IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem/Stromal Cell Death Via RIPK1-Independent Necroptosis

Interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) are two vital inflammatory factors elevated in many diseases (e.g. aplastic anemia), and an inflammatory microenvironment can cause cell damage and may be involved in the pathogenesis of certain diseases. However, the precise mechanisms of cell dysfunction or impairment during the inflammatory process are not fully understood. IFN-γ alone or in combination with TNF-α have distinct effects on the immunoregulatory properties of bone marrow derived mesenchymal stem cells (MSCs), and they have been raised to be optimal prime factors to enhance the immunosuppressive capacity of MSCs engineered in vitro. Yet controversies remain with regards to the normal function maintenance of cells as they may be impaired after exposure to inflammatory factors. Here, we find that IFN-γ and TNF-α can synergistically induce cell dysfunction and death of MSCs via a RIPK1-independent manner of necroptosis. When MSCs are exposed to both IFN-γ and TNF-α at a broad range of concentrations used in previous studies for about 24 hours or more, its morphological features, biological functions, differentiation capacity and immunoregulatory properties are totally injured. First of all, IFN-γ and TNF-α synergistically induce distinct morphological changes of MSCs. When primed with both IFN-γ and TNF-α, MSCs show a gradual change from a fibroblastic-like spindle shape to an enlarged widespread morphology and the cells grow in a disordered way. And from a high power view, the cells looked like a flat spider web and the blurred cell membrane hints that these cells may have “ruptured”, but most notably, the immunofluorescence staining shows that the nuclei were intact. Secondly, MSCs are functionally impaired after exposure in regard to their capacities of proliferation and migration, which, however, are not associated with increased cell apoptosis or senescence. Furthermore, the adipocytes, osteocytes and chondrocytes differentiation capacities of MSCs are entirely impaired and the immunosuppressive efficacies of MSCs on T cells are also attenuated by the synergistic effect of IFN-γ and TNF-α, though these impaired MSCs retain normal capacity of releasing indoleamine 2,3-dioxygenase (IDO), an immunosuppressive mediator of human MSCs. Mechanistically, we initially find that the signaling of IFN-γ might be enhanced by TNF-α through increasing the abundance of its receptor IFN-γ receptor1 on the surface of MSCs, which is possible to be the interplay mechanism of the two cytokines. And when the injured cells are subjected to RNA-Sequencing, it reveals that those MSCs have undergone a unique cell death, which is necroptosis, a form of programmed cell death characterized by a necrotic morphology including cell swelling, membrane rupture, and releasing of cellular contents, such as inflammatory cytokines, damage-associated molecular patterns (DAMPs), and chemokines, thereby leading to a variety of inflammatory responses subsequently. Compared with the control groups, IFN-γ and TNF-α synergistically increased the expression level of major necroptotic signaling cascade RIPK1, RIPK3 and MLKL into a significant level, and further analysis reveals that all other genes associated with necroptosis (TRADD, TICAM1, ZBP1, TLR3, and TLR4) are significantly upregulated as well. Finally, the rescue experiments targeting signaling pathways of necroptosis show that the necroptotic process of MSCs induced by IFN-γ & TNF-α cannot be reversed by RIPK1 inhibitor (Necrostatin-1), yet RIPK3 and MLKL inhibitor (GSK872 and Necrosulfonamide) hold individual or collaborative effects of protection against it, suggesting the undergoing necroptosis in this setting is independent of RIPK1, and targeting downstream proteins of necroptotic signaling pathway is a promising strategy for inhibiting such a cell death. Collectively, our results disclose an inflammatory injury mechanism of human bone marrow derived MSCs based on its morphological and functional alterations in combination with RNA-Seq identification, and a firstly found necroptotic manner of cell death in MSCs sheds new light on revealing the MSC deficits in some inflammatory or immune-mediated diseases with expectations to innovate some potential therapeutics. Our findings also suggest that cell therapy based on MSCs primed with IFN-γ and TNF-α should take this inflammatory injury effect into consideration.