IL-22-binding protein exacerbates influenza, bacterial super-infection

Abstract

Secondary bacterial pneumonia is a significant complication of severe influenza infection and Staphylococcus aureus and Streptococcus pneumoniae are the primary pathogens of interest. IL-22 promotes S. aureus and S. pneumoniae host defense in the lung through epithelial integrity and induction of antimicrobial peptides and is inhibited by the soluble decoy receptor IL-22-binding protein (IL-22BP). Little is known about the effect of the IL-22/IL-22BP regulatory pathway on lung infection, and it has not been studied in the setting of super-infection. We exposed wild-type and IL-22BP^-/- mice to influenza A/PR/8/34 for 6 days prior to infection with S. aureus (USA300) S. pneumoniae. Super-infected IL-22BP^-/- mice had decreased bacterial burden and improved survival compared to controls. IL-22BP^-/- mice exhibited decreased inflammation, increased lipocalin 2 expression, and deletion of IL-22BP was associated with preserved epithelial barrier function with evidence of improved tight junction stability. Human bronchial epithelial cells treated with IL-22Fc showed evidence of improved tight junctions compared to untreated cells. This study revealed that IL-22BP^-/- mice are protected during influenza, bacterial super-infection, suggesting that IL-22BP has a pro-inflammatory role and impairs epithelial barrier function likely through interaction with IL-22.

Figure 1.. The impact of influenza infection on the IL-22 pathway.

C57BL/6 mice were infected with 100 PFU of influenza A/PR/8/34 or PBS for 14 days and lungs were harvested every 2 days post-infection. A – Il22 gene expression in lung tissue (n=4/time point). B – Il22bp gene expression in lung tissue (n=4/time point). C – Il22ra1 gene expression in lung tissue (n=4/time point). * p < 0.05 Significance was tested by one-way ANOVA.

Figure 2.. IL-22BP^−/− mice have improved bacterial clearance and decreased mortality.

C57BL/6 and IL-22BP^−/− mice were infected with 100 PFU of influenza A/PR/8/34 or 7 4vehicle for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours or 10⁴ CFU/mL S. pneumoniae for 48 hours. SA = S. aureus, FS = influenza, S. aureus, F = influenza, FS.pneumo = influenza, S. pneumoniae infection throughout the figures. A – MRSA bacterial colony counts in the lung (n = 7−10). B – Weight loss observed during influenza or super-infection (n = 6−18). C – Influenza, MRSA survival curve (n = 7−10). D – S. pneumoniae bacterial colony counts in the lung (n = 8−9). E – Bacterial dissemination to other organs (n = 3−4). F – Influenza matrix protein gene expression measured by RTPCR (n = 6−8). * p < 0.05 Significance was tested by unpaired t test or one-way ANOVA or Gehan-Brislow-Wilcoxon test. Each experiment was independently performed twice and the data shown are from combined experiments with the exception of the survival curve in panel C and dissemination in panel E that are single representative studies.

Figure 3.. IL-22BP deficiency decreases inflammation.

C57BL/6 and IL-22BP^−/− mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours or 10⁴ CFU/mL S. pneumoniae for 48 hours. A, B – Total cell count in BAL fluid (n = 6−10). C, D – Differential cell counts in BAL fluid (n = 6−10). E – Protein production of pro-inflammatory cytokines in lung homogenate measured by Lincoplex (n = 4−6). * p < 0.05 Significance was tested by unpaired t test or one-way ANOVA. Each experiment was independently performed twice and the data shown are from combined experiments.

Figure 4.. Deletion of IL-22BP did not impact monocyte subpopulations in the lung.

C57BL/6 and IL-22BP^−/− mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours. Mouse lungs were homogenized to single cell suspensions and stained for analysis of monocyte populations by flow cytometry. A – Alveolar macrophages. B – Interstitial macrophages. C – Inflammatory monocytes. D – Monocyte derived dendritic cells E – TRAIL positive monocytes (n = 6−8). Data are shown from a single representative study.

