CC16 drives VLA-2-dependent SPLUNC1 expression

Abstract

Rationale: CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated.

Objectives: We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity.

Methods: We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge.

Measurements and main results: We identified 8 antimicrobial proteins significantly decreased by CC16^-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden.

Conclusion: Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.

Keywords: CC16; SPLUNC1; airway epithelia; mass spectrometry; mycoplasma pneumoniae.

Figure 1

Apical expression of SPLUNC1 is decreased in CC16^-/- MTECs. (A) Schematic of the experimental design for the mass spectrometry and quantitative proteomics approach to identify apically secreted proteins by WT and CC16^-/- MTECs. (B) Volcano plot illustrating the apically secreted proteins identified. Above the horizontal black line represents the cut-off for a p value of <0.05, while the two vertical lines represent the cut-off values of 2-fold change in either the positive or negative direction. Selected antimicrobial proteins are highlighted in red and labeled. (C) Spectral counts of SPLUNC1 protein secreted by WT and CC16^-/- MTECs by LC-MS/MS. ^***P<0.001 Unpaired t test. Data are presented as mean ± SEM from six independent experiments. (D) SPLUNC1 apical protein expression was confirmed for control-treated WT and CC16^-/- MTECs by western blotting. The same volume of protein (20μl) was loaded for each sample. ^**P<0.01 Unpaired t test. Data are presented as mean±SEM. (E) WT (n=8) and CC16^-/- (n=7) MTECs were treated with media (control) for 48 hours, after which BPIFA1 expression was measured by RT-PCR with GAPDH as a housekeeping control. ^****P<0.0001 Unpaired t test. Data are presented as mean±SEM.

Figure 2

rCC16 treatment increases SPLUNC1 expression in vitro and in vivo. (A) WT (n=3) and CC16^-/- (n=3) MTECs were treated with media (control) or rCC16 (25μg/ml) for 24 hours, after which BPIFA1 expression in cell lysates was measured by RT-PCR with GAPDH as a housekeeping control. ^*P<0.05, ^{* *}P<0.01, ^{* **}P<0.001, ^{* ***}P<0.0001 by One-Way ANOVA Tukey’s multiple comparison test. SPLUNC1 apical protein expression was confirmed for control- and rCC16-treated WT (B) and CC16^-/- (C) MTECs by western blotting (n=3 per group). The same volume of protein (20μl) was loaded for each sample. ^**P<0.01, ^***P<0.001 by Unpaired t test. Data are presented as mean±SEM. (D) CC16^-/- mice were treated intravenously with saline (n=10) and rCC16 (n=10) for 3 days, after which BPIFA1 expression in lung tissue was measured by RT-PCR with GAPDH as a housekeeping control. ^*P<0.05 by Unpaired t test. (E) SPLUNC1 protein expression was measured in the BALF from the 3-day saline (n=4) and rCC16 (n=4) treated CC16^-/- mice. The same volume of protein (20μl) was loaded for each sample. ^**P<0.01 by Unpaired t test. Data are presented as mean±SEM.

Figure 3

CC16 deficiency does not affect epithelial cell differentiation. (A) Dot plot showing the average normalized expression of BPIFA1 and SCGB1A1 (rows) across cell types (columns) generated from single-cell RNA sequencing data of healthy adult tracheal epithelial cells described in Goldfarburen et al. (33). The color of the dot represents the average gene expression in the cell type and the diameter of the dot indicates the fraction of cells that express the gene within the cell type. (B, C) Major epithelial cell type expression was measured in WT (n=6) and CC16^-/- (n=6) mice (B) and in WT (n=3) and CC16^-/- (n=3) MTECS (C) using flow cytometry. Indirect FACs staining was used to identify club (CYP2F2), ciliated (TUBA1A), goblet (MUC5AC), and basal (KRT5) expression. No significant differences in epithelial cell expression were observed between WT and CC16^-/- mice and MTECs by Unpaired t test. NS=not significant. Data are presented as mean±SEM.

