hPMSCs Regulate the Level of TNF-α and IL-10 in Th1 Cells and Improve Hepatic Injury in a GVHD Mouse Model via CD73/ADO/Fyn/Nrf2 Axis.

Mesenchymal stem cells (MSCs) ameliorate graft-versus-host disease (GVHD)-induced tissue damage by exerting immunosuppressive effects. However, the related mechanism remains unclear. Here, we explored the therapeutic effect and mechanism of action of human placental-derived MSCs (hPMSCs) on GVHD-induced mouse liver tissue damage, which shows association with inflammatory responses, fibrosis accompanied by hepatocyte tight junction protein loss, the upregulation of Bax, and the downregulation of Bcl-2. It was observed in GVHD mice and Th1 cell differentiation system that hPMSCs treatment increased IL-10 levels and decreased TNF-α levels in the Th1 subsets via CD73. Moreover, hPMSCs treatment reduced tight junction proteins loss and inhibited hepatocyte apoptosis in the livers of GVHD mice via CD73. ADO level analysis in GVHD mice and the Th1 cell differentiation system showed that hPMSCs could also upregulate ADO levels via CD73. Moreover, hPMSCs enhanced Nrf2 expression and diminished Fyn expression via the CD73/ADO pathway in Th1, TNF-α+, and IL-10+ cells. These results indicated that hPMSCs promoted and inhibited the secretion of IL-10 and TNF-α, respectively, during Th1 cell differentiation through the CD73/ADO/Fyn/Nrf2 axis signaling pathway, thereby alleviating liver tissue injury in GVHD mice.

Human placental mesenchymal stromal cells modulate IFN-γ and IL-10 secretion by CD4+T cells via CD73, and alleviate intestinal damage in mice with graft-versus-host disease.

BACKGROUND: Intestinal inflammatory damage is an important factor in the development of graft-versus-host disease (GVHD). IFN-γ and IL-10 play key roles in gastrointestinal inflammation, and human placental mesenchymal stromal cells (hPMSCs) can alleviate inflammatory damage during GVHD. CD73 is highly expressed by hPMSCs. We aimed to study whether hPMSCs could alleviate intestinal damage in GVHD mice by modulating IFN-γ and IL-10 in CD4+T cells by CD73. METHODS: A GVHD mouse model was induced using 8-week-old C57BL/6J and BALB/c mice, which were treated with regular hPMSCs (hPMSCs) or hPMSCs expressing low level of CD73 (shCD73). Then, the levels of IFN-γ and IL-10 in CD4+T cells were determined using flow cytometry. Transmission electron microscopy, western blotting, and morphological staining were employed to observe the intestinal damage. RESULTS: hPMSCs ameliorated pathological damage and inhibited the reduction of the tight junction molecules occludin and ZO-1. They also downregulated IFN-γ and upregulated IL-10 secretion in CD4+T cells via CD73. Moreover, IL-10 mitigated the inhibitory effects of IFN-γ on the expression of occludin in both Caco-2 and NCM460 cells in vitro, but did not affect ZO-1. In addition, hPMSCs upregulated the level of AMPK phosphorylation in CD4+T cells by CD73, which is positively associated with the proportion of CD4+IFN-γ+IL-10+T, and CD4+IFN-γ-IL-10+T cells. CONCLUSIONS: Our findings suggested that hPMSCs may balance the levels of IFN-γ and IL-10 in CD4+T cells by promoting the phosphorylation of AMPK via CD73, which alleviates the loss of occludin and ZO-1 in intestinal epithelial cells and, in turn, reduces inflammatory injury in GVHD mice.

Targeting the Immunoglobulin IGSF9 Enhances Antitumor T-cell Activity and Sensitivity to Anti-PD-1 Immunotherapy.

