Th1, Th2, Th17 cell subsets in two different immunosuppressive protocols in renal allograft recipients (Sirolimus vs mycophenolate mofetil): A cohort study

Long-term use of calcineurin inhibitors (CNI) is associated with nephrotoxicity, which is an important cause of renal dysfunction. Therefore, CNI-minimization strategies which decrease the CNI nephrotoxicity under the protection of additional immunosuppressant drugs have been developed. The aim of current cohort study was to compare the effect of two immunosuppressive protocols [tacrolimus (TAC) in combination with mycophenolate mofetil (MMF) and prednisolone (PRED) versus TAC in combination with sirolimus (SRL) and prednisolone] on the frequency of T helper cell subsets (Th1, Th2 and Th17 cells) and their associated cytokine (IFN-γ, IL-4 and IL-17A) levels in renal allograft recipients.In this study, renal transplant recipients who received induction therapy (Antithymocyte globulin) and were also on triple immunosuppressive therapy were included and divided in to two groups: Group A was comprised 14 patients who received TAC, MMF and PERD whereas group B was composed of 10 patients who received TAC, SRL and PERD. The frequency of Th1, Th2 and Th17 cells in the peripheral blood mononuclear cells (PBMCs) of the patients was analyzed by flow cytometry before and 4 months after transplantation. In addition, IFN-γ, IL-4 and IL-17A concentrations in PBMC culture supernatants of patients before and 4 months after transplantation were quantified by ELISA.The results of our study showed that TAC, MMF and PRED protocol did not diminish the frequency of Th17 cells at 4 months post-transplantation (5% ± 2.5) compared with pre-transplantation (2.3% ± 1; P < 0.05). However, Th17 (3.6% ± 1.5 pre-transplantation vs 2.2% ± 0.9 at 4 months post-transplantation; P < 0.05), Th2 (1.4% ± 0.3 pre-transplantation vs 0.8% ± 0.4 at 4 months post-transplantation; P < 0.05) cell subsets and IL-4 concentration (71.5 pg/ml ± 12 pre-transplantation vs 62.5 pg/ml ±4.4 at 4 months post-transplantation; P < 0.05) were significantly decreased after transplantation in patients who had received SRL, TAC and PRED.In conclusion, the data of the current study suggest that using reduced dose of TAC in SRL, TAC and PRED protocol is in favor of allograft survival; however a cohort study with larger sample size is needed for confirming our results.     

Affects of immunosuppression on circulating dendritic cells: an adjunct to therapeutic drug monitoring after heart transplantation

OBJECTIVES: Recent evidence emerges dendritic cells (DCs) as pharmacological targets of immunosuppressive drugs. Therefore, in this study we monitored DCs in peripheral blood to compare the effects of calcineurin inhibitors (CNI: cyclosporine, tacrolimus) and mammalian target of rapamycin inhibitors (sirolimus, SRL, everolimus, ERL) basis-immunosuppressive therapies in human heart transplanted (HTx) recipients. METHODS: We compared HTx recipients which were converted from either CNI to ERL (severe renal dysfunction, n=8), or from SRL to ERL (approval of ERL for HTx, n=8) with 20 healthy human controls. Twenty four after the last CNI or SRL dose recipients were treated with ERL/BID on days 1-3. Peripheral blood was collected at trough in the morning before and on day 4 after conversion. Percentages of positive myeloid and plasmacytoid DC (m and pDC) subsets in peripheral blood were analysed by flow cytometry. The status of maturation was further characterised by flow cytometry analysis of % expression of CD83 and % expression of various intracellular cytokines (IL-1beta, TNF-alpha, IL-8, IL-12), respectively. RESULTS: HTx recipients had higher % positive mDCs regardless the immunosuppressive therapy compared to controls (p<0.05). Whereas, % positive pDCs were only significantly lower in recipients converted from CNI to ERL compared to controls (p<0.05). The data consolidate the finding that the subset ratio pDCs/mDCs was lower in recipients compared to controls. But after conversion from CNI or SRL to ERL the ratio increased towards pDCs. Percentages of expression of CD83 on mDCs were not different among the recipient groups and controls. Recipients with CNI and SRL had higher % expression of IL-12 and lower % expression of IL-1beta compared to controls (p<0.05). However, after conversion to ERL % expression of both IL-12 and IL-1beta returned to control values in both groups. CONCLUSIONS: The results showed that analysis of immunosuppression of circulating DCs in peripheral blood may be an adjunct to therapeutic drug monitoring to optimize immunosuppressive therapy after HTx.     

