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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Sep 15, 2012.
Published in final edited form as:
PMCID: PMC3304096
NIHMSID: NIHMS313484

Multiple CD4+ T cell subsets produce immunomodulatory interleukin-10 during respiratory syncytial virus infection

Abstract

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10 producing cells in the lung during acute RSV infection were CD4+ T cells. The IL-10-producing CD4+ T cells included Foxp3+Tregs, Foxp3 CD4+ T cells that co-produce IFN-γ, and Foxp3 CD4+ T cells that do not co-produce IFN-γ. RSV infection of IL-10-deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared to control mice. We also observed an increase in the magnitude of the RSV-induced CD8+ and CD4+ T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4+ T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4+ T cells and a concomitant decrease in Foxp3+ regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.

Keywords: Virus, Lung, T cells, interleukin-10, immunopathology

Introduction

The lung environment with its constant exposure to inert foreign antigens must preserve a fine balance between tolerance and inflammation to effectively clear respiratory pathogens while at the same time maintaining tissue homeostasis. Improper regulation of a pathogen-induced immune response can result in excessive immune-mediated injury to the lung tissue, termed immunopathology. Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in young children, the elderly and immunocompromised individuals (16). The host immune response is thought to contribute to the severity of RSV-induced respiratory disease (7). In addition to RSV, other respiratory viral pathogens, such as pandemic influenza strains and human coronaviruses that cause severe acute respiratory syndrome (SARS), are believed to induce a poorly regulated immune response resulting in immune-mediated disease (8, 9).

The lung environment contains a number of mechanisms that regulate the immune response (10), including the production of immunosuppressive cytokines such as IL-10. Both innate and adaptive immune cells have the capacity to produce IL-10, including mast cells, NK cells, neutrophils, eosinophils, macrophages, dendritic cells (DC), CD4+ T cells, CD8+ T cells, and B cells (11). In addition, almost all immune cells express the IL-10R1 and IL-10R2 subunits (1214), which together comprise the IL-10 receptor (IL-10R), and are thus capable of responding to IL-10. Due to the broad expression of IL-10 and the potential responsiveness of most immune cells, it is clear that IL-10 can influence both innate and adaptive immunity.

IL-10 mediates pleiotropic effects on the immune response, including suppressing the activation and/or effector functions of immune cells. In macrophages, IL-10-induced signaling results in inhibition of pro-inflammatory cytokines and reduced production of chemoattractants required for the recruitment of T cells, monocytes, DCs, and neutrophils to the site of infection (15). In addition, IL-10 suppresses the surface expression of MHC class II and costimulatory molecules on macrophages and immature DCs, resulting in the decreased activation and proliferation of T cells (1618). IL-10 can also directly inhibit T cell cytokine production (e.g. IL-2, IL-5, and TNF) and induce T cell anergy (1921).

The critical role of IL-10 during persistent infections has been well established (15, 22), however its capacity to modulate the immune response during an acute viral infection resulting in immunopathology remains unknown. In the murine model of RSV infection, it is evident that RSV-induced disease is immune-mediated since depletion of T cells ameliorates disease even in the presence of prolonged viral replication (23, 24). This makes RSV an ideal model for studying dysregulation of an immune response in the lung mucosal environment. To disect the role of IL-10 during a respiratory viral infection resulting in immunopathology, we first identified the IL-10-producing cells during acute RSV infection and examined the effects of IL-10 deficiency on the adaptive immune response. We demonstrate that CD4+ T cells account for the majority of IL-10 production during the peak of the virus-specific T cell response. Furthermore, we show multiple CD4+ T cell subsets produce IL-10. In the absence of IL-10 or following IL-10R blockade we observed a significant increase in RSV-induced disease severity correlating with the kinetics of the RSV-specific adaptive immune response. Finally, we show IL-10R blockade results in an increase in the number of TH1 and TH17 cells with a concomitant decrease in the number of T regulatory cells (Treg). Taken together, our data suggest that IL-10 plays a critical role in modulating the adaptive immune response to acute RSV infection and in the prevention of immune-mediated pulmonary injury.

Materials and Methods

Mice, Infection, and Antibody Treatment

Six to eight week-old female BALB/cAnNCr mice were purchased from the National Cancer Institute (Frederick, MD). BALB/c IL-10-deficient mice (IL-10 KO) were obtained from Dr. Daniel Berg (University of Iowa, Iowa City, IA) (25, 26). VERT-x mice, expressing an IL-10-eGFP reporter, were obtained from Dr. Christopher Karp (Cincinnati Children’s Hospital Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH) (27). The A2 strain of RSV was a gift from Dr. Barney Graham (National Institues of Health, Bethesda, MD) and was propagated on HEp-2 cells (American Type Culture Collections; ATCC, Manassas, VA). For infections, mice were anesthetized with isoflurane and infected intranasally (i.n.) with of 1–3 × 106 PFU of RSV. In some experiments, mice were administered either 0.5 mg rat IgG (MP Biomedicals, Inc., Aurora, OH) or 0.5 mg anti-IL-10R mAb (clone 1B1.3) via i.p. injections starting one day prior to infection and every other day thereafter. CD4+ and/or CD8+ T cells were depleted via i.p. injection of 0.25 mg of anti-CD4 (clone GK1.5) and/or anti-CD8 (clone 2.43) antibody at days −2 and +2 relative to infection. All experimental procedures involving mice were approved by the University of Iowa Animal Care and Use Committee.

