BACKGROUND: G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). METHODS: The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. RESULTS: In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (T regs ), an increase in the cluster of differentiation 8 positive (CD8 + ) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8 + T cells, and induced the proportion of T regs . Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. CONCLUSION Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cells in RA.
G-Protein-Coupled Receptor Kinase 2/metabolism* ; Arthritis, Rheumatoid/immunology* ; Animals ; Dendritic Cells/metabolism* ; Paroxetine/therapeutic use* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice ; Humans ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Lymphocyte Activation/drug effects* ; Female ; T-Lymphocytes/metabolism* ; Middle Aged

OBJECTIVE: Ginsenoside Rh1 (G-Rh1) has been confirmed to inhibit the growth of breast cancer and colon cancer, but its therapeutic effect on hepatocellular carcinoma (HCC) is unclear. This study investigates the therapeutic effect of G-Rh1 on HCC as well as the underlying mechanism. METHODS: Bioinformatics methods were used to analyze glucocorticoid receptor (GR) expression and the tumor microenvironment in HCC tissues from HCC patients. The effect of G-Rh1 on HCC cells was investigated in vitro using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The therapeutic effect of G-Rh1 was investigated in vivo using subcutaneous transplantation models in C57BL/6J and nude mice. Additionally, the proportion of infiltrating immune cells in tumors was analyzed using flow cytometry, the GR and major histocompatibility complex class-I (MHC-I) expression of HCC cells after G-Rh1 treatment was analyzed using Western blotting, and G-Rh1-treated Hepa1-6 cells were cocultured with bone marrow-derived dendritic cells and B3Z T cells to further analyze the ability of G-Rh1 to induce dendritic cell (DC) maturation and CD8⁺ T cell activation. RESULTS: GR expression was upregulated in HCC tissues, and high GR expression was associated with a worsened immune microenvironment. In vitro studies showed that G-Rh1 had no significant effect on the proliferation of HCC cells, while in vivo studies showed that G-Rh1 exerted antitumor effects in C57BL/6J mice but not in nude mice. Further research revealed that G-Rh1 ameliorated the immunosuppressive tumor microenvironment, thereby enhancing the antitumor effects of lenvatinib by increasing the infiltration of CD8⁺ T cells, mature DCs, and MHC-I-positive cells. MHC-I was upregulated by G-Rh1 via GR suppression. Moreover, overexpression of GR abolished the G-Rh1-mediated promotion of MHC-I expression in Huh7 cells, as well as the maturation of DCs and the activation of CD8⁺ T cells. CONCLUSION G-Rh1 can regulate the immune microenvironment of HCC by targeting GR, thus increasing the antitumor effect of lenvatinib. Please cite this article as: Wang XH, Fu YL, Xu YN, Zhang PC, Zheng TX, Ling CQ, Feng YL. Ginsenoside Rh1 regulates the immune microenvironment of hepatocellular carcinoma via the glucocorticoid receptor. J Integr Med. 2024; 22(6): 710-720.
Carcinoma, Hepatocellular/genetics* ; Ginsenosides/pharmacology* ; Animals ; Liver Neoplasms/genetics* ; Receptors, Glucocorticoid/genetics* ; Tumor Microenvironment/drug effects* ; Humans ; Mice, Inbred C57BL ; Mice ; Mice, Nude ; Cell Line, Tumor ; Male ; Dendritic Cells/drug effects*

BACKGROUNDDendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.

METHODSBone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.

RESULTSCompared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.

CONCLUSIONThese results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.

Animals ; Apoptosis ; drug effects ; physiology ; Bone Marrow Cells ; cytology ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Lipopolysaccharides ; pharmacology ; Matrix Metalloproteinase 13 ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering

OBJECTIVETo explore the effects of aspirin and inflammation on the maturation and function of dendritic cells (DC) on the supernatant of VX-2 squamous cell carcinoma.

METHODSThe rabbit buccal VX-2 squamous cell carcinoma models with inflammation were established by tumor particle implantation, mechanical trauma, and high sugar diet. The rabbits were divided into three groups. For the experimental group (rabbit buccal VX-2 squamous cell carcinoma with local inflammation), aspirin were given by gavage for three consecutive days. For the control group (rabbit buccal VX-2 squamous cell carcinoma with local inflammation), normal saline was given by gavage for three consecutive days. For the blank group (tumor without inflammation), normal saline was given by gavage for three consecutive days. Each tumor specimens were collected in three days and made into tissue homogenate. The supernatant was collected after centrifugation. Normal rabbit peripheral blood mononuclear cells were separated and co-cultured with different states of supernatant. The expression of DC surface markers CD83, CD86, and human leukocyte antigen-DR (HLA-DR) were detected by flow cytometry. The state of function of DC was tested by mixed lymphocyte reaction.

