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J Virol. May 2010; 84(10): 5007–5014.
Published online Mar 3, 2010. doi:  10.1128/JVI.02408-09
PMCID: PMC2863832

A Detrimental Effect of Interleukin-10 on Protective Pulmonary Humoral Immunity during Primary Influenza A Virus Infection [down-pointing small open triangle]

Abstract

Interleukin-10 (IL-10) is an important anti-inflammatory molecule that can cause immunosuppression and long-term pathogen persistence during chronic infection of mice with viruses such as lymphocytic choriomeningitis virus. However, its specific role in immunity to acute viral infections is not fully understood. We found that IL-10 plays a detrimental role in host responses to acute influenza A virus since IL-10−/− mice had improved viral clearance and survival after infection compared to wild-type mice. Enhanced viral clearance in IL-10−/− mice was not correlated with increased CD4+ or CD8+ T-cell recruitment into the lung but was correlated with increased pulmonary anti-influenza virus antibody titers, and this was dependent upon the presence of T cells, primarily CD4+ T cells. In addition, virus-specific antibody produced during the early stages of infection in the respiratory tract of IL-10−/− but not wild-type mice was sufficient to mediate passive protection against viral challenge of naïve mice. Complement was necessary for this antibody-mediated passive protection, but FcγR or neutrophil deficiency did not significantly influence viral clearance. Our results show that an absence of IL-10 at the time of primary infection leads to enhanced local virus-specific antibody production and, thus, increased protection against influenza A virus infection.

Interleukin-10 (IL-10) is known to play a critical immunoregulatory role during immune responses to microbial pathogens. Many bacterial and viral infections stimulate host IL-10 production, which is ultimately beneficial or detrimental, depending upon the type of infection. In animal models, IL-10 production by dendritic cells is proposed to be critical for the induction of tolerance that is induced by respiratory exposure to antigen (2). During the host defense against microbial infection, IL-10 can hamper pathogen clearance but can also improve immunopathology by regulating innate and adaptive immunity and limiting the magnitude of inflammatory responses. IL-10 can enhance chronic infections caused by Leishmania and lymphocytic choriomeningitis virus (LCMV) due to the suppression of immune responses to these pathogens (1, 3, 4, 8). On the other hand, IL-10 was shown to inhibit immunopathological consequences following infection with a wide variety of pathogens, including Toxoplasma gondii, Trypanosoma cruzi, and Helicobacter hepaticus (20).

With chronic viral infections, IL-10 can enhance microbial persistence through the induction of immunological anergy (13). Specifically, during LCMV infection of mice, IL-10 is responsible for the functional impairment and deletion of virus-specific CD8+ T cells as well as a more general immunosuppression (3, 4, 8). On the other hand, information regarding the role of IL-10 during acute influenza virus infection appears to be contradictory. Sun et al. (17) previously found that an inhibition of IL-10 signaling in the midst of an ongoing influenza virus infection resulted in increased inflammation and decreased survival. However, the influence of IL-10 during the early stages of immune response induction after viral infection was not examined. Conversely, a recent study by McKinstry et al. (14) reported that IL-10-deficient mice have significantly increased survival after influenza infection. Conclusions regarding the beneficial or detrimental role of IL-10 in these two studies were based entirely on survival studies, but no significant influence of IL-10 on viral persistence or clearance was reported.

Previously, we used C57BL/6 IL-10−/− mice to investigate the role of IL-10 during post-influenza virus bacterial infection (18). In those experiments, mice were first intranasally (i.n.) challenged with a sublethal dose (10 PFU) of influenza virus, followed approximately 1 week later with i.n. Streptococcus pneumoniae challenge. Compared to wild-type (WT) mice, IL-10−/− mice did not have notably improved survival from secondary bacterial infection in this coinfection model. Remarkably, however, IL-10−/− mice had a significantly decreased viral burden at the recovery stage of sublethal influenza virus infection (18). To our knowledge, this was the first evidence that IL-10 actually influenced the kinetics of viral clearance during acute influenza infection. Importantly, the use of viral burden as a readout provided a tremendous advantage for studying the underlying immune mechanisms responsible for microbial synergy while minimizing the nonspecific effects of a lethal viral burden. We have now used IL-10−/− mice to further investigate the regulatory role of IL-10 and have found that IL-10 has a detrimental role during initial responses to primary influenza virus infection regardless of the challenge dose. Our results indicate that IL-10 inhibits CD4+ T-cell-helper function during the induction of initial virus-specific antibody responses and thereby leads to impaired resistance to primary influenza virus infection.