Figure 4

Inhibition of VLA-2 decreases SPLUNC1 expression in WT MTECs and healthy HNECs. (A) Dot plot of α2, β1, and α4 integrin subunit (gene names: ITGA2, ITGA4, and ITGB1, respectively) expression across healthy human adult tracheal epithelial cells from single-cell RNA sequencing data as described in Goldfarbmuren et al. (B) Expression of α2 and β1 integrin subunits was verified in WT MTECs (n=3) and CC16^-/- MTECs (n=3) by western blotting. The same amount of protein (10 μg) was loaded for each sample. α2 and β1 protein levels are normalized to GAPDH. (C) Protein-protein interactions between CC16 and the integrin receptor components α2β1 using protein 3D structure modeling in the Protein Data Bank. The strength of interactions were measured by the Fiberdock energy scoring. The negative scores reflect the strength of binding. (D) WT MTECs (n=3) were treated with media, rCC16 (25 μg/mL), or rCC16 in combination with BTT3033 (VLA-2 inhibitor) (130 nM) for 24 hrs. Following the treatments, BPIFA1 gene expression was measured by RT-PCR with GAPDH as a housekeeping control. ^*P<0.05 by One-Way ANOVA Tukey’s multiple comparison test. (E) SPLUNC1 apical protein expression was assessed in the WT MTECs treated with media, rCC16, and rCC16 in combination with BTT3033 by western blotting (n=3/group). The same volume of protein (20μl) was loaded for each sample. ^*P<0.05, ^**P<0.01 by One-Way ANOVA Tukey’s multiple comparison test. (F) SPLUNC1 apical protein expression was assessed by western blotting in healthy human nasal epithelial cells (HNECs) treated with media, rCC16 (25 μg/mL), or rCC16 in combination with BTT3033 (VLA-2 inhibitor) (130 nM) for 24 hrs (n=5-6 per group). ^*P<0.05, ^**P<0.01 by One-Way ANOVA Tukey’s multiple comparison test.

Figure 5

Inhibition of VLA-2 results in decreased SPLUNC1 expression and increased Mp burden. (A) BPIFA1 gene expression was measured by RT-PCR in WT MTECs treated with media only (n=3), Mp (n=3), Mp+rCC16 (n=4), Mp+rCC16+BTT3033 (n=4), or Mp+BTT3033 (n=4). GAPDH was used as a housekeeping control. ^*P<0.05, ^**P<0.01 by One-Way ANOVA Tukey’s multiple comparison test. (B) Apical SPLUNC1 protein expression was measured in the same samples from panel A, as well as additional experimental samples to confirm reproducibility, by western blotting. A representative blot is shown. ^*P<0.05, ^**P<0.01 by One-Way ANOVA Tukey’s multiple comparison test. (C) Mp burden was measured in the same samples from panels A by RT-PCR. GAPDH was used as a housekeeping control. ^*P<0.05, ^**P<0.01 by One-Way ANOVA Tukey’s multiple comparison test. Data are presented as mean±SEM.

Figure 6

D67A rCC16 does not induce SPLUNC1 expression and results in increased Mp burden. (A) Bpifa1 expression was measured by RT-PCR in WT and CC16^-/- MTECs that were treated with media, WT rCC16, and mutant D67A rCC16 for 24 hrs. Gapdh was used as a housekeeping control. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001 by Two-Way ANOVA Šídák’s multiple comparisons test. (B) SPLUNC1 expression was measured in the same samples as panel (A) by western blotting. ^*P<0.05 by Two-Way ANOVA Šídák’s multiple comparisons test. (C) Mp burden was measured by RT-PCR in WT and CC16^-/- MTECs that were infected with Mp or treated with media during rCC16 and/or mutant D67A rCC16 treatment for 24 hrs. Gapdh was used as a housekeeping control. ^*P<0.05, ^**P<0.01, ^****P<0.0001 by Two-Way ANOVA Šídák’s multiple comparisons test.

Figure 7

rSPLUNC1 treatment decreases Mp burden in CC16^-/- mice. (A) Mp burden was measured by RT-PCR in lung tissue from CC16^-/- infected with Mp, followed by rescue with rSPLUNC1 treatment. GAPDH was used as a housekeeping control. ^**P<0.01 by Unpaired t-test. (B) Mp burden was measured in the BALF from these same mice in panel (A); however, Mp burden was measured by plating samples on PPLO agar, followed by counting colonies, to determine Mp CFU/mL. ^***P<0.001 by Unpaired t-test.