Disease Measurements

Disease was assessed daily by monitoring weight loss as well as pulmonary function using an unrestrained whole body plethysmograph (Buxco Electronics, Wilmington, NC) as previously described (28, 29). Pressure and volume differences in the chamber caused by respiration were monitored for 5 mins and recorded to obtain a daily average in order to calculate the dimensionless parameter enhanced pause (Penh) as previously described (30). Penh positively correlates to airway hyper-reactivity (30), which is narrowing of the small airways in response to stimuli.

Tissue Processing

Lungs and bronchoalveolar lavage (BAL) fluid from mice were harvested as previously described (31). Lung tissue and lymph nodes were digested in 4 ml or 1 ml, respectively, of HBSS with CaCl2 and MgCL2 (Gibco, Grand Island, NY) supplemented with 60 U/ml DNase I (Sigma, St. Louis, MO) and 125 U/ml collagenase (Invitrogen, Carlsbad, CA) for 30 minutes at 37°C. After digestion, lungs where pressed through a wire mesh screen (Cellector; Bellco Glass, Inc., Vineland, NJ) to create a single-cell suspension. Mediastinal lymph nodes (medLN) were disrupted by gentle pressing between the ends of frosted glass slides (Surgipath, Richmond, IL).

Extracellular Staining

1–2 × 106 cells were placed in a 96-well round-bottom plate (Corning Inc., Corning, NY). Cells were incubated with mAbs for extracellular proteins—CD90.2 (Thy1.2; clone 53-2.1), CD4 (clone RM4.5), CD8 (clone 53-6.7), CD11a (clone M17/4), and CD44 (clone IM7)—and simultaneously blocked with anti-FcγRII/III mAb (clone 93) (all mAbs obtained from eBioscience, San Diego, CA) in FACS buffer (PBS, 2% FCS, 0.02% sodium azide) for 30 mins at 4°C. Cells were subsequently washed three times with FACS buffer at 4°C, fixed with FACS lysing solution (BD Biosciences, San Diego, CA) for 15 mins at room temperature, washed three times with FACS buffer at 4°C and resuspended in FACS buffer for analysis. Samples were collected on a BD FACSCanto flow cytometer and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Ex vivo cell stimulations

Cells were incubated at 1–2 × 106 cells/well in a 96-well round-bottom plate (Corning Inc.). For peptide stimulations, cells were incubated with or without 1 μM of M282–90 peptide (Biosynthesis, Inc., Lewisville, TX)and 10 μg/ml brefeldin A (Sigma) in RPMI 1640 supplemented with 10% FCS (Atlanta Biologicals, Lawrenceville, GA), 5 nM 2-mercaptoethanol (Sigma), 2 mM L- glutamine, 10 U/ml penicillin, 10 μg/ml streptomycin sulfate, 10 mM HEPES, 1mM Sodium Pyruvate, and 0.1 mM MEM non-essential amino acids (all obtained from Gibco) for 5 h at 37°C (31). Alternatively, cells were stimulated in the presence or absence of 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of 10 μg/ml brefeldin A in 10% FCS complemented RPMI 1640 for 5 h at 37°C.

Intracellular Cytokine Staining

Briefly after ex vivo stimulation, cells were stained for extracellular proteins and fixed in FACS lysing solution as described above. Cells were subsequently incubated in FACS buffer containing 0.5% saponin (Sigma) and intracellular cytokine antibodies—IL-10 (clone JES5-16E3), IFN-γ (clone XMG1.2), TNF-α (clone MP6-XT22), IL-17A (clone TC11-18H10.1) (all mAbs obtained from eBioscience)—for thirty minutes at 4°C. Cells were washed twice with FACS buffer containing saponin, once with FACS buffer, and resuspended in FACS buffer prior to analysis on a BD FACSCanto.

Foxp3 Staining

After staining extracellular proteins as described above, cells were stained for Foxp3 using the mouse regulatory T cell staining buffer kit (eBioscience) according to the manufacturer’s instructions. Cells were stained with mAbs against Foxp3 (clone FJK-16s), Helios (clone 22F6), IL-10 (clone JES5-16E3), and IFN-γ (clone XMG1.2) (all mAbs obtained from eBioscience). After staining, cells were resuspended in FACS buffer prior to analysis.