Figure 5.. IL-22BP deficiency alters histopathology during super-infection.

C57BL/6 and IL-22BP^−/− mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours or 10⁴ CFU/mL S. pneumoniae for 48 hours. A, B – Histology score (n = 6−10). C – Representative hematoxylin-eosin staining of lungs 24 hours after MRSA or 48 hours after S. pneumoniae (40x magnification). * p < 0.05 Significance was tested by unpaired t test or one-way ANOVA. Each experiment was independently performed twice and the data shown are from combined experiments.

Figure 6.. IL-22BP deletion improves airway epithelial barrier function during super-infection.

C57BL/6 and IL-22BP^−/− mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours or 10⁴ CFU/mL S. pneumoniae for 48 hours. A, C – Protein in BAL fluid (n = 4−10). B – Absorbance of Evans Blue dye in BAL fluid (n = 11−14). D – Absorbance of lactate dehydrogenase in BAL fluid (n = 6−10). * p < 0.05 Significance was tested by unpaired t test or one-way ANOVA. Each experiment was independently performed twice and the data shown are from combined experiments, except for panel E which is a single representative study.

Figure 7.. IL-22BP and IL-22 are important for airway epithelial tight junction integrity.

A,B – Claudin and Tjp1 gene expression measured by RT-PCR in C57BL/6 and IL-22BP^−/− mice that were infected with 100 PFU of influenza A/PR/8/34 for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours (A, n = 8−10) or influenza alone (B, n = 6−9). C – Transepithelial electrical resistance of air-liquid differentiated human bronchial epithelial cells at 0 through 24 hours after inoculation with S. aureus and treatment with IL-22Fc or vehicle (n = 3 independent cultures). D – Immunofluorescence staining of polarized human bronchial epithelial cells treated with IL-22Fc or vehicle for 24 hours. Inset numbers reflect ratio of overlap staining. E – Colocalization of ZO-1 and occluden in cells treated with IL-22Fc for 0–72 hours (n = 3 independent cultures, 6 fields per culture). * p < 0.05 Significance was tested by unpaired t test or two-way ANOVA. Each experiment was independently performed two to three times and the data shown are from combined experiments except for panel D which is a single representative study.

Figure 7.. IL-22BP and IL-22 are important for airway epithelial tight junction integrity.

A,B – Claudin and Tjp1 gene expression measured by RT-PCR in C57BL/6 and IL-22BP^−/− mice that were infected with 100 PFU of influenza A/PR/8/34 for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours (A, n = 8−10) or influenza alone (B, n = 6−9). C – Transepithelial electrical resistance of air-liquid differentiated human bronchial epithelial cells at 0 through 24 hours after inoculation with S. aureus and treatment with IL-22Fc or vehicle (n = 3 independent cultures). D – Immunofluorescence staining of polarized human bronchial epithelial cells treated with IL-22Fc or vehicle for 24 hours. Inset numbers reflect ratio of overlap staining. E – Colocalization of ZO-1 and occluden in cells treated with IL-22Fc for 0–72 hours (n = 3 independent cultures, 6 fields per culture). * p < 0.05 Significance was tested by unpaired t test or two-way ANOVA. Each experiment was independently performed two to three times and the data shown are from combined experiments except for panel D which is a single representative study.

Figure 8.. Deletion of IL-22BP results in increased lipocalin 2 expression.

C57BL/6 and IL-22BP^−/− mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days prior to infection with 5 × 10⁷ CFU MRSA for 24 hours. A – Il22bp expression during single or super-infection (n = 6−10). B – Il22ra1 expression in WT and IL-22BP^−/− mice (n = 6−10). C,D – Il17 and Il22 expression during super-infection (n = 7−8). E – IL-10 protein concentration in lung homogenate (n = 4−6). F – AMP expression during influenza single infection or super-infection (n = 4−6). Significance was tested by unpaired t test or two-way ANOVA. Each experiment was independently performed twice and the data shown are from combined experiments.