RESULTSThe positive rate of CD83, CD86, and HLA-DR of the experimental and control groups were both lower than that of the blank group (P<0.05). In addition, the ability to stimulate T cell proliferation of the experimental and control groups were weaker than that of the blank group (P<0.05). No significant difference was observed between the experi- and HLADR of DC. The short-term administration of aspirin is not conducive to the phenoty and function of DC in a rabbit mental and control groups (P>0.05).

CONCLUSIONInflammation may inhibit the function and expression of CD83, CD86, buccal VX-2 squamous cell carcinoma inflammatory environment

Animals ; Aspirin ; pharmacology ; Carcinoma, Squamous Cell ; Coculture Techniques ; Dendritic Cells ; drug effects ; Flow Cytometry ; Humans ; Inflammation ; Leukocytes, Mononuclear ; Organothiophosphorus Compounds ; Rabbits

OBJECTIVETo investigate the inducing effect of 'modified' cytokine cocktail on the dendritic cell maturation and migration capability.

METHODSPBMNC were isolated from human peripheral blood stem cell (PBSC) by using density gradient centrifugation, the immature DC (imDC) were induced by using GM-CSF and IL-4 in vitro. Total A549 RNA was transfected into imDC by using electroporation, which was stimulated to matuation by the "gold standard" cytokine cocktail and "modified" cytokine cocktail, respectively. The expression of DC surface markers (CD11c, HLA-DR, CD80, CD83 and CD86) and chemokine receptor (CCR5, CCR7 and CXCR4) were detected by flow cytometry; the mRNA expression levels of DC chemokine receptor (CCR2, CCR5, CCR7, CXCR3 and CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12) were detected by RT-PCR.

RESULTSAs compared with "gold standard cytokine cocktail", the "modified" cytokine cocktail-induced DC expressed higher levels of surface markers (CD11c, HLA-DR, CD80, CD83 and CD86), chemokine receptors (CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12).

CONCLUSIONThe "modified" cytokine cocktail can more effectively induce the DC maturation, enhace the migratory capability of DC and more generate the immunostimulatory DC, when compared with the "gold standard" cytokine cocktail effect.

Antigens, CD ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Chemokines ; metabolism ; Cytokines ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Receptors, Chemokine ; metabolism

OBJECTIVETo test the effect of bifunctional molecule IL2-GMCSF in promoting the activation of dendritic cells (DCs) cultured in tumor conditioned medium.

METHODSWe prepared a tumor conditioned medium using mouse melanoma cell line B16F10 supplemented with IL2-GMCSF, GM-CSF, IL-2, or the combination of the latter two. After culturing mouse DC cell line DC2.4 in the conditioned medium for 24 h, the DCs were examined for phagocytosis, proliferation, maturation phenotype, cytokine secretion, and signal pathway activation.

RESULTSDC2.4 cells displayed characteristics of immature DCs. After cell culture in the conditioned medium, the cells showed enhanced phagocytosis but significantly suppressed cell proliferation activity. Culture in the conditioned medium also promoted DC cell maturation and secretion of macrophage-derived chemokine (MDC), but inhibited IL-12 secretion. Supplementation of the conditioned medium with IL2-GMCSF promoted phagocytosis, proliferation, maturation, and cytokine (including both IL-12 and MDC) secretion of DC2.4 cells. Compared with GM-CSF, IL2-GMCSF induced a higher level of NF-κB signal pathway activation but suppressed STAT3 activation.

CONCLUSIONCompared with GM-CSF, IL2-GMCSF can better promote DC activation in the context of tumor-induced immune suppression, and thus shows potentials in anti-tumor therapy.

Animals ; Cell Differentiation ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Chemokine CCL22 ; metabolism ; Culture Media, Conditioned ; chemistry ; Dendritic Cells ; cytology ; drug effects ; Gene Expression Regulation, Neoplastic ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Immune Tolerance ; Interleukin-12 ; metabolism ; Interleukin-2 ; pharmacology ; Melanoma, Experimental ; pathology ; Mice ; NF-kappa B ; metabolism ; Phagocytosis ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

OBJECTIVETo investigate the anti-atherosclerosis mechanism of Astragalus mongholicus polysaccharides (APS) by examining its effect on gene expression profiles of the dendritic cells (DCs) from healthy donors.

METHODSPeripheral blood DCs from healthy donors were incubated with 200 mg/L APS overnight, and changes in the gene expression profiles were investigated using microarray technique and RT-PCR.

RESULTSCompared with the control cells, APS-treated DCs showed significantly up-regulated expressions of CD36 (0.97 ± 0.23 vs 5.45 ± 1.14) and IL-27 (1.08 ± 0.22 vs 2.97 ± 0.61) and down-regulated expression of expression of IFI16 (0.98 ± 0.18 vs 0.46 ± 0.11).

CONCLUSIONSAPS can promote the maturation and differentiation of DCs by up-regulating CD36 and IL-27 and down-regulating IFI16, and thus positively affects the occurrence and progression of the atherosclerosis.