Plaque assays

Lungs were harvested on days 4 and 7 post-infection and processed for plaque assays as previously described (32).

ELISAs

Lungs from RSV infected mice were disrupted using a tissue homogenizer (Ultra-Turrax T25; IKA Works, Inc., Wilmington, NC) in RPMI 1640 containing 10% FCS and a 1/200 dilution of a protease inhibitor cocktail (Sigma). Lung homogenates were centrifuged at 2000 rpm for 10 min. Cytokine protein levels in the supernatant were determined for IL-10, IFN-γ by ELISA as previously described (33) (IL-10 and IFN-γ Abs were obtained from eBioscience). Detection limits for cytokine protein levels were 62.5 pg/ml for IL-10 and 1250 pg/ml for IFN-γ.

Multiplex Bead Assays

Lungs from RSV infected IL-10 KO and WT mice were harvested and supernatants were collected as described above. A Milliplex kit was used to determine the protein levels of 27 different cytokines and chemokines by following the manufacture’s instructions (Millipore, Billerica, MA). The assay was run on a BioPlex instrument (Bio-Rad, Hercules, CA).

Histology

Whole lungs from naïve and RSV infected rIgG and anti-IL-10R mAb treated mice on day 6 p.i. were fixed in 10 percent formalin (Thermo Fisher Scientific, Waltham, MA). Fixed lungs were embedded in paraffin, sectioned at 4 μm thickness and stained by the University of Iowa Comparative Pathology Laboratory. Slides were either H&E or periodic acid Schiff (PAS) stained and randomized and blinded for analysis by a board-certified veterinary pathologist (Dr. D. Meyerholz, University of Iowa). Sectioned slides were scored for perivascular aggregates of leukocytes (PVA) (1, within normal parameters; 2, small numbers of solitary cells with uncommon aggregates; 3, multifocal small to moderate aggregates; and 4, moderate to high cellularity with multifocal large cellular aggregates that may be expansive into adjacent tissues), interstitial disease (ID) (1, within normal parameters; 2, mild, detectable focal to multifocal congestion with uncommon to small numbers of leukocytes and some atelectasis; 3, moderate, multifocal to coalescing congestion, leukocyte cellularity, and atelectasis with rare luminal leakage of cellular and fluid debris; and 4, severe, coalescing interstitial congestion, leukocytes, and atelectasis with admixed extensive loss of airspace and luminal accumulation of cellular and fluid debris), edema (1, none; 2, rare with alveoli with wispy to partial pools of eosinophilic seroproteinaceous fluid; 3, focal microscopic fields with contiguous alveoli partially filled by pools of fluid; and 4, multiple fields having coalescing alveoli filled by pools of fluid), and mucus (1, none; 2, epithelial mucinous hyperplasia with none to rare luminal mucus; 3, epithelial mucinous hyperplasia with luminal mucus accumulation in airways; and 4, severe mucinous change with some airways completely obstructed by mucus). The total score is the sum of the PVA, ID, edema, and mucus scores.

Statistical analysis

Data was graphically compiled using Prism software (Graphpad Software Inc., San Diego, CA) with bars representing the standard error of the mean (SEM). Statistical analysis was completed using InStat software (Graphpad Software Inc.); unpaired, two-tailed Student t tests and analysis of variances with a Dunnett’s post test were calculated to determine statistical significance at a level of α=0.05.

Results

CD4+ T cells account for the majority of IL-10-producing cells during the peak of the virus-specific T cell response

To determine the role of IL-10 during acute RSV infection we first examined the kinetics and cellular source of IL-10 production. Cells from the lung, medLN, and lung airways (i.e. BAL) were stimulated in vitro with PMA and ionomycin and stained for intracellular IL-10. The total number of IL-10-producing cells peaked on day 6 p.i. in the lung and medLN and on day 8 p.i. in the BAL (Fig. 1A). CD4+ T cells comprised the majority of IL-10-producing cells (Fig. 1B). In contrast, IL-10-producing CD8+ T cells did not accumulate in large numbers in any of the tissues analyzed (Fig. 1C).

Figure 1
The kinetics of IL-10-producing cells during acute RSV infection. At the indicated time points, cells from the lung, medLN, and BAL of RSV-infected mice were isolated and stimulated with PMA and ionomycin followed by intracellular cytokine staining for ...

