β-sitosterol ameliorates inflammation and Pseudomonas aeruginosa lung infection in a mouse model

New Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulators of defective protein are having a positive impact on Cystic Fibrosis (CF) care []. F508del-CFTR pwCF treated with the potent modulator Trikafta (Kaftrio) feel significant benefits of lung function with reduction of inflammatory markers []. Whether mutant CFTR modulators are sufficient to completely halt and report to healthy baseline levels lung inflammation in pwCF adults and adolescents with advanced lung disease is presently under scrutiny and novel anti-inflammatory strategies continue to be proposed (for update see Editorial [] and the related Research Topic collection of articles). Conversely, whether CF lung infection and inflammation augment or reduce the efficacy of mutant CFTR modulators is also controversial, considering the heterogeneity of the CF airway surface pathophysiology (for recent review see []). We previously demonstrated that β-sitosterol (BSS) extracted from Nigella arvensis L. seeds, broadly used as anti-inflammatory remedies in traditional medicine of Northern Africa, inhibits the expression of the pro-inflammatory neutrophil chemokine Interleukin (IL)−8 in CF bronchial epithelial cells exposed to P.aeruginosa []. BSS is one of the most abundant sterols deriving from plants, widely tested for efficacy and safety in clinical pharmacology with many potential applications as anti-microbial, anti-inflammatory, or immunomodulatory agent []. Very importantly, BSS is one of the bioactive compounds of traditional Chinese herbal medicine against respiratory diseases []. We hypothesized that BSS can be used to reduce the inflammatory response in CF. Preclinical evaluation can be performed in models with varying degrees of disease severity (acute and chronic). First, BSS was tested in the mouse model of lung acute infection showing that this treatment significantly improved the health status of mice (supplementary figure 1A). Total and differential cell counts in the bronchoalveolar lavage fluid (BALF) showed a trend for reduction (supplementary figure 1B), which was not statistically significant after a single treatment, indicating that repeated administrations may be required to demonstrate therapeutic efficacy. Significant reduced bacterial burden in the airways was also observed (supplementary figure 1C). Next BSS was tested in the mouse model of lung chronic infection that mimics the advanced stage of lung pathology in humans [,]. Chronic infection is usually established by including CF-adapted bacterial variants in immobilizing agents, where they grow in microcolonies under micro-aerobic/anaerobic conditions, as in the mucus of pwCF [,]. P. aeruginosa persistence in this model has a greater effect on inflammation and damage profile rather than CFTR mutation itself and can be used in pre-clinical studies for CF. For the chronic infection, P. aeruginosa MDR-RP73 clinical strain embedded in agar beads was prepared as described [] and detailed in online data supplement. Twenty-four hours before infection, C57Bl/6NCr male mice were treated with 5 mg/Kg or 25 mg/Kg BSS by oral gavage. Mice were monitored for body weight and treatment was extended daily for 6 days. BSS treated mice exhibited significant faster recovery of body weight than vehicle-treated mice, thus suggesting an improvement of health status (Fig. 1A). When mice were analysed six days after infection, a reduction of leukocyte recruitment in BALF of mice treated with BSS was observed (Fig. 1B). Both neutrophils and alveolar macrophages were decreased in BSS treated mice. To verify that reducing inflammatory cells in chronically infected mice does not impair host defense or exacerbate infection, bacterial load was evaluated in the airways of mice, including BALF and lung. A decrease of bacteria recovered in the airways of mice was found, thus indicating an ameliorating effect of BSS on infection (Fig. 1C). As we have previously shown that addition of BSS in broth culture of P. aeruginosa strain PAO1 does not result in bactericidal and bacteriostatic effects [], the decrease of infection in BSS-treated murine lungs could be related to a more effective host immune response. Independently of the mechanism, our findings indicate that BSS improves the ability of mice to fight against chronic infection. Since the modulatory response of inflammatory cascade is crucial for clearance of microbial agents and equally important to ensure that infection does not cause excessive tissue injury, the effect of BSS on production of chemokines, cytokines and growth factors was evaluated in lung homogenate. BSS-treated mice showed a reduction of the typical chemokines activated by bacterial infection such as KC, MCP-1, MIP-1α and MIP-1β in the lung (Fig. 1D). IL-1α, the pro-inflammatory cytokine, playing a central role during infection was also decreased in BSS-treated mice (Fig. 1E). Moreover, the cytokines IL-6 and IL-12(p70) and the growth factor of granulocytes G-CSF were lowered (Fig. 1F). These data demonstrate that treatment with BSS reduces the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in mice chronically infected with P. aeruginosa. The precise mechanism of anti-inflammatory action of BSS is presently not understood. What we previously found in a CF bronchial epithelial cell model exposed to P.aeruginosa is that BSS inhibits the activation of the calcium-dependent classical isoform alpha of Protein Kinase C (PKC) []. This is consistent with the interaction of P.aeruginosa with bronchial epithelial cells inducing the release of "danger signals", as ATP and UTP that, ligating PY2 purinergic receptor, activates the release of calcium from endoplasmic reticulum and PKC-dependent activation of transcription factors like NF-kB, finally promoting expression of several chemokines and pro-inflammatory cytokines. Whether this represents the main mechanism of anti-inflammatory action of BSS requires further investigation. Most importantly, this anti-inflammatory activity is accompanied by a beneficial protecting activity against infection. Anti-infective effect of BSS, not necessarily dependent on its unlikely bacteriostatic or bactericidal effect, has been reported in different models [,], including those from our group []. Although this effect has not been yet extensively investigated, it has been shown to influence virulence factors by inactivating bacterial toxins or inducing anti-bacterial responses of the host, as it protects mice from lethal infection by Streptococcus pneumonie, by interacting with pneumolysin [], regulates invasion and survival of Brucella abortus via nitric oxide and cytokine production [] or alleviates Salmonella typhimurium induced colitis by increasing expression of antimicrobial peptides []. In addition, BSS has been shown to modulate the adaptive immune response against infection in different experimental models, e.g., activation of M1 macrophages through Th1 response [] and activation of dendritic cells []. All these reports encourage future investigations on mechanism of protection driven by BSS on murine model of P. aeruginosa chronic lung infection presented here. Anti-inflammatory effect of phytosterols has been demonstrated in several animal models [], and promising immune modulating properties have been found in clinical trials in patients with chronic infections such as pulmonary tuberculosis, by HIV or Human Papilloma Virus suggesting them as adjuvants to conventional pharmacological treatments or alternative drugs []. The doses used here, reflecting potential dosages in humans, are compatible with those used by Racette [], showing no adverse effects up to a daily intake of 2 gs BSS daily. Although pre-treatment before infection and single daily dosing could represent a limitation of our findings to be reassessed in future experiments, BSS has the potential to undergo pharmaceutical development to be applied in chronic lung inflammatory diseases sustained by P.aeruginosa infection with huge amount of neutrophil infiltrates. Specific pre-clinical and clinical validation in CF lung models is required to test its application as a complementary drug to new generation mutant CFTR modulators.