MATERIALS AND METHODS

Murine model of viral infection.

Specific-pathogen-free, 6- to 8-week old, C57BL/6 WT mice were purchased from Taconic Laboratories (Germantown, NY) and Charles River Laboratories (Wilmington, MA). C57BL/6 IL-10−/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred at Albany Medical College according to IACUC guidelines.

Viral challenge was performed with A/PR8/34 (PR8) influenza virus (Charles River Laboratories) administered i.n. to anesthetized mice in 50 μl of sterile phosphate-buffered saline (PBS). Titers of virus stocks and viral levels in bronchoalveolar lavage fluid (BALF) samples and lungs of infected mice were determined by plaque assays on MDCK cell monolayers. For determinations of morbidity, mice were weighed on the day of infection and daily thereafter.

Expression of alveolar and lung inflammatory cells.

BALF samples were collected by making an incision in the trachea and washing the lung twice with 0.8 ml PBS (pH 7.2). Total leukocyte counts were determined by using a hemacytometer. BALF cell populations were further evaluated by using Diff-Quick-stained cytospin preparations.

For pulmonary B-cell analysis, single-cell suspensions were obtained from lungs by digestion with 2.5 mg/ml collagenase D, 0.25 mg/ml DNase I (Roche Diagnostics, Mannheim, Germany), and 1 mM MgCl2 for 1 h at 37°C under constant agitation, followed by passage through nylon mesh and density gradient centrifugation on Lympholyte M (Cedarlane Laboratories Limited, Ontario, Canada).

For flow cytometric analysis, BALF cells or lung lymphocytes were fixed with 2% paraformaldehyde, incubated with monoclonal antibody (MAb) 2.4G2 against Fc0γRII/III, and stained with phycoerythrin (PE)-conjugated anti-CD11c (Caltag Laboratories, Burlingame, CA), allophycocyanin (APC)-Cy7-conjugated anti-CD11b (BD Biosciences), and Tri-color-conjugated anti-Ly6G MAb (eBioscience, San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD3, APC-conjugated anti-CD4, and PE-conjugated anti-CD8 MAbs were used for T-cell analysis. PE-Cy5-conjugated anti-CD19 MAb (eBioscience) was used for B-cell analysis. The stained cells were analyzed with a BD FACSCanto apparatus using BD FACSDiva software.

Neutrophil depletion.

Anti-Ly6G MAb was purified from culture supernatants of RB6-8C5 hybridomas by use of anti-rat IgG agarose columns (Sigma). Mice were injected intraperitoneally (i.p.) with 100 μg of MAb RB6-8C5 daily for 2 days before influenza infection, followed by inoculation every 3 days after infection (three to five mice/group). The efficiency of neutrophil depletion in BALF of virus-infected mice was confirmed by flow cytometry.

Quantitative reverse transcription (RT)-PCR.

Alveolar macrophages and BALF dendritic cells were purified with CD11c microbeads by an autoMACS separator system (18). Total RNA derived from naïve or day 7 post-influenza-virus-infected mice was characterized by using the Mouse Innate and Adaptive Immune Response PCR array with RT2 SYBR green/fluorescein PCR master mix (SABiosciences, Frederick, MD) on a Bio-Rad iCycler.

Determination of cytokine production by ELISA.

BALF was harvested and assayed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA) using commercially available kits from R&D Systems (Minneapolis, MN) and PBL Interferon Source (Piscataway, NJ).

