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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: J Immunol. 2013 Oct 2;191(10):10.4049/jimmunol.1301237. doi: 10.4049/jimmunol.1301237

Influenza A Exacerbates Staphylococcus aureus Pneumonia by Attenuating IL-1β Production in Mice1

Keven M Robinson *,#, Sun Mi Choi , Kevin J McHugh *, Sivanarayana Mandalapu *, Richard I Enelow §, Jay K Kolls , John F Alcorn *
PMCID: PMC3827735  NIHMSID: NIHMS524809  PMID: 24089191

Abstract

Pneumonia is a leading cause of death worldwide. Staphylococcal aureus can be a cause of severe pneumonia alone or as a common pathogen in secondary pneumonia following influenza. Recently, we have reported that preceding influenza attenuated the Type 17 pathway, increasing the lung’s susceptibility to secondary infection. IL-1β is known to regulate host defense including playing a role in TH17 polarization. We examined if IL-1β signaling is required for S. aureus host defense and whether influenza infection impacted S. aureus induced IL-1β production and subsequent Type 17 pathway activation. Mice were challenged with S. aureus (USA300) with or without preceding influenza A/PR/8/34 H1N1 infection. IL-1receptor1 −/− mice had significantly higher S. aureus burden, increased mortality, and decreased Type 17 pathway activation following S. aureus challenge. Co-infected mice had significantly decreased IL-1β production versus S. aureus alone at early time points following bacterial challenge. Preceding influenza did not attenuate S. aureus induced inflammasome activation, but there was early suppression of NF-κB activation, suggesting an inhibition of NF-κB dependent transcription of pro- IL-1β. Furthermore, overexpression of IL-1β in influenza, S. aureus co-infected mice rescued the induction of IL-17 and IL-22 by S. aureus and improved bacterial clearance. Finally, exogenous IL-1β did not significantly rescue S. aureus host defense during co-infection in IL-17RA −/− mice or in mice in which IL-17 and IL-22 activity were blocked. These data reveal a novel mechanism by which influenza A inhibits S. aureus induced IL-1β production resulting in attenuation of Type 17 immunity and increased susceptibility to bacterial infection.

Introduction

Influenza is a significant cause of morbidity and mortality worldwide. In the United States, influenza affects 5 – 20% of the population yearly and results in 30,000 deaths. Despite improvements in medical care, influenza pandemics continue to occur. While most cases of influenza do not result in death, secondary bacterial infections can lead to increased mortality, particularly in previously healthy individuals. Increased intensive care admission, cost, and mortality have been described in children and young adults with influenza and S. aureus co-infection compared to those with either influenza or S. aureus infection alone (1). In addition, a primary cofactor associated with mortality in community-acquired methicillin-resistant S. aureus2 is preceding influenza- like illness (2).

An important role for T cells in host defense against bacterial infections has recently emerged with the discovery that patients with hyperimmunoglobulin E syndrome have mutations in STAT3, a key transcription factor in the development of TH17 cells (3). Clinically, these patients have increased susceptibility to S. aureus infections of the lungs and skin. They are unable to produce TH17 cells and IL-17A, suggesting that the Type 17 pathway plays a critical role in the immune response against S. aureus (4). TH17 cells are a subset of CD4+ T cells that produce high levels of the cytokines IL-17 and IL-22 (5–7). They have high expression of the transcription factors retinoid orphan receptor (ROR)α and RORγT driven by IL-6, TGF-β, and IL-1β signaling through STAT3, SMAD, and NF-κB pathways, respectively (6, 8–10). IL-23, a cytokine produced by antigen presenting cells, is also important in TH17 cell regulation, proliferation, and cytokine production (11–12).

We have previously shown that mice co-infected with Influenza A and S. aureus have worsened bacterial burden and mortality compared to mice infected with S. aureus alone (13). Co-infected mice exhibit influenza A-induced attenuation of S. aureus driven Type 17 immunity and increased susceptibility to bacterial pneumonia. We have also demonstrated that influenza suppressed S. aureus-induced IL-23 production by CD11c+ cells, however exogenous IL-23 only partially rescued S. aureus host defense. The specific additional mechanisms by which influenza increases the lung’s susceptibility to S. aureus infection remains unknown. Prior studies have shown that IL-1β plays a role in host defense against influenza A and S. aureus through activation of the inflammasome (14–17). Because IL-1β is known to influence polarization of TH17 cells, we hypothesized that inhibition of S. aureus induced IL-1β activation by preceding influenza infection may play a critical role in attenuation of Type 17 immunity and host defense against S. aureus.

