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Spinal cord injury (SCI) causes motor, sensory, and autonomic dysfunctions. The gut microbiome has an important role in SCI, while short-chain fatty acids (SCFAs) are one of the main bioactive mediators of microbiota. In the present study, we explored the effects of oral administration of exogenous SCFAs on the recovery of locomotor function and tissue repair in SCI. Allen's method was utilized to establish an SCI model in Sprague-Dawley (SD) rats. The animals received water containing a mixture of 150 mmol/L SCFAs after SCI. After 21 d of treatment, the Basso, Beattie, and Bresnahan (BBB) score increased, the regularity index improved, and the base of support (BOS) value declined. Spinal cord tissue inflammatory infiltration was alleviated, the spinal cord necrosis cavity was reduced, and the numbers of motor neurons and Nissl bodies were elevated. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (qPCR), and immunohistochemistry assay revealed that the expression of interleukin (IL)-10 increased and that of IL-17 decreased in the spinal cord. SCFAs promoted gut homeostasis, induced intestinal T cells to shift toward an anti-inflammatory phenotype, and promoted regulatory T (Treg) cells to secrete IL-10, affecting Treg cells and IL-17+ γδ T cells in the spinal cord. Furthermore, we observed that Treg cells migrated from the gut to the spinal cord region after SCI. The above findings confirm that SCFAs can regulate Treg cells in the gut and affect the balance of Treg and IL-17+ γδ T cells in the spinal cord, which inhibits the inflammatory response and promotes the motor function in SCI rats. Our findings suggest that there is a relationship among gut, spinal cord, and immune cells, and the "gut-spinal cord-immune" axis may be one of the mechanisms regulating neural repair after SCI.
Subject(s)
Animals , Rats , Interleukin-17 , Rats, Sprague-Dawley , Recovery of Function , Spinal Cord Injuries/drug therapy , T-Lymphocytes, Regulatory , Receptors, Antigen, T-Cell, gamma-delta/immunologyABSTRACT
AbstractObjective: To investigate the effect of γδ T cells on the proliferation, apoptosis and autophagy of multiple myeloma cells.@*METHODS@#Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteers, and stimulated with zoledronic acid (Zol) in combination with rhIL-2. Flow cytometry analysis was used to detected the purity of γδ T cells. γδ T cells were collected and co-cultured with RPMI-8226 or U-266 cells at different effector target ratios. The proliferation of RPMI-8226 or U-266 cell lines were detected by CCK-8. Cell cycle and cell apoptosis were detected by flow cytometry and Western blot．The expressions of autophagy-related proteins were detected by Western blot.@*RESULTS@#γδ T cells can be expanded in vitro. γδ T cells could inhibit the proliferation of RPMI-8226 or U-266 cells, induced cell cycle arrest and promoted apoptosis in an effector target-dependent manner. In addition, γδ T cells could induce autophagy of myeloma cells, inhibited the expression of autophagy-related PI3K, P-AKT and P-mTOR, while increased the expression of AMPK and Beclin-1.@*CONCLUSION@#γδ T cells can inhibit the proliferation of RPMI-8226 and U-266 myeloma cells, induce cell cycle arrest, promote apoptosis, and enhance autophagy in vitro. The mechanism may be related to inhibition of PI3K/AKT/mTOR signaling pathway and/or activation of AMPK/Beclin-1 signaling pathway.
