day 10, the numbers of IKDC have returned to similar levels as those observed in mock-infected mice (1.5 ± 0.4 × 105 cells/lung). On the other hand, in RSV-infected animals, IKDC peaked at day 3 (4.7 ± 0.7 × 105 cells/lung) and returned to control levels by day 8 after infection (Fig. 3,B). Recruitment of IKDC to LN was similar in both hMPV and RSV infections. As shown in Fig. 3 C, IKDC started to be recruited by day 2 with a peak of 4.2 × 104 cells at day 6 after infection, to rapidly decline to 0.7 × 104 cells by day 8 after infection. Thus, IKDC trafficking after hMPV and RSV infection resembles that of pDC. Overall, these data indicate that IKDC is the smallest DC population recruited to the airways upon paramyxovirus infection, followed by pDC and being the cDC the predominant DC subset recruited to the lung.

FIGURE 3.

IKDC recruitment to the lung in hMPV- and RSV-infected mice. Three-color flow cytometry studies using anti-B220-PerCP, anti-CD11c-PE-Cy7, and anti-DX5-PE were performed on lung and LN cells from hMPV-, RSV-, or mock-infected BALB/c mice. IKDC were identified as DX5+B220+CD11cint by flow cytometry analysis using FlowJo software. A, Sorted IKDC were stained with Wright-Giemsa staining. Scale bar, 10 μm. B, Total lung IKDC. C, Total LN IKDC. Graphs represent mean ± SEM (n = 4–11 mice/group/time point). ∗, p < 0.05 and ∗∗, p < 0.01, compared with mock-infected mice. Data are representative of four independent experiments.

FIGURE 3.

IKDC recruitment to the lung in hMPV- and RSV-infected mice. Three-color flow cytometry studies using anti-B220-PerCP, anti-CD11c-PE-Cy7, and anti-DX5-PE were performed on lung and LN cells from hMPV-, RSV-, or mock-infected BALB/c mice. IKDC were identified as DX5+B220+CD11cint by flow cytometry analysis using FlowJo software. A, Sorted IKDC were stained with Wright-Giemsa staining. Scale bar, 10 μm. B, Total lung IKDC. C, Total LN IKDC. Graphs represent mean ± SEM (n = 4–11 mice/group/time point). ∗, p < 0.05 and ∗∗, p < 0.01, compared with mock-infected mice. Data are representative of four independent experiments.

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We have previously shown that hMPV and RSV infections induce a distinct profile of cytokines in human peripheral blood pDC and monocyte-derived DC (29). Since the response of lung DC to these paramyxoviruses has not been investigated, we next examined the ability of lung pDC and cDC to produce cytokines upon hMPV and RSV infection. We isolated lung pDC (CD11cintB220+Gr-1+mPDCA1+) and cDC (CD11chighMHC-IIhigh) from naive mice (Figs. 4,A and 5,A) by cell sorting. Isolation of sufficient numbers of lung IKDC from naive mice to carry out this set of experiments was not feasible. Cells were infected for 24 h and cell-free supernatants were tested for the presence of various cytokines including IFN-α and IFN-β. A highly purified pDC population was isolated from lungs after cell sorting (Fig. 4,A). Exposure of pDC to hMPV resulted in production of a significant amount of IFN-α, while very low to undetectable levels were observed upon RSV infection (Fig. 4 B). In addition, hMPV but not RSV was able to induce TNF-α and CCL5 production, although their concentrations did not reach statistical significance. Production of IL-12p40 was induced by both viruses, with hMPV being a stronger inducer than RSV. Similar levels of CCL3 and CCL4 were produced after infection with hMPV or RSV. Overall, RSV failed to induce cytokine production by lung pDC, with the exception of CCL3 and CCL4. Concentrations of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, GM-CSF, IFN-γ, G-CSF, KC, CCL11, and CCL2 were at the lower limit of detection in control or virus-infected lung pDC (data not shown). These results collectively indicate that pDC produce a limited repertoire of cytokines in response to viral infection and that hMPV is a stronger inducer of cytokines compared with RSV.

FIGURE 4.

