IL-15 Prevents the Development of T-ALL from Aberrant Thymocytes with Impaired DNA Repair Functions and Increased NOTCH1 Activation.

We previously reported that NOD.Scid mice lacking interleukin-15 (IL-15), or IL-15 receptor alpha-chain, develop T-acute lymphoblastic leukemia (T-ALL). To understand the mechanisms by which IL-15 signaling controls T-ALL development, we studied the thymocyte developmental events in IL-15-deficient Scid mice from NOD and C57BL/6 genetic backgrounds. Both kinds of mice develop T-ALL characterized by circulating TCR-negative cells expressing CD4, CD8 or both. Analyses of thymocytes in NOD.Scid.Il15-/- mice prior to T-ALL development revealed discernible changes within the CD4-CD8- double-negative (DN) thymocyte developmental stages and increased frequencies of CD4+CD8+ double-positive cells with a high proportion of TCR-negative CD4+ and CD8+ cells. The DN cells also showed elevated expressions of CXCR4 and CD117, molecules implicated in the expansion of DN thymocytes. T-ALL cell lines and primary leukemic cells from IL-15-deficient NOD.Scid and C57BL/6.Scid mice displayed increased NOTCH1 activation that was inhibited by NOTCH1 inhibitors and blockers of the PI3K/AKT pathway. Primary leukemic cells from NOD.Scid.Il15-/- mice survived and expanded when cultured with MS5 thymic stromal cells expressing Delta-like ligand 4 and supplemented with IL-7 and FLT3 ligand. These findings suggest that IL-15 signaling in the thymus controls T-ALL development from aberrant thymocytes with an impaired DNA repair capacity and increased NOTCH1 activation.

NLRX1 inhibits the early stages of CNS inflammation and prevents the onset of spontaneous autoimmunity.

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1-/- mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity.

Essential role of suppressor of cytokine signaling 1 (SOCS1) in hepatocytes and macrophages in the regulation of liver fibrosis.

The hepatic fibrogenic response is a protective mechanism activated by hepatocyte damage and is resolved upon elimination of the cause. However, persistent injuries cause liver fibrosis (LF) to evolve into cirrhosis, which promotes the development of hepatocellular carcinoma (HCC). Development of efficient treatments for LF requires better understanding the underlying molecular pathogenic mechanisms. The loss of suppressor of cytokine signaling 1 (SOCS1) expression promotes LF and HCC in human and mice, but the underlying mechanisms remain unclear. SOCS1 is a key regulator of immune cell activation. To investigate the anti-fibrogenic functions of SOCS1 in hepatocytes and macrophages, we generated mice lacking SOCS1 in hepatocytes (Socs1fl/flAlbCre) or macrophages (Socs1fl/flLysMCre) and evaluated hepatic fibrogenic response to carbon tetrachloride (CCl4). Socs1fl/flAlbCre and Socs1fl/flLysMCre mice showed severe LF characterized by increased collagen deposition, hydroxyproline content, myofibroblast accumulation along with elevated expression of Acta2 and Col1a1 genes. CCl4 treatment triggered significant damage to hepatocytes in Socs1fl/flAlbCre mice but not in Socs1fl/flLysMCre mice. In both mice CCl4 treatment reduced the expression of Mmp2 and increased the expression of Timp1. SOCS1 deficiency in hepatocytes or macrophages did not affect Il6, Tnfa or Tgfb, but diminished Infg and augmented Pdgfb expression. Both Socs1fl/flAlbCre and Socs1fl/flLysMCre livers showed increased mononuclear cell infiltration accompanied by elevated Ccl2 expression. Our findings show that SOCS1 exerts non-redundant functions in hepatocytes and macrophages to regulate the hepatic fibrogenic response possibly through limiting hepatocyte damage and the inflammatory response of macrophages, and support the idea of exploiting SOCS1 in LF treatment.

Attenuation of MET-mediated migration and invasion in hepatocellular carcinoma cells by SOCS1.