To verify our results that were obtained using in vitro stimulation, we utilized mice that express an IL-10-eGFP reporter to examine IL-10-producing cells directly ex vivo during an acute RSV infection. In agreement with our in vitro restimulation data discussed above, the frequency of IL-10-eGFP+ cells peaked on day 8 p.i. in the lung and BAL (supplemental Fig. 1). Approximately 80 to 90 percent of IL-10-eGFP+ cells on day 6 and 8 p.i. were T cells as identified by Thy1.2 staining in the lung and BAL (supplemental Fig. 1). Within the Thy1.2+ population, CD4+ T cells represented the majority of IL-10-eGFP+ T cells in lungs and BAL at all time points examined. Taken together, these data demonstrate CD4+ T cells account for the majority of IL-10-producing cells during the peak of the virus-specific T cell reponse.

To confirm that CD4+ T cells produce the majority of IL-10 in vivo during RSV infection, we quantified IL-10 protein levels in the lungs of mice depleted of CD4+ and/or CD8+ T cells. Our results indicate that CD4+ T cells were responsible for the majority of IL-10 protein produced in the lung during an acute RSV infection (Fig. 2A). Elimination of CD8+ T cells resulted in increased IL-10 production suggesting that CD8+ T cells may suppress IL-10 production by CD4+ T cells. IFN-γ production was also measured using the above approach and as expected CD8+ T cells produced significantly more IFN-γ than the CD4+ T cells in response to RSV infection (Fig. 2B). Taken together, these data demonstrate that CD4+ T cells are the major producers of IL-10 in vivo during an acute RSV infection. The capacity of CD4+ T cells to produce IL-10, suggests their potential to significantly modulate the immune response via anti-inflammatory cytokine production.

Figure 2
CD4+ T cells are the primary in vivo source of IL-10 during acute RSV infection. Wild-type mice were treated with depleting anti-CD4 and/or anti-CD8 mAbs or control rat IgG and subsequently infected with RSV. On day 6 p.i., lungs were analyzed for (A) ...

Multiple CD4+ T cell subsets produce IL-10 in response to RSV infection

Since CD4+ T cells accounted for the majority of RSV-induced IL-10-producing cells, we next determined the CD4+ T cell subsets responsible for the IL-10 production. On day 6 p.i., lung, medLN, and BAL cells were stimulated in vitro with PMA and ionomycin and stained for intracellular IL-10, IFN-γ, and Foxp3. In all tissues analyzed, we observed equal proportions of IL-10+ CD4+ T cells that only produced IL-10, co-produced IFN-γ (i.e. TH1 cells) or co-expressed Foxp3 (i.e. Tregs) (Fig. 3). Interestingly, in the BAL we observed a small population of Foxp3+ Tregs that co-produced IL-10 and IFN-γ. These data demonstrate that multiple CD4+ T cell subsets contribute to IL-10 production during an acute RSV infection.

Figure 3
Multiple CD4+ T cell subsets produce IL-10 during acute RSV infection. At day 6 p.i., cells from the lung, medLN, and BAL were stimulated with PMA and ionomycin. Cells were subsequently stained for CD4, Thy1.2, IL-10, Foxp3, and IFN-γ. Gated IL-10 ...

In addition, we determined the frequency of IL-10 producing cells within these CD4+ T cell subsets (Table 1). On day 6 p.i., we observed the highest frequency of IL-10-only producing conventional and regulatory CD4+ T cells (5.1 ± 1.6 and 36.7 ± 6.0 percent, respectively) in the BAL. Furthermore, in the BAL we observe the highest proportion of conventional and regulatory CD4+ T cells that co-produce IL-10 and IFN-γ (11.1 ± 4.4 and 8.8 ± 4.1 percent, respectively) when compared to other tissues. In all tissues analyzed IL-10 producing conventional and regulatory CD4+ T cells were observed as summarized in Table 1. This data demonstrates the distribution of IL-10 producing cells in the conventional and regulatory CD4+ T cell compartments following acute RSV infection.

Table I
Frequencies of cytokine producing CD4+ T cells on day 6 following RSV infection.

Increased RSV disease severity in the absence of IL-10 or following IL-10R blockade

To examine the role of IL-10 during acute RSV infection, we utilized either IL-10 deficient (IL-10 KO) mice or anti-IL-10R mAb to determine disease severity in the absence of IL-10 or following IL-10R blockade. IL-10 KO mice exhibited greater weight loss and increased Penh compared to control mice following acute RSV infection (Fig. 4A). Similar results were obtained from mice treated with anti-IL-10R mAb prior to and during RSV infection as compared to IgG-treated controls (Fig. 4B). These data indicate that IL-10 plays a critical role in limiting disease severity at the peak of the adaptive immune response during RSV infection.

Figure 4
IL-10 influences RSV-induced disease severity. Weight loss depicted as the percent of initial weight and airway function (Penh) were assessed daily following RSV infection of (A) IL-10-deficient or (B) anti-IL-10R mAb treated mice. Cumulative data from ...