Ex vivo cytokine production by lung pDC upon hMPV and RSV infection. Pulmonary pDC were isolated from naive BALB/c mice by collagenase digestion. Enriched mPDCA1+ populations were stained with anti-B220-PerCP, anti-CD11c-PE-Cy7, and anti-Gr-1-PE and sorted by FACS. pDC (105 cells/well) were infected with hMPV or RSV at a MOI of 3 for 24 h. Concentration of IFN-α was determined by ELISA and other cytokines by Bio-Plex assay. A, Upper dot plot represents lung cells before FACS sorting indicating R1 region as mPDCA1+B220+Gr-1+ cells. Lower dot plots show R1 region after FACS sorting where pDC were identified as mPDCA1+Gr-1+B220+CD11cint. Sorted pDC were stained with Wright-Giemsa. Scale bar, 10 μm. B, Cytokine production by lung pDC after hMPV and RSV infection. Bar graphs represent mean ± SEM for n = 3 independent experiments. ∗, p < 0.05. Uninf, Uninfected.

FIGURE 4.

Ex vivo cytokine production by lung pDC upon hMPV and RSV infection. Pulmonary pDC were isolated from naive BALB/c mice by collagenase digestion. Enriched mPDCA1+ populations were stained with anti-B220-PerCP, anti-CD11c-PE-Cy7, and anti-Gr-1-PE and sorted by FACS. pDC (105 cells/well) were infected with hMPV or RSV at a MOI of 3 for 24 h. Concentration of IFN-α was determined by ELISA and other cytokines by Bio-Plex assay. A, Upper dot plot represents lung cells before FACS sorting indicating R1 region as mPDCA1+B220+Gr-1+ cells. Lower dot plots show R1 region after FACS sorting where pDC were identified as mPDCA1+Gr-1+B220+CD11cint. Sorted pDC were stained with Wright-Giemsa. Scale bar, 10 μm. B, Cytokine production by lung pDC after hMPV and RSV infection. Bar graphs represent mean ± SEM for n = 3 independent experiments. ∗, p < 0.05. Uninf, Uninfected.

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FIGURE 5.

Ex vivo cytokine production by lung cDC upon hMPV and RSV infection. cDC were isolated from lungs of naive mice. Enriched CD11c+ populations were stained with anti-MHC-II-FITC and anti-CD11c-PE-Cy7 and sorted by FACS. cDC (105 cells/well) were infected with hMPV or RSV at a MOI of 3 for 24 h. Concentration of IFN-β was determined by ELISA and other cytokines by Bio-Plex assay. A, Dot plot represents cDC (CD11chighMHC-IIhigh) after FACS sorting. Sorted cDC were stained with Wright-Giemsa. Scale bar, 10 μm. B, Cytokine production in lung cDC after hMPV and RSV infection. Bar graphs represent mean ± SEM for n = 3 independent experiments. ∗, p < 0.05. Uninf, Uninfected.

FIGURE 5.

Ex vivo cytokine production by lung cDC upon hMPV and RSV infection. cDC were isolated from lungs of naive mice. Enriched CD11c+ populations were stained with anti-MHC-II-FITC and anti-CD11c-PE-Cy7 and sorted by FACS. cDC (105 cells/well) were infected with hMPV or RSV at a MOI of 3 for 24 h. Concentration of IFN-β was determined by ELISA and other cytokines by Bio-Plex assay. A, Dot plot represents cDC (CD11chighMHC-IIhigh) after FACS sorting. Sorted cDC were stained with Wright-Giemsa. Scale bar, 10 μm. B, Cytokine production in lung cDC after hMPV and RSV infection. Bar graphs represent mean ± SEM for n = 3 independent experiments. ∗, p < 0.05. Uninf, Uninfected.

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When we determined the response of cDC to hMPV and RSV, we observed that hMPV but not RSV was able to induce significant levels of IFN-β (Fig. 5 B). Both viruses were able to induce significant amounts of IL-1α, IL-6, IL-10, KC, and CCL11. Other cytokines including IL-12p40, IL-12p70, and TNF-α and chemokines CCL3, CCL4, and CCL5 were also similarly induced by hMPV and RSV but did not reach statistical significance when compared with uninfected cells. Levels of IFN-α were below the lower limit of detection in control or viral-infected cDC (data not shown). These data indicate that, with the exception of IFN-β production, hMPV induces a similar profile of cytokines than RSV by pulmonary cDC.

