Roles of lung epithelium in neutrophil recruitment during pneumococcal pneumonia

Abstract

Epithelial cells line the respiratory tract and interface with the external world. Epithelial cells contribute to pulmonary inflammation, but specific epithelial roles have proven difficult to define. To discover unique epithelial activities that influence immunity during infection, we generated mice with nuclear factor-κB RelA mutated throughout all epithelial cells of the lung and coupled this approach with epithelial cell isolation from infected and uninfected lungs for cell-specific analyses of gene induction. The RelA mutant mice appeared normal basally, but in response to pneumococcus in the lungs they were unable to rapidly recruit neutrophils to the air spaces. Epithelial cells expressed multiple neutrophil-stimulating cytokines during pneumonia, all of which depended on RelA. Cytokine expression by nonepithelial cells was unaltered by the epithelial mutation of RelA. Epithelial cells were the predominant sources of CXCL5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas nonepithelial cells were major sources for other neutrophil-activating cytokines. Epithelial RelA mutation decreased whole lung levels of CXCL5 and GM-CSF during pneumococcal pneumonia, whereas lung levels of other neutrophil-recruiting factors were unaffected. Defective neutrophil recruitment in epithelial mutant mice could be rescued by administration of CXCL5 or GM-CSF. These results reveal a specialized immune function for the pulmonary epithelium, the induction of CXCL5 and GM-CSF, to accelerate neutrophil recruitment in the infected lung.

Figure 1.

Air space neutrophil emigration and air space edema requires lung epithelial nuclear factor-κB (NF-κB) RelA. Neutrophils in (A) bronchoalveolar lavage fluid (BALF) and (B) blood were counted in the presence or absence of lung epithelial RelA. (n = 7 mice/group in uninfected; n = 8–9 mice in the pneumonia group, collected over three independent experiments). *P < 0.05 compared with uninfected mice of same genotype. ^†P < 0.05 compared with wild type (WT) mice. EpRelA^Δ/Δ = lung epithelial RelA mutant. (C) Ly6G mRNA in left lung lobe was measured by quantitative RT-PCR. Data were expressed as fold-induction compared with uninfected WT mice. Means ± SEM were calculated by combining data from two independent experiments (n = 4–5 mice in the uninfected; n = 6–8 mice in the pneumonia group). Data were collected after 0 hours (uninfected) and 9 hours (pneumonia) of pneumococcal pneumonia. *P < 0.05 compared with uninfected mice of same genotype. (D) Airspace neutrophils were counted (n = 5–9 mice/group, collected over five independent experiments). ^†P < 0.05 compared with WT mice. (E) Airspace albumin was measured by ELISA with BALF (n = 4–8 mice/group, collected over five independent experiments). (F) Lung bacterial burden was measured by whole lung CFU (n = 5–7 mice/group, collected over three independent experiments). ^†P < 0.05 compared with WT mice. Data were statistically analyzed by using a two-way ANOVA followed by a Bonferroni’s post hoc test.

Figure 2.

Lung epithelial NF-κB RelA is necessary for elevated concentrations of CXCL5, granulocyte-macrophage colony-stimulating factor (GM-CSF), and CCL20 protein during pneumococcal pneumonia. Concentrations of CXCL1 (A), CXCL2 (B), CXCL5 (C), G-CSF (D), GM-CSF (E), and CCL20 (F) were measured in whole lung homogenate using ELISA at 0 (uninfected) and 9 hours (pneumonia) after intratracheal pneumococcus (n = 6 mice in the uninfected; n = 6–7 mice in the pneumonia group, collected over two independent experiments). *P < 0.05 compared with uninfected mice of same genotype. ^†P < 0.05 compared with WT mice. Data were statistically analyzed by using a two-way ANOVA followed by a Bonferroni’s post hoc test.

Figure 3.

Neutrophil recruitment could be rescued by CXCL5 or GM-CSF but not by CCL20 in the lungs of epithelial RelA mutant mice with pneumococcal pneumonia. Neutrophil counts in BALF (A) and blood (B) were assessed in samples collected 9 hours after intratracheal instillation of pneumococcus plus recombinant murine (rm) CXCL5, GM-CSF, CCL20, or vehicle (PBS + 1% BSA) (n = 6 mice in the vehicle group; n = 4 mice in the recombinant protein group, collected over two independent experiments). *P < 0.05 compared with WT mice with vehicle. ^†P < 0.05 compared with RelA mutant mice with vehicle. Data were statistically analyzed by using a one-way ANOVA, followed by a Bonferroni post hoc test.