MicroRNA-10a-3p mediates Th17/Treg cell balance and improves renal injury by inhibiting REG3A in lupus nephritis

BackgroundThe therapeutic approaches guided toward microRNAs (miRNAs) have been extensively explored in lupus nephritis (LN), but the precise position of miR-10a-3p posted in disease is not translated thoroughly. Therein, this work pivoting on miR-10a-3p was launched with the involvement of regenerating islet-derived 3 α (REG3A).MethodsPeripheral blood samples from LN patients and healthy controls (n = 132) were collected. miR-10a-3p and REG3A expression in peripheral blood mononuclear cells were tested. Mice were injected with miR-10a-3p agomir, miR-10a-3p antagomir and/or REG3A low expression vector for presentation of their roles in renal function, T helper cell 17 (Th17)/regulatory cell (Treg) balance, renal pathological damage, JAK2/STAT3 pathway activation and renal injury in LN. The relation between miR-10a-3p and REG3A was tested.ResultsMiR-10a-3p was down-regulated while REG3A was up-regulated in LN. Restoring miR-10a-3p or silencing REG3A decreased Th17/Treg ratio in CD4⁺ T cells, inhibited JAK2/STAT3 pathway activation, ameliorated renal function, improved renal pathological damage and alleviated renal injury in LN. REG3A depletion negated the effects of down-regulated miR-10a-3p on LN. MiR-10a-3p targeted REG3A.ConclusionThe work elucidates that miR-10a-3p restoration decreases Th17/Treg ratio and attenuates renal injury in LN via inhibiting REG3A and the activation of JAK2/STAT3 pathway, which renews the therapeutic reference for LN management.     

LncRNA XLOC_003810 modulates thymic Th17/Treg balance in myasthenia gravis with thymoma

Imbalance of T helper 17 (Th17)/regulatory T (Treg) cells is involved in the pathogenesis of myasthenia gravis with thymoma (MG-T). Long non-coding RNAs (lncRNAs) are implicated in the regulation of Th17/Treg balance. This study was designed to explore the role of XLOC_003810, a novel lncRNA, in regulating the Th17/Treg balance in MG-T. The thymic CD4⁺ T cells were isolated from control subjects and MG-T patients. The Th17/Treg balance was evaluated by determining proportions of Th17 and Treg cells and expression of Th17- and Treg- associated molecules. Lentivirus-mediated silencing and overexpression of XLOC_003810 in CD4⁺ T cells were performed. The results showed that XLOC_003810 expression was higher in MG-T thymic CD4⁺ T cells than that in the control group. Furthermore, the ratio of Th17/Treg cells, proportion of Th17 cells and levels of Th17-associated molecules were significantly increased, whereas the proportion of Treg cells and levels of Treg-associated molecules were decreased in MG-T thymic CD4⁺ T cells. Importantly, the Th17/Treg imbalance in MG-T thymic CD4⁺ T cells was aggravated by XLOC_003810 overexpression, whereas it was attenuated by XLOC_003810 silencing. Collectively, XLOC_003810 modulates thymic Th17/Treg balance in MG-T patients, providing the scientific basis for the clinical targeted therapy of MG-T.     