Evidence is accumulating that viral pathogens can use DC as a conduit for subverting the immune response and establishing infection in the host (30). We and others have previously shown that both RSV and hMPV have the ability to inhibit the production of cytokines such as IFN-α by human pDC in response to TLR9 agonists (29, 31). However, it is unknown whether hMPV and RSV are capable of inhibiting cytokine production in lung pDC after infection in vivo. Therefore, BALB/c mice were infected with hMPV, RSV, or mock for 3 days (when sizable numbers of pDC are recruited at the site of infection; see Fig. 1,B). Lung pDC were isolated by cell sorting as described in Materials and Methods. Cells (105 pDC/well) were stimulated with 1 μg/ml CpG ODN 1826 and cell-free supernatant was collected after 24 h. As shown in Fig. 6, infection with hMPV or RSV significantly inhibited the capacity of lung pDC to produce IFN-α, IL-6, TNF-α, CCL3, CCL4, and CCL5 (mostly by RSV infection) in response to CpG ODN. We also observed a modest reduction in the production of CCL11, CCL5 (by hMPV infection), and CCL2 and a marginal change in IL-12p70 by any of the two infections in vivo. No change was observed in the production of IL-12p40 in any of the three cultures. These data demonstrate that both hMPV and RSV are able to interfere with the ability of lung pDC to respond to a secondary stimulus for the production of IFN-α and other cytokines.

FIGURE 6.

Cytokine production by lung pDC in response to TLR9 stimulation. pDC were isolated from lungs of hMPV-, RSV-, or mock-infected mice at day 3 after infection. pDC (105 cells/well) were stimulated with 1 μg/ml CpG ODN 1826 for 24 h. Concentration of IFN-α was assessed by ELISA and other cytokines by Bio-Plex assay. Bar graphs represent mean ± SEM for n = 3 independent experiments. ∗, p < 0.05.

FIGURE 6.

Cytokine production by lung pDC in response to TLR9 stimulation. pDC were isolated from lungs of hMPV-, RSV-, or mock-infected mice at day 3 after infection. pDC (105 cells/well) were stimulated with 1 μg/ml CpG ODN 1826 for 24 h. Concentration of IFN-α was assessed by ELISA and other cytokines by Bio-Plex assay. Bar graphs represent mean ± SEM for n = 3 independent experiments. ∗, p < 0.05.

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We have previously shown that RSV and hMPV are able to inhibit the ability of human monocyte-derived DC (moDC) to stimulate T cell proliferation in vitro (29). To investigate the effect of hMPV and RSV on the Ag-presenting capacity of pulmonary cDC, we isolated lung cDC from virus-infected and mock-infected mice at different time points after infection. cDC isolated from lungs at 1, 2, 3, and 6 wk after hMPV, RSV, or mock infection were loaded with OVA peptide and cocultured with purified CD4+ T cells from DO11.10 mice. Cocultures of pulmonary cDC from mock-infected mice with DO11.10 CD4+ T cells resulted in a robust T cell proliferation (Fig. 7). At the 1-wk time point, lung cDC from hMPV- or RSV-infected mice were more efficient in stimulating CD4+ T cell proliferation than cDC isolated from mock mice. However, at week 2, we observed that infection with RSV significantly decreased the ability of cDC to stimulate CD4+ T cell proliferation by ∼50%. cDC from hMPV-infected mice were also deficient in the induction of OVA-specific T cell proliferation but in a lower extent (∼20%). At week 3 after infection, both viruses were able to decrease in a similar degree (∼70%) the cDC stimulatory capacity when compared to cDC from mock-infected mice. By week 6 after infection, cDC from both RSV- and hMPV-infected mice were able to stimulate T cell proliferation as efficient as those from mock mice. When expression of RSV or hMPV Ags was assessed in lung cDC at 2 or 3 wk after infection, RT-PCR assays indicated that cDC isolated from infected mice did not express viral Ag (data not shown). These findings indicate that RSV and hMPV infection transiently impairs the ability of pulmonary cDC to present Ag to T cells.

