IL-17 Receptor Signaling in the Lung Epithelium Is Required for Mucosal Chemokine Gradients and Pulmonary Host Defense against K. pneumoniae

Abstract

The cytokine IL-17, and signaling via its heterodimeric IL-17RA/IL-17RC receptor, is critical for host defense against extracellular bacterial and fungal pathogens. Polarized lung epithelial cells express IL-17RA and IL-17RC basolaterally. However, their contribution to IL-17-dependent pulmonary defenses in vivo remains to be determined. To address this, we generated mice with conditional deletion of Il17ra or Il17rc in Scgb1a1-expressing club cells, a major component of the murine bronchiolar epithelium. These mice displayed an impaired ability to recruit neutrophils into the airway lumen in response to IL-17, a defect in bacterial clearance upon mucosal challenge with the pulmonary pathogen Klebsiella pneumoniae, and substantially reduced epithelial expression of the chemokine Cxcl5. Neutrophil recruitment and bacterial clearance were restored by intranasal administration of recombinant CXCL5. Our data show that IL-17R signaling in the lung epithelium plays a critical role in establishing chemokine gradients that are essential for mucosal immunity against pulmonary bacterial pathogens.

Fig. 1. Characterization of the IL17R^fl/fl mice following IL-17 intranasal administration

(A) C57BL/6 mice with indicated genotypes were challenged with recombinant IL-17 intranasally (300ng/mouse) for 24h and BAL cells were harvested. Representative FACS plots of neutrophil (PMN) and Macrophage (Mac) determined by Gr-1 and F4/80 staining were shown. (B) Il17ra^fl/fl and Il17rc^fl/fl mice with indicated genotypes were challenged with IL-17 and total numbers of BAL neutrophils and macrophages were enumerated. G-CSF and IL-6 in the BAL fluid were measured by Luminex (C). Data are represented as mean +/− SEM. See also supplementary Fig. 1–2 for further characterization of these mice.

Fig. 2. Defective PMN responses and bacterial clearance in the IL-17RA epithelial conditional KO mice

Il17ra^fl/fl Scgb1a1-Cre⁻ and Il17ra^fl/fl Scgb1a1-Cre⁺ mice were challenged with recombinant IL-17 intranasally (300ng/mouse) for 24h and BAL neutrophils and macrophage numbers were determined by FACS (A). Scgb1a1-Cre mediated recombination was shown in supplementary Fig. 3. Il17ra^fl/fl Scgb1a1-Cre⁻ and Il17ra^fl/fl Scgb1a1-Cre⁺ mice were challenged with recombinant IL-1β (10ng/mouse) plus IL-23 (500ng/mouse) intranasally for 24h and BAL neutrophils and macrophage numbers were determined by FACS (B). Gene expression of Th17 canonical cytokines from the lungs of IL-1β+IL-23 treated mice were determined by real-time RT-PCR (C). Il17ra^fl/fl Scgb1a1-Cre⁻, Il17ra^fl/fl Scgb1a1-Cre⁺, and Il17ra^fl/fl E2a-Cre⁺ mice were infected with 10⁴ KP-43816 intranasally and sacrificed 24h post infection. Bacterial burden in the lungs were determined by standard CFU assay (D). Data are represented as mean +/− SEM.

Fig. 3. Histopathology of the IL-17RA epithelial conditional KO mice after K. pneumoniae infection

Il17ra^fl/fl Scgb1a1-Cre⁻ and Il17ra^fl/fl Scgb1a1-Cre mice were infected with 10⁴ KP-43816 intranasally and sacrificed 24h post infection. Representative H&E stainings were shown (A) and pathology scores were graphed (B). Data are represented as mean +/− SEM.

Fig. 4. RNA-seq analysis on mouse bronchial brushes from the IL-17RC epithelial conditional KO mice

Il17rc^fl/fl Scgb1a1-Cre⁻ and Il17rc^fl/fl Scgb1a1-Cre⁺ mice were challenged with recombinant IL-17 intranasally (300ng/mouse) for 6h and bronchial brushings were harvested for RNA extraction and RNA-seq analysis. (A). Heat map of selected known IL-17R-dependent genes. (B). Enriched KEGG pathways from the gene set enrichment analysis (GSEA). (C). Enriched pathways using the Upstream Regulator Analytic from the Ingenuity Pathway Analysis software. Real-time RT-PCR validation of these RNA-seq findings was shown in supplementary Fig. 4.

Fig. 5. Loss of Cxcl5 expression in the IL-17RA epithelial conditional KO mice

Il17ra^fl/fl Scgb1a1-Cre⁻ and Il17ra^fl/fl Scgb1a1-Cre⁺ mice were challenged with recombinant IL-17 intranasally (300ng/mouse) for 24h and IL-17 downstream chemokines and cytokines in the BAL fluids were measured by Luminex (A). Gene expression in whole lung homogenates and bronchial brushings were determined by real-time RT-PCR (B). Il17ra^fl/fl Scgb1a1-Cre⁻ and Il17ra^fl/fl Scgb1a1-Cre⁺ mice were infected with 10⁴ KP43816 intranasally. 2h post infection, a subgroup of Il17ra^fl/fl Scgb1a1-Cre⁺ the mice received 1ug recombinant CXCL5 (LIX). Mice were sacrificed at 4h after infection for BAL cell enumeration by FACS (C). A separate cohort of mice were sacrificed at 24h after infection and bacterial burden in the lungs were determined by CFU (D). See also the pathology of these mice in supplementary Fig. 5. Data are represented as mean +/− SEM.

Fig. 6. Defective PMN responses and bacterial clearance in the IL-17RC epithelial conditional KO mice

WT or Il17re+/− as well as littermates Il17re−/− mice were infected with 10⁴ KP-43816 intranasally and sacrificed 24h post infection. Bacterial burden in the lungs were determined by standard CFU assay (A). See also supplementary Fig. 6. C57BL/6 mice were challenged with recombinant IL-17C (500ng/mouse) with or without IL-17RE Fc (500ng/mouse) intranasally and sacrificed at 24h. BAL neutrophils and macrophage numbers were determined by FACS (B). C57BL/6 mice were infected with 10⁴ KP43816 intranasally with or without IL-17RE Fc (500ng/mouse) intranasally and sacrificed at 24h. Bacterial burden in the lungs were determined by CFU (C) and gene expression in the lungs were analyzed by real-time RT-PCR (D). Littermate controls as well as Il17rc^fl/fl and Il17rc^fl/fl Scgb1a1-Cre⁺ mice were infected with 10⁵ KP-2146 intranasally and sacrificed 24h post infection. Bacterial burden in the lungs were determined by CFU (E). Data are represented as mean +/− SEM.