Circulating Treg cells from patients with cerebral aneurysms displayed deficiency in ICOS expression and function

Inducible costimulator (ICOS) is a member of the CD28 family. When activated, ICOS signalling promotes FOXP3 CNS2 gene demethylation and stabilizes Treg differentiation. Cerebral aneurysm (CA) is the local ballooning of the cerebral vasculature, characterized by higher levels of inflammation mediators and tissue remodelling. FOXP3⁺ Treg cell dysfunction may contribute to CA pathogenesis. In this study, the expression and function of ICOS in Treg cells was investigated. Circulating CD4⁺CD25^hi T cells from CA subjects demonstrated significantly lower levels of ICOS expression than circulating CD4⁺CD25^hi T cells from healthy subjects. In both healthy subjects and CA subjects, FOXP3⁺ Treg cells were highly concentrated in the ICOS⁺ fraction of CD4⁺CD25^hi T cells. Anti-ICOS costimulation, in combination with anti-CD3 and IL-2, significantly increased FOXP3 expression in CD4⁺CD25^hiICOS⁺ T cells but not in CD4⁺CD25^hiICOS^- T cells. In addition, anti-CD3/IL-2 and anti-ICOS costimulation significantly elevated the expression of IL-10 and TGF-β, decreased the expression of IL-17, and enhanced CD4⁺CD25^hiICOS⁺ T cell-mediated suppression of autologous CD4⁺CD25^- Tconv proliferation. Interestingly, CD4⁺CD25^hiICOS⁺ T cells from CA subjects presented lower responsiveness toward anti-ICOS costimulation than CD4⁺CD25^hiICOS⁺ T cells from healthy subjects. Overall, these results demonstrated that ICOS signalling could significantly improve FOXP3 expression and enhance Treg functional potency. However, circulating Treg cells from CA patients displayed reduced ICOS expression and lower responsiveness toward anti-ICOS stimulation.     

Increased IL-10/IL-17 ratio is aggravated along with the prognosis of patients with chronic lymphocytic leukemia

ObjectiveThis study is to investigate the association between the Treg/Th17 cells and prognosis of chronic lymphocytic leukemia (CLL).MethodsTotally 50 CLL patients and 20 Health controls were included in this study. Regulatory T (Treg) cells and the cell subset secreting IL-17 (Th17) in peripheral blood were detected with flow cytometry. Serum levels of IL-10 and IL-17 were determined with ELISA, and expression of Foxp3 and RORγt was assessed with quantitative real-time PCR.ResultsTreg and Th17 cell proportions in peripheral blood in the CLL patients were significantly higher than control. Serum levels of IL-10 and IL-17, and expression of Foxp3 and RORγt, were significantly increased in the CLL patients. Ratios of Treg/Th17 and IL-10/IL-17 were significantly elevated in the CLL patients. Compared with those before treatment, Treg/Th17 and IL-10/IL-17 ratios were declined in the CLL patients in remission. Compared with the non-remission group, Treg cells were significantly decreased, while Th17 cells were significantly increased, resulting in decreased Treg/Th17 ratio, in the remission group. Moreover, the serum IL-10 level was significantly decreased, while the serum IL-17 level was significantly increased, resulting in declined IL-10/IL-17 ratio, in the remission group. Correlation analysis showed that, Treg and Th17 cell counts were significantly associated with CD38 and ZAP-70 expression in the CLL patients. Moreover, the IL-10/IL-17 ratio was also significantly associated with CLL prognostic factors.ConclusionAltered Treg/Th17 and IL-10/IL-17 ratios in CLL would be aggravated along with the disease progression, which might be used as indicators for the disease prognosis.     