FIGURE 7.

CD4+ T cell proliferation induced by cDC from hMPV- and RSV-infected mice. cDC were isolated from lungs of hMPV-, RSV-, or mock-infected mice at different time points. cDC were loaded with 1 μM OVA peptide for 1 h before coculture with CD4+ T cells from D011.10 mice at a ratio 1:10. T cell proliferation was analyzed on day 3 of culture by [3H]thymidine incorporation. Data are expressed as cpm (mean ± SEM). A representative experiment from three similar experiments is shown. ∗, p < 0.05 and ∗∗, p < 0.01.

FIGURE 7.

CD4+ T cell proliferation induced by cDC from hMPV- and RSV-infected mice. cDC were isolated from lungs of hMPV-, RSV-, or mock-infected mice at different time points. cDC were loaded with 1 μM OVA peptide for 1 h before coculture with CD4+ T cells from D011.10 mice at a ratio 1:10. T cell proliferation was analyzed on day 3 of culture by [3H]thymidine incorporation. Data are expressed as cpm (mean ± SEM). A representative experiment from three similar experiments is shown. ∗, p < 0.05 and ∗∗, p < 0.01.

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The observed inhibition of T cell proliferation by lung cDC isolated from infected mice may be the result of altered expression of APC costimulatory molecules. Positive and negative costimulatory molecules individually or collectively regulate T cell activation thresholds. To examine the possibility that viral infection altered the surface molecule expression, lung cDC (CD11chighMHC IIhigh) were isolated from hMPV-, RSV-, and mock-infected mice and were stained with anti-CD40, anti-CD80, anti-CD86, anti-PD-L1, and anti-PD-L2 Abs. As shown in Fig. 8, expression of CD80 gradually increased over a 3-wk period after RSV or hMPV infection compared with mock-infected mice. Similarly, PD-L1 expression increased progressively over the time of infection. Eight weeks after infection, expression of both CD80 and PD-L1 by cDC was comparable to that observed in mock-infected mice. Expression of CD86 was slightly increased by week 2 after RSV infection but not hMPV infection compared with cDC from mock-infected mice. CD40 and PD-L2 were not affected by viral infection. These findings indicate that both RSV and hMPV are able to induce activation of pulmonary cDC.

FIGURE 8.

Cell surface molecule expression by lung cDC from hMPV- and RSV-infected mice. Three-color flow cytometry studies using anti-CD11c and anti-MHC-II in combination with anti-CD40, anti-CD80, anti-CD86, anti-PD-L1, or anti-PD-L2 were performed on lung cells from hMPV-, RSV-, or mock-infected BALB/c mice. Gated cDC (CD11chighMHC-IIhigh) were analyzed for the expression of each additional surface molecule expression. Isotype control (dotted histograms), cDC from mock-infected mice (shaded histograms), and cDC from virus-infected mice (open histograms) are shown. A representative experiment from three similar experiments is shown.

FIGURE 8.

Cell surface molecule expression by lung cDC from hMPV- and RSV-infected mice. Three-color flow cytometry studies using anti-CD11c and anti-MHC-II in combination with anti-CD40, anti-CD80, anti-CD86, anti-PD-L1, or anti-PD-L2 were performed on lung cells from hMPV-, RSV-, or mock-infected BALB/c mice. Gated cDC (CD11chighMHC-IIhigh) were analyzed for the expression of each additional surface molecule expression. Isotype control (dotted histograms), cDC from mock-infected mice (shaded histograms), and cDC from virus-infected mice (open histograms) are shown. A representative experiment from three similar experiments is shown.