To determine if IL-10 had an effect on viral titers, we performed plaque assays using whole lung homogenates to measure viral load at the peak of RSV replication (i.e. day 4 p.i.) and following viral clearance (i.e. day 7 p.i.). There were no significant differences in viral titer at either time point in IL-10 KO mice (Fig. 5A) or anti-IL-10R mAb treated mice (Fig. 5B). These results show the effect of IL-10 deficiency on disease severity is not due to either altered viral replication or delayed viral clearance, but instead may be the result of alterations of the RSV-specific immune response.

Figure 5
IL-10 does not affect RSV replication and clearance kinetics. Lungs were harvested on day 4 and 7 p.i. from RSV-infected (A) IL-10 KO or (B) anti-IL-10R mAb treated mice. RSV titers were determined by plaque assay. The limit of detection (L.O.D.) is indicated ...

Increased inflammatory environment in the absence of IL-10

Given the increased disease severity observed during RSV infection in the absence of IL-10, we next examined the overall inflammatory environment in the lung following acute RSV infection. Using a multiplex bead assay, we quantified cytokine and chemokine protein levels from whole lung homogenates at days 0, 4, 6, 8, and 10 p.i. from control and IL-10 KO mice infected with RSV. In the absence of IL-10, we observed a marked increase in the levels of pro-inflammatory cytokines and chemokines following RSV infection (Fig. 6). In the lungs of RSV-infected IL-10 KO mice we observed a significant increase in IL-6 (p < 0.01) and TNF-α (p < 0.001) on day 6 p.i., and a significant increase in IFN-γ on days 6 (p < 0.001) and 8 p.i. (p < 0.05) as compared to control mice. In addition, CXCL1 was significantly increased in the lungs of IL-10 KO mice at days 4 (p < 0.05) and 6 p.i. (p < 0.01) and slightly increased on day 8 p.i. when compared to control mice. CXCL9 was increased and CCL2 was significantly increased (p < 0.001) on day 6 p.i. in the absence of IL-10 following RSV infection. Overall, in the absence of IL-10 we observed increased levels of pro-inflammatory cytokines and chemokines that can enhance inflammation and the infiltration of immune cells responsible for mediating immunopathology. Taken together, these data support the hypothesis that IL-10 plays a critical role in regulating the severity of RSV-induced pulmonary disease by regulating inflammation and limiting immunopathology.

Figure 6
Increase in pro-inflammatory cytokines and chemokines in the absence of IL-10. At days 0, 4, 6, 8, and 10 p.i., lungs from IL-10 KO mice were harvested and cytokine and chemokine protein levels within the lung supernatants were determined by multiplex ...

IL-10R blockade results in an increase in the magnitude of the RSV-specific CD8+ T cell response

Since the increase in RSV-induced disease severity (days 6–8 p.i.) in the absence of IL-10 or following IL-10R blockade occurred during the rise of the RSV-specific adaptive immune response, we next investigated the impact of IL-10 deficiency on the magnitude of the RSV-specific CD8+ T cell response. In mice treated with anti-IL-10R mAb, there is a significant increase (p < 0.01) in the total number of CD8+ T cells in the medLN on day 6 p.i. (Fig. 7A). Furthermore, there is a significant increase (p < 0.001) in the total number of CD11ahi CD8lo T cells in the medLN of mice treated with anti-IL-10R mAb on day 6 p.i. (Fig. 7B), indicating the presence of antigen-experienced cells (34).

Figure 7
The magnitude of the CD8+ T cell response is increased following IL-10R blockade. Mice were treated with anti-IL-10R mAb as described in the materials and methods. (A) The total number of CD8+ T cells at day 6 (left) and 8 (right) p.i. in the lung and ...

Using the M282–90 peptide to stimulate RSV-specific CD8+ T cells ex vivo, we observed a significant increase in the magnitude of the M282–90 RSV-specific CD8+ T cell response in the medLN at day 6 p.i. (p < 0.001) and in the lung on day 8 p.i. (p < 0.01) (Fig. 7C, 7D). In the medLN of mice treated with anti-IL-10R, we observed a significant increase in the total number of M282–90-specific CD8+ T cells producing IFN-γ (p < 0.01) or co-producing IFN-γ and TNF-α (p < 0.0001) on day 6 p.i. In the lungs of anti-IL-10R mAb treated mice, there is a significant increase in the total number of M282–90-specific CD8+ T cells that produce IFN-γ (p < 0.001) or co-produce IFN-γ and TNF-α (p < 0.01) on day 8 p.i. These data indicate an increase in both the magnitude and effector function of RSV-specific CD8+ T following IL-10R blockade demonstrating a role for IL-10 in limiting the RSV-specific CD8+ T cell response.