Therapeutic anti-psoriatic effects of myeloid-derived suppressor cells in combination with systemic tacrolimus (FK-506) in an imiquimod-induced mouse model of psoriasis

Although tacrolimus (FK-506) has been shown to be an effective monotherapy for psoriasis, it does not always work well. Currently, combination therapy is frequently used to manage psoriasis because clinical trials have shown it may provide additive or synergistic benefits and reduce risks of adverse effects. Myeloid-derived suppressor cells (MDSCs) have potent immunomodulatory and anti-inflammatory properties in autoimmune diseases. We previously reported that MDSCs had protective effects in a murine model of imiquimod (IMQ)-induced psoriasis. The present study was undertaken to investigate the systemic immunomodulatory and therapeutic efficacy effects of MDSC plus FK-506 in an IMQ-induced mouse model of psoriasis and to investigate the immunomodulatory mechanisms involved. Systemic MDSC plus FK-506 therapy was found to have a significant anti-psoriatic effect in the murine model, to reduce levels of pro-inflammatory cytokines Th1 cytokines (TNF-α and IFN-γ) and Th17 cytokines (IL-17A and IL-23) in serum and skin. However, treatment with MDSCs or FK-506 alone had little impact. Furthermore, the anti-psoriatic effects of MDSC plus FK-506 were associated with histopathological reductions in inflammatory infiltration, epidermal hyperplasia, and hyperkeratosis. In addition, this combined treatment also attenuated IMQ-induced splenomegaly, and increased the proportion of CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells and decreased the proportions of CD4⁺IFN-γ⁺ Th1 cells and CD4⁺IL-17⁺ Th17 cells in spleen. Taken together, our results show systemic combination therapy with MDSCs and FK-506 had a better therapeutic effect in our IMQ-induced psoriasis model than either agent alone, and suggest that this combinatorial therapy might be useful for the management of autoimmune skin diseases like psoriasis.     

Peripheral CD4+ CD25+ Treg cell expansion in lung transplant recipients is not affected by calcineurin inhibitors

CD4+CD25+ regulatory T (Treg) cells have been shown to play a role in allograft tolerance and their peripheral counts vary according to the degree of graft acceptance in lung transplant recipients (LTR). Recent studies demonstrate that certain drugs might modulate generation, expansion and activity of Treg cells. Aim of this study was to evaluate the effect of therapeutic regimens used in our institution on peripheral CD4+CD25(high)CD69- Treg cell numbers in a group of 51 LTR with stable clinical conditions. They were treated with standard immunosuppression: calcineurin inhibitor (CNI)+azathioprine (AZA)+steroids (n=28) or with CNI+mycophenolate mofetil (MMF)+steroids (n=11) or with CNI+steroids (n=12). These stable LTR were compared with age-matched healthy controls (n=35) and with 19 LTR who developed bronchiolitis obliterans syndrome (BOS) and were treated analogously. Stable LTR showed higher peripheral Treg cell counts with respect to age-matched healthy controls (59.9+/-31.8/mul versus 42.1+/-16.9/mul, respectively; p<0.05). This increase was detectable in all patients treated with CNI either in association with AZA or MMF. During these treatments a significant expansion of Treg cell counts was detectable during acute rejection (AR) episodes (86.03+/-26.6/mul during AR versus 36.34+/-7.6 before AR; p<0,05). Moreover, the development of BOS was associated to a significant decrease of Treg cell counts irrespective to the immunosuppressive regimen used. In conclusion, therapeutic regimens based on CNI seem to allow a certain degree of peripheral Treg cell expansion in stable LTR.     

Immunoregulatory Cordyceps sinensis increases regulatory T cells to Th17 cell ratio and delays diabetes in NOD mice

Cordyceps sinensis (CS) is a parasitic fungus, and it has been used widely in traditional Chinese medicines (TCM) for centuries. Many studies have shown that CS has immunoregulatory activity in many disease models, but the underlying mechanism remains elusive. We studied whether CS could suppress the onset of diabetes by altering T lymphocyte subsets in non-obese diabetic (NOD) mice. We found that the onset of type1 diabetes in NOD mice was associated with an imbalance of CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells and IL-17 producing Th17 cells. Oral administration of CS resulted in reduction in the overall incidence of diabetes, and this was due to an increase in the ratio of Treg cells to Th17 in the spleen and pancreatic lymph nodes (PLNs). Taken together, these data imply that CS is able to modulate Treg to Th17 cell ratio in vivo, thus contributing to the inhibition of diabetes.     