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In addition to the surface molecules described above, we examined whether RSV or hMPV infection altered the phenotype of lung cDC subsets based on CD103 expression. This marker associates with two major cDC populations in mice, CD103+CD11blow and CD103CD11bhigh (32), which differ in their ability to prime CD4+ and CD8+ T cells (33), production of proinflammatory cytokines, and generation of Foxp3-mediated regulatory function of naive T cells (34). CD103 (αE) is the α-chain of the αEβ7 integrin, which has been reported to be essential for the adhesion of human and mice intestinal lymphocytes to epithelial cells through the interactions with E-cadherin (35). CD103+ DC in the lung express high amounts of CD11c and MHC-II and low levels of CD11b (32). It is currently unknown whether respiratory viral infections alter the phenotype of cDC based on expression of CD103. Analysis of the cDC CD103+ population was performed by three-color flow cytometry analysis in total lung cells from virus- or mock-infected mice. Gated CD11chighMHC-IIhigh cells were further analyzed for the expression of CD103 (Fig. 9,A, upper panels). Consistent with a previous report, we found that the lung cDC (CD11chighMHC-IIhigh) compartment in uninfected mice is composed by a mixed population of CD103+ and CD103 cells (32). Moreover, CD11b expression was inversely correlated to CD103 expression since we observed CD103+CD11b and CD103CD11b+ cells in the cDC subset (data not shown). However, we observed that the percentage of CD103+ cDC decreased from ∼50 to ∼20% after 7 days of infection, as shown in Fig. 9,A (lower panels). Percentage of CD103+ cDC was consistently decreased in infected mice at weeks 2 and 3 but returned to normal levels at week 8 after infection (Fig. 9 B).

FIGURE 9.

CD103 (αE) expression by lung cDC upon hMPV and RSV infection. Expression of CD103 by lung cDC from hMPV-, RSV-, or mock-infected mice was assessed by three-color flow cytometry studies using anti-CD11c-PE-Cy5 and anti-MHC-II-FITC in combination with anti-CD103-PE. A, Upper dot plots correspond to total lung cells 7 days after infection: lineage-negative cells, MHC-II+ cells, CD11c+ cells, and CD103+ cells. Gated cDC (CD11chighMHC-IIhigh) were analyzed for CD103 expression. Lower dot plots show two major subpopulations of lung cDC as CD103+ and CD103 cells. B, Percentage of lung cDC CD103+ at different time points. Bar graph represents mean ± SEM (n = 4–16 mice/group/time point). ∗, p < 0.05 and ∗∗, p < 0.01, compared with mock-infected mice. Data are representative of three independent experiments.

FIGURE 9.

CD103 (αE) expression by lung cDC upon hMPV and RSV infection. Expression of CD103 by lung cDC from hMPV-, RSV-, or mock-infected mice was assessed by three-color flow cytometry studies using anti-CD11c-PE-Cy5 and anti-MHC-II-FITC in combination with anti-CD103-PE. A, Upper dot plots correspond to total lung cells 7 days after infection: lineage-negative cells, MHC-II+ cells, CD11c+ cells, and CD103+ cells. Gated cDC (CD11chighMHC-IIhigh) were analyzed for CD103 expression. Lower dot plots show two major subpopulations of lung cDC as CD103+ and CD103 cells. B, Percentage of lung cDC CD103+ at different time points. Bar graph represents mean ± SEM (n = 4–16 mice/group/time point). ∗, p < 0.05 and ∗∗, p < 0.01, compared with mock-infected mice. Data are representative of three independent experiments.

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This study analyzed the effect of paramyxovirus infections on the immune function of lung DC. Viruses have developed many ways to evade the host immune response, including interference with the antiviral effects of IFNs, interference with Ag processing and presentation, and suppression of the maturation and migration of DC (36). Although previous work has shown that RSV and hMPV are able to impair the DC-mediated immune responses in vitro (29, 31, 37), this is the first report which demonstrates that RSV and hMPV are able to impair the function of the DC compartment in vivo. Following RSV and hMPV infection, we observed an impaired capacity of lung pDC to respond to TLR activation as indicated by the reduced production of IFN-α as well as other cytokines. We also observed an impaired Ag-presenting function, as measured by decreased T cell proliferation following stimulation of T cells with cDC from infected mice.