Figure 4.

Induction of CXCL5, GM-CSF, and CCL20 mRNA requires lung epithelial RelA during pneumococcal pneumonia. mRNA levels of CXCL1 (A), CXCL2 (B), G-CSF (C), CXCL5 (D), GM-CSF (E), and CCL20 (F) were measured using quantitative RT-PCR in left lung lobes collected 0, 6, or 9 hours after intratracheal pneumococcus. Data were expressed as fold-induction compared with WT uninfected (0 h) mice. Means ± SEM were calculated by combining data from two independent experiments (n = 4–5 mice in the uninfected group; n = 5–10 mice in the pneumonia group, collected over four independent experiments). *P < 0.05 compared with uninfected mice of same genotype. ^†P < 0.05 compared with WT mice. Data were statistically analyzed by using a two-way ANOVA, followed by a Bonferroni’s post hoc test.

Figure 5.

Epithelial and myeloid cytokines are not interdependent. Concentrations of TNF-α (A), IL-1α (B), and IL-1β (C) were measured in whole lung homogenate by ELISA at 0 (uninfected) and 9 hours (pneumonia) after intratracheal pneumococcus infection, using WT and EpiRelA^Δ/Δ mice (n = 4 mice in the uninfected group; n = 7 mice in the pneumonia group, collected over two independent experiments). Concentrations of TNF-α (D), IL-1α (E), IL-1β (F), CXCL5 (G), GM-CSF (H), and CCL20 (I) were measured in whole lung homogenate by ELISA at 0 (uninfected) and 9 hours (pneumonia) after intratracheal pneumococcus, using WT and AM RelA^Δ/Δ (myeloid RelA mutant, with targeted alveolar macrophages) mice (n = 4 mice in the uninfected group; n = 10 mice in the pneumonia group, collected over four independent experiments). *P < 0.05 compared with uninfected mice of same genotype. ^†P < 0.05 compared with WT mice. Data were statistically analyzed by using a two-way ANOVA followed by a Bonferroni’s post hoc test.

Figure 6.

Flow-assisted cell sorting collection of epithelial cells from infected lungs. (A) Single-cell suspensions from the elastase-digested left lung lobe separate into three distinct populations on the basis of relative expression of CD45 and CD326. Results are representative of n = 6 mice collected over two independent experiments after 9 hours of pneumococcal pneumonia. Sorted lung cells from each of the three populations were measured for mRNA expression of SP-C, aquaporin 5 Aqp-5, Clara cell CC10, and Foxj1 (B); Ly6G and MARCO (macrophage receptor with collagenous structure) (C); and E-selectin and PECAM-1 (D) by quantitative RT-PCR. Data were expressed as fold-induction compared with the cell population expressing the least of that marker. Means ± SEM were calculated by combining data from two independent experiments (n = 3 mice/group). *P < 0.05 compared with cells expressed least. Data were statistically analyzed by using a one-way ANOVA followed by a Bonferroni’s post hoc test. WBCs = white blood cells.

Figure 7.

Epithelial cells are the major sources of CXCL5, GM-CSF, and CCL20 during pneumococcal pneumonia, and all cytokine induction in epithelial cells depends on RelA. (A) RelA is effectively targeted by transgenesis. RT-PCR of WT RelA and mutant RelA allele products in sorted CD45⁻CD326⁺ (Epithelial), CD45⁺ (WBCs), and CD45⁻CD326⁻ (Others) cells from left lung lobe. Results are representative of n = 6 mice collected over two independent experiments. These cell populations were sorted from the uninfected (Ctrl) and 9 hours pneumonic (PN) lungs of WT and mutant mice, and cytokines were measured using quantitative RT-PCR for CXCL5 (B), GM-CSF (C), CCL20 (D), CXCL1 (E), CXCL2 (F), and G-CSF (G). Data were expressed as fold-induction compared with epithelial cells from uninfected WT mice. Means ± SEM were calculated by combining data from two independent experiments (n = 3 mice/group in each uninfected and pneumonia). *P < 0.05 compared with uninfected mice of the same genotype. ^†P < 0.05 compared with WT mice. Data were statistically analyzed by using a two-way ANOVA followed by a Bonferroni’s post hoc test.