Immune checkpoints modulate the immune response and represent important immunotherapy targets for cancer treatment. However, as many tumors are resistant to current immune checkpoint inhibitors, the discovery of novel immune checkpoints could facilitate the development of additional immunotherapeutic strategies to improve patient responses. Here, we identified increased expression of the adhesion molecule immunoglobulin superfamily member 9 (IGSF9) in tumor cells and tumor-infiltrating immune cells across multiple cancer types. IGSF9 overexpression or knockout in tumor cells did not alter cell proliferation in vitro or tumor growth in immunocompromised mice. Alternatively, IGSF9 deficient tumor cells lost the ability to suppress T-cell proliferation and exhibited reduced growth in immunocompetent mice. Similarly, growth of tumor cells was reduced in IGSF9 knockout syngeneic and humanized mice, accompanied by increased tumor-infiltrating T cells. Mechanistically, the extracellular domain (ECD) of IGSF9 bound to T cells and inhibited their proliferation and activation, and the tumor-promoting effect of IGSF9 ECD was reversed by CD3+ T-cell depletion. Anti-IGSF9 antibody treatment inhibited tumor growth and enhanced the antitumor efficacy of anti-programmed cell death protein 1 immunotherapy. Single-cell RNA sequencing revealed tumor microenvironment remodeling from tumor promoting to tumor suppressive following anti-IGSF9 treatment. Together, these results indicate that IGSF9 promotes tumor immune evasion and is a candidate immune checkpoint target. SIGNIFICANCE: IGSF9 is an immune checkpoint regulator that suppresses T-cell activation in cancer and can be targeted to stimulate antitumor immunity and inhibit tumor growth.

Highly Efficient Near-Infrared Thermally Activated Delayed Fluorescent Emitters in Non-Doped Electroluminescent Devices.

Constructing organic near-infrared (NIR) luminescent materials to confront the formidable barrier of "energy gap law" remains challenging. Herein, two NIR thermally activated delayed fluorescence (TADF) molecules named T-ß-IQD and TIQD were developed by connecting N,N-diphenylnaphthalen-2-amine and triphenylamine with a novel electron withdrawing unit 6-(4-(tert-butyl)phenyl)-6H-indolo[2,3-b]quinoxaline-2,3-dicarbonitrile. It is confirmed NIR-TADF emitters concurrent with aggregation-induced emission effect, J-aggregate with intra- and intermolecular CN⋅⋅⋅H-C and C-H⋅⋅⋅π interactions, and large center-to-center distance in solid states can boost the emissive efficiencies both in thin films and non-doped organic light-emitting diodes (OLEDs). Consequently, the T-ß-IQD-based non-doped NIR-OLED achieved the maximum external quantum efficiency (EQEmax ) of 9.44 % with emission peak at 711 nm, which is one of the highest efficiencies reported to date for non-doped NIR-OLEDs.

hPMSCs-Derived Exosomal miRNA-21 Protects Against Aging-Related Oxidative Damage of CD4+ T Cells by Targeting the PTEN/PI3K-Nrf2 Axis.

Mesenchymal stem cells (MSCs)-derived exosomes were considered a novel therapeutic approach in many aging-related diseases. This study aimed to clarify the protective effects of human placenta MSCs-derived exosomes (hPMSC-Exo) in aging-related CD4+ T cell senescence and identified the underlying mechanisms using a D-gal induced mouse aging model. Senescent T cells were detected SA-ß-gal stain. The degree of DNA damage was evaluated by detecting the level of 8-OH-dG. The superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities were measured. The expression of aging-related proteins and senescence-associated secretory phenotype (SASP) were detected by Western blot and RT-PCR. We found that hPMSC-Exo treatment markedly decreased oxidative stress damage (ROS and 8-OH-dG), SA-ß-gal positive cell number, aging-related protein expression (p53 and γ-H2AX), and SASP expression (IL-6 and OPN) in senescent CD4+ T cells. Additionally, hPMSC-Exo containing miR-21 effectively downregulated the expression of PTEN, increased p-PI3K and p-AKT expression, and Nrf2 nuclear translocation and the expression of downstream target genes (NQO1 and HO-1) in senescent CD4+ T cells. Furthermore, in vitro studies uncovered that hPMSC-Exo attenuated CD4+ T cell senescence by improving the PTEN/PI3K-Nrf2 axis by using the PTEN inhibitor bpV (HOpic). We also validated that PTEN was a target of miR-21 by using a luciferase reporter assay. Collectively, the obtained results suggested that hPMSC-Exo attenuates CD4+ T cells senescence via carrying miRNA-21 and activating PTEN/PI3K-Nrf2 axis mediated exogenous antioxidant defenses.

