Mesenchymal stem cells preconditioned with a TLR5 agonist enhanced immunoregulatory effect through M2 macrophage polarization in a murine graft-versus-host disease model.

Graft-versus-host disease (GVHD) is a common complication following hematopoietic stem cell transplantation and can be life-threatening. Mesenchymal stem cells (MSCs), adult stem cells with immunomodulatory properties, have been used as therapeutic agents in a variety of ways and have demonstrated efficacy against acute GVHD (aGVHD); however, variability in MSC pro- and anti-inflammatory properties and the limitation that they only exhibit immunosuppressive effects at high levels of inflammation have prevented their widespread clinical use. The outcomes of GVHD treated with MSCs in the clinic have been variable, and the underlying mechanisms remain unclear. Therefore, the unique biological effects of Toll-like receptor 5 (TLR5) agonists led us to compare and validate the efficacy of MSCs primed with KMRC011, a TLR5 agonist. KMRC011 is a stimulant that induces the secretion of cytokines, which play an important role in immune regulation. In this study, we found that MSCs pretreated with KMRC011 increased the secretion of immunosuppressive cytokines indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX2) and increased the expression of M2 macrophage polarizing cytokines macrophage colony-stimulating factor (M-CSF) and interleukin 10 (IL-10) in vitro. We investigated the immunosuppressive effects of TLR5 agonist (KMRC011)-primed MSCs on lymphocytes and their preventive and therapeutic effects on an in vivo mouse aGVHD model. In vitro experiments showed that KMRC011-primed MSCs had enhanced immunosuppressive effects on lymphocyte proliferation. In vivo experiments showed that KMRC011-primed MSCs ameliorated GVHD severity in a mouse model of induced GVHD disease. Finally, macrophages harvested from the spleens of mice treated with KMRC011-primed MSCs showed a significant increase in the anti-inflammatory M2 phenotype. Overall, the results suggest that KMRC011-primed MSCs attenuated GVHD severity in mice by polarizing macrophages to the M2 phenotype and increasing the proportion of anti-inflammatory cells, opening new horizons for GVHD treatment.

The IL-6 signaling pathway contributes critically to the immunomodulatory mechanism of human decidua-derived mesenchymal stromal cells.

Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been proposed as a treatment for graft-versus-host disease (GVHD), which is a major complication following allogeneic hematopoietic cell transplantation. However, clinical trials have not yielded good results, and human decidua-derived mesenchymal stromal cells (DSCs) have been proposed as an alternative. In addition, the mechanism by which DSCs exert their immunomodulatory effects is still unknown. We found that knockdown of IL-6 in DSCs reduced the expression of PD-L1 and PD-L2, which are known as classical immune checkpoint inhibitors. Expression of PD-L1 and PD-L2 was restored by adding recombinant IL-6 to the DSCs. When DSCs and IL-6-knockdown DSCs were administered as treatment in a murine GVHD model, the group receiving IL-6-knockdown DSCs had significantly higher mortality and clinical scores compared to the group receiving DSCs. Taken together, these data suggest that the IL-6 signaling pathway is a crucial contributor to the immunosuppressive capacity of DSCs.

TLR5 agonist in combination with anti-PD-1 treatment enhances anti-tumor effect through M1/M2 macrophage polarization shift and CD8+ T cell priming.

Immune checkpoint inhibitors have revolutionized anti-tumor therapy, notably improving treatment responses in various tumors. However, many patients remain non-responsive and do not experience benefits. Given that Toll-like receptors (TLRs) can counteract tumor immune tolerance by stimulating both innate and adaptive immune responses, TLR agonists are being explored as potential immune adjuvants for cancer treatment. In this study, we assessed the potential of enhancing the efficacy of immune checkpoint inhibitors by activating innate immunity with a TLR5 agonist. In a mouse tumor model, combination therapy with TLR5 agonist and anti-PD-1 significantly inhibited tumor growth. The TLR5 agonist shifted the balance from M2-like to M1-like macrophages and upregulated the expression of co-stimulatory molecules in macrophages. Furthermore, TLR5 agonist promoted the activation and tumor infiltration of CD8+ T cells. As a result, the TLR5 agonist augmented the anti-tumor efficacy of anti-PD-1, suggesting its potential in modulating the tumor microenvironment to enhance the anti-tumor response. Our findings point toward the possibility of optimizing immune checkpoint inhibitor therapy using TLR5 agonists.

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin ß-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor.

To identify the specific region of eCG involved in FSH-like activity, the following mutant expression vectors were constructed targeting the amino acid residues 102-104 of the eCG ß-subunit: single mutants, eCGßV102G/α, eCGßF103P/α, and eCGßR104K/α; double mutants, eCGßV102G;F103P/α, eCGßV102G;R104K/α, and eCGßF103P;R104K/α; triple mutant, eCGßV102G;F103P;R104K/α. The LH-like and FSH-like activities of eCG mutants were examined in CHO-K1 cells expressing rat LH/CG receptor and rat FSH receptor. The levels of eCGßV102G/α, eCGßR104K/α, and eCGßV102G;R104K/α in the culture supernatant were markedly lower than those of eCGß/α-wt. The other mutants and rec-eCGß/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGßV102G;R104K/α, and eCGßF103P;R104K/α were approximately 61%, 52%, and 54%, respectively, of those of eCG-wt. The Rmax values of the mutants were 58.9%-78.8% those of eCG-wt with eCGßR104K/α exhibiting the lowest value. The FSH-like activities of single mutants were only 16%-20% of those of eCG-wt. Additionally, the FSH-like activity of double mutants was less than 10% of that of eCG-wt. In particular, the FSH-like activities of ßV102G;R104K/α and ßF103P;R104K/α were 2.5-2.9% of that of eCG-wt. These results suggest that the amino acid residues 102-104 of the eCG ß-subunit are dispensable and that the residue 104 of the eCG ß-subunit plays a pivotal role in signal transduction through the rat FSH receptor. Thus, these mutants may aid future studies on eCG interactions with mammalian FSH receptors in vitro and in vivo.