IL-10R blockade results in an increased TH1 CD4+ T cell response following acute RSV infection

Since the RSV-specific CD8+ T cell response was increased following IL-10R blockade, we next evaluated the impact of IL-10 on the magnitude of the effector CD4+ T cell response during acute RSV infection. IL-10R blockade resulted in a significant increase in the total number of CD4+ T cells in the lung (p < 0.01) and BAL (p < 0.05) on day 8 p.i. (Fig. 8A). Intracellular cytokine staining for IFN-γ and TNF-α production by CD4+ T cells from the lung and medLN stimulated with PMA and ionomycin revealed a significant increase (p <0.01) in the total number of effector CD4+ T cells in the lung on day 8 p.i. in mice administered anti-IL-10R mAb (Fig. 8B). Furthermore, on day 8 p.i. in the lung, mice treated with anti-IL-10R mAb exhibited a significant increase in the number of CD4+ T cells that produce IFN-γ (p < 0.001) or co-produce IFN-γ and TNF-α (p < 0.05). Thus, similar to the increase in the number of CD8+ T cells described above, we observed an increase in the magnitude of the TH1 CD4+ T cell response in the lungs of mice administered anti-IL-10R mAb during acute RSV infection. This increase in the number of virus-specific IFN-γ-producing CD8+ and CD4+ T cells following IL-10R blockade during a RSV infection suggests a critical role for IL-10 in limiting the magnitude of the RSV-specific adaptive immune response.

Figure 8
Increased TH1 response following IL-10R blockade. (A) The total number of CD4+ T cells in the lung, medLN, and BAL at day 6 (left) and 8 (right) p.i. (B) The total number of PMA and ionomycin stimulated CD4+ T cells producing only IFN-γ (closed ...

Increased lung pathology following IL-10R blockade

To determine if the increased disease severity and effector CD8+ and CD4+ T cell response results in increased lung pathology following IL-10R blockade, histological analysis of sections from whole lungs were stained with either H&E or PAS and analyzed. On day 6 p.i., a significant increase (p <0.001) was observed in lung pathology when scoring the perivascular aggregation of leukocytes (PVA), interstitial disease (ID), eosinophilic seroproteinaceous fluid in the airspaces (edema), and luminal mucus accumulation in the airways (Fig. 9A). Histology of rIgG-treated mice following RSV infection revealed tissue inflammation with minimal mucus in the luminal space of the lung airways (Fig. 9B). The histological sections from mice treated with anti-IL-10R mAb demonstrate the increased pathology with increased areas of edema, tissue inflammation and increased mucus in the luminal space of the lung airways (Fig 9C). Together these data indicate that IL-10R blockade results in increased lung pathology that is associated with increased disease severity following RSV infection.

Figure 9
Increased lung pathology following IL-10R blockade. (A) Cumulative pathological scoring analysis of sections from whole lungs of rIgG and anti-IL-10R mAb treated mice following RSV infection. Representative hematoxylin and eosin (H&E) stained ...

Alteration of additional CD4+ T cell subsets following IL-10R blockade

With the increased TH1 CD4+ T cell response during RSV infection in the absence of IL-10, we wanted to investigate the effect of IL-10 deficiency on additional CD4+ T cell subsets. IL-10R blockade resulted in alterations in the Treg and TH17 CD4+ T cell subsets following acute RSV infection (Fig. 10). To determine changes in the natural and inducible Treg compartments, we identified natural Tregs by their expression of the transcription factor Helios (35, 36). On day 8 p.i., there is a significant decrease in Foxp3+Helios+ CD4+ T cells in the lung (p < 0.01), medLN (p < 0.001), and BAL (p < 0.05) in mice administered anti-IL-10R mAb (Fig. 10A). However there is no difference in the total number of Foxp3+Helios CD4+ T cells between mice treated with the anti-IL-10R mAb and controls. These data suggest that IL-10, directly or indirectly, supports the maintenance or induction of natural Tregs during an acute RSV infection.

Figure 10
Increased TH17 response and decreased in T regulatory cell response following IL-10R blockade. (A) At day 8 p.i., natural Tregs (Foxp3+Helios+) and inducible Tregs (Foxp3+Helios) were quantified in the lung, medLN, and BAL of RSV-infected mice ...

Coincident with the decrease in the total number of natural Tregs, we observed a significant increase in the total number of IL-17A-producing CD4+ T cells in mice treated with anti-IL-10R mAb (Fig. 10B). On day 8 p.i., IL-17A+ CD4+ T cells are significantly increased following IL-10R blockade in the lung (p < 0.01) and BAL (p < 0.05) during acute RSV infection. Similar results were obtained using IL-10 KO mice (data not shown). We did not observe IL-17A-producing CD4+ T cells in the medLN or IL-17F-producing cells in the lung, medLN, and BAL of experimental or control mice (data not shown). Thus, IL-10R blockade leads to decreased numbers of Tregs in the lung with a concomitant increase in the number of TH17 cells.