Subject(s)
Humans , AMP-Activated Protein Kinases/pharmacology , Apoptosis , Autophagy , Beclin-1/pharmacology , Cell Proliferation , Leukocytes, Mononuclear/metabolism , Multiple Myeloma/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , T-Lymphocytes , TOR Serine-Threonine Kinases/metabolismABSTRACT
Objective:To investigate the correlation between preterm infants with brain injury and the proportion of lymphocyte subsets, especially γδ-T cells in the postnatal peripheral blood, and to determine the predictive potential of γδ-T cells in the early peripheral blood in brain injury.Methods:It was a prospective study involving 106 preterm infants with gestational age less than 34 weeks who were delivered in the Department of Neonatology, the Third Affiliated Hospital of Zhengzhou University from January 1, to June 1, 2021.Relative levels of γδ-T , CD4 + T, CD8 + T, CD3 + T and total lymphocyte subsets in peripheral blood collected within the first 24 hours after birth were measured by flow cytometry.Recruited infants were divided into brain injury group (36 cases) and non-brain injury group (70 cases) according to serial cranial ultrasound and magnetic resonance imaging(MRI) at the corrected gestational age of 36-37 weeks.Differences in general conditions and the proportion of lymphocyte subsets between groups were compared by the t-test or Chi- square test.Patients in brain injury group were further divided into intracranial hemorrhage(ICH) group(8 cases), periventricular leukomalacia (PVL) group (6 cases)and diffuse white matter damage (WMD) group(22 cases). The proportion of lymphocyte subsets among the different groups was compared by One- Way ANOVA, followed by the LSD- t test. Results:The proportion of γδ-T cells in postnatal peripheral blood of preterm infants at 24 hours after birth in brain injury group was significantly lower than that of non-brain injury group [(0.09±0.12)% vs.0.15±0.13)%, t=-2.445, P=0.016]. No significant differences in the proportion of the CD4 + and CD8 + T cell subsets were found between them.Both preterm infants in PVL group and WMD group had a significantly lower proportion of γδ-T cells at 24 hours after birth compared to that of the non-brain injury group [(0.03±0.05)%, (0.07±0.09)% and (0.15±0.13)%], respectively, ( t=-2.190, -2.659, all P<0.05). Conclusions:γδ-T cells in early postnatal peripheral blood may be involved in the development of brain injury in preterm infants and they had early predictive value for preterm infants at high risk of brain injury, especially the leukomalacia and diffuse white matter injury.
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Background: The occurrence of tumor is closely related to the function of immune system. As an effector cell of innate immunity, the function of γδ T cells is reported to be regulated by co-stimulatory molecules. T-cell immunoglobulin mucin-3 (Tim-3) and programmed death-1 (PD-1), two critical inhibitory co-stimulatory molecules, may affect the immune function of T lymphocytes via binding with their ligands, thus mediating the immune escape of tumor cells. Aims: To investigate the expressions and clinical significance of Tim-3 and PD-1 on γδ T cells in peripheral blood of colon cancer patients. Methods: Peripheral blood samples of 44 colon cancer patients were collected preoperatively at the First Affiliated Hospital of Soochow University from Dec. 2017 to Jun. 2018. Forty healthy volunteers were served as controls. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Expressions of Tim-3 and PD-1 on γδ T cells were detected by flow cytometry, and their correlations with tumor clinicopathological characteristics were analyzed. Results: The proportions of Tim-3+, PD-1+ and Tim-3+PD-1+ γδ T cells in peripheral blood of colon cancer patients were significantly higher than those of healthy volunteers (P0.05). Conclusions: Tim-3 and PD-1 are highly expressed on γδ T cells in peripheral blood of colon cancer patients and associated with the clinicopathological stage of tumor. Expressions of Tim-3 and PD-1 on peripheral blood γδ T cells might be the promising objective indicators for evaluating the development and progression of colon cancer.