Intracellular cytokine staining.

BALF cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS) for 5 h at 37°C in the presence of 50 ng/ml phorbol myristate acetate (PMA), 500 ng/ml ionomycin, and 10 μg/ml brefeldin A. The cells were then fixed with 2% paraformaldehyde and stained with PE-Cy7-conjugated anti-gamma interferon (IFN-γ) (BD Biosciences), PE-conjugated anti-tumor necrosis factor alpha (TNF-α), and APC-conjugated anti-CD4 (BD Biosciences) MAbs. Fixed cells were also stained with PE-conjugated anti-IL-4 (BD Biosciences), APC-conjugated anti-IL-13 (eBioscience), and FITC-conjugated anti-CD4 (BD Biosciences) MAbs. Pulmonary lymphocytes isolated from asthmatic mice were used as positive controls for cytokine staining. The stained cells were stored in the dark at 4°C and analyzed within 24 h with a FACSCanto apparatus using FACSDiva software.

Determination of virus-specific antibodies by ELISA.

Concentrations of H1N1-specific antibodies in serum and BALF samples were measured by ELISA. Briefly, Maxisorp ELISA plates (Nalge Nunc International, Rochester, NY) were coated overnight at 4°C in PBS containing 2 μg/ml Fluvirin (Chiron Vaccines, Liverpool, United Kingdom). After washing, 2-fold dilutions of serum or BALF samples were incubated in the plates at 37°C for 2 h. Detection was performed by using biotin-labeled goat anti-mouse IgM, IgG, IgG1, IgG2a, or IgG3 antibodies (Southern Biotechnology Associates, Birmingham, AL). Finally, an avidin-biotin-horseradish peroxidase (HRP) complex (BD Biosciences) was added, and OptEIA substrate solution (BD Biosciences) was used for signal development.

T-cell depletion.

Anti-CD4 and anti-CD8 MAbs were purified from culture supernatants of GK1.5 and 53-6-72 hybridomas, respectively, by using anti-rat IgG agarose columns (Sigma). C57BL/6 mice were injected i.p. with 500 μg of MAb daily for 3 days before influenza virus infection, followed by i.p. inoculation every 3 days after infection (18). The efficiency of pulmonary CD4 and CD8 cell depletion after influenza virus infection was confirmed by flow cytometric analysis of isolated BALF cells and splenocytes.

Passive protection.

WT and IL-10−/− mice were challenged i.n. with 200 PFU of A/PR/8, and BALF samples were isolated 9 days later. Pooled BALF samples were incubated at 55°C for 1 h to inactivate complement. A total of 2 × 104 PFU/ml PR8 virus was mixed with 10% BALF diluted in PBS at 4°C immediately before infection. Mice were then infected i.n. with 1,000 PFU as described above. The mice were either sacrificed 24 h later to determine lung viral burden or monitored daily for survival. For BALF antibody depletion, a 5-fold dilution of immune BALF was incubated with goat anti-mouse Ig-coated protein A beads at 4°C for 4 h. The efficiency of antibody depletion was confirmed by ELISA.

Histology.

Mice were sacrificed 7 days after i.n. infection, and the lungs were removed for histological analyses. Paraffin-embedded tissues were sectioned to a thickness of 5 μm and stained with hematoxylin and eosin (H&E) by standard methods.

Statistical analysis.

The data are expressed as means ± standard deviations (SD). A Student's t test (to compare two samples) and analysis of variance (ANOVA) (to compare multiple samples) were used for statistical analysis. Survival analyses were performed by using the Kaplan-Meier log-rank test. A P value of <0.05 was considered to be significant.

RESULTS

Increased resistance to influenza virus infection in IL-10-deficient mice.

During influenza virus infection, numerous cells can be found to express IL-10 (14, 17). We similarly detected increased levels of IL-10 in murine BALF after i.n. inoculation of A/PR/8 influenza virus, which peaked on day 7 of infection (Fig. (Fig.1a).1a). A significant source of this IL-10 in BALF appeared to be the CD11c+ cell population, which contained mainly alveolar macrophages and some dendritic cells (Fig. (Fig.1b1b).

