Epithelial host defense proteins comprise a critical component of the pulmonary innate immune response to infection. The short palate, lung, nasal epithelium clone (PLUNC) 1 (SPLUNC1) protein is a member of the bactericidal/permeability-increasing (BPI) fold-containing (BPIF) protein family, sharing structural similarities with BPI-like proteins. SPLUNC1 is a 25 kDa secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been implicated in airway host defense against Pseudomonas aeruginosa and other organisms. SPLUNC1 is reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to assess the importance of SPLUNC1 surfactant activity in airway epithelial secretions and to explore its biological relevance in the context of a bacterial infection model. Using cultured airway epithelia, we confirmed that SPLUNC1 is critically important for maintenance of low surface tension in airway fluids. Furthermore, we demonstrated that recombinant SPLUNC1 (rSPLUNC1) significantly inhibited Klebsiella pneumoniae biofilm formation on airway epithelia. We subsequently found that Splunc1(-/-) mice were significantly more susceptible to infection with K. pneumoniae, confirming the likely in vivo relevance of this anti-biofilm effect. Our data indicate that SPLUNC1 is a crucial component of mucosal innate immune defense against pulmonary infection by a relevant airway pathogen, and provide further support for the novel hypothesis that SPLUNC1 protein prevents bacterial biofilm formation through its ability to modulate surface tension of airway fluids.

The airway epithelium is the first line of host defense against pathogens. The short PLUNC 1 (SPLUNC1) protein is secreted in respiratory tracts and a member of the BPI fold-containing (BPIF) protein family, which shares structural similarities with bactericidal/permeability-increasing (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, 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 to those of their wild-type (WT) 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 deficiencya 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.

Background Cigarette smoking constitutes a major lifestyle risk factor for osteoporosis and hip fracture. It is reported to impair the outcome of many clinical procedures, such as wound infection treatment and fracture healing. Importantly, although several studies have already demonstrated the negative correlation between cigarette consume and impaired bone homeostasis, there is still a poor understanding of how does smoking affect bone health, due to the lack of an adequately designed animal model. Our goal was to determine that cigarette smoke exposure impairs the dynamic bone remodeling process through induction of bone resorption and inhibition of bone formation. Methods We developed cigarette smoke exposure protocols exposing mice to environmental smoking for 10 days or 3 months to determine acute and chronic smoke exposure effects. We used these models, to demonstrate the effect of smoking exposure on the cellular and molecular changes of bone remodeling and correlate these early alterations with subsequent bone structure changes measured by microCT and pQCT. We examined the bone phenotype alterations in vivo and ex vivo in the acute and chronic smoke exposure mice by measuring bone mineral density and bone histomorphometry. Further, we measured osteoclast and osteoblast differentiation gene expression levels in each group. The function changes of osteoclast or osteoblast were evaluated. Results Smoke exposure caused a significant imbalance between bone resorption and bone formation. A 10-day exposure to cigarette smoke sufficiently and effectively induced osteoclast activity, leading to the inhibition of osteoblast differentiation, although it did not immediately alter bone structure as demonstrated in mice exposed to smoke for 3 months. Cigarette smoke exposure also induced DNA-binding activity of nuclear factor kappaB (NFκB) in osteoclasts, which subsequently gave rise to changes in bone remodeling-related gene expression. Conclusions Our findings suggest that smoke exposure induces RANKL activation-mediated by NFκB, which could be a “smoke sensor” for bone remodeling.

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