AIM: To investigate the role of suppressor of cytokine signaling 1 (SOCS1) in regulating MET-mediated invasive potential of hepatocellular carcinoma (HCC) cells. METHODS: Stable derivatives of mouse (Hepa1-6) and human (hep3B, HepG2) HCC cell lines expressing SOCS1 or control vector were evaluated for their ability to migrate towards hepatocyte growth factor (HGF) in the transwell migration assay, invade extracellular matrix in response to HGF stimulation in a 3-D invasion assay by confocal microscopy, and to undergo anchorage-independent proliferation in semisolid agar. Following intravenous and intrasplenic inoculation into NOD.scid.gamma mice, the ability of Hepa cells to form othotopic tumors was evaluated. Following HGF stimulation of Hepa and Hep3B cells, expression of proteins implicated in epithelial-to-mesenchymal transition was evaluated by western blot and qRT-PCR. RESULTS: SOCS1 expression in mouse and human HCC cells inhibited HGF-induced migration through matrigel. In the 3-D invasion assay, HGF stimulation induced invasion of HCC cells across type-I collagen matrix, and SOCS1 expression significantly reduced the depth of invasion. SOCS1 expression also reduced the number and size of colonies formed by anchorage-independent growth in semisolid agar. Following intravenous inoculation, control Hepa cell formed large tumor nodules that obliterated the liver whereas the SOCS1-expressing Hepa cells formed significantly smaller nodules. Tumors formed by SOCS1-expressing cells showed reduced phosphorylation of STAT3 and ERK that was accompanied by reduced levels of MET protein expression. HGF stimulated Hepa cells expressing SOCS1 showed increased expression of E-cadherin and decreased expression of EGR1, SNAI1 and ZEB1. Comparable results were obtained with Hep3B cells. SOCS1 expressing HCC cells also showed reduced levels of EGR1 and SNAI1 transcripts. CONCLUSION: Our findings indicate that loss of SOCS1-dependent control over epithelial-to-mesenchymal transition may contribute to MET-mediated migration, invasion and metastatic growth of HCC.

SOCS1 regulates senescence and ferroptosis by modulating the expression of p53 target genes.

The mechanism by which p53 suppresses tumorigenesis remains poorly understood. In the context of aberrant activation of the JAK/STAT5 pathway, SOCS1 is required for p53 activation and the regulation of cellular senescence. In order to identify p53 target genes acting during the senescence response to oncogenic STAT5A, we characterized the transcriptome of STAT5A-expressing cells after SOCS1 inhibition. We identified a set of SOCS1-dependent p53 target genes that include several secreted proteins and genes regulating oxidative metabolism and ferroptosis. Exogenous SOCS1 was sufficient to regulate the expression of p53 target genes and sensitized cells to ferroptosis. This effect correlated with the ability of SOCS1 to reduce the expression of the cystine transporter SLC7A11 and the levels of glutathione. SOCS1 and SOCS1-dependent p53 target genes were induced during the senescence response to oncogenic STAT5A, RasV12 or the tumor suppressor PML. However, while SOCS1 sensitized cells to ferroptosis neither RasV12 nor STAT5A mimicked the effect. Intriguingly, PML turned cells highly resistant to ferroptosis. The results indicate different susceptibilities to ferroptosis in senescent cells depending on the trigger and suggest the possibility of killing senescent cells by inhibiting pathways that mediate ferroptosis resistance.

SOCS1: Regulator of T Cells in Autoimmunity and Cancer.

SOCS1 is a negative feedback regulator of cytokine and growth factor receptor signaling, and plays an indispensable role in attenuating interferon gamma signaling. Studies on SOCS1-deficient mice have established a crucial role for SOCS1 in regulating CD8+ T cell homeostasis. In the thymus, SOCS1 prevents thymocytes that had failed positive selection from surviving and expanding, ensures negative selection and prevents inappropriate developmental skewing toward the CD8 lineage. In the periphery, SOCS1 not only controls production of T cell stimulatory cytokines but also attenuates the sensitivity of CD8+ T cells to synergistic cytokine stimulation and antigen non-specific activation. As cytokine stimulation of CD8+ T lymphocytes increases their sensitivity to low affinity TCR ligands, SOCS1 likely contributes to peripheral T cell tolerance by putting brakes on aberrant T cell activation driven by inflammatory cytokines. In addition, SOCS1 is critical to maintain the stability of T regulatory cells and control their plasticity to become pathogenic Th17 and Th1 cells under the harmful influence of inflammatory cytokines. SOCS1 also regulates T cell activation by dendritic cells via modulating their generation, maturation, antigen presentation, costimulatory signaling, and cytokine production. The above control mechanisms of SOCS1 on T cells, T regulatory cells and dendritic cells collectively contribute to immunological tolerance and prevent autoimmune manifestation. On other hand, silencing SOCS1 in dendritic cells or CD8+ T cells stimulates efficient antitumor immunity. Thus, even though SOCS1 is not a cell surface checkpoint inhibitor, its regulatory functions on T cell responses qualify SOCS1as a "non-classical" checkpoint blocker. SOCS1 also functions as a tumor suppressor in cancer cells by regulating oncogenic signal transduction pathways. The loss of SOCS1 expression observed in many tumors may have an impact on classical checkpoint pathways. The potential to exploit SOCS1 to treat inflammatory/autoimmune diseases and elicit antitumor immunity is discussed.

