ABSTRACT
Mesenchymal Stromal Cells (MSCs) are investigated as cellular therapeutics for Inflammatory Bowel Diseases and associated Perianal Fistula, although consistent efficacy remains a concern. Determining host factors that modulate MSCs' potency including their secretion of angiogenic & wound healing factors, immunosuppression and anti-inflammatory properties are important determinants of their functionality. We investigated the mechanisms that regulate the secretion of angiogenic & wound healing factors and immune suppression of human bone marrow MSCs. Secretory analysis of MSCs focusing on eighteen angiogenic & wound healing secretory molecules identified the most abundancy of Vascular Endothelial Growth Factor-A(VEGF-A). MSC viability and secretion of other angiogenic factors are not dependent on VEGF-A secretion which exclude the autocrine role of VEGF-A on MSC's fitness. However, combination of inflammatory cytokines IFNγand TNFαreduces MSC's VEGF-A secretion. To identify the effect of intestinal microvasculature on MSCs' potency, coculture analysis was performed between Human Large Intestine Microvascular Endothelial Cells(HLMVECs) and human bone marrow derived MSCs. HLMVECs do not attenuate MSCs' viability despite blocking their VEGF-A secretion. In addition, HLMVECs neither attenuate MSC's IFNγmediated upregulation of immunosuppressive enzyme Indoleamine 2,3-dioxygenase(IDO) nor abrogate suppression of T cell proliferation despite the attenuation of VEGF-A secretion. We found that HLMVECs express copious amounts of endothelial nitric oxide synthase (eNOS) and mechanistic analysis showed that pharmacological blocking reverses HLMVEC mediated attenuation of MSC's VEGF-A secretion. Together these results suggest that secretion of VEGF-A and immunosuppression are separable functions of MSCs which are regulated by distinct mechanisms in the host.
ABSTRACT
Introduction: Mesenchymal Stromal/Stem cells (MSCs) are an essential component of the regenerative and immunoregulatory stem cell compartment of the human body and thus of major importance in human physiology. The MSCs elicit their beneficial properties through a multitude of complementary mechanisms, which makes it challenging to assess their phenotype and function in environmental toxicity screening. We here employed the novel combinatorial assays matrix approach/technology to profile the MSC response to the herbicide Atrazine, which is a common environmental xenobiotic, that is in widespread agricultural use in the US and other countries, but banned in the EU. Our here presented approach is representative for screening the impact of environmental xenobiotics and toxins on MSCs as an essential representative component of human physiology and well-being. Methods: We here employed the combinatorial assay matrix approach, including a panel of well standardized assays, such as flow cytometry, multiplex secretome analysis, and metabolic assays, to define the phenotype and functionality of human-donor-derived primary MSCs exposed to the representative xenobiotic Atrazine. This assay matrix approach is now also endorsed for characterization of cell therapies by leading regulatory agencies, such as FDA and EMA. Results: Our results show that the exposure to Atrazine modulates the metabolic activity, size, and granularity of MSCs in a dose and time dependent manner. Intriguingly, Atrazine exposure leads to a broad modulation of the MSCs secretome (both upregulation and downmodulation of certain factors) with the identification of Interleukin-8 as the topmost upregulated representative secretory molecule. Interestingly, Atrazine attenuates IFNγ-induced upregulation of MHC-class-II, but not MHC-class-I, and early phosphorylation signals on MSCs. Furthermore, Atrazine exposure attenuates IFNγ responsive secretome of MSCs. Mechanistic knockdown analysis identified that the Atrazine-induced effector molecule Interleukin-8 affects only certain but not all the related angiogenic secretome of MSCs. Discussion: The here described Combinatorial Assay Matrix Technology identified that Atrazine affects both the innate/resting and cytokine-induced/stimulated assay matrix functionality of human MSCs, as identified through the modulation of selective, but not all effector molecules, thus vouching for the great usefulness of this approach to study the impact of xenobiotics on this important human cellular subset involved in the regenerative healing responses in humans.
Subject(s)
Atrazine , Mesenchymal Stem Cells , Humans , Atrazine/toxicity , Interleukin-8 , Xenobiotics , Bone MarrowABSTRACT
Mesenchymal Stromal Cells (MSCs) derived from bone marrow are widely tested in clinical trials as a cellular therapy for potential inflammatory disorders. The mechanism of action of MSCs in mediating immune modulation is of wide interest. In the present study, we investigated the effect of human bone-marrow-derived MSCs in modulating the circulating peripheral blood dendritic cell responses through flow cytometry and multiplex secretome technology upon their coculture ex vivo. Our results demonstrated that MSCs do not significantly modulate the responses of plasmacytoid dendritic cells. However, MSCs dose-dependently promote the maturation of myeloid dendritic cells. Mechanistic analysis showed that dendritic cell licensing cues (Lipopolysaccharide and Interferon-gamma) stimulate MSCs to secret an array of dendritic cell maturation-associated secretory factors. We also identified that MSC-mediated upregulation of myeloid dendritic cell maturation is associated with the unique predictive secretome signature. Overall, the present study demonstrated the dichotomy of MSC functionality in modulating myeloid and plasmacytoid dendritic cells. This study provides clues that clinical trials need to investigate if circulating dendritic cell subsets in MSC therapy can serve as potency biomarkers.
