Therapeutic potential of mesenchymal stromal cells in later phase antimicrobial resistant bacterial pneumosepsis: effects on the innate and adaptive immune system
Byrnes, Declan
Byrnes, Declan
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Publication Date
2023-08-25
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Thesis
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Abstract
Introduction: Sepsis is a dysfunctional immune response to infection and remains a major source of mortality worldwide. Sepsis can be divided into three stages: early, transitionary, and late phase with the early phase characterised by potent inflammatory responses to infection and organ failure, while late phase sepsis is characterised by immunoparalysis and exhaustion, leading to long-term immunosuppression. Of all infection sources, those of the lower respiratory tract are the most lethal and have remained as such since 1990. These lower respiratory tract infections (LRTIs) can lead to acute respiratory distress syndrome (ARDS) which is most often caused by primary pneumonia and severe sepsis. Even with advances in diagnostic tools, research has shown that due to the heterogeneity of early sepsis symptoms and signs, the difficulty in identifying the source of infection, and the delays in obtaining culture results, there are delays in appropriate treatments which leads to increased mortality risks. The average 90-day mortality for sepsis is 32.2%, with septic shock reaching 38.5%, showing a prominent societal issue. Should early-goal directed therapy be implemented and rapid identification of the infection occur, it can greatly improve patient outcomes, however, with the rise of antimicrobial resistant (AMR) bacteria, conventional antibiotic treatments are becoming ineffective. ESKAPE pathogens are the primary source of concern as they have enhanced rates of antibiotic resistances leading to ineffective treatment strategies for sepsis patients. This delays the clearance of infection and further exacerbates patient risk factors, with infections due to AMR pathogens increasing mortality rates in sepsis by 2.1%. Mesenchymal stromal cells (MSCs) represent an ideal cell therapy for the treatment of inflammatory disorders, with the EU approving their use in the treatment of perianal fistulas in Crohn’s disease. This is due to their immunomodulatory properties and enhanced wound healing facilitated by their secretory factors containing extracellular vesicles (EVs), cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). However, issues remain in regard to their therapeutic function as clinical trials have not been able to match the efficacy shown in pre-clinical models. As such, studies have now begun to focus on enhancing the efficacy of the MSCs through the use of pre-activation strategies, optimising the timing of administration and the dosage. Hypothesis and Aims: The therapeutic efficacy of MSCs is dependent on activation state, administration timing, and dosing strategy which can be optimised in an animal model of antimicrobial resistant (AMR) pneumosepsis to provide pre-clinical data to overcome the difficulties faced in translating research into the clinical setting. The purpose of these studies was to (1) determine the optimal source of MSC from either the bone marrow (BM), adipose (AD), or umbilical cord (UC) in vitro and in vivo; (2) enhance the functionality of the MSCs using activation strategies; (3) assess the therapeutic efficacy of the cells in a new model of antimicrobial resistant pneumosepsis; (4) determine the optimal dosing regimen of the MSCs in the animal model. Methods: In vitro models of lung injury and immune cell dysfunction were used to determine the optimal source of MSCs through assessing their conditioned media (CM), and the efficacy of the naïve, hypoxia-grown, and activated MSC-CM on specific indices of pneumosepsis. NF-κB activation in the presence of inflammatory cytokines and LPS, cytokine production, cell viability, wound healing, activation markers, apoptosis, immunoparalysis, T-cell proliferation, and antibacterial capacity was analysed using cell lines, primary cells, and Gram-negative and Gram-positive bacteria. The in vivo prolonged infection model constituted of rodents being intratracheally inoculated with a bolus of antimicrobial resistant Klebsiella pneumoniae, with whole cell MSC therapy; BM-, AD-, or UC- MSCs occurring at 48-hours post-infection. Physiological indices, blood, bronchoalveolar lavage (BAL), and tissues were obtained 120-hours after bacterial administration for analysis. The in vivo concurrent cell administration animal model constituted of rodents being intratracheally inoculated with a bolus K. pneumoniae with a subsequent naïve, cytomix pre-activated (combination of TNF-α, IL-1β, and IFN-γ for 24-hours), or hypoxia-grown UC-MSC administration at 1-hour post infection. While the in vivo optimal dosing regimen animal model constituted of rodents being intratracheally inoculated with a bolus k. pneumoniae with a single dose of naïve or cytomix pre activated UC-MSCs at 24-hours, or a single and repeated dose of UC-MSCs at 24- and 48-hours post infection. Physiological indices, blood, bronchoalveolar lavage (BAL), and tissues were obtained 72- hours after bacterial administration for analysis. Key indices of injury and recovery that were assessed included arterial oxygenation, lung compliance, lung inflammation, cytokine response, histologic morphology, immune cell function and activation, and fibrosis assessment. Results: MSC-CM was demonstrated to reduce NF-κB activation in pulmonary and monocytic cell lines as indicated by reduced luciferase, AP-1-inducible secreted embryonic alkaline phosphatase, and inflammatory cytokines. Their secreted factors also improved wound healing, increased cell viability, a restoration of a pro-apoptotic phenotype in neutrophil-like cells, enhanced activation and cell function of innate immune cells, reduced T-cell proliferation, restoration of cytokine response to LPS in immunoparalysed cells, and an antibacterial effect. These effects were dependent on cell source and activation state with cytomix pre-activated UC-MSC-CM showing enhanced efficacy compared to either naïve or cytomix pre-activated BM- and AD-MSC-CM. Hypoxia-grown MSC-CM showed enhanced immunosuppression, reducing the activation state and phagocytic capacity of neutrophil like cells indicating their potential as a potent suppressor of inflammatory responses. The prolonged animal model of pneumosepsis did not show MSC efficacy due to the infection having been resolved by the experimental endpoint. There was improved lung morphology, reduced indices of fibrosis, and enhanced macrophage/monocyte cell function but this was dependant on MSC source and activation state. In the concurrent UC-MSC administration series comparing naïve, hypoxia-grown, and cytomix pre activated MSCs, it was shown that hypoxia-grown MSCs impaired lung function and did not modulate macrophage/monocyte cell function. Cytomix pre-activated MSCs were superior in attenuating K. pneumoniae pneumosepsis, improving lung compliance, enhancing non-neutrophil cell survival, reducing bronchioalveolar lavage (BAL) bacteria, and attenuating histologic injuries in lungs. In the BAL, both naïve and cytomix pre-activated MSCs reduced inflammatory cytokines which correlated to decreases in BAL neutrophils, increased CD4:CD8 T-cell ratios, enhanced macrophage/monocyte cell function, and restored a pro-apoptotic phenotype in neutrophils. There was reduced indices of immunoparalysis after naïve and cytomix pre-activated MSC administration. Determining the optimal dosing regimen showed that a single dose of naïve MSCs at 24-hours post infection was largely ineffective, whereas a repeated administration at 48-hours attenuated K. pneumoniae pneumosepsis. While a single dose of cytomix pre-activated MSCs was effective in the animal model, this effect could be enhanced with repeated administrations. There was similar efficacy in this series when compared to concurrent MSC administration, however, only repeated doses of cytomix pre-activated MSCs was able to improve lung function by increasing oxygenation. Cytomix pre-activated MSC administration also showed enhanced systemic responsiveness to an endotoxin injury, priming the immune system to combat potential secondary infections.
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NUI Galway