determination-regulatory-t-cell-subsets-murine-thymus-pancreatic

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Uppsala University

We recently reported that administration of 9-[(1R, 3R)-trans-cyclopentan-3-ol] adenine (MDL 201,449A), a transcriptional inhibitor of TNFalpha, decreased hyperglycemia in murine diabetes induced by multiple low doses of streptozotocin (MLDSTZ). In the present study, we first investigated if in vivo administration of MDL 201,449A in the MLDSTZ model affects cytokine release from cultured spleen cells. Secondly, we studied how MDL 201,449A affects cytokine release from normal cultured spleen cells. In all experiments, the mitogen concanavalin A (2 micro g/ml) was added to the cultured spleen cells in order to enhance cytokine release. MLDSTZ treatment in vivo caused increased IFNgamma secretion, a decreased/retarded rate of increased TNFalpha accumulation, whereas IL-10 production was not altered compared to vehicle-treated mice. MDL 201,449A treatment of MLDSTZ mice did not affect cytokine release from spleen cells subsequently cultured in the absence of MDL 201,449A. We also studied cytokine release from normal spleen cells in the presence or absence of MDL 201,449A. Production of TNFalpha, IFNgamma and IL-10 was all suppressed by the drug. In groups where exposure to MDL 201,449A was discontinued, cytokine levels increased promptly and in the case of TNFalpha secretion, it exceeded the production from control cells. Our data suggest an enhanced Th1 cytokine secretion from spleen cells derived from MLDSTZ-treated mice. MDL 210,449A may be a potent inhibitor of cytokine secretion, albeit not completely selective for TNFalpha. However, when MDL 201,449A is withdrawn, there may be a rebound phenomenon of increased TNFalpha secretion.

Cytokine release by murine spleen cells following multiple low dose streptozotocin-induced diabetes and treatment with a TNFalpha transcriptional inhibitor.

Cytokines may be involved in islet destruction during Type 1 diabetes. Exposure to interleukin-1beta (IL-1beta) or IL-1beta plus interferon-gamma (IFN-gamma) of rodent islets induces expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent formation of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) may impair beta-cell function. Using iNOS deficient (iNOS -/-) islets, we have further investigated the relation between NO formation and PGE(2) induction. We found that iNOS -/- islets responded with a reduced PGE(2) formation following IL-1beta or (IL-1beta + IFN-gamma) treatment compared to wild-type (wt) islets, while COX-2 mRNA or protein content were unchanged. By the addition of an NO donor together with IL-1beta, PGE(2) formation could be stimulated from iNOS -/- islets. We conclude that the lowered capacity of PGE(2) formation observed from cytokine exposed iNOS -/- islets is due to a decreased stimulation of PGE(2) formation by the COX-2 enzyme in the absence of NO, rather then differences in expressed COX-2 protein.

Cytokine-induced PGE(2) formation is reduced from iNOS deficient murine islets.

Elevated levels of glucose and free fatty acids as well as changes in the cytokine production are common features of both type 1 and type 2 diabetes. Especially regarding type 1 diabetes, immunological factors are believed to be responsible for much of the disease pathology. The aim of this study was to investigate whether the diabetic environment in itself could affect cytokine production. Spleen cells from normal mice were cultured for 96 h with addition of different concentrations of glucose (2.8, 5.6, 11.1, 28 mM) or the free fatty acid palmitate (50-100 microM). Cytokine supernatant secretions and mRNA expressions were determined. The cytokine production was highest in cells cultured at 11.1mM glucose. TNFalpha and IFNgamma secretion was decreased by high glucose. Palmitate and/or the ethanol used to dissolve it had a suppressive effect on the secretion of all the investigated cytokines. This effect was counteracted by an elevated glucose concentration for TNFalpha and IFNgamma, but not IL-10. In conclusion, our data suggest that metabolic aberrations characterizing a diabetic environment can have a direct impact on cytokine production by immune cells.