Discussion

Here we examined the kinetics of IL-10 production during acute RSV infection and found that the highest frequency of IL-10-producing cells occurred between days 6 through 10 post-infection, a time frame that overlaps with the virus-specific adaptive immune response (Fig. 1, supplemental Fig. 1) (37, 38). We found that three major subsets of CD4+ T cells were responsible for the majority of IL-10 production at the peak of the immune response following RSV infection (Fig. 3). Approximately one third of the IL-10-producing CD4+ T cells expressed Foxp3, representing a portion of the Treg population, whereas the remainder were conventional Foxp3 CD4+ T cells that produced either IL-10 alone or co-produced IFN-γ. In addition, we observed a small frequency of IL-10+ IFN-γ+ regulatory (Foxp3+) CD4+ T cells only in the lung airways following RSV infection. We hypothesize that the environment in the lung airways is unique causing these CD4+ T cells to alter their phenotype. We are currently investigating the plasticity of these IL-10+ IFN-γ+ regulatory (Foxp3+) CD4+ T cells and their role during acute RSV infection.

TH1 cells have been previously reported to be the major producers of IL-10 in other pathogenic models including influenza A virus (IAV), Leishmania species, mycobacterial and Borrelia burgdorferi infections (3943). The observaton that other CD4+ T cell subsets are capable of producing IL-10 and that IL-10 can regulate T cell function, has led to the hypothesis that IL-10 acts in either an autocrine manner to directly suppress T cell activation or a paracrine manner through DCs to control immunopathology (11, 4446). Our results support the conclusion that in the absence of IL-10 and its regulation of T cells, increased RSV-induced disease severity is a consequence of increased immunopathology. However, the molecular mechanisms that induce IL-10 production by effector T cells during RSV infection remains to be determined.

We demonstrate that CD4+ T cells produce the majority of IL-10 in vivo during an acute RSV infection. In contrast, we observe only a small number of IL-10-producing CD8+ T cells (Fig. 1). IL-10 production by T cells has been examined in a number of other respiratory virus infections. Consistent with our results, McKinstry et al. recently reported that CD4+ T cells make the majority of IL-10 during a high dose IAV infection (43). Virus-specific CD8+ T cells that co-produce IL-10 and IFN-γ in the lung have been reported during acute SV5 infection (47). However these studies did not assess IL-10 production by CD4+ T cells during SV5 infection. Therefore it is unclear if either CD4+ or CD8+ T cells represent the dominant cell source of IL-10 during acute SV5 infection. In contrast to our findings with RSV, Sun et al. report that CD8+ T cells are responsible for the majority of IL-10 production during an acute low dose IAV infection (48). It is currently unclear what accounts for the reported discrepancies in the proportion of CD4+ and CD8+ T cells that make IL-10 during respiratory viral infections. A more recent study by Sun et al. indicates that IL-10 production by CD8+ T cells during an acute IAV infection requires the expression of Blimp-1 and is synergistically promoted by IL-2 produced by helper CD4+ T cells as well as IL-27 produced by innate immune cells (49). It remains to be determined if the reduced number of IL-10-producing CD8+ T cells during an acute RSV infection is the result of a lack of CD4+ T cell help and/or decreased IL-27 production.

Exacerbated disease severity and increased levels of pro-inflammatory cytokines and chemokines were observed in the absence of IL-10 following RSV infection (Figs. 4 and and6).6). However, we did not observe any changes in peak viral titers (day 4 p.i.) or viral clearance (day 7 p.i.) between IL-10 KO or anti-IL-10R mAb treated mice compared to their controls (Fig. 5). These data support the idea that increased disease severity is independent of RSV replication and clearance kinetics (23). Instead, alterations in the RSV immune response (i.e. increased inflammatory conditions) result in increased immunopathology. Similarly, Haeberle et al. reported that treatment with recombinant IL-10 significantly decreased the production of chemokines, such as CCL2, CCL3 and CCL5 in the lung airways when analyzed 24 h after RSV infection (50). CCL5 has been positively associated with RSV-induced disease severity and blocking CCL5 results in the increased production of IL-12 and decreased airway hyperreactivity (51). Consistent with its effect on chemokine production, treatment with recombinant IL-10 significantly reduced the number of cellular infiltrating lymphocytes and decreased alveolar inflammation following acute RSV infection (50). Exogenous addition of IL-10 did not effect viral replication (50). We demonstrate that IL-10 deficiency significantly increases weight loss and airway resistance following RSV infection (Fig. 4). The above results combined with our findings suggest that IL-10 acts by inhibiting pro-inflammatory cytokine production in the lung, which in turn reduces RSV-induced pulmonary inflammation and immunopathology.