FIG. 1.
Influenza virus infection induces IL-10 production. (a) IL-10 protein levels in BALF 5, 7, and 9 days after i.n. infection with 50 PFU A/PR/8/34 (PR8) virus (four to six mice per group). (b) Quantitative RT-PCR analysis of IL-10 transcripts in CD11c+ ...

IL-10−/− mice were next used to investigate the potential regulatory role of IL-10 in antiviral immune responses. WT and IL-10−/− mice were infected i.n. with various doses (sublethal to lethal) of influenza virus. The viral burden in the infected mice was then monitored by collecting BALF and lung tissue at various times after infection. It was found that the viral burden peaked at around day 5 after the onset of primary infection. Interestingly, regardless of the inoculation dose, an increased clearance of virus was detected thereafter in IL-10−/− mice compared to WT mice (Fig. 2a and b). After sublethal virus inoculation, enhanced clearance in IL-10−/− mice was most obvious beginning around day 9. However, after a lethal inoculation (500 PFU), when the majority of WT mice died before day 9, significantly increased viral clearance was found for IL-10−/− mice on day 7. Increased viral clearances in IL-10−/− mice were comparable in lung tissue and BALF (Fig. (Fig.2b).2b). In conclusion, these results show that the deletion of IL-10 enhances pulmonary immune recovery from influenza virus infection regardless of the inoculation dose.

FIG. 2.
IL-10−/− mice are more resistant to influenza virus infection. (a) C57BL/6 mice were infected i.n. with 50 PFU (left), 200 PFU (middle), or 500 PFU (right) of PR8 influenza virus. Numbers of viral PFU in the BALF were then determined on ...

The weights of animals infected with 200 PFU of A/PR/8 were also measured as a sign of morbidity. Twenty-five percent of WT mice died at this challenge dose, but all IL-10−/− mice survived (data not shown). Among surviving animals, weight losses during the first 6 days of infection were essentially identical between WT and IL-10−/− mice (Fig. (Fig.2c).2c). Thereafter, IL-10−/− mice showed less weight loss and an earlier recovery of weight, suggesting substantially reduced morbidity and enhanced recovery after influenza virus infection compared to WT animals. Finally, and of considerable note, at a high dose of virus (500 PFU), 90% of IL-10−/− mice survived, while all WT mice succumbed to infection (Fig. (Fig.2d).2d). Collectively, these results indicate that the absence of IL-10 at the time of infection enhances resistance to influenza virus infection, an effect that is particularly noticeable at the time of viral clearance, i.e., about 1 week after initial infection, when adaptive immunity is typically induced. This observation prompted further studies to understand the mechanism responsible for the detrimental effects of IL-10 on adaptive antiviral immune responses.

Pulmonary cell recruitment during influenza virus infection of WT and IL-10−/− mice.

IL-10 is known to be an important anti-inflammatory cytokine, so it is possible that a dysregulated balance between immunopathology and viral clearance would account for the difference in the severities of illness observed for WT and IL-10−/− mice (16). To investigate this, WT and IL-10−/− mice were infected with 50 or 500 PFU of A/PR/8 virus, and lung histology was performed on day 7 after infection. Lungs isolated from WT and IL-10−/− mice exhibited discrete regions of peribronchial and perivascular inflammation at this time point (Fig. (Fig.3a).3a). However, there were no obvious differences among histological sections from WT and IL-10−/− mice after either sublethal or lethal challenge. These data indicate that the absence of IL-10 does not significantly increase the extent of general inflammation observed within the lung following influenza virus infection.

FIG. 3.
Cell recruitment after influenza virus infection. (a) H&E-stained sections of lungs harvested 7 days after infection with 50 PFU of influenza virus. The results are representative of data from three mice per group. Similar results were obtained ...

