Subcutaneous delivery of mesenchymal stromal cells induces immunoregulatory effects in the lymph node prior to their apoptosis

Background: Mesenchymal stromal cell (MSC) therapy commonly involves systemic infusion of MSCs, which undergo apoptosis in the lung and induce immunoregulatory macrophages that reduce disease. The relevance of this mode of action, however, is yet to be determined for MSCs administered via other routes. Here, we administered MSCs via subcutaneous (SC) injection into inflamed tissue and investigated the immunomodulatory effects on the local lymph node (LN), which is a major site for the initiation and regulation of immune responses. Methods: A mouse model of localised skin inflammation was established with low-dose lipopolysaccharide (LPS) to in vivo prime adipose-derived MSCs delivered via SC injection. We then analysed the immunomodulatory changes in the LN draining the inflamed tissue, as well as the neutrophil TNF response to LPS re-exposure. Results: When administered directly into the inflamed skin tissue, SC MSC injection induced an expansion of IL-10-producing MerTK⁺ subcapsular sinus macrophages and T cell zone macrophages, as well as activated CD44⁺ regulatory T cells (Tregs), in the draining LN, which was not observed in the non-draining LN. SC injection of viable, but not apoptotic, MSCs dampened TNF production by inflammatory cells in the draining LN when re-exposed to the inflammatory stimulus. SC injection of MSCs remote to the site of inflammation also did not attenuate the LN response to subsequent inflammatory challenge. Conclusions: MSCs delivered directly into inflamed skin activated immunoregulatory cells in the local LN and inhibited LN responsiveness to subsequent inflammatory challenge. The immunoregulatory effects of SC-injected MSCs in the LN require priming by inflammatory cytokines in the local milieu. Furthermore, SC-injected MSCs exert anti-inflammatory effects in the draining LN prior to their apoptosis, in contrast to intravenously delivered MSCs, where anti-inflammatory effects are linked to their apoptosis.