Increased susceptibility to pulmonary Pseudomonas infection in Splunc1 knockout mice

Abstract

The airway epithelium is the first line of host defense against pathogens. The short palate, lung, and nasal epithelium clone (SPLUNC)1 protein is secreted in respiratory tracts and is a member of the bacterial/permeability increasing (BPI) fold-containing protein family, which shares structural similarities with BPI-like proteins. On the basis of its homology with BPIs and restricted expression of SPLUNC1 in serous cells of submucosal glands and surface epithelial cells of the upper respiratory tract, SPLUNC1 is thought to possess antimicrobial activity in host defense. SPLUNC1 is also reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to determine the in vivo functions of SPLUNC1 following Pseudomonas aeruginosa infection and to elucidate the underlying mechanism by using a knockout (KO) mouse model with a genetic ablation of Splunc1. Splunc1 KO mice showed accelerated mortality and increased susceptibility to P. aeruginosa infection with significantly decreased survival rates, increased bacterial burdens, exaggerated tissue injuries, and elevated proinflammatory cytokine levels as compared with those of their wild-type littermates. Increased neutrophil infiltration in Splunc1 KO mice was accompanied by elevated chemokine levels, including Cxcl1, Cxcl2, and Ccl20. Furthermore, the expression of several epithelial secretory proteins and antimicrobial molecules was considerably suppressed in the lungs of Splunc1 KO mice. The deficiency of Splunc1 in mouse airway epithelium also results in increased biofilm formation of P. aeruginosa. Taken together, our results support that the ablation of Splunc1 in mouse airways affects the mucociliary clearance, resulting in decreased innate immune response during Pseudomonas-induced respiratory infection.

Figure 1. Splunc1 KO mice exhibit increased susceptibility to Pseudomonas aeruginosa-induced acute pneumonia

Splunc1 KO and WT mice were intranasally inoculated with 10⁹ CFU PAO1 per mouse. Survival is represented by Kaplan-Meier survival curves (p = 0.0056, 95% confidence interval, log-rank test). Results were obtained from two independent experiments (n=19 mice in each group).

Figure 2. Splunc1 KO mice are susceptible to Pseudomonas aeruginosa induced respiratory infection

Age-matched Splunc1 KO and WT mice were intranasally inoculated with 1.5×10⁷ CFU PAO1 per mouse. A. CFU in lung homogenates from left lobes were determined at 6h and 24h after PAO1 infection. B. At 24h post-infection, Splunc1 KO mice exhibited significantly increased bacterial load in their BALF. Results are mean ± SEM from three independent experiments; n = 4-6 mice for each treatment group. **p < 0.01 for Splunc1 KO to WT comparisons at each time point.

Figure 3. Splunc1 KO mice show increased inflammatory cell recruitment following Pseudomonas aeruginosa infection

A. Total inflammatory cells were significantly higher in Splunc1 KO mice than in WT mice at both 6h and 24h after PAO1 challenge. B. Macrophages in BAL from WT mice increased more than those in Splunc1 KO mice after 6h bacterial exposure. C. Total neutrophils in BAL from Splunc1 KO mice increased more than those from WT mice. Results are mean ± SEM from three independent experiments; n = 4-6 mice for each group. *p < 0.05, **p < 0.01 for Splunc1 KO to WT comparisons at each time point.

Figure 4. Splunc1 KO mice display more severe lung injury after Pseudomonas aeruginosa infection

Lung tissues were harvested without (A,B) or with PAO1 challenge at 6h (C,D) and 24h (E,F) after infection, fixed and H&E stained for histological evaluation. Splunc1 KO mice exhibited more significant peribronchial inflammation, airway lumen leukocyte accumulation, and severe pneumonia after PAO1 infection. Black arrows indicate alveolar inflammation while blue arrows point to peribronchial inflammation. Alveolar space and bronchial lumen are labeled as AS and BL.

Figure 5. Increased pro-inflammatory cytokine production in Splunc1 KO mice following PAO1 bacterial challenge

Splunc1 KO and WT mice were infected as described. Cytokine concentrations in BALF were measured using a Luminex assay and are reported in pg/ml. Results are mean ± SEM from three independent experiments; n = 4-6 mice for each group. *p < 0.05, **p < 0.01 for Splunc1 KO to WT comparisons at each time point.

Figure 6. Increased chemokine expression in Splunc1 KO mice following PAO1 bacterial challenge

Splunc1-deficiency selectively enhances production of a distinct set of chemokines in response to PAO1 infection. Lungs were collected from Splunc1 KO and WT mice. Gene expressions in lungs of mice were quantified by real time PCR. The expression of chemokine ligands Cxcl1 (A), Cxcl2 (B), and Ccl20 (C) all increased in Splunc1 KO mice while Cxcl5 (D) showed no difference compared to expression in WT mice. Results are mean ± SEM from three independent experiments; n = 4-6 mice for each group. *p < 0.05, **p < 0.01 for Splunc1 KO to WT comparisons at each time point.

Figure 7. Splunc1 KO mice exhibit decreased expression of mucociliary clearance-related genes

Splunc1-deficiency influences the expression of major airway secretory proteins, including Muc5ac (A), Muc5b (B), CCSP (C), and ciliated cell marker Foxj1 (D) both before and after PAO1 infection. Results are mean ± SEM from three independent experiments; n = 4-6 mice for each group. *p < 0.05, **p < 0.01 for Splunc1 KO to WT comparisons at each time point.

Figure 8. Splunc1 KO mice display diminished expression of antimicrobial molecules

Splunc1-deficiency attenuates expression of major airway antimicrobial peptide LL37 (A) and antimicrobial proteins lactoferrin (B) and lysozyme (C), but has no effect on β-defensin 2 (D) after PAO1 infection. Results are mean ± SEM from three independent experiments; n = 4-6 mice for each group. *p < 0.05, **p < 0.01 for Splunc1 KO to WT comparisons at each time point.

Figure 9. rSPLUNC1 significantly inhibits biofilm formation of Gram-negative bacteria

Determination of anti-biofilm effects of SPLUNC1 on P. aeruginosa. Quantifications were carried out by counting CFU representing biofilm-forming bacteria recovered from the surface of cultured epithelial cells or by measuring the biofilm biomass on abiotic surfaces via crystal violet staining (OD₅₉₀). A, CFU of biofilm-forming P. aeruginosa growth on the primary cultured tracheal epithelial cells derived from WT (Splunc1+/+), heterozygous (Splunc1+/−), and KO (Splunc1−/−) mice. B, P. aeruginosa biofilm biomass measurement after treatment with increasing concentrations of rSPLUNC1. Kanamycin and ampicillin were used as positive controls to disrupt PAO1 biofilm formation. Results are mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 for the comparison between groups.

Figure 10. Mechanisms underlying SPLUNC1 antibacterial function

SPLUNC1 may regulate the expression of other epithelial antimicrobial peptides/proteins and modulate airway secretion; SPLUNC1 is a surfactant protein, which decreases airway surface tension and enhances mucociliary clearance as well as interferes with bacterial biofilm establishment. SPLUNC1 may also exert antibacterial activity by inhibiting bacterial growth. Following P. aeruginosa exposure, the deficiency of epithelial SPLUNC1 results in compromised host defense and increased susceptibility to bacterial infection.