The importance of DC during the immune response to RSV and hMPV is supported by our analysis of infected mice. First, we studied the trafficking of pDC, cDC, and IKDC after hMPV or RSV infection, as the three types of DC so far described in the mouse respiratory tract (38). The flow cytometry data show that pDC, cDC, and IKDC are rapidly recruited to the lung and draining mediastinal LN after intranasal instillation of RSV and hMPV. In line with these results there is a study demonstrating that both myeloid DC and pDC numbers were increased in nasal washes of RSV-infected children (39). As demonstrated here, the recruitment of pDC was different between hMPV and RSV infection. RSV induced lung pDC recruitment that peaked at day 3 and returned to basal levels by day 6, while pDC reached the maximum numbers at day 8 in hMPV-infected mice to return to normal numbers by day 12. In contrast with this report, previous work indicate that infection of mice with RSV led to sustained pDC recruitment in the lung of infected mice (40). However, these experiments differ from ours in that pDC were identified as CD11c+B220+ while we used a more comprehensive set of markers to identify mouse pDC. Specifically, we used a three-color flow cytometry analysis to identify pDC as CD11cintB220+Gr-1+ since the expression of CD11c and B220 is shared by IKDC (21, 22, 41). The present data were confirmed by identifying pDC by the combined expression of mPDCA1, a specific marker for mouse pDC (42, 43), and B220 (data not shown). In fact, a recent report has demonstrated that the heterogeneous population of CD11c+B220+ can be resolved into three distinct subsets based on the expression of Siglec-H and NK1.1. Siglec-H+/NK1.1 cells are the only true pDC that produce IFN-α in response to TLR stimulation. A second Siglec-H-/NK1.1 subset mostly consists of CD19+Ig+ B cells. Finally, Siglec-H-/Nk1.1+ cells correspond to IKDC (44). Thus, is possible that the sustained pDC recruitment after RSV infection reported by others may represent recruitment of B cells to the lung. No differences were observed in the recruitment of pDC to the LN between RSV and hMPV infection.

In regard to cDC analysis, our data showed that this DC subset is the most prominent among DC subsets in the lung after RSV and hMPV infection. The increase in the number of cDC in the lung was sustained up to day 18 after infection, when lungs from infected mice still contained significantly more cDC compared with mock-infected mice. These data are in agreement with those previously reported by Beyer et al. (45) in the lung after RSV infection, where cDC, also identified as CD11chighMHC-IIhigh, were increased and sustained until day 21 (45). In addition, we show that hMPV induce a similar pattern of cDC recruitment than that of RSV infection in both lung and LN.

IKDC was the smallest DC subset recruited to the lung in infected mice. We observed a pattern that resembles that of pDC, including a biphasic and more prolonged recruitment after hMPV infection compared with RSV. Although the classification (21, 22, 24, 44, 46), origin (47), as well as the physiological role of IKDC (21, 23, 41, 48, 49) remain controversial, our data suggest that this DC subset may play a role in both hMPV and RSV infections. Altogether, these data demonstrate a distinct pattern of DC trafficking to the lung in hMPV or RSV infections. To the best of our knowledge, this is the first study that analyzes the DC response in a mouse model of hMPV infection.

The kinetics of DC recruitment demonstrates one more feature of the immune response which is differentially altered by RSV and hMPV, as indicated in previous studies, in which regardless of the amount of infectious dose, virus-specific response was observed in regard to clinical disease (body weight loss), lung neutrophilia, profile of bronchoalveolar lavage cytokines/chemokines, and viral clearance. Also, we have recently reported that the contribution of T cell subsets to the pathogenesis of RSV and hMPV infection is also clearly distinct, as CD4+ and CD8+ cells appear to be more important in hMPV and RSV disease, respectively (50).