We also examined the composition of infiltrating BALF leukocytes in WT and IL-10−/− mice by cytospin and fluorescence-activated cell sorter (FACS) analysis (Fig. 3b and c). Regardless of the time after infection, there were no significant differences in total BALF cell counts between WT and IL-10−/− mice (data not shown). In response to infection, numbers of leukocytes in the airway increased in both strains of mice, and neutrophils and monocytes were the most abundant cell types found on days 5 and 7 after infection. This was confirmed by FACS analysis, which showed that Ly6G+ cells accounted for up to 60% of the total cell counts. Although IL-10−/− mice had a greater percentage of Ly6G+ granulocytes on day 7 after low-dose viral infection (50 PFU) than WT mice, this relative increase was not observed after high-dose viral infection (500 PFU) (data not shown). By day 9, T cells were the predominant cell type, while B cells (CD19+) and NK cells (NK1.1+) accounted for <1% of the BALF cells. Similar levels of CD4+ and CD8+ T cells were recruited into the lungs of both WT and IL-10−/− mice after influenza virus infection.

To verify that the observed increases in viral clearance by IL-10−/− mice were not due to differences in Ly6G+ cell influx, neutrophils in WT and IL-10−/− mice were depleted by anti-Ly6G antibody treatment before and after infection with 50 PFU of influenza virus. It was found that the enhanced viral clearance in IL-10−/− mice compared to WT mice was not affected by neutrophil depletion (Fig. (Fig.3d).3d). Thus, neutrophils do not play a significant role in the recovery process following sublethal influenza virus infection. Taken together, the results show that the detrimental role of IL-10 is not associated with decreased or altered kinetics of immune cell recruitment after influenza virus infection.

Pulmonary cytokine and antibody responses during influenza virus infection of WT and IL-10−/− mice.

The functional activity of recruited immune cells following influenza virus infection was next measured. BALF cytokine levels in virus-infected WT and IL-10−/− mice were assayed by ELISA. Naïve IL-10−/− mice tended to have increased basal levels of the proinflammatory cytokines TNF-α and IL-1β early in infection (data not shown), although the expression level of these cytokines was not significantly different from that of naïve WT mice. In addition, levels of these cytokines were decreased at the recovery phase of influenza virus infection for both groups of mice (Fig. (Fig.4).4). Low levels of IL-17 were present after influenza virus infection, and again, no significant differences were detected between WT and IL-10−/− mice. An increased level of production of IFN-α in the lungs of WT mice compared to the level in the lungs of IL-10−/− mice on day 5 after infection was observed, but these levels subsequently decreased in both groups of mice. IFN-β was barely detectable in any experimental group. Of note, however, IL-10−/− mice had significantly increased levels of IFN-γ on days 7 to 9 following influenza virus infection compared to levels in WT mice (Fig. (Fig.44).

FIG. 4.
Cytokine responses after influenza virus infection. TNF-α, IL-1β, IL-17A, IFN-α, IFN-β, and IFN-γ levels in BALF 5, 7, and 9 days after i.n. infection with 50 PFU of PR8 virus (six mice/group) were determined. **, ...

Antibodies against both viral hemagglutinin and neuraminidase are involved in protection against influenza (9, 15). To determine whether IL-10 influenced the induction of antibody expression, H1N1-specific antibody levels in BALF were determined by ELISA. Similar H1N1-specific IgM levels were found for WT and IL-10−/− mice on day 7 after influenza virus infection, and there was only a low level of production of IgG (Fig. (Fig.5a).5a). However, increased levels of both H1N1-specific IgM and IgG were found in IL-10−/− mice compared to WT mice on day 9 after infection. IgG3 appeared to be the major IgG isotype expressed, while there were only low levels of IgG1 or IgG2a (Fig. (Fig.5b).5b). The significant increase in levels of antibody expression by IL-10−/− mice was consistent with the increase in levels of CD19+ lung B cells that occurred in these mice on days 7 to 9 after influenza virus infection; no significant differences in WT lung B-cell numbers were observed during this time frame after viral infection (Fig. (Fig.5c5c).