Methods

Mice

Six to eight week old male wild type (WT) C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). IL-1R1 −/− and WT control mice were purchased from Jackson Laboratories (Bar Harbor, ME). IL-17RA −/− were generated as previously described (18). Mice were maintained under pathogen-free conditions at the Children’s Hospital of Pittsburgh of UPMC and conducted with approval from the University of Pittsburgh Institutional Animal Care and Use Committee. All of the studies used age- and sex-matched mice.

S. aureus infection

Methicillin-sensitive S. aureus3 (American Type Culture Collection4 49775) producing γ-hemolysin and Panton-Valentine leukocidin was purchased from the ATCC. MRSA (USA 300) was provided as a gift by Dr. Alice Prince, Columbia University. S. aureus was cultured as detailed by ATCC instructions in casein hydrolysate yeast extract containing-modified medium overnight for 18 hours to stationary growth phase. Mice were inoculated with either MSSA (1x108 or 4x108 cfu) or MRSA (5x107) in 50 µl sterile PBS by oropharyngeal aspiration, and lungs were harvested 30 minutes to 120 hours later. Mice in all studies received S. aureus 6 days following influenza infection as described below.

Influenza A/PR/8/34 H1N1 infection

Influenza A/PR/8/34 H1N1 was propagated in chicken eggs as previously described (19). Mice were infected with 100 pfu of Influenza A PR/8/34 H1N1 (in 40 µl sterile PBS) from a frozen stock or control PBS by oropharyngeal aspiration. Infected mice were incubated for 6 days, then received S. aureus inoculum or control PBS. After an additional 30 minutes to 120 hours, lungs were harvested.

Adenoviral IL-1β infection

E1- and E3-deleted adenoviral vector encoding enhanced green fluorescent protein (Ad-eGFP) was constructed as described (20–21) by Cre-lox recombination with reagents generously provided by S. Hardy (Somatix, Alameda, CA). Briefly, a SnaBI-HpaI fragment containing part of the cytomegalovirus promoter, the eGFP cDNA, and part of the SV40 poly (A) sequence was inserted in the pAdlox shuttle plasmid. E1-substituted recombinant adenovirus was generated by cotransfection of SfiI-digested pAdlox-EGFP and ψ5 helper virus DNA into the adenoviral packaging cell line CRE8, propagated, and purified as described (21). The E1- and E3-deleted adenovirus encoding mouse interleukin 1 beta (Ad-mIL1beta) was constructed cloning the mIL1beta cDNA as a SalI-NotI fragment in the pAdlox shuttle plasmid. E1-substituted recombinant adenovirus was generated by cotransfection as described above. Mice were infected with adenovirus expressing IL-1β (2.5 x108 pfu in 50 µl sterile PBS) or enhanced GFP (EGFP) (1 x1010 pfu in 50 µl sterile PBS) (control) by oropharyngeal aspiration 3 days after influenza A infection.

Analysis of lung inflammation

At indicated time points, mouse lungs were lavaged with 1 ml sterile PBS for inflammatory cell differential counts. The cranial lobe of the right lung was homogenized in sterile PBS by mechanical grinding. The resulting lung homogenate was used for bacterial colony counting and cytokine analysis by Lincoplex (Millipore) or by ELISA for IL-18 (R&D Systems) and IL-1β (Biolegend). The middle and caudal lobes of the right lung were snap-frozen and homogenized under liquid nitrogen for RNA extraction by either standard TRIzol extraction or RNA isolation kit (Agilent Techonologies). RNA analysis was performed by standard RT-PCR using Assay on Demand Taqman probes and primers (Applied Biosystems).

