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

  • Alice Rossi
    Affiliations
    Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
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  • Alessandra Bragonzi
    Affiliations
    Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
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  • Melessike Medede
    Affiliations
    Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
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  • Ida De Fino
    Affiliations
    Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
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  • Giuseppe Lippi
    Affiliations
    Section of Clinical Biochemistry, Department of Neurosciences, Biomedicine and Movement, University of Verona, Italy

    Section of Molecular Pathology, Department of Pathology and Diagnostics, University Hospital of Verona, Verona, Italy
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  • Marco Prosdocimi
    Affiliations
    Rare Partners srl Impresa Sociale, Milano, Italy
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  • Anna Tamanini
    Affiliations
    Section of Molecular Pathology, Department of Pathology and Diagnostics, University Hospital of Verona, Verona, Italy
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  • Author Footnotes
    # Giulio Cabrini and Maria Cristina Dechecchi share senior authorship.
    Giulio Cabrini
    Footnotes
    # Giulio Cabrini and Maria Cristina Dechecchi share senior authorship.
    Affiliations
    Section of Clinical Biochemistry, Department of Neurosciences, Biomedicine and Movement, University of Verona, Italy

    Center on Innovative Therapies for Cystic Fibrosis, Department of Life Sciences and Biotechnology, University of Ferrara, Italy
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  • Author Footnotes
    # Giulio Cabrini and Maria Cristina Dechecchi share senior authorship.
    Maria Cristina Dechecchi
    Correspondence
    Corresponding author: University of Verona, Piazzale Stefani 1, 37126 Verona, Italy.
    Footnotes
    # Giulio Cabrini and Maria Cristina Dechecchi share senior authorship.
    Affiliations
    Section of Clinical Biochemistry, Department of Neurosciences, Biomedicine and Movement, University of Verona, Italy
    Search for articles by this author
  • Author Footnotes
    # Giulio Cabrini and Maria Cristina Dechecchi share senior authorship.
Open AccessPublished:August 13, 2022DOI:https://doi.org/10.1016/j.jcf.2022.08.005

      Highlights

      • Despite new CFTR modulators give a great hope to patients, research on anti-inflammatories and antibiotics remains a priority in CF. We previously demonstrated that β-sitosterol (BSS), inhibits the expression of IL-8 in CF bronchial epithelial cells exposed to Pseudomonas aeruginosa. We hypothesized that BSS can be used to reduce the inflammatory response in CF.
      • In the mouse model of lung chronic infection BSS reduced leukocyte recruitment in the bronchoalveolar lavage fluid. Both neutrophils and alveolar macrophages were decreased. Moreover, decreased bacteria recovered in the airways of mice treated with BSS was found. Treatment with BSS reduced the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis. This activity is accompanied by an improvement of health status.
      • BSS could become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.

      Abstract

      We previously demonstrated that β-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.