Effects of a diabetes-like environment in vitro on cytokine production by mouse splenocytes.

Many experimental islet studies compare the effect of allogeneic transplantations with either syngeneically transplanted or sham-operated animals. Presently we examined multiple "control" treatments to be able to distinguish effects by the operating procedures themselves versus reactions induced by islet graft rejection. Herein, we have studied untreated, sham-operated, syngeneically or allogeneically (C57BL/6) islet transplanted BALB/c mice, and subsequently examined cytokine production (TNFalpha, IFNgamma, IL-4, IL-10, IL-17 and TGF-beta) in vitro by RT-PCR and ELISA in spleen cells and transplants. To investigate if the strain of the recipient mice influences cytokine production we also performed allogeneic islet transplantations in the reverse direction. So-called control treatments such as sham operations and syngeneic transplantations had a distinct effect on cytokines in spleen cells, possibly induced by surgery and/or anaesthesia. This seems to decrease the regulatory T cells, thereby leading to increased cytokine expression. Furthermore, spleen cells from surgically manipulated animals seem to have a decreased responsive capacity to con A stimulation in culture. Cytokine generation, FoxP3 mRNA expression and COX-2 mRNA expression in the two investigated mouse strains were sometimes altered in opposite directions by the treatments. In conclusion, the genetic background of both the islet donor and recipient has a major impact on both the magnitude and skewing of a cytokine response. Moreover, factors not directly related to allorejection influences systemic cytokine production in connection to islet transplantation.

Factors influencing the regulation of cytokine balance during islet transplantation in mice.

Regulatory T (Treg) cells are characterized by the expression of CD4, CD25 and the intracellular Foxp3. However, these markers do not indicate whether Treg cells are thymic derived Treg (tTreg) cells or peripherally induced Treg (pTreg) cells. Recently, Helios and Neuropilin-1 (Nrp1) has been reported as potential markers for tTreg cells. Herein, we used flow cytometry to examine the proportion of CD4(+)CD8(-)CD25(+) Treg cells expressing Helios, Nrp1 and Foxp3 in thymus, pancreatic draining lymph nodes (PDLNs) and spleen of CD-1 mice, and thymus of NOD and C57BL/6 mice. The frequency of Helios(+) cells was higher than that of Nrp1(+) cells in CD4(+)CD8(-)CD25(+) and CD4(+)CD8(-)CD25(+)Foxp3(+) Treg cells in thymus. Interestingly, the proportion of IL-10(+), Ebi3(+)and CTLA-4(+) cells was higher in Helios(+) than Nrp1(+) tTreg cells. The anti-apoptotic activity of Helios(+) tTreg cells was higher in thymus compared to Nrp1(+) tTreg cells. Nrp1 seems to be expressed at a later developmental stage compared to Helios and Foxp3. Furthermore, the expression of Nrp1 in CD4(+)CD25(+) T cells of younger mice did not increase after stimulating them in vitro with anti-CD3 and -CD28. Thus, under these conditions, Helios could be considered a more reliable marker for distinguishing tTreg cells from pTreg cells than Nrp1. 

Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naïve mice.

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases. 

Article	Year
Cytokine release by murine spleen cells following multiple low dose streptozotocin-induced diabetes and treatment with a TNFalpha transcriptional inhibitor. International immunopharmacology\| PubMed ID: 14555286	2003
Cytokine-induced PGE(2) formation is reduced from iNOS deficient murine islets. Molecular and cellular endocrinology\| PubMed ID: 15196696	2004
Effects of a diabetes-like environment in vitro on cytokine production by mouse splenocytes. Cytokine\| PubMed ID: 18455422	2008
Factors influencing the regulation of cytokine balance during islet transplantation in mice. Transplant immunology\| PubMed ID: 18835356	2009
Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naïve mice. Scientific reports\| PubMed ID: 25586548	2015
Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells. Scientific reports\| PubMed ID: 26224624	2015

Lina Thorvaldson

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