Protein Quantification

Whole - cell lysates were extracted from homogenized liquid N2 snap - frozen middle and caudal lobes of the right lung. Equal amounts of protein (20 µg) were separated on Nupage 4–12% BisTris gels (Novex) and transferred to a nitrocellulose membrane. Target proteins were detected by Western Blot and immunostaining with specific primary antibody followed by horseradish peroxidase-labeled secondary antibody. The specific immunoreactive bands were detected by chemiluminescense. The caspase-1 p20 and NFκB-p65 antibodies were purchased from Santa Cruz Biotechnology and the Phospho-NFκB-65 antibody from Cell Signaling. Quantification of band intensity (pixel density) was performed using NIH Image J software.

Caspase-1 Activity

Caspase-1 activity was measured by using Abcam’s (Cambridge, MA) Caspase-1 Colorimetric assay kit. The assay measures spectrophotometric detection (at 405 nm) of chromophore p-nitroanilide after cleavage from labeled substrate YVAD-p-NA.

Inhibition of IL-17R and IL-22

Mice received 10 µg of recombinant mouse IL-17 R Fc Chimera (R&D Systems) and 50 µg of monoclonal anti-mouse IL-22 antibody (R&D Systems) in 100 µl of sterile PBS by oropharyngeal aspiration 24 hours prior to receiving S. aureus.

Statistical Analysis

All of the data presented as the mean +/− standard error of the mean (SEM). Significance was tested by unpaired t test (for two means) or one-way ANOVA (for multiple data groups) followed by Tukey posthoc test. Significance of survival data was tested by the Gehan-Brislow-Wilcoxin test. Data was analyzed using the GraphPad Prism and/or Microsoft Excel software package.

Results

Type 17 pathway activation and efficient clearance of S. aureus from the lung requires IL-1β signaling

To further examine mechanisms of influenza A inhibition of Type 17 immunity and host defense against S. aureus, we investigated the requirement for IL-1β signaling during S. aureus pneumonia. We challenged WT or IL-1R1 −/− mice with 4x108 cfu of S. aureus (ATTC 49775) and assessed bacterial clearance and lung inflammation 24 hours following challenge. There was significantly decreased clearance of S. aureus in the IL-1R1−/− mice compared to WT (Figure 1A). Abrogation of IL-1R1 signaling resulted in decreased gene expression of Type 17 pathway effector cytokines, IL-17A and IL-17F, and IL-17/IL-22 associated antimicrobial peptide, RegIIIβ (Figure 1B). In addition, IL-1R1 deletion resulted in decreased pro-neutrophil cytokine production (Figure 1C). To further demonstrate the importance of IL-1β signaling during S. aureus pneumonia, we challenged WT and IL-1R1−/− mice with 1x109 cfu of S. aureus (ATTC 49775) and found significantly increased mortality in the IL-1R1−/− mice (Figure 1D). These data suggest IL-1β is crucial to host defense against S. aureus through activation of the Type 17 pathway during the acute phase of S. aureus infection. Since preceding influenza infections results in worsened secondary S. aureus infection and Type 17 pathway inhibition, we proposed that influenza may attenuate IL-1β production in response to S. aureus. C57BL/6 mice were challenged with 100 pfu of Influenza APR/8/34 H1N1 for 6 days followed by 108 cfu of S. aureus (ATTC 49775) and were harvested after 6, 24, 48, 72, 96, and 120 hours. At 6 to 24 hours following bacterial challenge, there was significant attenuation of IL-1β protein production in the co-infected mice compared to mice that received S. aureus alone (Figure 1E). Similarly, levels of IL-1α, another IL-1R1 ligand, were significantly attenuated in co-infected mice versus S. aureus only infected (846.6 ± 139.7 vs. 224.5 ± 16.1 pg/ml at 6 hours and 754.9 ± 142.9 vs. 351.1 ± 57.4 pg/ml at 24 hours). We have previously shown that IL-1β levels are lower than in S. aureus infection in influenza alone infected mice at the 24 hour (7 days post-influenza) time point (20.3 ± 1.2 pg/ml) (13). These data suggest that suppression of IL-1β by preceding influenza may be a novel mechanism of increased susceptibility to secondary bacterial infections.

Figure 1. IL-1β signaling is critical to Type 17 pathway activation and host defense against S. aureus.