Similar to these observations, findings from the avian H5N1 outbreaks in Hong Kong revealed that patients with severe disease exhibited high serum cytokine levels of IL-6, TNF-α and IFN-γ and increased serum chemokine levels of CCL2, CXCL9 and CXCL10 when compared to less severely ill controls (8, 5255). The levels of IL-6, IFN-α, IFN-γ, CCL2 and CXCL10 were also significantly elevated in the serum of SARS patients with more severe disease (5658). Overall, in the absence of IL-10 our data support the notion that a strong pro-inflammatory response contributes to increased RSV disease severity. Thus therapeutic approaches aimed at increasing IL-10 production may offer a means to decrease RSV-induced immunopathology without affecting viral clearance.

IL-10R blockade resulted in an increase in the magnitude and effector function of RSV-specific CD8+ T cells and CD4+ T cells (Fig. 7, ,8).8). A similar observation was reported by Sun et al. where acute IAV infection resulted in an increased total number of IFN-γ producing CD8+ and CD4+ T cells in mice treated with anti-IL-10R mAb (48). In addition, we observed a significant increase in the number of TH17 CD4+ T cells during an acute RSV infection in the absence of IL-10 (Fig. 10). In agreement with our findings, McKinstry et al. recently reported a significant increase in IL-17 production and the accumulation of TH17 cells in the lungs of IL-10 KO mice following an acute high dose IAV infection (43).

In contrast to the increased number of TH17 cells discussed above we observed a decrease in the total number of Helios+ Foxp3+ Tregs in the lung following IL-10R blockade in RSV infected mice. Recent work suggests that Helios expression may selectively identify natural Tregs (35, 36). Thus, the decreased number of natural Tregs may directly contribute to increased disease severity. In recent studies, depletion of natural Tregs results in exacerbated RSV-induced disease and an increased number of IFN-γ+ TNF-α+ CD8+ T cells (29, 59). In a model of colitis, IL-10 was reported to be necessary to maintain Foxp3 expression of CD4+ T cells under inflammatory conditions (60). Murai et al. demonstrated that the suppressive function of Tregs was maintained by IL-10R expression and the capacity to directly respond to IL-10. However, the environment in the intestinal mucosa is unique with different regulatory factors than the lung, and the findings of Murai et al. may not necessarily translate to lung mucosal tissue. Therefore, the role of IL-10 in the regulation of Foxp3 expression will need to be further evaluated in the lung environment. We are currently examining the role of IL-10 in the induction and/or maintenance of pulmonary Tregs during acute RSV infection.

IL-10 polymorphisms have been associated with RSV-induced disease severity in humans (61, 62). Here we demonstrate that IL-10 plays a critical role in regulating the immune response and preventing exacerbated disease due to immune-mediated pulmonary injury during acute RSV infection. Multiple respiratory viruses, such as pandemic IAV strains and the human coronavirus that causes SARS, are believed to induce immunopathology resulting in severe morbidity and mortality (8, 9, 5658). Therefore, identifying novel approaches to modulate virus-induced immunopathology would be beneficial in treating acute respiratory viral infections. Exogenous IL-10 treatment has been shown to be safe and well tolerated in clinical trials for several chronic inflammatory diseases including Crohn’s disease, Psoriasis, and chronic hepatitis C (6368). In patients with chronic hepatitis C who were non-responsive to IFN-based therapy, IL-10 treatment did not affect viral titers but reduced chronic liver inflammation and fibrosis (66). Using IL-10 as an immunosuppressive treatment during a respiratory viral infection resulting in immunopathology may be feasible to prevent severe morbidity and mortality. Our data indicate that IL-10 is an attractive target based on its ability to regulate RSV-induced innate and adaptive immune responses without affecting viral clearance.

Supplementary Material

Acknowledgments

This work was supported by the NIH grant R01 AI063520 (to S.M.V.) and Training in Statistics in Microbiology, Infectious Diseases, and Bioinformatics training grant T32 GM077973 (to K.A.W.).

The authors would like to thank Drs. John Harty and Stanley Perlman for their critical review of the manuscript. We would also like to thank Stacey Hartwig for her excellent technical assistance.

The multiplex cytokine assay data presented was obtained at the Flow Cytometry Facility, which is a Carver College of Medicine Core Research Facilities/Holden Comprehensive Cancer Center Core Laboratory at the University of Iowa. The Facility is funded through user fees and the generous financial support of the Carver College of Medicine, Holden Comprehensive Cancer Center, and Iowa City Veteran’s Administration Medical Center.

Abbreviations used in this paper

RSV
respiratory syncytial virus
SARS
severe acute respiratory syndrome
DC
dendritic cells
Treg
T regulatory cells
i.n
intranasal
BAL
bronchoalveolar lavage
PVA
perivascular aggregates of leukocytes
ID
interstitial disease
medLN
mediastinal lymph node
p.i
post-infection
IAV
influenza A virus

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