hPMSCs inhibit the expression of PD-1 in CD4+IL-10+ T cells and mitigate liver damage in a GVHD mouse model by regulating the crosstalk between Nrf2 and NF-κB signaling pathway.

BACKGROUND: The activation of T cells and imbalanced redox metabolism enhances the development of graft-versus-host disease (GVHD). Human placenta-derived mesenchymal stromal cells (hPMSCs) can improve GVHD through regulating T cell responses. However, whether hPMSCs balance the redox metabolism of CD4+IL-10+ T cells and liver tissue and alleviate GVHD remains unclear. This study aimed to investigate the effect of hPMSC-mediated treatment of GVHD associated with CD4+IL-10+ T cell generation via control of redox metabolism and PD-1 expression and whether the Nrf2 and NF-κB signaling pathways were both involved in the process. METHODS: A GVHD mouse model was induced using 6-8-week-old C57BL/6 and Balb/c mice, which were treated with hPMSCs. In order to observe whether hPMSCs affect the generation of CD4+IL-10+ T cells via control of redox metabolism and PD-1 expression, a CD4+IL-10+ T cell culture system was induced using human naive CD4+ T cells. The percentage of CD4+IL-10+ T cells and their PD-1 expression levels were determined in vivo and in vitro using flow cytometry, and Nrf2, HO-1, NQO1, GCLC, GCLM, and NF-κB levels were determined by western blotting, qRT-PCR, and immunofluorescence, respectively. Hematoxylin-eosin, Masson's trichrome, and periodic acid-Schiff staining methods were employed to analyze the changes in hepatic tissue. RESULTS: A decreased activity of superoxide dismutase (SOD) and a proportion of CD4+IL-10+ T cells with increased PD-1 expression were observed in GVHD patients and the mouse model. Treatment with hPMSCs increased SOD activity and GCL and GSH levels in the GVHD mouse model. The percentage of CD4+IL-10+ T cells with decreased PD-1 expression, as well as Nrf2, HO-1, NQO1, GCLC, and GCLM levels, both in the GVHD mouse model and in the process of CD4+IL-10+ T cell generation, were also increased, but NF-κB phosphorylation and nuclear translocation were inhibited after treatment with hPMSCs, which was accompanied by improvement of hepatic histopathological changes. CONCLUSIONS: The findings suggested that hPMSC-mediated redox metabolism balance and decreased PD-1 expression in CD4+IL-10+ T cells were achieved by controlling the crosstalk between Nrf2 and NF-κB, which further provided evidence for the application of hPMSC-mediated treatment of GVHD.

hPMSCs protects against D-galactose-induced oxidative damage of CD4+ T cells through activating Akt-mediated Nrf2 antioxidant signaling.

