Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense

Abstract

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Figure 1

Generation of IL-17R KO mice. (A) A gene targeting vector was constructed that replaces exons 4–11 with a PGK-neo cassette. A thymidine kinase cassette (MC-TK) was inserted into the 5′ end of the vector in the opposite orientation of the IL-17R gene. Exons are depicted as filled boxes. The initiation (ATG) and termination (TAG) codons as well as the transmembrane domain (TM) are indicated. The Asp718 restriction sites and probe used for genomic Southern blot analyses are indicated. (B) Genomic DNAs from wild-type (+/+), IL-17R^+/−, and IL-17R KO mice were digested with Asp718 and subject to Southern blot analysis using the depicted probe. The sizes of the mutant and wild-type alleles are indicated. (C) Analysis of IL-17 binding to wild-type and IL-17R–deficient cells. The binding of IL-17:Fc to wild-type (+/+) and IL-17R–deficient (−/−) spleen cells gated on expression of the T cell markers CD4 or CD8 or the B cell marker CD19 as well as thioglycollate elicited peritoneal exudate cells gated on expression of the granulocytic marker Gr-1 was determined by flow cytometry and shown in the shaded histograms. Background binding to an irrelevant Fc protein is shown in the open histograms.

Figure 1

Generation of IL-17R KO mice. (A) A gene targeting vector was constructed that replaces exons 4–11 with a PGK-neo cassette. A thymidine kinase cassette (MC-TK) was inserted into the 5′ end of the vector in the opposite orientation of the IL-17R gene. Exons are depicted as filled boxes. The initiation (ATG) and termination (TAG) codons as well as the transmembrane domain (TM) are indicated. The Asp718 restriction sites and probe used for genomic Southern blot analyses are indicated. (B) Genomic DNAs from wild-type (+/+), IL-17R^+/−, and IL-17R KO mice were digested with Asp718 and subject to Southern blot analysis using the depicted probe. The sizes of the mutant and wild-type alleles are indicated. (C) Analysis of IL-17 binding to wild-type and IL-17R–deficient cells. The binding of IL-17:Fc to wild-type (+/+) and IL-17R–deficient (−/−) spleen cells gated on expression of the T cell markers CD4 or CD8 or the B cell marker CD19 as well as thioglycollate elicited peritoneal exudate cells gated on expression of the granulocytic marker Gr-1 was determined by flow cytometry and shown in the shaded histograms. Background binding to an irrelevant Fc protein is shown in the open histograms.

Figure 1

Generation of IL-17R KO mice. (A) A gene targeting vector was constructed that replaces exons 4–11 with a PGK-neo cassette. A thymidine kinase cassette (MC-TK) was inserted into the 5′ end of the vector in the opposite orientation of the IL-17R gene. Exons are depicted as filled boxes. The initiation (ATG) and termination (TAG) codons as well as the transmembrane domain (TM) are indicated. The Asp718 restriction sites and probe used for genomic Southern blot analyses are indicated. (B) Genomic DNAs from wild-type (+/+), IL-17R^+/−, and IL-17R KO mice were digested with Asp718 and subject to Southern blot analysis using the depicted probe. The sizes of the mutant and wild-type alleles are indicated. (C) Analysis of IL-17 binding to wild-type and IL-17R–deficient cells. The binding of IL-17:Fc to wild-type (+/+) and IL-17R–deficient (−/−) spleen cells gated on expression of the T cell markers CD4 or CD8 or the B cell marker CD19 as well as thioglycollate elicited peritoneal exudate cells gated on expression of the granulocytic marker Gr-1 was determined by flow cytometry and shown in the shaded histograms. Background binding to an irrelevant Fc protein is shown in the open histograms.

Figure 2

Reduced survival of mice deficient in IL-17R signaling. (A) IL-17R KO, IL-17R^+/−, or control mice (n = 10 each group) were challenged with intranasal K. pneumoniae and survival was recorded every 12 h. To confirm that the reduced survival phenotype was due to a lack of IL-17R signaling and not due to a developmental defect, 6–8-wk-old IL-17R KO mice or C57BL/6 mice infused with AdIL-17R Fc (Western blot of IL-17R/Fc fusion protein depicted in B) or a control virus were challenged with intranasal K. pneumoniae and survival was recorded every 12 h (C; n = 10 each group).

