Epithelial Cell-Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia

Abstract

Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6G(bright)CD11b(bright) neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia.

Keywords: CXCL2; lung epithelium; neutrophil activation; pneumococcal pneumonia; type I IFNs.

Figure 1.

Genes induced by pneumonia selectively in epithelial cells. (A) Heatmap of 1,166 genes with significantly higher expression in epithelial cells from pneumonic lung compared with both other groups during pneumonia (false discovery rate [FDR] q < 0.05). Epi CTRL, epithelial cells from uninfected mouse lungs; Epi PN, epithelial cells from pneumonic mouse lungs; Non-Epi PN, nonepithelial cells from pneumonic mouse lungs (n = 3 mice per group, each column from an independent mouse). Red intensity represents the degree of higher level of expression, and blue intensity the degree of lower level of expression. (B) To identify secreted proteins induced especially in epithelial cells owing to pneumonia, the genes with significant (FDR q < 0.05) and strong (>twofold) up-regulation in epithelial cells from mice with pneumonia compared with both other groups (epithelial cells from uninfected mice and nonepithelial cells from pneumonic mice) were probed using Database for Annotation, Visualization and Integrated Discovery Bioinformatics. A total of 32 genes were identified as secreted proteins based on the SP_PIR_Secretion annotation. Expression of each transcript in the indicated cell population is shown normalized to the relative expression of that transcript in epithelial cells sorted from uninfected mouse lungs.

Figure 2.

Pneumonia can induce secreted and transmembrane (Sectm) 1 transcription in epithelial cells by mechanisms other than RelA. (A and B) C57BL/6 mice were infected with Streptococcus pneumoniae serotype 19F. mRNAs were measured in left-lung homogenates using quantitative RT-PCR (qRT-PCR) at indicated time points. Values represent fold induction relative to 0 hours and were expressed as means (±SEM). Significance was determined by one-way ANOVA with Sidak’s post hoc test (n = 4–8 per group). *P < 0.05 versus 0 hours. (C and D) NK2 homeobox 1–Cre Rela-floxed mice in which RelA was mutated throughout the lung epithelium, or wild-type (WT) control littermates with no Cre expression, were infected with pneumococcus and killed 9 or 24 hours after infection, after which left-lung epithelial cells, identified as CD45⁻EpCAM⁺, were isolated by FACS. Values represent fold induction relative to epithelial cells isolated from 0-hour Cre-negative mice and were expressed as mean (±SEM). Significance was determined by two-way ANOVA with Sidak’s post hoc test (n = 3–4 per group). *P < 0.05 versus 0 hour.

Figure 3.

Type I IFN signaling mediates Sectm1 induction through signal transducer and activator of transcription (STAT) 1. (A) Alveolar epithelial cell–like E10 cells were stimulated with IFN-β (1,000 U/ml) for 6 hours. Expression of Sectm1a was measured using qRT-PCR. Values represent fold induction relative to vehicle group and were expressed as means (±SEM). Significance was determined by Student’s t test. Results reflect data from three independent experiments. *P < 0.05 versus vehicle. (B) Immunoblot analysis of E10 cells 48 hours after transfection with either nontargeting (NT) or STAT1 small interfering RNA (siRNA). (C) qRT-PCR of Sectm1a expression in siRNA transfected cells treated with vehicle or IFN-β (1,000 U/ml) for 6 hours. Values represent fold induction compared with nontargeting and vehicle-treated group and were expressed as means (±SEM). Significance was determined by two-way ANOVA with Sidak’s post hoc test. Results reflect data from three independent experiments. *P < 0.05 versus nontargeting siRNA. (D) C57BL/6 mice were infected with S. pneumoniae serotype 19F. Expressions of IFN-β were measured in left-lung homogenates using qRT-PCR at indicated time points. Values represent fold induction relative to 0 hours and are expressed as means (±SEM). Significance was determined by one-way ANOVA with Sidak’s post hoc test (n = 4–8 per group). *P < 0.05 versus 0 hour. (E and F) WT and IFN-α/β receptor (IFNAR)-deficient mice were infected with S. pneumoniae serotype 19F and killed 24 hours after infection. mRNAs were measured in lung homogenates using qRT-PCR. Values represent fold induction relative to 0 hour, WT, and are expressed as means (±SEM). Significance was determined by two-way ANOVA with Sidak’s post hoc test (n = 4–5 in each experimental group). *P < 0.05 versus WT.

Figure 4.