Analysis of the role of palmitoleic acid in acute anterior uveitis

PurposeTo study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU).MethodsPA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4⁺T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA.ResultsThe fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4⁺T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4⁺T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls.ConclusionAn increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4⁺T cells and attenuated disease severity in EAU mice.     

CD4+ Foxp3+ regulatory T cells induced by TGF-β, IL-2 and all-trans retinoic acid attenuate obliterative bronchiolitis in rat trachea transplantation

Obliterative bronchiolitis (OB) is the major obstacle for long-term allograft survival in lung transplantation, and the underlying mechanism is still not well understood. Regulatory T cells (Tregs) have been shown to be essential in the maintenance of immune tolerance. In this study we investigated the role of Tregs in protecting OB in rat. We show that the combination of TGF-β, Interleukin (IL)-2, and all-trans retinoic acid (atRA) could induce naïve rat CD4⁺CD25⁻ T cells to differentiate into CD4⁺CD25⁺Foxp3⁺ T cells in vitro, and they acquired suppressive function. In a rat orthotopic tracheal transplantation OB model, the adoptive transfer of the induced Tregs reduced symptoms of airway obliteration and fibrication of grafts when compared with adoptive transfer of control cells without suppressive property. Moreover, recipients treated with the induced Tregs secreted high level of immunosuppressive cytokine TGF-β and IL-10, and low level of pro-inflammatory cytokines IL-17, IFN-γ, IL-6, and MCP-1, and had fewer effector T cells including Th17 cells and Th1 cells in the graft. Taken together, these findings suggest that in vitro induced Tregs by the combination of TGF-β, IL-2, and atRA are effective in protecting rat trachea allograft rejection through the inhibition of effector T cells and their function. These datas implicate new therapies to prevent OB and allograft rejection in human lung transplantation.     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

Combined therapy of CD4+CD25+ regulatory T cells with low-dose sirolimus,but not calcineurin inhibitors,preserves suppressive function of regulatory T cells and prolongs allograft survival in mice

Our previous study proved that sirolimus is a potent immunosuppressant which induces long-term allograft survival depends on persistence of alloantigens. CD4⁺CD25⁺ regulatory T (Treg) cells are potent suppressors in transplantation. Our objectives focus on whether combined-therapy of Tregs with immunosuppressants could prolong allograft survival in mice.The study showed that inhibition of Tregs was maintained by co-cultured with sirolimus (1 nM) in vitro, but not tacrolimus (1 nM) or CsA (1 nM). When the concentration was increased > 100 nM, suppression was fallen. Based on the ability of sirolimus to target effector T cells, but retaining the inhibition of Tregs, an adoptive infusion of donor alloantigen specific Tregs combined with 30-day sirolimus (1 mg/kg) and 3-day ATG (20 mg/kg) was found to prolong heart allograft survival in mice. Even though the cell numbers of CD4⁺ T cells were found to decrease in sirolimus-treated mice, sirolimus selectively enhanced the numbers of CD4⁺CD25⁺ cells and increased the expression of Foxp3 in spleens and lymph nodes, respectively, in recipients. However, combined therapy with low-dose CsA (5 mg/kg) or tacrolimus (1 mg/kg) reduced significantly the expression of Foxp3 and failed to prolong the allograft survival.In summary, expanded Tregs exposed to sirolimus can survive, proliferate, and preserve inhibition in vitro. Tregs are more resistant to sirolimus than other T cells. Combined with Tregs, sirolimus rather than calcineurin inhibitors, prolongs the allograft survival. Sirolimus may be the best copartner for Tregs therapy. It also suggests calcineurin-dependent signals may be required in the development of Tregs.