Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions. METHODS: Human, bone marrow derived MSCs were exposed to human plasma +/- heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry. RESULTS: Plasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14+ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells. CONCLUSIONS: This study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.

Phenotypic and functional alterations of myeloid-derived suppressor cells during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system involving dysregulated encephalitogenic T cells. Myeloid-derived suppressor cells (MDSCs) have been recognized for their important function in regulating T-cell responses. Recent studies have indicated a role for MDSCs in autoimmune diseases, but their significance in MS is not clear. Here, we assessed the frequencies of CD14+ HLA-DRlow monocytic MDSCs (Mo-MDSCs) and CD33+ CD15+ CD11b+ HLA-DRlow granulocytic MDSCs (Gr-MDSCs) and investigated phenotypic and functional differences of Mo-MDSCs at different clinical stages of MS and in healthy subjects (HC). Increased frequencies of Mo-MDSCs (P < 0.05) and Gr-MDSCs (P < 0.05) were observed in relapsing-remitting MS patients during relapse (RRMS-relapse) compared to stable RRMS (RRMS-rem). Secondary progressive MS (SPMS) patients displayed a decreased frequency of Mo-MDSCs and Gr-MDSCs compared to HC (P < 0.05). Mo-MDSCs within RRMS patients expressed significantly higher cell surface protein levels of CD86 and CD163 compared to SPMS patients. Mo-MDSCs within SPMS exhibited decreased mRNA expression of interleukin-10 and heme oxygenase 1 compared to RRMS and HC. Analysis of T-cell regulatory function of Mo-MDSCs demonstrated T-cell suppressive capacity in RRMS and HCs, while Mo-MDSCs of SPMS promoted autologous T-cell proliferation, which aligned with a differential cytokine profile compared to RRMS and HCs. This study is the first to show phenotypic and functional shifts of MDSCs between clinical stages of MS, suggesting a role for MDSCs as a therapeutic target to prevent MS disease progression.

Mesenchymal stromal cells modulate tissue repair responses within the injured vocal fold.

OBJECTIVES/HYPOTHESIS: This study aimed to determine whether local injection of human mesenchymal stromal cells (MSC) could modulate the early inflammatory response within injured vocal folds (VFs) to promote wound-healing processes. STUDY DESIGN: Experimental xenograft model. METHODS: VF injury was surgically induced by bilateral resection of the lamina propria of rabbits, and MSC were immediately injected into the injured area of both VFs. Animals were sacrificed on days 2, 4, and 24. Histological analyses were performed by hematoxylin and eosin, Masson's Trichrome, and elastin staining. Cell death was visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and the M2 macrophage marker, CD163, detected by immunohistochemistry. Persistence of injected MSC was evaluated by fluorescent in situ hybridization (FISH). Quantitative polymerase chain reaction was performed on the contralateral VF. RESULTS: Histological examination at days 2 and 4 indicated that MSC were able to reduce tissue inflammation, with gene expression analysis confirming a significant reduction of proinflammatory markers, interleukin (IL)-1ß, and IL-8. FISH demonstrated low-level persistence of injected MSC at both time points, and TUNEL confirmed localized cell death at the injury site. Increased levels of CD163+ anti-inflammatory macrophages indicated a change in the immune milieu, supporting wound resolution. Evidence of a more organized collagen matrix suggests that MSC may enhance the production of a functional repair tissue after injury, despite their low-level persistence within the tissue. CONCLUSIONS: This study demonstrates that MSC are able to positively modulate the early wound-healing response through resolution of the inflammatory phase and promotion of tissue repair. LEVEL OF EVIDENCE: NA Laryngoscope, 130:E21-E29, 2020.

Tissue immune profiles supporting response to mesenchymal stromal cell therapy in acute graft-versus-host disease-a gut feeling.

Acute graft-versus-host disease (aGvHD), post-allogeneic hematopoietic stem cell transplantation, is associated with high mortality rates in patients not responding to standard line care with steroids. Adoptive mesenchymal stromal cell (MSC) therapy has been established in some countries as a second-line treatment.Limitations in our understanding as to MSC mode of action and what segregates patient responders from non-responders to MSC therapy remain. The principal aim of this study was to evaluate the immune cell profile in gut biopsies of patients diagnosed with aGvHD and establish differences in baseline cellular composition between responders and non-responders to subsequent MSC therapy.Our findings indicate that a pro-inflammatory immune profile within the gut at the point of MSC treatment may impede their therapeutic potential. These findings support the need for further validation in a larger cohort of patients and the development of improved biomarkers in predicting responsiveness to MSC therapy.