Conducting airway epithelial cells produce Sectm1 during pneumonia. (A and B) Surfactant protein (SP) C–green fluorescent protein (GFP) transgenic mice were infected with S. pneumoniae serotype 19F. Left lungs were harvested 24 hours after infection and digested to single-cell suspensions with elastase. Four cell populations consisting of CD45⁺, CD45⁻GFP^bright, CD45⁻GFP⁻EpCAM⁺T1α^bright, and CD45⁻GFP⁻EpCAM⁺T1α⁻ were sorted by flow cytometry. Expression of SPC, caveolin-1, CC10, and Forkhead box protein J1 (Foxj1) were measured across these four cell populations using qRT-PCR. Values represent fold induction relative to CD45⁺ cells and are expressed as means (±SEM). Significance was determined by one-way ANOVA with Sidak’s post hoc test (n = 4 in each group). *P < 0.05 versus CD45⁺. (C) Sectm1 transcripts were measured using qRT-PCR across the four cell populations. Values represent fold induction relative to CD45⁺ cells and are expressed as means (±SEM). Significance was determined by one-way ANOVA with Sidak’s post hoc test (n = 4 in each group). *P < 0.05 versus CD45⁺. EpCAM, epithelial cell adhesion molecule; SSC, side scatter.

Figure 5.

Sectm1a interacts with myeloid cells in the pneumonic lung. (A) C57BL/6 mice were infected with S. pneumoniae serotype 19F, and left lungs were harvested 24 hours after infection. Single-cell suspensions from collagenase-digested left lung were incubated with vehicle (data not shown), recombinant Fc (25 μg/ml, stoichiometrically matched to recombinant mouse Sectm1a-Fc), or recombinant Sectm1a-Fc (40 μg/ml). The cells were washed and stained with phycoerythrin (PE)-conjugated anti-mouse IgG2a, followed by staining with anti-mouse CD45, Ly6G, CD11b, and CD19 antibodies. Binding of Sectm1a to cells was detected as PE signal. Results are representative of three independent experiments. Gates were determined based on cells incubated with vehicle (data not shown). (B) Neutrophils (CD45⁺Ly6G⁺) of the pneumonic lungs were further divided into two subpopulations based on the relative expression of CD11b and Ly6G, and binding of Sectm1a was determined as in (A). Cells represented in blue or red histograms reflect those incubated with Fc or Sectm1a-Fc, respectively. (C) Uninfected lungs were harvested and Sectm1a binding assay was performed using the same doses of Fc (25 μg/ml) and Sectm1a-Fc (40 μg/ml) as in (A) to determine the binding of Sectm1a-Fc to neutrophils (CD45⁺Ly6G⁺). Gates were determined based on vehicle group (data not shown). Results are representative of three independent experiments. (D) Blood was collected 20 hours after intrabronchial instillation of S. pneumoniae serotype 19F. Sectm1a binding assay was performed using the same doses of Fc (25 μg/ml) and Sectm1a-Fc (40 μg/ml) to determine the binding of Sectm1a-Fc to blood neutrophils (CD45⁺Ly6G⁺). Gates were determined based on vehicle group (data not shown). Results are representative of three independent experiments. Ly6G, Ly6g lymphocyte antigen 6 complex, locus G; rm, recombinant murine.

Figure 6.

Sectm1a promotes chemokine (C-X-C) motif ligand (CXCL) 2 production by neutrophils from the pneumonic lung. C57BL/6 mice were infected with S. pneumoniae serotype 19F, and bronchoalveolar lavage fluid was collected 20 hours after infection, from which neutrophils were isolated. Isolated neutrophils were cultured and stimulated with vehicle, Fc (39 μg/ml, stoichiometrically matched to recombinant mouse Sectm1a-Fc), or Sectm1a-Fc (60 μg/ml) for 2 hours (A–E), 8 hours (F and G), or 4 hours (H–J). (A–E) qRT-PCR was performed on total cellular RNA to determine the gene expressions of CXCL1, CXCL2, TNF-α, IL1-β, and CXCL10. Values represent fold induction relative to vehicle group and were expressed as means (±SEM). Significance was determined by one-way ANOVA with Sidak’s post hoc test (n = 9 per group). *P < 0.05 versus vehicle. (F and G) CXCL2 and TNF-α levels were measured by ELISA from culture supernatants of neutrophils. Values represent fold induction relative to vehicle and were expressed as means (±SEM). Significance was determined by one-way ANOVA with Sidak’s post hoc test (n = 9 per group). *P < 0.05 versus vehicle. (H–J) After stimulation, neutrophils were washed and stained with anti-mouse CD45, Ly6G, CD11b, CD18, and CD62L antibodies. The expression of surface markers, including CD11b, CD18, and CD62L, was determined in each group using flow cytometry. Cells represented in red, blue, or orange histograms reflect those stimulated with vehicle, Fc, or Sectm1a-Fc, respectively. Results are representative of four samples.