Figure 1

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C57BL/6 and IL-1R1 −/− mice were infected with 4x108 cfu of S. aureus for 24 hrs. A, Bacterial colony counts in lung homogenate (n=5). B-C, Type 17 pathway gene expression in lung RNA (n=5). D, Mortality curve for C57BL/6 and IL-1R1 −/− mice infected with 1x109 cfu of S. aureus (n=8). C57BL/6 mice were infected with 100 pfu of Influenza A/PR/8/34 or vehicle for 6 days, mice were then challenged with 108 cfu of S. aureus for 6 – 120 hrs. E, IL-1β cytokine concentrations in lung homogenate (n=6–7). * p < 0.05 versus S. aureus alone, ** p < 0.05 versus WT, *** p < 0.10 versus WT

Preceding influenza A infection does not impact S. aureus induced inflammasome activation in the lung

In order to determine how influenza may inhibit S. aureus induced IL-1β, we investigated the well known IL-1β regulatory pathways. IL-1β is produced following the activation of pattern recognition receptors by microbial products, which initiates IL-1β gene expression by induction of NF-κB and synthesis of inactive pro-IL-1β. (22). Pro- IL-1β is converted into active IL-1β by several mechanisms including activation of caspase-1 by protein complexes known as the inflammasome (23). IL-18 is a proinflammatory cytokine produced by macrophages and other cells through the inflammasome pathway, similar to IL-1β. We examined whether preceding influenza has similar inhibitory effects on IL-18 levels in our model. C57BL/6 mice were challenged with 100 pfu of Influenza A PR/8/34 H1N1 for 6 days followed by 5x107 cfu of S. aureus (USA 300). At 6 and 24 hours following bacterial challenge, there was no decrease in IL-18 protein levels or gene expression (Figure 2A,B) in co-infected mice compared to mice that received S. aureus alone. In contrast, there was increased gene expression of IL-18 in co-infected mice at 6 hours following bacterial challenge and increased protein levels in co-infected mice at 24 hours following bacterial challenge. These data suggest that influenza does not cause overall inflammasome suppression. To examine this further, caspase-1 expression, cleavage, and activity were assessed. C57BL/6 mice were challenged with 100 pfu of Influenza A PR/8/34 H1N1 for 6 days followed by 5x107 cfu of S. aureus (USA 300). Caspase-1 expression in the lung was not reduced in co-infected mice compared to S. aureus challenge alone, in fact it was significantly increased (Figure 2C). Next, we examined caspase-1 cleavage (an activation marker) and activity in lung homogenate. There were no differences in caspase-1 p20 protein levels or enzymatic activity (Figure 2D,E) between S. aureus or co-infected mice. These data suggest that caspase-1 suppression is not playing a role in influenza A inhibition of IL-1β.

Figure 2. Influenza A does not impact S. aureus induced inflammasome activation.

Figure 2

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C57BL/6 mice were infected with 100 pfu of Influenza A/PR/8/34 or vehicle for 6 days, mice were then challenged with 5x107 cfu of S. aureus for 6–24 hrs. A, IL-18 protein concentration in lung homogenate as measured by ELISA (n=6). B, IL-18 gene expression in lung RNA (n=7–8). C, Caspase-1 gene expression in lung RNA (n=7–8). D, Western blot analysis for anti-caspase-1 p20 cleaved product. E, Caspase-1 enzyme activity (n=6). * p < 0.05 versus S. aureus alone

Preceding influenza A infection suppresses S. aureus induced NF-κB activation leading to attenuation of IL-1β production in the lung

Since inflammasome activation did not appear to be impaired during co-infection, we investigated if influenza was inhibiting transcriptional activation of IL-1β. We measured IL-1β mRNA in the lung and found there was significant inhibition of expression at 6 hours following bacterial challenge in co-infected mice (Figure 3A). In order to determine if NF-κB dependent transcription of pro-IL-1β is the mechanism by which influenza A inhibits IL-1β production, C57BL/6 mice were challenged with influenza and S. aureus as described in the previous section and after 1 and 2 hours, protein levels were assessed. There was decreased phosphorylated NF-κB Rel A (p65) in the co-infected mice versus S. aureus alone (Figure 3 B,C). Of note, naïve mice (labeled as control) had decreased phosphorylated NF-κB compared to mice receiving either influenza and S. aureus or S. aureus alone. There was no difference in non-phosphorylated NF-κB protein levels between naïve and infected mice (Figure 3B,C). These data support a mechanism by which ongoing influenza A infection suppresses subsequent NF-κB activation triggered by S. aureus, leading to inhibition of IL-1β transcription and subsequent attenuation of Type 17 immunity in the lung.

