Targeting the IL-22/IL-22BP axis enhances tight junctions and reduces inflammation during influenza infection

Abstract

The seasonal burden of influenza coupled with the pandemic outbreaks of more pathogenic strains underscore a critical need to understand the pathophysiology of influenza injury in the lung. Interleukin-22 (IL-22) is a promising cytokine that is critical in protecting the lung during infection. This cytokine is strongly regulated by the soluble receptor IL-22-binding protein (IL-22BP), which is constitutively expressed in the lungs where it inhibits IL-22 activity. The IL-22/IL-22BP axis is thought to prevent chronic exposure of epithelial cells to IL-22. However, the importance of this axis is not understood during an infection such as influenza. Here we demonstrate through the use of IL-22BP-knockout mice (il-22ra2^-/-) that a pro-IL-22 environment reduces pulmonary inflammation during H1N1 (PR8/34 H1N1) infection and protects the lung by promoting tight junction formation. We confirmed these results in normal human bronchial epithelial cells in vitro demonstrating improved membrane resistance and induction of the tight junction proteins Cldn4, Tjp1, and Tjp2. Importantly, we show that administering recombinant IL-22 in vivo reduces inflammation and fluid leak into the lung. Taken together, our results demonstrate the IL-22/IL-22BP axis is a potential targetable pathway for reducing influenza-induced pneumonia.

Fig. 1

Influenza induces a pro-IL-22 environment. Mice were infected with PR8 (A/PR/8/34, 100 PFU) via oropharyngeal administration. Whole lung mRNA was purified. a mRNA expression of Il22 is induced by day 4 and peaks by day 6. b Il22ra2 mRNA is expressed constitutively in the lung and expression is reduced after infection. c Lung cells from naive mice and influenza-infected mice were isolated from whole lung and sorted at day 5 post infection. Il22ra2 mRNA expression was measured by RT-qPCR. Data are presented as the mean and standard deviation and is representative of three experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. **p < 0.01 compared to day 0 and ****p < 0.0001 compared to day 0. n = at least 5 for all samples and is representative of two experiments. Axes are clearly labeled to denote what each shape means in each figure, respectively. In a and b, each shape denotes the transcript levels found at different days following influenza infection as labeled on the x axis

Fig. 2

Il22ra2^−/− mice recover more quickly after influenza infection. Mice were infected with PR8 (A/PR/8/34, 100 PFU) via oropharyngeal administration. a Morbidity as measured by weight loss. Each time point is representative of eight mice from two experiments. b Lung viral load was measured by quantitative qRT-PCR for the influenza M1 gene. Each point represents an individual mouse. n = 8 mice per group from two independent experiments. c Histopathology from day 10 after infection demonstrates extensive inflammation in wild-type mice in comparison to very mild, very focal inflammation in the Il22ra2^−/− mice. d Total affected vs. unaffected areas in infected wild-type and il-22ra2^−/− mice. e Slides were scored blindly for bronchiolitis and alveolitis as described in Materials and methods. n = at least 8 for each figure, with each experiment being reproduced at least twice. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (a, b) or Student’s T test. ***p < 0.001 and ****p < 0.0001. In a and b, shapes are defined on the right y axis. In d–f shapes are defined on the x axis. The original magnification is 10x

Fig. 3

Il22ra2^−/− mice have decreased inflammation 5 and 10 days after influenza infection. Mice were infected with PR8 (A/PR/8/34, 100 PFU) via oropharyngeal administration. Bronchoalveolar lavage was collected and cells were collected. a Total inflammation from day 5. b Total inflammation from day 10 after infection. c Differential counts from day 5. d Differential counts from day 10. e Ratio of M2/M1 macrophages in wild-type vs. il-22ra2^−/− mice. f Cytokine analysis for IL-6, IL-17, Ifnγ, and RANTES from lung homogenate. n = 8 mice from two independent experiments for a–d. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test or Student’s T test (e). *p < 0.05, **p < 0.01, and ****p < 0.0001

Fig. 4

Il22ra2^−/− mice have decreased pulmonary injury and increased tight junctions during influenza infection. BAL fluid was collected and analyzed from influenza-infected mice 5 days after infection. a Cell cytotoxicity was detected by measuring lactate dehydrogenase. b Total protein was measured using the BCA Protein Assay Kit. c Evan’s blue dye (0.1% in sterile PBS) was administered i.v. 30 min prior to collecting BAL. Evan’s blue is measured by absorbance at 640 on a spectrophotometer. d–f Bronchial brushings were performed to collect epithelial cells and RNA was purified and quantitative RT-PCR was performed for d Tjp1, Tjp2, Cldn4, and Ocln. Data are presented as gene expression over uninfected controls. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (a, b) or Student’s T test (c, d). *p < 0.05, **p < 0.01, and ****p < 0.0001

Fig. 5

rIL-22 administration induces tight junctions in vitro during infection. a Human airway cells were grown at air–liquid interface. Cells were infected with 2009 H1N1 (MOI = 100) and treated with human rIL-22:Fc or vehicle (PBS) basolaterally. Transepithelial resistance was measured daily. Each point represents four separate experiments from cells from four different donors. n = 4 wells per experiment. b, c NHBE cells monolayers grown on collagen and infected (A/PR/8/34, MOI = 50) and treated with 30 ng/ml rhIL-22 (R&D Systems) 1 day after infection. RNA was collected and quantitative RT-PCR was performed for b CLDN4 and c TJP2. d A549 cells were infected and treated with IL-22 (30 ng/ml). Shown is representative immunofluorescence staining for ZO-1 and DAPI from two independent experiments. n = 4 wells per experiment. e Comparative quantification of ZO-1 mean fluorescent intensity by group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. n = 5 and is representative of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 6

rIL-22 administration reduces inflammation and lung leak in vivo during infection. a mIL-22 (100 ng) and IL-22:Fc were administered to mouse c10 STAT3 luciferase reporter cells, respectively. Luciferase activity was measured following treatment with each. C57Bl/6 mice were infected oropharyngeally with 100 PFU H1N1 (Pr8) and then treated with 5 µg IL-22:Fc or IgG 6 days later. b IL-22:Fc treatment leads to a significant reduction in lung leak. c, d To confirm this was not due to off-target effects of human IL-22 or the Fc, ella-cre^+/+ and ella-cre^+/+ × il-22ra1^fl/fl were infected oropharyngeally with 100 PFU H1N1 (Pr8). Six and eight days later, mice were treated oropharyngeally with 5 µg IL-22:Fc. IL-22:Fc reduced c weight loss and d total inflammation in control (ella-cre^+/+) mice. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. n = 6 and is representative of two experiments. **p < 0.01 and ***p < 0.001