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Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction.

ABSTRACT: Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE₂) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.

SUBMITTER: Skibber MA

PROVIDER: S-EPMC9297916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mesenchymal stromal cells (MSC) are sensors of inflammation, and they exert immunomodulatory properties through the secretion of cytokines and exosomes and direct cell-cell interactions. MSC are routinely used in clinical trials and effectively resolve inflammatory conditions. Nevertheless, inconsistent clinical outcomes necessitate the need for more robust therapeutic phenotypes. The immunomodulatory properties of MSC can be enhanced and protracted by priming (aka licensing) them with IFNγ and TNFα. Yet these enhanced properties rapidly diminish, and prolonged stimulation could tolerize their response. Hence a balanced approach is needed to enhance the therapeutic potential of the MSC for consistent clinical performance. Here, we investigated the concentration-dependent effects of IFNγ and TNFα and developed gelatin-based microgels to sustain a licensed MSC phenotype. We show that IFNγ treatment is more beneficial than TNFα in promoting an immunomodulatory MSC phenotype. We also show that the microgels possess integrin-binding sites to support adipose tissue-derived MSC (AD-MSC) attachment and a net positive charge to sequester the licensing cytokines electrostatically. Microgels are enzymatically degradable, and the rate is dependent on the enzyme concentration and matrix density. Our studies show that one milligram of microgels by dry mass can sequester up to 641 ± 81 ng of IFNγ. Upon enzymatic degradation, microgels exhibited a sustained release of IFNγ that linearly correlated with their degradation rate. The AD-MSC cultured on the IFNγ sequestered microgels displayed efficient licensing potential comparable to or exceeding the effects of bolus IFNγ treatment. When cultured with proinflammatory M1-like macrophages, the AD-MSC-seeded on licensing microgel showed an enhanced immunomodulatory potential compared to untreated AD-MSC and AD-MSC treated with bolus IFNγ treatment. Specifically, the AD-MSC seeded on licensing microgels significantly upregulated Arg1, Mrc1, and Igf1, and downregulated Tnfα in M1-like macrophages compared to other treatment conditions. These licensing microgels are a potent immunomodulatory approach that shows substantial promise in elevating the efficacy of current MSC therapies and may find utility in treating chronic inflammatory conditions.

Project description:Ruxolitinib is a JAK2/JAK1 inhibitor that blocks the inflammatory JAK-STAT signaling pathway. Ruxolitinib has been demonstrated to be effective in the treatment of steroid-resistant acute graft-versus-host disease (GVHD). Ruxolitinib's effect on inflammatory cells of hematopoietic origin is known. However, its effect on nonhematopoietic cell types with immune-modulating and antigen-presenting cell competency plausibly involved in pathogenesis of GVHD has not been explored. Mesenchymal stromal cells (MSCs) are CD45- nonhematopoietic cells of the bone marrow with immune modulatory functions in vivo. MSCs' immunobiology largely depends on their responsiveness to IFNγ. We aimed to define the effect of ruxolitinib on the immunobiology of MSCs that are modulated by IFNγ. Human bone marrow derived MSCs, peripheral blood mononuclear cells (PBMCs), and primary bone marrow aspirates were analyzed for their sensitivity to ruxolitinib-mediated blocking of IFNγ-induced STAT-1 phosphorylation and downstream effector molecules, utilizing Western blot, flow cytometry, secretome analysis, and phosflow techniques. IFNγ-induced cytostatic effects on MSCs are reversed by ruxolitinib. Ruxolitinib inhibits IFNγ and secretome of activated peripheral PBMC-induced STAT-1 phosphorylation on human bone marrow derived MSCs. In addition, ruxolitinib inhibits IFNγ-induced pro-GVHD pathways on MSCs, which includes HLAABC(MHCI), HLADR(MHCII), CX3CL1, and CCL2. IFNγ-induced immunosuppressive molecules IDO and PDL-1 were also inhibited by ruxolitinib on MSCs. Comparative analysis with PBMCs has demonstrated that MSCs are as equal as to HLADR+ PBMC populations in responding to ruxolitinib-mediated inhibition of IFNγ-induced STAT-1 phosphorylation. Ex vivo analysis of human marrow aspirates has demonstrated that ruxolitinib blocks IFNγ-induced STAT-1 phosphorylation in CD45+/-HLADR+/- populations at different levels, which is depending on their sensitivity to IFNγ responsiveness. These results inform the hypothesis that ruxolitinib's immune-modulatory effects in vivo may pharmacologically involve marrow and tissue-resident MSCs. Ruxolitinib affects the immunobiology of MSCs equivalent to professional HLADR+ antigen presenting cells, which collectively mitigate GVHD.

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Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction.

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