The effects of inflammatory microenvironments on MSC viability, phenotype, secretome and  function

Introduction: Acute respiratory distress syndrome (ARDS) is a severe clinical syndrome which is characterised by acute hypoxaemia and diffuse lung inflammation. The estimated incidence of ARDS (Berlin definition) is 10.4 % among intensive care unit (ICU) admissions and the estimated hospital mortality rate is around 40 % across mild, moderate and severe ARDS. Despite the high incidence and mortality, no efficient therapeutics have been identified for ARDS treatment. The pathogenesis of ARDS involves infectious and non-infectious aetiology, dysfunction of alveolar-capillary barrier and dysregulation of immune response. Mesenchymal stromal cell (MSC)-based therapies have shown therapeutic benefits in the restoration of alveolar-capillary barriers and immunomodulatory effects on neutrophils, macrophages, T cells and other immune cells in ex vivo and in vivo preclinical ARDS models. Clinical trials using different types of MSCs have shown the safety of MSC-based therapies, yet significant efficacy remains elusive. The variable clinical outcomes of MSC-therapies in ARDS are attributed to the tissue sources, donor variability, doses, administration route of MSCs and host microenvironment. A growing body of evidence suggests that inflammatory microenvironments play pivotal roles in evoking MSC therapeutic benefits in ARDS. In this project, inflammatory cytokines and lung homogenate were applied to investigate the effects of inflammatory microenvironments in ARDS on MSC phenotype, secretome and functions in vitro. In addition, healthy and Escherichia coli (E. coli)-induced ARDS models were used to investigate the interaction between MSCs and lung microenvironment in vivo. Methods: 1) A cocktail of inflammatory cytokines including interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α (cytomix) was used to mimic the inflammatory microenvironment. Three types of MSCs including bone marrow-derived MSC (BM-MSC), human wild type induced pluripotent stem cell-derived MSC (iMSC WT) and beta-2 microglobulin knockout iMSC (iMSC B2M KO) were cultured with or without cytomix. Naïve and cytomix-primed MSCs and conditioned media (CM) were harvested to analyse the therapeutic effects of MSCs, including their expression of immunomodulatory surface markers, their release of cytokines, their anti-microbial properties and their anti-inflammatory effects on pulmonary epithelia, macrophages and T cells. 2) Lung homogenates from sham and E. coli-induced ARDS animals were used to mimic healthy and ARDS lung microenvironments. BM-MSCs were exposed to lung homogenate for 24 h, and BM-MSCs and CM were harvested to analyse the apoptosis, immunophenotype, secretome, anti-microbial properties and anti-inflammatory effects on T cells. 3) BM-MSCs were administered to healthy and E. coli-induced ARDS animals and retrieved at 0.5 h, 1 h, 3 h, 6 h and 24 h post cell administration. The morphology, apoptosis and surface biomarkers of the retrieved BM-MSCs were analysed. The composition of immune cell populations and cytokines in healthy and ARDS lung microenvironment were analysed at the time points post cell administration. Results: 1) Compared to naïve BM-MSC, iMSC WT and iMSC B2M KO, cytomix increased the expression of HLA-ABC on BM-MSC and iMSC WT, CD54 and CD200 on BM-MSC, iMSC WT, iMSC B2M KO, CD120b on BM-MSC and CD119 on two types of iMSC, respectively; cytomix increased the release of IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 from three types of MSCs while decreasing the release of TNF receptor 1 (TNFR1), transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) from iMSC WT; cytomix enhanced three types of MSC inhibition on E. coli proliferation and BM-MSC inhibition on Klebsiella pneumoniae (K. pneumoniae); cytomix enhanced three types of MSC promotion on macrophage phagocytosis and three types of CM inhibition on T cell proliferation. 2) Compared to sham lung homogenate, E. coli-instilled ARDS lung homogenate induced lower levels of apoptosis in BM-MSCs, higher expression levels of CD54, CD200 and CD119 on BM-MSCs and higher levels of IL-6, IL-8, MCP-1 and VEGF in CM. Both sham and ARDS lung homogenate retained BM-MSC inhibition on E. coli, K. pneumoniae and Staphylococcus aureus (S. aureus) and enhanced BM-MSC inhibition on T cell expansion. 3) BM-MSCs were retrievable at 0.5 h and 1 h from healthy lungs and until 6 h from E. coli-induced ARDS lungs post cell administration. BM-MSCs underwent apoptosis in ARDS lung microenvironment. The expression levels of CD105, CD90, CD73, HLA-ABC, CD54 and CD200 on BM-MSCs were decreased in both healthy and ARDS lung microenvironment. On the other hand, BM-MSCs altered both healthy and ARDS lung microenvironment by increasing the percentage of non-classical monocytes in mononuclear phagocytes (MNPs) and decreasing the percentage of B cells in lymphocytes. In addition, BM-MSCs transiently increased the levels of cytokine-induced neutrophil chemoattractant 1 (CINC1), IL-6, MCP-1 and matrix metalloproteinase (MMP)-9 in ARDS lung proteome at 3 h post cell administration. Conclusion: 1) Inflammatory cytokines enhance MSC therapeutic effects by increasing the immunomodulatory surface markers on MSCs, promoting MSC anti-microbial effects and anti-inflammatory effects on macrophages and T cells. 2) Both iMSC WT and iMSC B2M KO show similar therapeutic benefits as BM-MSCs, and iMSC B2M KO has no immunogenicity, providing promising alternatives to MSC-based therapies in ARDS. 3) BM-MSCs underwent apoptosis in ex vivo lung microenvironment. The inflammatory ARDS microenvironment improves BM-MSC therapeutic benefits by inducing higher expression levels of immunomodulatory biomarkers on BM-MSCs and more release of inflammatory cytokines and VEGF in CM, compared to healthy lung microenvironment. 4) BM-MSCs live a longer lifespan in the inflammatory ARDS lung microenvironment than in the healthy lung microenvironment. BM-MSCs undergo apoptosis and phenotype shifting in both healthy and ARDS lung microenvironment in vivo. BM-MSCs modulate the composition of immune cells and cytokines in inflammatory ARDS lung microenvironments. These findings provide new insights into MSC behaviours in healthy and ARDS microenvironment, implying inflammatory microenvironment as a key mediator for MSC therapeutic effects. Further research is required to identify different subtypes of ARDS microenvironment and to optimise the source, dose, functional characterisation and pre-activation strategies of MSC-based therapies in ARDS treatment.

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University of Galway

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University of Galway Theses (PhD Theses)