Figure 2

Reduced survival of mice deficient in IL-17R signaling. (A) IL-17R KO, IL-17R^+/−, or control mice (n = 10 each group) were challenged with intranasal K. pneumoniae and survival was recorded every 12 h. To confirm that the reduced survival phenotype was due to a lack of IL-17R signaling and not due to a developmental defect, 6–8-wk-old IL-17R KO mice or C57BL/6 mice infused with AdIL-17R Fc (Western blot of IL-17R/Fc fusion protein depicted in B) or a control virus were challenged with intranasal K. pneumoniae and survival was recorded every 12 h (C; n = 10 each group).

Figure 2

Reduced survival of mice deficient in IL-17R signaling. (A) IL-17R KO, IL-17R^+/−, or control mice (n = 10 each group) were challenged with intranasal K. pneumoniae and survival was recorded every 12 h. To confirm that the reduced survival phenotype was due to a lack of IL-17R signaling and not due to a developmental defect, 6–8-wk-old IL-17R KO mice or C57BL/6 mice infused with AdIL-17R Fc (Western blot of IL-17R/Fc fusion protein depicted in B) or a control virus were challenged with intranasal K. pneumoniae and survival was recorded every 12 h (C; n = 10 each group).

Figure 3

K. pneumoniae growth curves in control and IL-17R KO mice (n = 4–6, *P < 0.05).

Figure 4

Reduced ANCs in the blood and lungs of IL-17R KO mice in response to K. pneumoniae infection. (A) Attenuation of increase in blood ANC in IL-17 R KO mice (n = 5–8, * denotes P < 0.05). (B) Reduction in lung neutrophil recruitment as measured as ANCs in BAL fluid (BALF) in IL-17R KO mice (n = 5–8, * denotes P < 0.05). (C) The reduced lung neutrophil recruitment was associated with a significant decrease in lung MPO activity in IL-17 R KO mice (n = 5–8, * denotes P < 0.05).

Figure 4

Reduced ANCs in the blood and lungs of IL-17R KO mice in response to K. pneumoniae infection. (A) Attenuation of increase in blood ANC in IL-17 R KO mice (n = 5–8, * denotes P < 0.05). (B) Reduction in lung neutrophil recruitment as measured as ANCs in BAL fluid (BALF) in IL-17R KO mice (n = 5–8, * denotes P < 0.05). (C) The reduced lung neutrophil recruitment was associated with a significant decrease in lung MPO activity in IL-17 R KO mice (n = 5–8, * denotes P < 0.05).

Figure 4

Reduced ANCs in the blood and lungs of IL-17R KO mice in response to K. pneumoniae infection. (A) Attenuation of increase in blood ANC in IL-17 R KO mice (n = 5–8, * denotes P < 0.05). (B) Reduction in lung neutrophil recruitment as measured as ANCs in BAL fluid (BALF) in IL-17R KO mice (n = 5–8, * denotes P < 0.05). (C) The reduced lung neutrophil recruitment was associated with a significant decrease in lung MPO activity in IL-17 R KO mice (n = 5–8, * denotes P < 0.05).

Figure 5

Representative lung histology of control and IL-17R KO mice after K. pneumoniae challenge. At 0 h, the lungs of control wild-type mice and IL-17R KO mice were essentially normal with no inflammatory infiltrate and mild edema. 18 h after infection there is minimal perivascular infiltration of neutrophils and moderate edema in the lungs of wild-type mice at 18 h after infection. In contrast, IL-17R KO mice had perivascular, peribronchiolar, and intraalveolar neutrophils at 18 h after infection associated with moderate diffuse perivascular edema. At 24 h after infection, wild-type mice had minimal perivascular infiltration of neutrophils and moderate edema in the lungs. In contrast, IL17R^−/− mice had severe perivascular edema that partially compressed and distorted adjacent alveoli. Within, and marginating the edema were large numbers of degenerate neutrophils and large numbers of bacterial rods compared with few visible bacteria in control mice (all images 40×).

Figure 6

Analysis of lung gene expression by RPA. (A) Representative gel of lung G-CSF and SCF gene expression after K. pneumoniae challenge. (B) Densitometric analysis of G-CSF and SCF gene expression (n = 3–4, *P < 0.05). (C) Representative gel of lung chemokine gene expression after K. pneumoniae challenge. (D) Densitometric analysis of MIP-2 gene expression (n = 3–4, * P < 0.05). bact., bacteria.

Figure 7

BAL fluid levels of G-CSF, MIP-2, and IL-17 after K. pneumoniae challenge.