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Background Pseudomonas aeruginosa (PA) is one of the most common Gram-negative bacteria causing hospital-acquired pulmonary infection, with high drug resistance and mortality. Therefore, it is urgent to introduce new non-antibiotic treatment strategies. Mesenchymal stem cells (MSCs), as important members of the stem cell family, were demonstrated to alleviate pathological damage in acute lung injury. However, the potential mechanism how MSC alleviate acute lung infection caused by PA remains unclear. Objective The purpose of this study was to investigate the effects of Adipose-derived mesenchymal stem cells (ASCs) on acute pulmonary infections and the possible mechanisms how ASCs reduce pulmonary inflammation induced by PA. Methods The therapeutic and mechanistic effects of ASCs on PA pulmonary infection were evaluated respectively in a murine model as well as in an in vitro model stimulated by PA and co-cultured with ASCs. Results 1. ASCs treatment significantly reduced the bacterial load, inflammation of lung tissue and histopathological damage by PA. 2. PA infection mainly activated Nod-like receptor containing a caspase activating and recruitment domain 4 (NLRC4) inflammasome in the lung of mice. ASCs attenuated acute lung infection in mice by inhibiting NLRC4 inflammasome activation. 3. NLRC4−/− mice showed a significant improvement in survival rate and lung bacterial load after PA infection. 4. ASCs mainly increased expression and secretion of STC‐1 in response to PA‐stimulated NLRC4 inflammasome activation. Conclusions PA infection attenuated macrophage phagocytosis through activation of NLRC4 inflammasome in macrophages, which eventually led to pulmonary inflammatory damage in mouse; ASCs reduced the activation of NLRC4 inflammasome in macrophages induced by PA infection, thereby increasing the phagocytic ability of macrophages, and ultimately improving lung tissue damage in mouse; ASCs may inhibit NLRC4 inflammasome through the secretion of STC-1.

Objetivo: Avaliar as concentrações pulmonares e brônquicas de ciprofloxacino (CIP) utilizando modelos animais de pneumonia aguda e crônica causada por Pseudomonas aeruginosa formadora de biofilme. Metodologia: Pneumonia aguda (2 dias) foi desenvolvida pela inoculação intratraqueal de ratos Wistar com 10⁸ UFC/mL de P. aeruginosa formadora de biofilme não-mucóide (PA14), enquanto pneumonia crônica (14 dias) foi desenvolvida pela inoculação de beads de alginato impregnados com P. aeruginosa mucóide ATCC 27853 (10⁷ UFC/mL). A investigação farmacocinética plasmática, pulmonar e brônquica (fluido de revestimento epitelial pulmonar – PELF) foi conduzida após dose i.v. bolus de CIP 20 mg/kg. Sondas de microdiálise (CMA 20®, 4 mm, 20 kDa) foram utilizadas para avaliar as concentrações livres pulmonares e brônquicas. CIP foi quantificado nas amostras biológicas por CLAE/fluorescência previamente validado. Análise farmacocinética não-compartimental foi conduzida com o software Phoenix® e os parâmetros comparados estatisticamente por ANOVA (Graphpad®) (α = 0,05). Resultados e Discussão: A infecção aguda gera parâmetros farmacocinéticos plasmáticos do CIP intermediários entre os previamente relatados para animais sadios e infectados cronicamente (TORRES et al. AAC, 61, 2017), com redução de clearance devido ao comprometimento das rotas de eliminação do CIP. A infecção aguda mostrou uma tendência de redução das concentrações livres intersticiais de CIP, com redução de 1,5 vezes no fator de penetração pulmonar em relação do grupo sadio (fT = 1,69 para fT = 1,02), intermediária em comparação com o grupo cronicamente infectado (fT = 0,44). No PELF as concentrações livres de CIP não foram alteradas pela infecção. O fator de penetração pulmonar/brônquico mostrou que animais sadios e agudamente infectados têm concentrações livres no PELF equivalentes a 46% das concentrações no fluido intersticial pulmonar, enquanto nos animais cronicamente infectados esta razão aumenta para 65%. Conclusões: A microdiálise brônquica é uma ferramenta importante para elucidar a penetração de fármacos no PELF. Concentrações plasmáticas livres de CIP aumentam com a cronificação da infecção não se mostrando adequadas para ajustes posológicos do fármaco no tratamento de pneumonias. Purpose: To evaluate ciprofloxacin (CIP) pulmonary and bronchial concentrations using animal models of acute and chronic biofilm-forming Pseudomonas aeruginosa pneumonia. Methods: Acute pneumonia (2 days) was developed by intratracheal inoculation of Wistar rats with 10⁸ CFU/mL of non-mucoid biofilm-forming P. aeruginosa (PA14) while chronic infection (14 days) was developed by inoculation of beads impregnated with mucoid P. aeruginosa ATCC 27853 (10⁷ CFU/mL). Plasma, pulmonary and bronchial (Pulmonary Epithelial Lining Fluid – PELF) pharmacokinetics investigation was conducted following CIP 20 mg/kg i.v. bolus dosing. Microdialysis probes (CMA 20® 4 mm, 20 kDa) were used to evaluate free pulmonary and bronchial concentrations. For plasma concentrations, the carotid artery was cannulated. CIP concentrations were determined by a HPLC/fluorescence validated method. Non-compartmental analysis was conducted using Phoenix® software and pharmacokinetic parameters statistically compared by ANOVA (Graphpad®) (α = 0.05). Results and Discussion: Acute infection resulted in CIP intermediate plasma pharmacokinetic parameters, between those previously reported for healthy and chronically infected animals (TORRES et al., AAC, 61, 2017), with reduction of clearance due to compromised drug’ elimination routes. A tendency of lower free interstitial pulmonary concentrations due to acute infection was observed, with a 1.5 times reduction of pulmonary penetration factor in comparison with healthy animals (fT = 1.60 to fT = 1.02), intermediate in comparison with that observed in chronically infected animals (fT = 0.44). Free CIP concentrations were not altered in PELF due to infection. The pulmonary/bronchial penetration factor showed that healthy and acutely infected animals had free PELF concentrations equivalent to 46% of pulmonary interstitial fluid concentrations, while in chronically infected animals this ratio increases to 65%. Conclusions: Bronchial microdialysis is an important tool to elucidate drug’s concentrations in PELF. CIP free plasma concentrations augment with infection chronicity and are not adequate for posological adjustment to treat pneumonias.