Objectives: Proprotein convertase subtilisin/kexin type 9 is a central regulator of lipid metabolism and has been implicated in regulating the host response to sepsis. Proprotein convertase subtilisin/kexin type 9 loss-of-function is associated with improved sepsis outcomes in the adult host through increased hepatic bacterial clearance. Thus, there is interest in leveraging proprotein convertase subtilisin/kexin type 9 inhibitors as a therapeutic strategy in adults with
Background
Pseudomonas aeruginosa (PA) is a major cause of morbidity and mortality among burn patients, despite antibiotic therapy. There is a need to identify innate immune defenses that prevent PA infection in injured adults in an effort to find therapeutic alternatives to antibiotics. Here, we tested our hypothesis that Microvesicles (MVs) in bronchoalveolar (BAL) fluid have a role in the immunity of the lung in response to pathogens.
Study Design
MVs were isolated from murine BAL fluid, quantified using nanoparticle tracking analysis, and injected into burn injured mice prior to PA infection. Survival was assessed and BAL bacterial loads enumerated. Neutrophil number and IL-6 activity were determined. Lungs were harvested and sphingosine (SPH) content analyzed via immunohistochemistry. Antimicrobial effects of MV and SPH enriched MVs were assessed in an in vitro assay.
Results
Burn injured mice have reduced BAL MV number and SPH content as compared to sham. When BAL MVs from healthy mice are administered to injured mice, survival and bacterial clearance are robustly improved. We further observed that intranasal administration of MVs restores SPH levels after burn injury, MVs directly kill bacteria, and this bacterial killing is increased when the MVs supplemented with SPH.
Conclusion
Using a pre-clinical model, BAL MVs are reduced after scald injury and BAL MV restoration to injured mice improves survival and bacterial clearance. The antimicrobial mechanisms leading to improved survival includes the quantity and SPH content of BAL MVs.
Cell-derived nanoparticles (CDNPs) containing cytosolic proteins and RNAs/DNAs can be isolated from stressed eukaryotic cells. Previously, CDNPs isolated from cultured cells, exerted immunomodulatory activities in different infections. Here, we sought to elucidate the role of CDNPs using a murine model of cecal ligation and puncture (CLP). We hypothesized that CDNPs influence the immune response at the site of infection, where severe cellular stress occurs. We observed early CDNP accumulation in the peritoneum after 4 hours and continued CDNP presence 24 hours after CLP. To determine whether CDNPs influence the host response to sepsis, we isolated CDNPs from a murine fibroblast cell line stressed by nutrient-deprivation, and injected them into septic mice. CDNP-treated mice demonstrated decreased peritoneal IL6-levels and an approximately 2-log lower bacterial load compared to control mice 24 hours after CLP. Additionally, a 20% CFU reduction was observed when incubating CDNPs with Pseudomona aeroginosa, indicating that CDNPs are bactericidal. To identify CDNP-responsive cells, CFSE-labeled CDNPs were injected into mice at the time of CLP. We observed that CDNPs were preferentially ingested by F4/80+ macrophages, and to a lesser degree, associated with inflammatory monocytes and neutrophils. Strikingly, CDNP-ingesting cells demonstrated elevated CD11b and MHCII expression compared to control cells. Altogether, our data indicate that CDNPs enhance the immune response at the site of infection and promote bacterial clearance, by 1) direct bacterial killing and 2) increasing phagocyte activation. Thus, CDNPs represent a novel, unexplored endogenous sepsis modulator with therapeutic potential.
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