BACKGROUND: Mesenchymal stem cells (MSCs) were considered a regenerative therapeutic approach in both acute and chronic diseases. However, whether MSCs regulate the antioxidant metabolism of CD4+ T cells and weaken immunosenescence remains unclear. Here, we reported the protective effects of hPMSCs in aging-related CD4+ T cell senescence and identified the underlying mechanisms using a D-gal-induced mouse aging model. METHODS: In vivo study, 40 male C57BL/6 mice (8 weeks) were randomly divided into four groups: control group, D-gal group, hPMSC group, and PBS group. In in vitro experiment, human naive CD4+ T (CD4CD45RA) cells were prepared using a naive CD4+ T cell isolation kit II and pretreated with the Akt inhibitor LY294002 and Nrf2 inhibitor ML385. Then, isolated naive CD4+ T cell were co-cultured with hPMSCs for 72 h in the absence or presence of anti-CD3/CD28 Dynabeads and IL-2 as a mitogenic stimulus. Intracellular ROS changes were detected by flow cytometry. The activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase were measured by colorimetric analysis. The senescent T cells were detected SA-ß-gal stain. The expression of aging-related proteins was detected by Western blotting, RT-PCR, and confocal microscopy. RESULTS: We found that hPMSC treatment markedly decreased the ROS level, SA-ß-gal-positive cells number, senescence-associated secretory phenotype (IL-6 and OPN) expression, and aging-related protein (P16 and P21) expression in senescent CD4+ T cells. Furthermore, hPMSC treatment effectively upregulated Nrf2 nuclear translocation and the expression of downstream target genes (HO-1, CAT, GCLC, and NQO1) in senescent CD4+ T cells. Moreover, in vitro studies revealed that hPMSCs attenuated CD4+ T cell senescence by upregulating the Akt/GSK-3ß/Fyn pathway to activate Nrf2 functions. Conversely, the antioxidant effects of hPMSCs were blocked by the Akt inhibitor LY294002 and Nrf2 inhibitor ML385 in senescent CD4+ T cells. CONCLUSIONS: Our results indicate that hPMSCs attenuate D-gal-induced CD4+ T cell senescence by activating Nrf2-mediated antioxidant defenses and that upregulation of Nrf2 by hPMSCs is regulated via the Akt/GSK-3ß/Fyn pathway.

Downregulated Recycling Process but Not De Novo Synthesis of Glutathione Limits Antioxidant Capacity of Erythrocytes in Hypoxia.

Red blood cells (RBCs) are susceptible to sustained free radical damage during circulation, while the changes of antioxidant capacity and regulatory mechanism of RBCs under different oxygen gradients remain unclear. Here, we investigated the changes of oxidative damage and antioxidant capacity of RBCs in different oxygen gradients and identified the underlying mechanisms using an in vitro model of the hypoxanthine/xanthine oxidase (HX/XO) system. In the present study, we reported that the hypoxic RBCs showed much higher oxidative stress injury and lower antioxidant capacity compared with normoxic RBCs. In addition, we found that the disturbance of the recycling process, but not de novo synthesis of glutathione (GSH), accounted for the significantly decreased antioxidant capacity of hypoxic RBCs compared to normoxic RBCs. We further elucidated the underlying molecular mechanism by which oxidative phosphorylation of Band 3 blocked the hexose monophosphate pathway (HMP) and decreased NADPH production aggravating the dysfunction of GSH synthesis in hypoxic RBCs under oxidative conditions.

Oligosaccharide attenuates aging-related liver dysfunction by activating Nrf2 antioxidant signaling.

Chitosan oligosaccharide (COS) is the depolymerized product of chitosan possessing various biological activities and protective effects against inflammation and oxidative injury. The aim of the present study was to investigate the antioxidant effects of COS supplements on aging-related liver dysfunction. We found that COS treatment significantly attenuated elevated liver function biomarkers and oxidative stress biomarkers and decreased antioxidative enzyme activities in liver tissues in D-galactose (D-gal)-treated mice. Furthermore, COS treatment significantly upregulated the expression of Nrf2 and its downstream target genes HO-1, NQO1, and CAT. Moreover, in vitro experiments showed that COS treatment played a vital role in protecting H2O2-exposed L02 cells against oxidative stress by activating Nrf2 antioxidant signaling. These data indicate that COS could protect against D-gal-induced hepatic aging by activating Nrf2 antioxidant signaling, which may provide novel applications for the prevention and treatment of aging-related hepatic dysfunction.