Astragalus Plant ; chemistry ; CD36 Antigens ; metabolism ; Cell Differentiation ; Dendritic Cells ; drug effects ; Humans ; Interleukins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphoproteins ; metabolism ; Polysaccharides ; pharmacology ; Transcriptome

The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1 × 10(6) units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3(+)/CD8(+) cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73% ± 12.51%) was dramatically higher than that before treatment (29.20% ± 8.34%, P < 0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.
Adolescent ; Antineoplastic Agents ; therapeutic use ; Bone Marrow ; drug effects ; immunology ; pathology ; Child ; Child, Preschool ; Cytokine-Induced Killer Cells ; cytology ; immunology ; transplantation ; Dendritic Cells ; cytology ; immunology ; transplantation ; Female ; Humans ; Immunotherapy, Adoptive ; methods ; Injections, Subcutaneous ; Interleukin-2 ; therapeutic use ; Leukemia, Myeloid, Acute ; immunology ; pathology ; therapy ; Male ; Neoplasm, Residual ; Primary Cell Culture ; Recurrence ; Remission Induction ; Treatment Outcome

OBJECTIVE: To explore the effect of nerve growth factor (NGF) on the  differentiation of murine bone marrow-derived dendritic cells (DCs) in vitro.
 METHODS: The bone marrow cells of femur and tibia from healthy C57B -L/6 mice were isolated and divided into 4 groups: a phosphate buffered saline (PBS) group (PBS group), a NGF group, a granulocyte monocyte colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) group (GM-CSF+IL-4 group), and a GM-CSF plus IL-4 and NGF group (n=6 in each group). The positive rate of CD11c+ and the proportion of CD8a- were compared at the 7th day among the different groups by flow cytometry. The immature DCs were acquired by classic methods with GM-CSF and IL-4. The purified DCs were obtained by magnetic bead positive selection for CD11c+ cells. The immature DCs were divided into 4 groups: a PBS group, a NGF group, a LPS group, and a NGF+LPS group (n=6 in each group), which were incubated with PBS, NGF, LPS and NGF+LPS, respectively. Cytokine levels of IL-6, IL-10 and IL-12 were detected by ELISA after 24 hours..
 RESULTS: 1) the percentage of CD11c+ DCs in the NGF group were more than that in the PBS group, and lower than that in the the GM- CSF+IL-4 group (both P<0.05). There was no difference between the GM-CSF + IL-4 group and the NGF+GM-CSF+IL-4 group (P>0.05). CD8a- DCs were dominant in these four groups; 2) NGF could further up-regulate the LPS-induced cytokine secretion from DCs, such as IL-6, IL-10, and IL-12 (all P<0.05), but NGF alone had no such effect (all P<0.05).
 CONCLUSION NGF can promote the murine bone-marrow cells differentiation into CD11c+ DCs, with CD8a-subset; NGF could enhance LPS-induced cytokine secretion from DCs (IL-6, IL-10 and IL-12).
Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Interleukin-10 ; analysis ; Interleukin-12 ; analysis ; Interleukin-4 ; pharmacology ; Interleukin-6 ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factor ; pharmacology

OBJECTIVETo explore the improvement of dendritic cells' (DCs) functions in chronic hepatitis B (CHB) patients by two different drugs plasma, i.e., Shen supplementing and detoxification (SSD) and Pi invigorating and detoxification (PID), thus comparing which method was more effective to activate DCs to improve T lymphocyte functions.

METHODSTotally 30 CHB outpatients were recruited. They were assigned to the immune tolerant group and the immune clearance group, 15 in each group. Totally 60 mL peripheral blood was extracted to isolate and develop mature DCs. Chinese compound containing (Liuwei Ganlu Syrup for SSD) and (Sijun Ganlu Syrup for PID) plasma were added to promote the maturation of DCs on the 7th day. Besides, non-drug plasma was taken as the control. On the ninth day, HBV core 18-27 loaded core peptide and its own T lymphocyte were co-cultivated for 72 h. Then T lymphocytes were collected. The expression levels of CD3, CD28, CD4, and CD8, programmed death-1 (PD-1) were detected using flow cytometry.

RESULTSCompared with non-drug plasma, the expression levels of CD3, CD4, and CD28 could be improved, and the expression levels of CD8 and PD-1 could be reduced by the two methods, showing statistical difference (P < 0.05). Besides, SSD containing plasma showed better effect in improving the molecular CD28 expression rate, and reducing the molecular PD-1 expression rate on the T cell surface, showing statistical difference when compared with that of PID containing plasma (P < 0.05).

CONCLUSIONSIn vitro intervention of DCs by SSD and PID containing plasmas combined co-cultivation of its own T lymphocytes could promote the activation of DCs to improve the function of T cells and the expression of T cell surface molecules. Besides, SSD showed more significant effect on infection immune of HBV patients in the tolerance stage.

Adult ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Middle Aged ; T-Lymphocytes ; immunology ; Young Adult