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AIM:To investigate the synergistic effect of toosendanin on regulating the cytotoxicity of γδ T cells to colorectal cancer cells. METHODS:γδ T cells amplified in vitro were identified by flow cytometry. Lactate dehydro-genase (LDH) release was detected to evaluate the cytotoxicity of γδ T cells and toosendanin to SW480 cells. The role of toosendanin in regulating the protein expression of Bcl-xL,Bcl-2 and MCL-1 was determined by Western blot. The effect of toosendanin on regulating the secretion of TNF-related apoptosis-inducing ligand(TRAIL) and Fas ligand(FasL) by γδ T cells was evaluated by ELISA. The mitochondrial membrane potential and apoptosis in SW480 cells treated with γδ T cells and toosendanin were analyzed by flow cytometry. The activation of caspase-9 and caspase-3 were determined by Western blot. RESULTS:CD3 and γδ T-cell receptor(TCR) were highly expressed in the γδ T cells amplified in vitro. Combina-tion with toosendanin significantly enhanced the cytotoxicity of γδ T cells to SW480 cells. Toosendanin did not influence the secretion of TRAIL and FasL secreted by γδ T cells. Toosendanin did not regulate the expression of Bcl-xL and Bcl-2 but suppressed the expression of MCL-1 in SW480 cells. In addition, enforced expression of MCL-1 obviously suppressed the synergistic effect of toosendanin on γδ T cell-induced cell death in SW480 cells. Meanwhile,co-treatment with toosendanin was able to enhance the γδ T cell-induced apoptosis and decrease of mitochondrial membrane potential. γδ T cell-depend-ent activation of caspase-9 and caspase-3 was significantly enhanced by toosendanin co-treatment in SW480 cells. CON-CLUSION:Toosendanin exerts synergistic effect on γδ T cell-induced cytotoxicity to colorectal cancer by suppressing the expression of MCL-1.
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Objective To compare the composition of γδ T cells in the peripheral blood of gestational diabetes mellitus(GDM) patients with those of normal pregnant women,so as to explore the association of γδ T cells with the onset and progression of GDM.Methods The peripheral blood mononuclear cells(PBMC) of GDM patients(case group) and normal pregnant women(con-trol group) were separated by Ficoll-Hypaque density gradient centrifugation.γδ T cells were labeled with FITC conjugated anti TCRγδ monoclonal antibody(McAb) and PE conjugated anti-CD3 McAb and analyzed by flow cytometry.Results γδ T cells in GDM patients' peripheral blood were about(6.89 ± 3.22) ％,which was higher than normal control group(4.26 ± 1.64) ％.The percentage of γδ T cells in non-treated GDM group was significantly increased to(8.79 ± 2.33)％,while in treated GDM group,the percentage of γδ T cells was drop to normal(3.76±1.62)％.Conclusion γδ T cells in GDM patients' peripheral blood and CD3+ T Cells were slightly increased.The alteration may be related to the onset of GDM.
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Objective To compare the composition of γδ T cells in the peripheral blood of gestational diabetes mellitus(GDM) patients with those of normal pregnant women,so as to explore the association of γδ T cells with the onset and progression of GDM.Methods The peripheral blood mononuclear cells(PBMC) of GDM patients(case group) and normal pregnant women(con-trol group) were separated by Ficoll-Hypaque density gradient centrifugation.γδ T cells were labeled with FITC conjugated anti TCRγδ monoclonal antibody(McAb) and PE conjugated anti-CD3 McAb and analyzed by flow cytometry.Results γδ T cells in GDM patients' peripheral blood were about(6.89 ± 3.22) ％,which was higher than normal control group(4.26 ± 1.64) ％.The percentage of γδ T cells in non-treated GDM group was significantly increased to(8.79 ± 2.33)％,while in treated GDM group,the percentage of γδ T cells was drop to normal(3.76±1.62)％.Conclusion γδ T cells in GDM patients' peripheral blood and CD3+ T Cells were slightly increased.The alteration may be related to the onset of GDM.