Figure 3. Influenza A suppresses NF-κB activation and IL-1β expression in the lung.

Figure 3

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C57BL/6 mice were infected with 100 pfu of Influenza A/PR/8/34 or vehicle for 6 days, mice were then challenged with 5x107 cfu of S. aureus for 1–24 hrs. A, IL-1β gene expression in lung RNA (n=7–8). B, Western Blot analysis for anti- phosphorylated NF-κB and anti-nonphosphorylated NF-κB p50 and p65. C, densitometry for the Western Blot bands. * p < 0.05 versus S. aureus alone

IL-1β overexpression rescues Type 17 activation and improves S. aureus clearance during co-infection

If the defect in early IL-1β observed in co-infected mice is indeed critical to impaired S. aureus clearance, then restoration of IL-1β production should provide a host defense benefit. To test this, we overexpressed IL-1β in influenza, S. aureus co-infected mice, proposing that it would rescue Type 17 immunity and aid bacterial clearance. Surprisingly, IL-1β overexpression resulted in decreased BAL inflammation, specifically macrophages, neutrophils, and lymphocytes (Figure 4A). However, IL-1β treatment did significantly increase the levels of IL-17A and IL-22 mRNA (Figure 4B) compared with those in control adenovirus infected mice. IL-1β also significantly increased lipocalin 2 expression (Figure 4C), an IL-17 associated antimicrobial peptide. In addition, overexpression of IL-1β also significantly increased production of the Type 17 cytokine-associated cytokines; G-CSF and KC (Figure 4D). There was no change in IL-6 levels between the two groups and decreased TNFα levels in the mice that received exogenous IL-1β, suggesting that exogenous IL-1β does not have generalized effects on inflammation (Figure 4E). As predicted by our hypothesis, IL-1β treatment significantly improved S. aureus clearance in co-infected mice (Figure 4F). In these mice, bacterial titers were similar to mice that received S. aureus alone (data not shown). In addition, we measured IL-1β levels at the time mice would have received S. aureus in our co-infection model. Mice that received IL-1β adenovirus had increased (375.2 ± 3.24 pg/mL versus 270.9 ± 42.56 pg/mL) IL-1β levels in lung homogenate compared to those that received control adenovirus. Influenza A attenuation of the Type 17 pathway was rescued by IL-1β, perhaps leading to improved bacterial immunity in influenza co-infection.

Figure 4. Overexpression of IL-1β rescues influenza-induced inhibition of Type 17 pathway activation and improves S. aureus clearance.

Figure 4

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C57BL/6 mice were infected with 100 pfu of Influenza A/PR/8/34, on day 3 received adenovirus (2.5 x 108 pfu in 50 µl sterile PBS) expressing IL-1β or enhanced GFP (EGFP) (control), and on day 6 were challenged with 5x107 cfu of S. aureus for 24 hrs. A, Bronchoalveolar lavage cell counts (n=11–12). B, Type 17 pathway expression in lung tissue measured by RT-PCR (n=8–10). C, Lipocalin 2 expression in lung tissue measured by RT-PCR (n=8–10). D-E, Type 17 associated cytokine production in lung homogenate (n=6). F, Bacterial colony counts in the lung (n=10). * p < 0.05 versus control

