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, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Kinetics of immune cell responses in the multiple low-dose streptozotocin mouse model of type 1 diabetes.

In type 1 diabetes (T1D), the insulin-producing ß cells are destructed by immune mechanisms. It has been hypothesized that the very first immune response in T1D onset comes from innate immune cells, which further activates the adaptive immune cells to attack the islets. Despite intensive research on characterization of islet-infiltrating immune cells, the kinetics of different immune cells in multiple low-dose streptozotocin (MLDSTZ)-induced T1D mouse model is still much unclear. Therefore, we investigated the proportions of innate immune cells such as neutrophils, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), macrophages, natural killer (NK) cells, and adaptive immune cells (T and B lymphocytes) in thymi, pancreatic-draining lymph nodes, and spleens of MLDSTZ mice on days 3, 7, 10, and 21 after the first injection of STZ by flow cytometry. The proportions of DCs and B cells were increased from day 3, while the proportions of B-1a lymphocytes and interferon-γ+ cells among NK cells were increased, but NK cells were decreased on day 10 in MLDSTZ-treated mice, illustrating that the initial immune response is induced by DCs and B cells. Later, the proportions of T helper 1 and cytotoxic T cells were increased from day 7, suggesting that the innate immune cells precede adaptive immune cell response in MLDSTZ mice. Altogether, our data demonstrate a possible sequence of events regarding the involvement of DCs, pDCs, NK cells, B-1a lymphocytes, B, and T cells at the early stage of T1D development.

Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells.

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-ß) 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.

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

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

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.

Impact of plastic adhesion in vitro on analysis of Th1 and Th2 cytokines and immune cell distribution from mice with multiple low-dose streptozotocin-induced diabetes.

Cytokines produced by Th1 or Th2 cells have been postulated to be important in the development of type 1 diabetes in humans and animal models, such as murine multiple low-dose streptozotocin (MLDSTZ)-induced diabetes. The aim of this study was to investigate cytokine production with or without in vitro depletion of plastic adherent cells from spleens isolated after MLDSTZ treatment. Spleen cells were prepared on day 14 from MLDSTZ- and saline-treated mice and divided into two fractions. One cell fraction was depleted of adherent cells by plastic adherence and the other was not. Both cell fractions were analysed by FACS for the distribution of immune cells. In other experiments, the cells were cultured for 48 h with concanavalin A stimulation. Supernatant samples were analysed by ELISA for TNFalpha, IFNgamma and IL-10 production. Either before or after the 48-h culture cytokine mRNA expression was determined by RT-PCR. Plastic adhesion decreased the macrophage numbers by approximately 30% and CD4(+)CD25(+) cells by about 60%. This was accompanied by increased medium levels of TNFalpha, IFNgamma and IL-10, which suggest that either CD4(+)CD25(+) cells, macrophages, or both, down-regulate production of both Th1 and certain Th2 cytokines. Depletion of adherent cells also decreased IL-4 mRNA amounts. MLDSTZ treatment increased the production of Th1 cytokines mainly at the protein level, and IL-10 mainly at the mRNA level. This indicates a sustained increase in Th1 production after MLDSTZ treatment and an increase in IL-10 that might reflect an attempt to counteract the MLDSTZ-induced immune damage. Plastic adhesion during cell preparation may affect the relative distribution of certain immune cells.

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

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

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.