FIG. 5.
Antibody responses in BALF and pulmonary B-cell expression after influenza virus infection. (a and b) BALF H1N1-specific IgM and IgG levels on days 7 and 9 (a) and H1N1-specific IgG1, IgG2a, and IgG3 levels on day 9 (b) after i.n. infection with 50 PFU ...

Enhanced pulmonary antibody production in IL-10−/− mice during influenza virus infection.

About 1 week following primary influenza virus infection of mice, there is a large influx of CD4+ and CD8+ T cells into the respiratory tract, and it is well established that these cells are important for protection against influenza virus infection (19). To define the potential roles of CD4 and CD8 T-cell subsets in the increased resistance of IL-10−/− mice to infection, in vivo cell depletion studies were performed. Significant levels of IL-10 were found in BALF of both T-cell-depleted and control WT mice on day 9 after infection (Fig. (Fig.6a).6a). Viral clearance was inhibited in both WT and IL-10−/− mice after the depletion of CD8+ cells, but CD8 cell-depleted IL-10−/− mice were still better able to clear virus than CD8 cell-depleted WT mice (Fig. (Fig.6b).6b). However, when CD4+ cells were depleted, levels of viral outgrowth were comparable for WT and IL-10−/− mice (Fig. (Fig.6b).6b). This finding suggests that the relative resistance of IL-10−/− mice to viral infection compared to WT mice is CD4+ cell dependent; i.e., the viral clearance advantage seen in IL-10−/− mice was lost upon CD4 cell depletion. Specific cytokine production by pulmonary CD4 T cells was then analyzed by flow cytometry. There were only very few TNF-α-expressing CD4 T cells in either WT or IL-10−/− mice on day 7 or 9 after influenza virus infection, and there were none producing IL-4 or IL-13. However, there were significant amounts of IFN-γ+ CD4+ T cells induced in the respiratory tract following influenza virus infection (Fig. (Fig.6c).6c). IFN-γ levels in BALF from CD4+ or CD8+ cell-depleted mice were also determined. In agreement with the data shown in Fig. Fig.4,4, control, rat IgG-treated, IL-10−/− mice expressed higher levels of IFN-γ than WT mice (Fig. (Fig.6d).6d). This IFN-γ production was associated with the presence of CD8+ cells but not CD4+ cells, since only CD8 T-cell depletion resulted in reduced IFN-γ levels (Fig. (Fig.5b).5b). The lack of enhanced IFN-γ expression by IL-10−/− CD4 T cells in CD8-depleted mice indicates that this is not the reason for the improved efficiency of viral clearance in IL-10−/− mice, which is CD4 T-cell dependent. This finding agrees with data from previous studies indicating that IFN-γ is not necessary for protection against influenza virus infection (10).

FIG. 6.
Influenza virus-induced immune responses in CD4+ and CD8+ T-cell-depleted mice. IL-10 levels (a), viral titers (b), percentages of IFN-γ+ CD4+ T cells in BALF on days 7 and 9 (c), and IFN-γ levels in BALF ...

H1N1-specific antibody levels in BALF of CD4+ or CD8+ cell-depleted mice were determined by ELISA. For control animals treated with normal rat IgG, there were increases in H1N1-specific IgM and IgG levels in the BALF of influenza virus-infected IL-10−/− mice compared to WT mice (Fig. (Fig.7a).7a). For IgM, the increases were not correlated specifically with the presence of either CD4 or CD8 T cells, but the increases in levels of IgG were clearly correlated with the presence of CD4 T cells (Fig. (Fig.7a).7a). It is worth noting that only minor differences in levels of serum IgG were observed for WT and IL-10−/− mice (Fig. (Fig.7b).7b). Taken together, the results suggest that increased levels of CD4-dependent antibody production in the respiratory tract of IL-10−/− mice are involved in the enhanced resistance of these animals to influenza virus infection.