IL-17 and IL-22 play an important role in IL-1β’s rescue of S. aureus clearance

To determine if IL-1β’s restoration of bacterial immunity was IL-17 dependent, we overexpressed IL-1β or control EGFP in influenza, S. aureus co-infected IL-17RA −/− mice. IL-1β failed to significantly improve bacterial clearance (Figure 5A), although there was a trend in improved bacterial clearance in the mice that received exogenous IL-1β (p = 0.38). In addition there was no decrease in lung inflammation in IL-17RA −/− mice (Figure 5B). Although there was no difference in the production of IL-17A and IL-23 mRNA, there was a significant increase of IL-22 mRNA in the mice that received exogenous IL-1β (Figure 5C). As expected, there was similar lipocalin 2 expression between the two groups (Figure 5D). Overexpression of IL-1β also significantly increased production of the Type 17 cytokine-associated chemokines, G-CSF and KC (Figure 5E). Similar to WT mice, there was no change in IL-6 between the two groups and decreased TNFα levels in the mice that received exogenous IL-1β (Figure 5F). Due to the trend in improved bacterial clearance in the IL-17RA −/− mice that received exogenous IL-1β and the significant increase of IL-22 mRNA in these mice, we proposed that IL-1β’s partial (non-significant) restoration of bacterial immunity may be dependent upon both IL-17 and IL-22. To test this, we overexpressed IL-1β or control EGFP in influenza, S. aureus co-infected WT mice that had received anti-IL-17 receptor and anti-IL-22 antibodies prior to bacterial challenge. In these mice, IL-1β failed to significantly improve bacterial clearance (p=0.27) (Figure 5G), although again we observed a trend in improved bacterial clearance in the mice that received exogenous IL-1β. In both IL-17RA −/− mice, and WT mice in which IL-17R and IL-22 were blocked, exogenous IL-1β did not prevent secondary bacterial pneumonia to the same degree compared to its effect on WT mice, suggesting that the TH17 axis plays an important role in IL-1β’s effect on influenza, S. aureus co-infection.

Figure 5. IL-17 and IL-22 play an important role in IL-1β’s rescue of influenza-induced S. aureus pneumonia.

Figure 5

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IL17R −/− mice were infected with 100 pfu of Influenza A/PR/8/34, on day 3 received adenovirus (2.5 x108 pfu in 50 ml sterile PBS) expressing IL-1β or enhanced GFP (EGFP) (control), and on day 6 were challenged with 5x107 cfu of S. aureus for 24 hrs. A, Bacterial colony counts in the lung (n=6). B, Bronchoalveolar lavage cell counts (n=6). C, Type 17 pathway expression in lung tissue measured by RT-PCR (n=6). D, Lipocalin 2 expression in lung tissue measured by RT-PCR (n=6). E-F, Type 17 associated cytokine production in lung homogenate (n=6). C57BL/6 mice were infected with 100 pfu of Influenza A/PR/8/34, on day 3 received adenovirus expressing IL-1β (2.5 x108 pfu in 50 µl sterile PBS) or enhanced GFP (EGFP) (1 x 1010 pfu in 50 µl sterile PBS) (control), on day 5 received 10 µg of IL-17R Fc chimera and 50 µg of anti-IL-22 (in 100 µl sterile PBS) and on day 6 were challenged with 5x107 cfu of S. aureus for 24 hrs. G, Bacterial colony counts in the lung (n=7–8). * p < 0.05 versus control

Discussion

These findings demonstrate a mechanism by which preceding influenza infection impairs Type 17 immunity and allows for increased susceptibility to secondary bacterial infection in the lung. We demonstrate that IL-1β signaling is required for S. aureus host defense and further, for activation of Type 17 cytokines and downstream target genes. Preceding influenza infection markedly attenuates acute IL-1β production induced by S. aureus. Our study shows that influenza A infection suppresses S. aureus induced NF-κB activation in mice, leading to inhibition of IL-1β production and subsequent attenuation of the Type 17 pathway. IL-1β overexpression rescued Type 17 pathway activation, enhancing the immune response to secondary bacterial challenge. In addition, the Type 17 axis was found to play an important role in IL-1β’s restoration of bacterial immunity. These data indicate a key mechanism by which influenza infection attenuates host defense against secondary infection and could provide a potential therapeutic target in the future.

