Differential Effects of Cytokine Versus Hypoxic Preconditioning of Human Mesenchymal Stromal Cells in Pulmonary Sepsis Induced by Antimicrobial-Resistant Klebsiella pneumoniae

Abstract

Background: Pulmonary sepsis is a leading cause of hospital mortality, and sepses arising from antimicrobial-resistant (AMR) bacterial strains are particularly difficult to treat. Here we investigated the potential of mesenchymal stromal cells (MSCs) to combat established Klebsiella pneumoniae pneumosepsis and further evaluated MSC preconditioning and pre-activation methods. Methods: The potential for naïve and preconditioned MSCs to enhance wound healing, reduce inflammation, preserve metabolic activity, and enhance bacterial killing was assessed in vitro. Rats were subjected to intratracheal K. pneumoniae followed by the intravenous administration of MSCs. Physiological indices, blood, bronchoalveolar lavage (BAL), and tissues were obtained 72 h later. Results: In vitro assays confirmed that preconditioning enhances MSC function, accelerating pulmonary epithelial wound closure, reducing inflammation, attenuating cell death, and increasing bacterial killing. Cytomix-pre-activated MSCs are superior to naïve and hypoxia-exposed MSCs in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, reducing bacteria, and attenuating histologic injuries in lungs. BAL inflammatory cytokines were reduced, correlating with decreases in polymorphonuclear (PMN) cells. MSCs increased PMN apoptosis and the CD4:CD8 ratio in BAL. Systemically, granulocytes, classical monocytes, and the CD4:CD8 ratio were reduced, and nonclassical monocytes were increased. Conclusions: Preconditioning with cytokines, but not hypoxia, enhances the therapeutic potential of MSCs in clinically relevant models of K. pneumoniae-induced pneumosepsis.

Multiple Dosing and Preactivation of Mesenchymal Stromal Cells Enhance Efficacy in Established Pneumonia Induced by Antimicrobial-Resistant Klebsiella pneumoniae in Rodents

“…After MSC treatment there was a significant reduction in the total number of T cells in the BAL which correlated with a significant reduction in cytotoxic CD8 + T cells. The CD4 + /CD8 + ratio was similar to that demonstrated after MSC administration during early infection [13]. During intermediate or established infection, T cell exhaustion and immune cell paralysis can be initiated.…”

“…By administering cytomix-licensed MSCs 24 and 48 h after the bacterial inoculation, lung function was restored, showing significantly improved arterial oxygenation and alveolar-arterial oxygen gradient. There was also a significant reduction in lung oedema with cytomix-licensed MSC administration that did not occur when administered during the acute phase of infection, as previously demonstrated [13]. This shows the importance of the therapeutic window for MSCs, whereby they can enhance the resolution of infection when administered before or during the peak of infection.…”

“…This 'translational failure' could be due to several factors including requirement for testing in more clinically relevant preclinical models, a more potent cell treatment, and/or better dosing regimens. We demonstrated in our previous study that the effects of naïve MSCs can be enhanced using cytomix activation in our preclinical pneumonia model, with a single MSC administration in presymptomatic animals, 1 h after bacterial infection [13].…”

“…MSCs have considerable promise as a potential treatment for bacterial pneumonia due to their immune modulation capacity [18], which extends to AMR strains [13]. The immunoevasive properties of MSCs have been well studied and documented, which allows for allogenic administered without adverse reactions, with xenogeneic administration of human MSCs into animals modelling the extreme end of this spectrum for host immune responses against nonself cells [19,20].…”

“…Multiple strategies have been employed for this including genetic modification, environmental stressors, and exposure to proinflammatory cytokines [12]. We previously demonstrated the enhanced therapeutic properties of MSCs after cytokine preactivation when administered early following K. pneumoniae infection [13]. Here, our hypothesis is that, when given at a later timepoint, the MSC therapy is less effective, but this can be rescued by administration of preactivated MSCs.…”