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Background Previous studies showed that the pathogenesis of uveitis is related to γδ T cells.However,it remains unclear that how these cells are involved in experimental autoimmune uveitis (EAU).Objective This study aimed to observe the dynamic changes of γδ T cells in EAU and explore the role of γδ T cells in the pathological process of EAU.Methods Forty-five C57BL/6(B6) mice were assigned to the normal control group (six mice) and EAU model group (thirty-nine mice).The mice were immunized subcutaneously at 6 spots on the footpads,tail base,and flank with emulsion containing human interphotoreceptor retinoid binding protein1-20 (IRBP1-20) emulsified in complete Freund's adjuvant.After immunization,the mice were examined for clinical signs of EAU by using a Genesis-D camera.The changes of histopathology were compared by hematoxylin and eosin staining.Mouse lymphocytes were isolated and purified from the spleens of IRBP1-20-immunized or normal B6 mice by using a γδ T-cell isolation kit.Flow cytometry was used to detect the changes of intracellular expression of interleukin-17A (IL-17A),and then transferred the activated γδ T cells into EAU models to analyze the changes of clinical signs and histopathology of EAU.Experimental study program as well as the use and feeding of the animals were authorized by the Animal Management and Use Committee of Shandong Traditional Chinese Medicine University.Results The inflammatory symptoms in mouse eyes appeared on day 12 after modeling.The initial changes were fundal blood vessel thickening and minimal inflammatory cell infiltration.Then,multifocal chorioretinal lesions,serious vasculitis and linear lesions were observed on days 16-20,along with abundant lymphocyte infiltration in the vitreous and retinal disorganization.The inflammation symptom scores and the pathological inflammation scores at different time points after modeling had statistically significant differences (F =51.399,P =0.000;F =47.342,P =0.000).The inflammatory symptoms in the eyes began to abate from day 28 onwards.The number of γδ T cells was obviously increased during the inflammation phase of EAU at day 16-20 after modeling,with the number of γδ T cells was (5.67 ±-0.49) ％ and (5.78 ±±0.55) ％,respectively,which was significantly higher than (1.53 ± 0.14) ％ before modeling,with significant differences between them (both at P＜0.05),meanwhile CD69 levels and the integrin lymphocyte function-associated antigen-1 (LFA-1) and secreted IL-17A were elavated.The secretion level of IL-17A was (13.40±0.50)％ and (17.80±2.37)％ on day 16 and day 20 after modeling,respectively,which was significantly higher than (1.53 ± 0.19) ％ before modeling,with significant differences between them (P =0.000,0.001).The activated γδ T cells were transferred into EAU model,the inflammation symptom scores were 1.00 (1.00,2.00) after activated γδ T cells were transferred into EAU model,which was significantly higher than 0.75 (0.05,1.00) of the untransferred group (Z =27.00,P =0.03),and the symptoms of EAU were aggravated.Conclusions The proportion of γδ T cells reaches peak in inflammation of EAU,and the cells are activated.The activated γδ T cells in the EAU model play a immune regulation role by secreting IL-17A.
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Objective:To explore the role of γδ T cells in the transdifferentiation of immature dendritic cells(imDC) into osteoclasts(OC). Methods:(1) Peripheral blood mononuclear cells(PBMNC) were cultured with zoledronate(Zol) and recombinant human interleukin-2(IL-2),and PBMNC from healthy volunteers were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and recombinant human interleukin-4(IL-4) to differentiate into imDC,which were then cultured with receptor activator nuclear factor к B ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) to differentiate into OC. The purity of γδ T cells,and phenotype changing of OC transdifferentiated from imDC were investigated by flow cytometry. (2) Co-culture system was es-tablished using millicell inserts.γδT cells isolated with immune magnetic bead were placed in the upper compartment and imDC in the lower compartment in the ratio of 10∶1. To explore the role of γδ T cells during differentiation of imDC into OC,tartrate resistant acid phosphatase( TRAP) staining and bone resorption observation staining were used. Tumor necrosis factor-alpha( TNF-α) of supernatant liquid from different cultures was measured using ELISA(Enzyme linked immunosorbent assay) kit. Results:(1) γδT cells can be ex-panded from PBMNC of MM patients, and the production capacity was similar to that of healthy volunteers ( 68. 87%± 20. 94% vs 69. 33%±16. 84%,P>0. 05 ) . ( 2 ) OC could be transdifferentiated from imDC when cultured with RANKL and M-CSF. ( 3 ) The number of TRAP+ multinuclear cell and the absorption area of dentine were significantly lower in the group of imDC indirectly co-cultured with γδ T cells than in the group of control imDC(5.67±0.58 vs 28.33±2.08,4.97%±4.3% vs 28.47%±12.8%, respectively). (4) Under the circumstance of γδ T cell-imDC indirect coculture,TNF-α got significantly higher. Conclusion: γδ T cells might inhibit the transdifferentiation of imDC into OC.γδ T cells-based immunotherapy is expected to be a new treatment for myeloma bone disease.