IL-1β is a pro-inflammatory cytokine that is produced following activation of pattern recognition receptors by microbial products, which initiates IL-1β gene expression and synthesis of pro-IL-1β. IL-1β induces recruitment of neutrophils and macrophages, activates the release of other cytokines important to host defense, TNFα and IL-6, and drives Type 17 pathway differentiation of naïve and innate T cells (8–9). Multiple studies have shown that IL-1β plays a role in host defense against influenza A through activation of the inflammasome and induction of IL-1β and IL-18 (14–16). Influenza has been shown to activate the NLRP3 and ASC inflammasome and the IL-1 receptor, caspase-1, and ASC are required for protection against influenza infection in mice. In addition, it has been reported that inflammasome-mediated IL-1β production is important to immunity against cutaneous S. aureus infection (17). S. aureus pneumonia activates the NLRP3 inflammasome leading to the release of IL-1β and IL-18, possibly contributing to pulmonary pathology. In our study, we observed that IL-1β is crucial to host defense against S. aureus in the lung through activation of the Type 17 pathway during the acute phase of S. aureus infection.

While the inflammasome activation of caspase-1 converts pro-IL-1β to IL-1β, IL-1β production is also regulated by induction of NF-κB dependent transcription of pro- IL-1β. Our co-infection model suggested that preceding influenza does not suppress S. aureus induced activation of the inflammasome, but alternatively suppresses NF-κB activation leading to inhibition of IL-1β. In our current study, exogenous IL-1β given 72 hours prior to S. aureus resulted in enhanced Type 17 pathway activation during influenza, S. aureus co-infection in WT mice. We observed an increase in expression of Type 17 effector cytokines and the Type 17 associated antimicrobial peptide, lipocalin 2. IL-1β promoted the clearance of S. aureus and reduced BAL inflammation in WT mice. Inflammatory cells and epithelial cells produce IL-1β during infection (24–25). IL-17 is secreted by both TH17 cells and γδ cells in response to IL-1β (26). In our experimental co-infection model, IL-1β acts quickly and within 24 hours following bacterial challenge to restore Type 17 pathway activation and restore bacterial immunity. IL-17 is likely being secreted from TH17 and γδT cells, as well as other innate T cell subsets in response to IL-1β.

IL-1β’s impact on S. aureus host defense was found to be partially regulated by IL-17 and IL-22, as evidence by decrease in restoration of bacterial immunity by exogenous IL-1β in IL-17RA −/− mice and WT mice in which IL-17R and IL-22 were blocked. IL-1β overexpression resulted in increased IL-22 production in IL-17RA −/− mice, but not IL-17 mRNA. IL-17 mRNA was likely not increased by IL-1β because expression is already elevated during lung infection in IL-17RA −/− mice as previously reported (18). Interestingly, there was increased production of the Type 17 cytokine-associated chemokines G-CSF and KC in IL-17RA −/− mice that received IL-1β adenovirus compared to control. Although G-CSF and KC are typically associated with IL-17 effector function, Numasaki et al showed that G-CSF can be induced by IL-1β in lung endothelial cells in the absence or presence of IL-17. In this study, the presence of IL-17 enhanced IL-1β’s induction of G-CSF (27). Unexpectedly, we also showed decreased production of TNFα in both WT and IL-17RA −/− mice that received IL-1β adenovirus compared to control. This may be due to decreased bacterial burden or lessened BAL inflammation in WT mice. Regardless, it is interesting that decreased TNFα was not associated with exacerbation of S. aureus.

Bacterial co-infection is a serious, often life threatening consequence of influenza infection. Influenza pandemic strains have proven to be consistently prevalent over the last century. With the recent emergence of antibiotic resistant bacteria strains, the severity of co-infection during an influenza epidemic or pandemic is a serious concern. Influenza A attenuation of S. aureus induced NF-κB activation, leading to inhibition of IL-1β production and subsequent diminution of Type 17 pathway activation may represent a novel pathway by which influenza predisposes to secondary bacterial infection. The identification of the mechanisms by which influenza alters host defense may lead to therapeutic immune targets, which would have broad clinical applications and potentially decrease morbidity and mortality associated with influenza and secondary bacterial pneumonia.

Footnotes

1

This work was supported by Parker B. Francis Fellowship (JFA), CHP Research Advisory Committee Start-up Grant (JFA), and NIH NHLBI R01 HL107380 (JFA).

2

methicillin-resistant S. aureus (MRSA)

3

methicillin-sensitive S. aureus (MSSA)

4

American Type Culture Collection (ATTC)

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