      Keywords

      New Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulators of defective protein are having a positive impact on Cystic Fibrosis (CF) care [
      • Barry P.J.
      • Taylor-Cousar J.L.
      Triple combination cystic fibrosis transmembrane conductance regulator modulator therapy in the real world - opportunities and challenges.
      ]. F508del-CFTR pwCF treated with the potent modulator Trikafta (Kaftrio) feel significant benefits of lung function with reduction of inflammatory markers [
      • Gabillard-Lefort C.
      • Casey M.
      • Glasgow A.M.A.
      • Boland F.
      • Kerr O.
      • Marron E.
      • Lyons A.M.
      • Gunaratnam C.
      • McElvaney N.G.
      • Reeves E.P.
      Trikafta Rescues CFTR and Lowers Monocyte P2X7R-induced Inflammasome Activation in Cystic Fibrosis.
      ]. 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 [
      • Ribeiro C.M.P.
      • McElvaney N.G.
      • Cabrini G.
      Editorial: novel anti-inflammatory approaches for cystic fibrosis lung disease: identification of molecular targets and design of innovative therapies.
      ] 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 [
      • Cabrini G.
      • Rimessi A.
      • Borgatti M.
      • Pinton P.
      • Gambari R.
      Overview of CF lung pathophysiology.
      ]). 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 [
      • Lampronti I.
      • Dechecchi M.C.
      • Rimessi A.
      • Bezzerri V.
      • Nicolis E.
      • Guerrini A.
      • Tacchini M.
      • Tamanini A.
      • Munari S.
      • D'Aversa E.
      • Santangelo A.
      • Lippi G.
      • Sacchetti G.
      • Pinton P.
      • Gambari R.
      • Agostini M.
      Cabrini G. β-sitosterol reduces the expression of chemotactic cytokine genes in cystic fibrosis bronchial epithelial cells.
      ]. 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 [
      • Marahatha R.
      • Gyawali K.
      • Sharma K.
      • Gyawali N.
      • Tandan P.
      • Adhikari A.
      • Timilsina G.
      • Bhattarai S.
      • Lamichhane G.
      • Acharya A.
      • Pathak I.
      • Devkota H.P.
      • Parajuli N.
      Pharmacologic activities of phytosteroids in inflammatory diseases: mechanism of action and therapeutic potentials.
      ]. Very importantly, BSS is one of the bioactive compounds of traditional Chinese herbal medicine against respiratory diseases [
      • Wang J.
      • Wu Q.
      • Ding L.
      • Song S.
      • Li Y.
      • Shi L.
      • Wang T.
      • Zhao D.
      • Wang Z.
      • Li X.
      Therapeutic Effects and molecular mechanisms of bioactive compounds against respiratory diseases: traditional chinese medicine theory and high-frequency use.
      ]. 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 [
      • Facchini M.
      • De Fino I.
      • Riva C.
      • Bragonzi A
      Long term chronic Pseudomonas aeruginosa airway infection in mice.
      ,
      • Bragonzi A.
      Murine models of acute and chronic lung infection with cystic fibrosis pathogens.
      ]. 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 [
      • Facchini M.
      • De Fino I.
      • Riva C.
      • Bragonzi A
      Long term chronic Pseudomonas aeruginosa airway infection in mice.
      ,
      • Bragonzi A.
      Murine models of acute and chronic lung infection with cystic fibrosis pathogens.
      ]. 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 [
      • Facchini M.
      • De Fino I.
      • Riva C.
      • Bragonzi A
      Long term chronic Pseudomonas aeruginosa airway infection in mice.
      ] 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 [
      • Lampronti I.
      • Dechecchi M.C.
      • Rimessi A.
      • Bezzerri V.
      • Nicolis E.
      • Guerrini A.
      • Tacchini M.
      • Tamanini A.
      • Munari S.
      • D'Aversa E.
      • Santangelo A.
      • Lippi G.
      • Sacchetti G.
      • Pinton P.
      • Gambari R.
      • Agostini M.
      Cabrini G. β-sitosterol reduces the expression of chemotactic cytokine genes in cystic fibrosis bronchial epithelial cells.
      ], 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) [
      • Lampronti I.
      • Dechecchi M.C.
      • Rimessi A.
      • Bezzerri V.
      • Nicolis E.
      • Guerrini A.
      • Tacchini M.
      • Tamanini A.
      • Munari S.
      • D'Aversa E.
      • Santangelo A.
      • Lippi G.
      • Sacchetti G.
      • Pinton P.
      • Gambari R.
      • Agostini M.
      Cabrini G. β-sitosterol reduces the expression of chemotactic cytokine genes in cystic fibrosis bronchial epithelial cells.
      ]. 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 [
      • Marahatha R.
      • Gyawali K.
      • Sharma K.
      • Gyawali N.
      • Tandan P.
      • Adhikari A.
      • Timilsina G.
      • Bhattarai S.
      • Lamichhane G.
      • Acharya A.
      • Pathak I.
      • Devkota H.P.
      • Parajuli N.
      Pharmacologic activities of phytosteroids in inflammatory diseases: mechanism of action and therapeutic potentials.
      ,
      • Wang J.
      • Wu Q.
      • Ding L.
      • Song S.
      • Li Y.
      • Shi L.
      • Wang T.
      • Zhao D.
      • Wang Z.
      • Li X.
      Therapeutic Effects and molecular mechanisms of bioactive compounds against respiratory diseases: traditional chinese medicine theory and high-frequency use.
      ], including those from our group [
      • Lampronti I.
      • Dechecchi M.C.
      • Rimessi A.
      • Bezzerri V.
      • Nicolis E.
      • Guerrini A.
      • Tacchini M.
      • Tamanini A.
      • Munari S.
      • D'Aversa E.
      • Santangelo A.
      • Lippi G.
      • Sacchetti G.
      • Pinton P.
      • Gambari R.
      • Agostini M.
      Cabrini G. β-sitosterol reduces the expression of chemotactic cytokine genes in cystic fibrosis bronchial epithelial cells.
      ]. 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 [
      • Li H.