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γδ T lymphocytes represent a minor subset of peripheral blood in humans (<10%). γδ T cells expressing Vγ9Vδ2 T cell receptor recognise the endogenous pool of isopentenyl pyrophosphate (IPP) that is overproduced in cancer cells as a result of dysregulated mevalonate pathway. Aminobisphosphonates increase the endogenous pool of IPP in cells by blocking the enzyme farnesyl pyrophosphate synthase (FPPS) of the mevalonate pathway. Activated γδ T cells release copious amounts of interferon (IFN)-γ and tumour necrosis factor (TNF)-α and exhibit potent anti-tumour activity. Combination of γδ T cells with therapeutic monoclonal antibodies can efficiently mediate antibody dependent cellular cytotoxicity against tumours. These features makes γδ T cells attractive mediator of cancer immunotherapy. We review here, the basic properties and importance of γδ T cells in tumour immunity, and highlight the key advances in anti-tumour effector functions of γδ T cells achieved over the last few years and also summarize the results of the clinical trials that have been done till date. Future immunotherapeutic approach utilizing γδ T cells holds considerable promise for treatment of different types of cancer.
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Objective: To investigate the variation of γδ T cells from healthy human peripheral blood(PB)and neonatus cord blood (CB)in proliferation and subtypes with isopentenyl pyrophosphate(IPP), and to acquire enough γδ T cells possessing distinct characteristics for experimental study.Methods: Mononuclear-cells from peripheral blood and cord blood induced by IPP were stained separately with monoclonal antibodies,which were fluorescein-labeled,and then used for assaying the expressing condition of surfaco molecules,as well as to evaluate the variation of γδ T cells on the percentage, subtypes and pbenotypes by FCM.Results:γδ T cells only account for a small proportion in both PB and CB.However,there was a significant difference in the heterogeneity between peripheral blood and cord blood γδ T cells.Vγ9Vδ2 T cells were dominant in peripheral blood γδ T cells.Most of Vγ9Vδ2 T cells in fresh isolated PBMC were central memory-type(CD27~+ CD45RA~-)and effector memory-type(CD27~-CD45RA~-)with IPP, PB γδ T cells proliferated strongly;The effector memory-typo(CD27~-CD45RA~-)turned into the main subtype in all Vγ9Vδ2 T cells,and HLA-DR and B7 molecules were highly expressed on the populations.But the cord blood γδ T cells showed rather complex subgroup heterogeneity,and Vγ9Vδ2 T cells were almost na(i)ve-type(CD27~+ CD45RA~+); Though γδ T cells were expanded(the percent of γδ T cells was increased),and Vγ9Vδ2 T cells were differentiated towards central memory-type and effector memory-type on day 14 with IPP,most of γδ T celLs still remained in the phase of na(i)ve-type(CD27~+ CD45RA~+).Conclusion:Tbere lies great differences of γδ T cells in quantity and subtypes between healthy person peripheral blood and neonatus cord blood.Peripheral blood γδ T cells can be activated and proliferated with IPP, while cord blood γδ T cells have the potential to deferentiate into director memory-type which can be used for experimental and clinical study with the synergy of corresponding cytokines.The immuno-regulation and effector function will be reported in other papers.