When we determined the effect of hMPV and RSV infection on lung DC, analysis of the cytokine responses of individual DC subsets (Figs. 4 and 5) revealed the potential contribution of pDC and cDC to the production of these mediators during hMPV and RSV infection. pDC isolated from lung were the major producers of IFN-α following ex vivo infection with hMPV but not RSV. This observation in murine cells differs from previous work in humans, as we and others have shown that RSV is able to stimulate human peripheral blood pDC to produce IFN-α as well as other cytokines (29, 51). Apart from potential species-specific differences (mouse vs human), we speculate that pDC in lung tissue may respond differently to hMPV or RSV than those from peripheral blood. Indeed, the observed lack of IFN-α production by pDC upon RSV stimulation is in line with recent findings indicating that depletion of pDC in mice did not alter the production of IFN-α in the lung upon RSV infection (52) and that alveolar macrophages rather than pDC are the major producers of IFN-α following RSV infection (D. Kolli and R. P. Garofalo, unpublished observations and Ref. 53). Our results also show that pDC responded to hMPV but not RSV to produce TNF-α and CCL5. Other chemokines were also induced in pDC by both viruses including CCL3 and CCL4. Analysis of cDC stimulation indicated a very similar profile of cytokines induced by RSV and hMPV. Both viruses were prominent inducers of IL-12p40, IL-10, IL-6, CCL3, CCL5, KC, CCL11, and CCL4, but they induced a modest production of IL-12p70, TNF-α, and IL-1α. Interestingly, only hMPV was able to induce IFN-β, which resembles our observation in human moDC (29), suggesting that hMPV is a stronger inducer of type I IFN than RSV in both human and mouse DC. These data are consistent with our previous reports where we have shown that the profile of cytokines/chemokines in bronchoalveolar lavage from mice infected with RSV or hMPV differs widely (26). Moreover, we have demonstrated that in human DC, RSV and hMPV induce a differential profile and abundance of cytokines (29). Overall, our results demonstrated that different subsets of isolated lung DC differ both qualitatively and quantitatively in cytokine and chemokine production in response to hMPV and RSV infection and suggest that pDC and cDC play a role as innate immune effectors critical in the host cytokine response to these paramyxovirus infections (26).

The ability of pDC to recognize and respond to viruses is critical to provide a first line of defense at mucosal surfaces. Functional analysis of the effect of RSV and hMPV on lung pDC demonstrated that these viral infections were able to impair the capacity of these cells to respond to TLR agonists. Indeed, infection of mice with hMPV and RSV reduced the ability of their lung pDC to produce IFN-α and other cytokines in response to ODN CpG. We and others have previously demonstrated that these two viruses inhibit the cytokine response of human DC in vitro (29, 31) and that prior infection of mice with hMPV or RSV inhibits the production of IFN-α in response to ODN CpG (54). Thus, this report provides novel evidence that the innate immune function of lung DC can be impaired by respiratory viral infections in vivo. These findings suggest that lung pDC may only weakly respond in situ to inflammatory stimuli, i.e., bacterial infection once they have been exposed to viral infections in the respiratory tract. In line with this report, a recent study has indicated that alveolar macrophages isolated after influenza infection have impaired NF-κB nuclear translocation to TLR ligation with reduced production of KC, MIP-2α, and TNF-α (55). The mechanistic basis for the impairment of lung pDC remains to be defined. It might be induced directly by viral interaction with pDC or via the interaction with infected epithelial cells or soluble mediators that are released by lung resident cells. However, no virus was detected in pDC isolated from infected mice (data not shown). Current studies are in progress to determine whether viral infections in vivo may alter the level of the expression of TLR9 in lung pDC.

It is well known that the immune memory against RSV and hMPV is incomplete even in healthy adult individuals (11, 56). T cell responses appear to be crucial to clear RSV or hMPV from the infected lung, because patients with immune deficiencies in the T cell arm of the adaptive immune response are unable to clear the virus affectively (57). In fact, recent clinical observations have suggested that T lymphocytes are virtually absent in lung tissue of fatal cases of RSV-induced lower respiratory tract infections (58), suggesting that certain viral pathogens have developed mechanisms to inhibit cell-mediated immune responses in the airway mucosa. Moreover, we have recently demonstrated in the mouse model that T cells are crucial for antiviral immunity against hMPV (50). The suppressive effects on T cell proliferation in vitro by RSV are well documented (59, 60, 61, 62). We and others have previously shown that RSV is able to inhibit the ability of human moDC to stimulate T cell proliferation in vitro (29, 37, 63), and a similar trend was observed in moDC infected with hMPV (29). In this work, we reported for the first time in an in vivo experimental model that infection with either RSV or hMPV impairs the capacity of lung cDC to present Ag to CD4+ T cells. cDC isolated from infected mice at weeks 2 and 3 have a reduced capacity to stimulate CD4+ T cell proliferation compared with cDC from mock-infected mice. Moreover, that inhibitory effect seems to be selective for lung cells since we did not observe any inhibition of the CD4+ T cell proliferation when we used cDC isolated from spleen of infected mice (data not shown). On the other hand, an increased stimulatory capacity of cDC was observed at week 1 after viral infection. These data are in agreement with previous work on RSV that has indicated that lung DC isolated as early as 10 days after infection led to a significant stimulation of T cells in an alloreaction (45).