FIG. 7.
Antibody responses in CD4+ and CD8+ T-cell-depleted mice. Shown are BALF H1N1-specific IgM and IgG levels (a) and serum H1N1-specific IgG levels (b) on day 9 after i.n. infection with 50 PFU of influenza virus. Mice were injected i.p. ...

Antibody-mediated passive protection against influenza virus infection.

Passive-protection studies were next performed to determine whether the increased levels of pulmonary antibodies in IL-10−/− mice were sufficient to protect naïve mice from influenza virus infection. For this purpose, WT and IL-10−/− mice were infected with 200 PFU of influenza virus, and BALF samples were then collected, diluted 1:10 in PBS, and administered i.n. to naïve mice together with 103 PFU of A/PR/8 influenza virus. It was found that immune BALF collected from IL-10−/− mice 9 days after infection again had increased H1N1-specifc antibody titers compared to immune BALF from WT mice (Fig. (Fig.8a),8a), in agreement with the results shown in Fig. Fig.6.6. Furthermore, while immune BALF from IL-10−/− mice provided 100% protection to naïve recipients, BALF from WT mice provided no protection (Fig. (Fig.8b).8b). Protection was mediated by specific antibody, since the depletion of immunoglobulins from the BALF using anti-mouse Ig-coated beads abrogated protection (Fig. (Fig.8c).8c). In addition, complement, but not FcγR, was required for passive protection, since immune BALF did not protect C3−/− mice but still mediated protection of FcγR−/− mice (Fig. (Fig.8d).8d). Of note, immune BALF from WT mice could provide 100% protection against challenge with 103 PFU A/PR/8 when given undiluted (data not shown); thus, the observed differences between IL-10−/− and WT BALF samples were quantitative. These results suggest that the elevated antibody titers in immune BALF from IL-10−/− mice enhanced effectiveness in mediating passive protection. Based on these data, we conclude that the improved production of influenza virus-specific antibody in the BALF of IL-10−/− mice increases viral clearance and mediates resistance to influenza virus infection in a complement-dependent manner.

FIG. 8.
Passive protection by immune BALF. (a) H1N1-specific IgM and IgG antibody (Ab) titers in immune BALF from WT and IL-10−/− mice infected with 200 PFU of PR8 virus. Titer values are expressed as the reciprocal BALF dilutions corresponding ...

DISCUSSION

IL-10 is known to play a central role in maintaining immune homeostasis at mucosal surfaces. In the current study, we found that IL-10 had an adverse effect on the regulation of protective immune responses to influenza virus infection. Mice lacking IL-10 were more resistant to influenza virus infection, as determined by enhanced viral clearance, decreased morbidity, and greater survival. Our results demonstrate a detrimental role for IL-10 during primary influenza virus infection of mice regardless of infectious dose. In contrast to chronic viral infection, such as infection with LCMV, the harmful role of IL-10 observed during influenza virus infection appears to be due mainly to the suppression of CD4+ T-cell function rather than CD8+ T-cell function. In addition, anti-influenza virus antibody produced in the respiratory tract of IL-10−/− mice was found to be highly efficient in mediating passive protection, which occurred in a complement-dependent manner.

For IL-10−/− mice, we found not only remarkably increased survival rates but also enhanced viral clearance compared to those of WT mice. Very recently, two other studies also addressed the potential role of IL-10 in influenza virus infection by challenging mice with lethal doses of A/PR/8 virus and monitoring survival. It was shown previously by Sun et al. (17) that a blockade of IL-10 signaling in the midst of an ongoing influenza virus infection decreased survival, which correlated with increased inflammation. On the other hand, McKinstry et al. (14) reported that IL-10-deficient mice had significantly increased rates of survival after influenza virus infection, which were correlated with enhanced IL-17 production. There is general agreement that by inhibiting both immunopathology and antiviral immune responses, IL-10 can act like a “double-edged sword” during infection, which might account for the opposite outcomes observed for these two survival studies, particularly since IL-10 activity was blocked at different times of infection. However, the nonspecific immune responses induced by lethal infectious doses might have somewhat obscured interpretations regarding the direct effect of IL-10 on antiviral immunity. Thus, by measuring both survival and viral burden in response to various doses of virus challenge, we were able to more closely investigate the mechanisms responsible for enhanced viral clearance in IL-10-deficient animals.

