Autoimmune disorders are becoming increasingly prevalent for unknown reasons. Among more than 80 know autoimmune disorders, Type 1 Diabetes (T1D) and Multiple sclerosis (MS) are top prevalent diseases affecting more than 2 million individuals in the United States and at least $40 billion costs per year. Preclinical studies revealed immunomodulatory and immunosuppressive properties of mesenchymal stem cell (MSC) to treat MS and T1D. However, lung entrapment, mal-differentiation, antibody production, phenotype change and potentially tumor formation are current challenges for stem cell therapy. In addition, there is a lack of mechanistic understanding regarding the molecular mechanisms of MSC therapy. Recent evidence suggests that MSCs exert their therapeutic efficacy partly through secreting active biomolecules and vesicles, which are collectively known as secretome. We therefore sought to harness the therapeutic potentials of secretome, study some of its subtypes including exosomes, and understand molecular pathways affected by MSC secretome and exosome.
MS is an inflammatory disease of the central nervous system (CNS) in which autoreactive T cells attack CNS, resulting in demyelination, neuronal injury and death, which account for the neurological disability. Here, using experimental autoimmune encephalomyelitis (EAE) as a MS mouse model, we show that systemic injection of MSC-Exo (30 g or 150 g) result in sustained recovery and improved motor function (p < 0.01) in a dose dependent manner. In vivo, injection of exosomes decreased neuroinflammation, and upregulated the number of CD4+CD25+FoxP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice, and reduced the infiltration and/or differentiation of Th1 and Th17 cells within the mice spinal cords. Co-culture of exosomes with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive enzyme indoleamine 2,3-dioxygenase. This recovery is associated reduced in neuroinflammation (p < 0.05).
We further harnessed the secretome and exosomes to improve the longevity of pancreatic islet xenotransplantation as a treatment for T1D. We first showed that alginate microcapsules loaded with processed conditioned media (pCM-Alg) reduces the infiltration and/or expression of CD68+ macrophages likely through the controlled release of pCM. In vitro cultures revealed that alginate could dose-dependently induce macrophages to secrete TNFα, IL-6, IL-1β, and GM-CSF. Addition of pCM to the cultures attenuated the secretion of TNFα (p = 0.023) and IL-6 (p < 0.0001) by alginate or lipopolysaccharide (LPS) stimulations. Mechanistically, pCM suppressed the NfκB pathway activation of macrophages in response to LPS (p < 0.0001) in vitro and cathepsin activity (p = 0.005) in response to alginate in vivo. Using this concept, we fabricated a hybrid alginate microcapsule (AlgXO) that controllably releases exosomes derived from Umbilical Cord Mesenchymal Stem Cells (XOs). Upon release, XOs suppress the local immune-microenvironment and mitigate the FBR against alginate microcapsules, where xenotransplantation of rat islets encapsulated in AlgXO led to > 5 months euglycemia in immunocompetent mouse model of Type 1 Diabetes. AlgXO significantly reduced the immune response in subcutaneous and intraperitoneal sites, while non-inflammatory fibrosis was observed in the subcutaneous space. In vitro analyses revealed that XOs suppressed the proliferation of CD3/CD28 activated splenocytes and CD3+ T cells. Comparing suppressive potency of XOs in purified CD3+ T cells versus splenocytes, we found XOs more profoundly suppressed T cells in the splenocytes coculture, where a heterogenous cell population presents. XOs also suppressed CD3/CD28 activated human peripheral blood mononuclear cells (PBMCs) and reduced their inflammatory cytokine secretion including IL-2, IL-6, IL-12p70, IL-22, and TNFα. We further confirmed that XOs mechanism of action is likely through myeloid cells and they suppress both murine and human macrophages partly through interfering with NfκB pathway. We believe that through its local controlled release of XOs, AlgXO is a platform that could alleviate the local immune response to implantable biomaterials.