      • Zhao X.
      • Wang J.
      • Dong Y.
      • Meng S.
      • Li R.
      • Niu X.
      Deng X. β-sitosterol interacts with pneumolysin to prevent Streptococcus pneumoniae infection.
      ], regulates invasion and survival of Brucella abortus via nitric oxide and cytokine production [
      • Reyes A.W.B.
      • Arayan L.T.
      • Huy T.X.N.
      • Vu S.H.
      • Min W.
      • Hur J.
      • Kim S.
      β-sitosterol contributes in the resistance to invasion and survival of Brucella abortus 544 within RAW264.7 cells, and cytokine production with reduced susceptibility to infection in BALB/c mice.
      ] or alleviates Salmonella typhimurium induced colitis by increasing expression of antimicrobial peptides [
      • Ding K.
      • Tan Y.Y.
      • Ding Y.
      • Fang Y.
      • Yang X.
      • Fang J.
      • Xu D.C.
      • Zhang H.
      • Lu W.
      • Li M.
      • Huang S.C.
      • Cai M.L.
      • Song Y.
      • Ding Y.J.
      Zhang SM. β-Sitosterol improves experimental colitis in mice with a target against pathogenic bacteria.
      ]. 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 [
      • Lee J.H.
      • Lee J.Y.
      • Park J.H.
      • Jung H.S.
      • Kim J.S.
      • Kang S.S.
      • Kim Y.S.
      • Han Y.
      Immunoregulatory activity by daucosterol, a beta-sitosterol glycoside, induces protective Th1 immune response against disseminated Candidiasis in mice.
      ] and activation of dendritic cells [
      • Fraile L.
      • Crisci E.
      • Córdoba L.
      • Navarro M.A.
      • Osada J.
      • Montoya M.
      Immunomodulatory properties of beta-sitosterol in pig immune responses.
      ]. 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 [
      • Marahatha R.
      • Gyawali K.
      • Sharma K.
      • Gyawali N.
      • Tandan P.
      • Adhikari A.
      • Timilsina G.
      • Bhattarai S.
      • Lamichhane G.
      • Acharya A.
      • Pathak I.
      • Devkota H.P.
      • Parajuli N.
      Pharmacologic activities of phytosteroids in inflammatory diseases: mechanism of action and therapeutic potentials.
      ], 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 [
      • Bouic P.J.
      Sterols and sterolins: new drugs for the immune system?.
      ]. The doses used here, reflecting potential dosages in humans, are compatible with those used by Racette [
      • Racette S.B.
      • Lin X.
      • Lefevre M.
      • Spearie C.A.
      • Most M.M.
      • Ma L.
      • Ostlund Jr., R.E.
      Dose effects of dietary phytosterols on cholesterol metabolism: a controlled feeding study.
      ], 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.
      Fig. 1
      Fig. 1Effect of BSS in a murine model of P. aeruginosa chronic infection. C57Bl/6NCr male mice were infected with P. aeruginosa MDR-RP73 embedded in agar beads by intratracheal inoculation (4–5 × 105 CFU). Treatment with 5 and 25 mg/Kg BSS or vehicle by gavage was started 24 h before and daily for 6 days after infection. A: percentage change from the initial body weight. Weight of mice were recorded before (t=−1) and after infection, daily for 6 days. Comparison between groups were made by two way ANOVA with Bonferroni post-test. Data reported are mean ± SEM of two different experiments. n = 5 (vehicle), 7 (5 mg/Kg) and 3 (25 mg/Kg). B: Inflammatory response in BALF. Total cells, neutrophils and alveolar macrophages recruited in BALF 6 days after infection were counted as detailed in Supplementary. n = 5 (vehicle), 7 (5 mg/Kg) and 3 (25 mg/Kg). C: Infection in BALF and lung. Total, lung and BALF CFUs are shown. n = 5 (vehicle), 7 (5 mg/Kg) and 3 (25 mg/Kg). D: Chemokine concentrations in supernatant of lung homogenate. E: cytokines. F: growth factors. Data are presented as the mean ± SEM pooled from two independent experiments. n = 6 (vehicle), 8 (5 mg/Kg) and 3 (25 mg/Kg). Comparisons between groups were made by non-parametric Kruskal Wallis test.
      Fig. 1
      Fig. 1Effect of BSS in a murine model of P. aeruginosa chronic infection. C57Bl/6NCr male mice were infected with P. aeruginosa MDR-RP73 embedded in agar beads by intratracheal inoculation (4–5 × 105 CFU). Treatment with 5 and 25 mg/Kg BSS or vehicle by gavage was started 24 h before and daily for 6 days after infection. A: percentage change from the initial body weight. Weight of mice were recorded before (t=−1) and after infection, daily for 6 days. Comparison between groups were made by two way ANOVA with Bonferroni post-test. Data reported are mean ± SEM of two different experiments. n = 5 (vehicle), 7 (5 mg/Kg) and 3 (25 mg/Kg). B: Inflammatory response in BALF. Total cells, neutrophils and alveolar macrophages recruited in BALF 6 days after infection were counted as detailed in Supplementary. n = 5 (vehicle), 7 (5 mg/Kg) and 3 (25 mg/Kg). C: Infection in BALF and lung. Total, lung and BALF CFUs are shown. n = 5 (vehicle), 7 (5 mg/Kg) and 3 (25 mg/Kg). D: Chemokine concentrations in supernatant of lung homogenate. E: cytokines. F: growth factors. Data are presented as the mean ± SEM pooled from two independent experiments. n = 6 (vehicle), 8 (5 mg/Kg) and 3 (25 mg/Kg). Comparisons between groups were made by non-parametric Kruskal Wallis test.

      CRediT authorship contribution statement

      Alice Rossi: Validation, Resources, Formal analysis, Writing – original draft. Alessandra Bragonzi: Conceptualization, Methodology, Resources, Formal analysis, Writing – original draft. Melessike Medede: Validation. Ida De Fino: Validation. Giuseppe Lippi: Writing – original draft. Marco Prosdocimi: Writing – original draft. Anna Tamanini: Conceptualization, Methodology. Giulio Cabrini: Conceptualization, Methodology, Writing – original draft. Maria Cristina Dechecchi: Conceptualization, Methodology, Writing – original draft.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Appendix. Supplementary materials

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