The mechanisms by which RSV and hMPV impair cDC function have not yet been identified. However, we observed that upon viral infection, several surface molecules were overexpressed in lung cDC from infected mice, including PD-L1. PD-L1 (B7-H1) and PD-L2 (B7-DC), members of the B7 family, are the ligands of PD-1, a member of the CD28 receptor family expressed on activated T and B cells (64). PD-L1 is expressed on activated T cells, B cells, monocytes and DC, as well as nonhematopoetic cells such as keratinocytes and endothelial cells. The expression of PD-L2 has only been described on monocytes and DC activated with cytokines, particularly IL-4, and on activated human endothelial cells (65, 66, 67). Both PD-1-PD-L1 and PD-1-PD-L2 interactions inhibit T cell proliferation, cytokine production, and CTL activity (65, 67, 68). Studies in vitro have shown that RSV infection is able to up-regulate PD-L1 and PD-L2 expression on epithelial cells (69) and human moDC (data not shown). In this work, however, the overexpression of PD-L1 upon infection suggests that inhibitory molecules may lead to a decreased capacity of lung DC to induce proliferation in Ag-specific CD4+T cells. Current work is in progress to determine whether the expression of PD-L1 is involved in the inhibitory effect of cDC during RSV and hMPV infection.

Alteration in the composition of lung CD103+ cDC was also observed after viral infection. The percentage of cDC CD103+ was reduced as early as day 2 (data not shown) and remained lower than in mock-infected mice until week 3 after infection and returned to control by week 8 (Fig. 9 B). Expression of αE(CD103)β7 is associated with important cellular activities of mucosal dendritic APCs, such as Ag presentation (32, 33, 70), production of proinflammatory cytokines, or stimulation of T regulatory cells (34) and some mucosal CD8+ T cells (71, 72). Overall, several evidences suggest an association of αE(CD103)β7 expression with important immune functions. It is, nevertheless, currently unclear whether and to what extent αE(CD103)β7 itself contributes directly to such cellular functions; therefore, the precise role of αE(CD103)β7 in immune regulation still remain largely elusive. Whether in our model of viral infection, the expression of CD103 in cDC plays an important role in the overall antiviral response against these two viruses remains to be determined.

In summary, our results demonstrate that respiratory infections by hMPV or RSV alter the pulmonary environment and the function of lung DC. These changes last beyond the period of acute infection are not apparently associated with persistent viral replication and may significantly alter the host ability to mount an effective immune response against pathogens or bystander Ags in this phase of relatively immunologic anergy.

We thank Mark Griffin at the Flow Cytometry and Cell Sorting Core Laboratory, UTMB, for help with cell sorting analysis and Giovanni Suarez for assistance with the Bio-Plex assays.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants P01 AI062885 and N01 AI30039 (to R.P.G.), an Unrestricted Research Grant from the American Thoracic Society, and a Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (to A.G.-P.).

3

Abbreviations used in this paper: hMPV, human metapneumovirus; RSV, respiratory syncytial virus; DC, dendritic cell; IKDC, IFN-producing killer DC; pDC, plasmacytoid DC; cDC, conventional DC; ODN, oligodeoxynucleotide; LN, lymph node; MOI, multiplicity of infection; PD-L, programmed death ligand; MHC-II, MHC class II.

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