IL-10 can be produced by many different cell types, including alveolar macrophages and dendritic cells. There was a significant induction of BALF IL-10 expression in WT mice on day 7 after influenza virus infection, suggesting a potential regulatory role for IL-10 in the adaptive immune response that begins to be induced at this time point. To gain insight into the role of IL-10 during influenza virus infection, we initially determined the magnitude and composition of the leukocyte infiltrates in the BALF of infected WT and IL-10−/− mice. However, no apparent differences in the numbers or compositions of leukocyte subsets were observed between WT and IL-10−/− mice. The mice were then examined for cytokine and antibody production in response to influenza virus infection. While a significantly increased level of production of IFN-γ by CD8+ T cells was found for IL-10−/− mice, this was not associated with enhanced viral clearance. The increased clearance in IL-10−/− mice was correlated with the presence of CD4+ T cells, but little IFN-γ production by these cells was observed at this time point. In addition, we found significantly lower levels of H1N1-specific antibodies in the BALF of CD4+ T-cell-depleted mice, which paralleled the increased viral burden. Taken together, the findings indicate that IL-10 inhibits the CD4+ T-helper function that is important for antibody-mediated viral control.

IL-10 is known to promote humoral immune responses by inducing immunoglobulin production and secretion, including T-cell-dependent IgA expression (7, 12). These properties might seem to contradict the increased pulmonary antibody levels that were observed for IL-10−/− mice in the current study. However, another major activity of IL-10 is the inhibition of macrophage and dendritic cell antigen presentation and proinflammatory cytokine production (6). The immunosuppressive effect of IL-10 on CD4+ T cells observed by us may thus be mediated through an indirect effect involving the IL-10 inhibition of antigen-presenting cell function. The lack of appropriate T-cell help might in turn limit the induction of antibody expression in the lung. Consistent with the data presented in this study, it was reported previously that the decreased antibody responses to intramuscular influenza virus vaccination seen in the elderly are associated with increased IL-10 production, even though the antigen-processing pathway is likely to be quite different from that of natural influenza virus infection (5). Thus, despite the fact that IL-10 can positively regulate B-cell activity, we found increased respiratory antibody levels following influenza virus infection of IL-10−/− mice, which were completely dependent on CD4+ T cells. While we did not detect high levels of IL-17 production in the BALF of either WT or IL-10−/− mice, McKinstry et al. (14) reported increased levels of CD4+ T-cell IL-17 production in lung tissues of IL-10−/− mice, which correlated with an increased resistance of IL-10−/− mice to influenza virus infection. One potential explanation for these differing results is that distinct mouse strains were utilized, i.e., we used C57BL/6 mice, while McKinstry and colleagues used BALB/c mice. Nevertheless, an important function for the Th17 pathway in enhancing the antiviral antibody production responsible for the increased resistance of IL-10-deficient animals to virus infection cannot be discounted. In fact, a role for IL-17 in increasing pulmonary antibody expression in mice was recently described (11). The functional significance of increased antibody levels in the BALF of IL-10−/− mice was shown in the current study by the ability of immune BALF to mediate protection in naïve mice. Passive protection was maintained in the absence of FcγR but was lost in the absence of C3, indicating a critical role for complement in mediating antibody-dependent protection in IL-10−/− mice.

In summary, we found that IL-10 impairs the initial T-helper-cell function that is required for effective virus-specific antibody production and thereby contributes to susceptibility to primary influenza virus infection.

Acknowledgments

This work was supported by NIH grants RO1 AI41715 and RO1 AI75312.

We have no conflicting financial interests.

Footnotes

[down-pointing small open triangle]Published ahead of print on 3 March 2010.

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