IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia

Abstract

Emerging evidence supports the concept that T helper type 17 (T(H)17) cells, in addition to mediating autoimmunity, have key roles in mucosal immunity against extracellular pathogens. Interleukin-22 (IL-22) and IL-17A are both effector cytokines produced by the T(H)17 lineage, and both were crucial for maintaining local control of the Gram-negative pulmonary pathogen, Klebsiella pneumoniae. Although both cytokines regulated CXC chemokines and granulocyte colony-stimulating factor production in the lung, only IL-22 increased lung epithelial cell proliferation and increased transepithelial resistance to injury. These data support the concept that the T(H)17 cell lineage and its effector molecules have evolved to effect host defense against extracellular pathogens at mucosal sites.

Figure 1

Primary HBE cells express IL-22R, and stimulation with IL-22 and IL-17A leads to the upregulation of host defense genes and increases clonogenic frequency. (a) Primary HBE cells were stained with a goat antibody to IL-22R. Fifteen percent of HBE cells were positive for IL-22R (red); controls with secondary antibody only are shown in purple. (b) Heat map of genes expressed in HBE cells after 24 h stimulation with media, 20 ng/ml IL-22, 10 ng/ml IL-17A or a combination of 20 ng/ml IL-22 and 10 ng/ml of IL-17A (n = 3 per condition). (c) G-CSF abundance in basolateral media of HBE cells after stimulation with IL-17A, IL-22 or both (n = 5–6 from three individual donors per condition). (d) IL6 abundance in basolateral media of HBE cells after stimulation with IL-17A, IL-22 or both (n = 5–6 from three individual donors per condition). (e) Primary HBE cells were seeded at specific densities into a 96-well plate containing media, 200 ng/ml of IL-22 or 100 ng/ml of IL-17A. IL-22 increased the clonogenicity of cells more than IL-17A or media alone did (n = 3–4 from three individual donors per condition). (f) We developed 10-μm wounds in primary HBE cells before stimulating them with media alone or 20 ng/ml of IL-22 (n = 5–6 from three individual donors per condition). For c–f, *P < 0.05 compared with media control and error bars represent means ± s.e.m.

Figure 2

IL-22 expression is elevated in mice infected with K. pneumoniae, and neutralizing IL-22 leads to decreased bacterial clearance from lung and spleen. Mice were infected with K. pneumoniae and killed at the designated time points. (a) IL-22 mRNA was measured by quantitative real-time PCR. Error bars represent means ± s.e.m. (b) IL-22 protein abundance was measured by ELISA and normalized per mg of protein. Error bars represent means ± s.e.m. (c) C57BL/6 and Il17a^−/− mice were infected with K. pneumoniae and evaluated for survival. Mice given antibody to IL-22 (anti–IL-22) were moribund at 24 h compared to WT or Il17a^−/− mice (n = 8–10, **P < 0.01 by log-rank test compared to Il17a^−/− or WT mice). (d,e) K. pneumoniae burden in the lung (d) or spleen (e) 24 h after infection in control or anti–IL-22–treated mice. (f) K. pneumoniae burden in the spleen in WT or Il17a^−/− mice at 24 and 48 h after infection. (g) K. pneumoniae burden in the lung 24 h after infection in control or anti–IL-22–treated Il17a^−/− mice. n = 5–6 per group, *P < 0.05 compared with controls for panels a,b,f,g and **P < 0.01 compared with controls for d,e. All values are mean ± s.e.m. (h) K. pneumoniae burden in the spleen 24 h after infection in control or anti–IL-22–treated Il17a^−/− mice (n = 5–6 per group and **P < 0.01 compared with controls).

Figure 3

IL-22 and IL-17A regulation of pulmonary cytokines and chemokines in K. pneumoniae infection. WT or Il17a^−/− mice were infected with K. pneumoniae and treated with 50 μg anti–IL-22 or control antibody intratracheally. At 24 h after antibody treatment, mice were killed and BAL was assayed for G-CSF (a), CXCL1 (b), IL6 (c) or CCL3 (d) by Luminex (n = 5–6 per group and *P < 0.05 or **P < 0.01 compared with controls. Error bars represent means ± s.e.m.).

Figure 4

IL-22 production in vivo requires IL-23. (a) WT and Il23a^−/− mice were infected with 1 × 10³ CFU K. pneumoniae and killed at the designated time points after infection. IL-22 mRNA was measured by quantitative real-time PCR and graphed as fold change over time (n = 5–6 per group. *P < 0.05 compared with Il23a^−/−-veh. Error bars represent means ± s.e.m.). WT and Il23a^−/− mice were infected with K. pneumoniae, and 12 h after infection, Il23a^−/− mice were treated with 1 μg of vehicle (PBS), IL-22, IL-17A or both cytokines. CFU were plated from lung homogenate (b) or spleen (c) at the 24-h time point (n = 5–6 per group. *P < 0.05 compared with all other groups. Error bars represent means ± s.e.m.).

Figure 5

Il23a^−/− mice rescued with IL-22, IL-17A and both cytokines have augmented Cxcl1, Cxcl2 and Cxcl9 expression in airways and alveolar epithelium. WT and Il23a^−/− mice were infected with K. pneumoniae, and 12 h after infection, Il23a^−/− mice were rescued with 1 μg of vehicle (PBS), IL-22, IL-17A or both cytokines. A representative in situ hybridization for Cxcl1, Cxcl2 and Cxcl9 is depicted. Il23a^−/− mice had reduced staining for Cxcl1, Cxcl2 and Cxcl9 in lung tissue compared with WT mice. After administration of IL-22, IL-17A or combination of IL-22 and IL-17A, Il23a^−/− mice had significantly increased expression of all three chemokines both in distal airway (black arrows) and alveolar epithelium (gray arrows).

Figure 6

IL-22 augments the antimicrobial activity of MTEC cells in vitro. IL-22 is produced abundantly from cells isolated from lymph node explants of individuals with cystic fibrosis. MTECs from WT mice were stimulated for 24 h with media (control), 20 ng/ml recombinant mouse IL-22, 10 ng/ml IL-17A or both cytokines. Csf3 (a) and Cxcl1 (b) abundance was measured in basolateral supernatants (n = 6 per condition, *P < 0.05 compared with media control. Error bars represent means ± s.e.m.). (c) Heat map of MTECs after 24 h stimulation with media, 20 ng/ml IL-22, 10 ng/ml IL-17A or both cytokines (n = 3 per condition). (d) MTECs harvested from Lcn2^−/− mice were stimulated for 24 h with media, IL-22, IL-17A or the combination of both and subsequently infected with 1 × 10⁴ CFU of K. pneumoniae apically for 8 h, and apical CFUs were then determined by plating several dilutions of the supernatants on tryptic soy agar (n = 3–5 per condition, *P < 0.05 compared with media control). (e) BAL samples from consenting subjects with cystic fibrosis and subjects without cystic fibrosis (control) were obtained. IL-23, IL-17A, IL-17F and IL-22 protein abundance was measured by ELISA (age range 2–18 years; n = 15 cystic fibrosis and n = 8 control; error bars represent means ± s.e.m.). T cells isolated from human explanted lymph nodes from individuals with cystic fibrosis (n = 5) and individuals without cystic fibrosis (n = 2) were unstimulated or stimulated with ConA, and IL-17A protein abundance (f) was measured by LINCOplex; IL-22 and IL-17F abundance (g) was measured by ELISA at 24–48 h.