Expression of SOCS1 and the downstream targets of its putative tumor suppressor functions in prostate cancer.

BACKGROUND: Suppressor of cytokine signaling 1 (SOCS1) is considered a tumor suppressor due to frequent epigenetic and micro-RNA-mediated repression of its gene expression in diverse cancers. In prostate cancer (PCa), elevated expression of miR-30d that targets SOCS1 mRNA is associated with increased risk of disease recurrence. SOCS1 can mediate its tumor suppressor functions by diverse mechanisms such as inhibiting the JAK-STAT signaling pathway, promoting the tumor suppressor functions of p53, attenuating MET receptor tyrosine kinase signaling and blocking the oncogenic potential of the cell cycle inhibitor p21CIP1 (p21). Here, we studied the expression of SOCS1 and the downstream targets of its putative tumor suppressor functions (p53, MET and p21) in human PCa specimens to evaluate their significance as markers of disease prognosis. METHODS: Tissue microarrays were constructed of 78 archived prostatectomy specimens that were grouped according to the recommendations of the International Society of Urological Pathology (ISUP) based on the Gleason patterns. SOCS1, p53, MET and p21 protein expression were evaluated by immunohistochemical staining alongside the common prostate cancer-related markers Ki67, prostein and androgen receptor. Statistical correlations between the staining intensities of these markers and ISUP grade groups, local invasion or lymph node metastasis were evaluated. RESULTS: SOCS1 showed diffuse staining in the prostatic epithelium. SOCS1 staining intensity correlated inversely with the ISUP grade groups (ρ = -0.4687, p <0.0001) and Ki67 (ρ = -0.2444, p = 0.031), and positively with prostein (ρ = 0.3511, p = 0.0016). Changes in SOCS1 levels did not significantly associate with those of p53, MET or p21. However, p21 positively correlated with androgen receptor expression (ρ = -0.1388, p = 0.0003). A subset of patients with regional lymph node metastasis, although small in number, showed reduced SOCS1 expression and increased expression of MET and p21. CONCLUSIONS: Our findings suggest that evaluating SOCS1 and p21 protein expression in prostatectomy specimens may have a prognostic value in identifying the aggressive disease. Hence, prospective studies with larger numbers of metastatic PCa specimens incorporating clinical correlates such as disease-free and overall survival are warranted.

Trans-presentation of interleukin-15 by interleukin-15 receptor alpha is dispensable for the pathogenesis of autoimmune type 1 diabetes.

Interleukin-15 (IL-15) is a pro-inflammatory cytokine that is required for the survival and activation of memory CD8+T cells, natural killer (NK) cells, innate lymphoid cells, macrophages and dendritic cells. IL-15 is implicated in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis and autoimmune type 1 diabetes (T1D). IL-15 receptor (IL-15R) consists of a specific α chain, the ß chain that is shared with IL-2R and the common γ chain. IL-15 is unique in the manner in which it binds and signals through its receptor subunits. IL-15 that is complexed with IL-15Rα binds to the ßγ receptor complex present on the responding cell to mediate its biological effects through a process referred to as trans-presentation. The trans-presented IL-15 is essential to mediate the biological effects on T lymphocytes and NK cells. Here we show that IL-15, but not IL-15Rα, is required for the development of spontaneous and virus-induced T1D, viral clearance and for antigen cross-presentation to CD8+ T lymphocytes. Our findings provide insight into the complexities of IL-15 signalling in the initiation and maintenance of CD8+ T cell-mediated immune responses.

NLRC5 elicits antitumor immunity by enhancing processing and presentation of tumor antigens to CD8(+) T lymphocytes.

Cancers can escape immunesurveillance by diminishing the expression of MHC class-I molecules (MHC-I) and components of the antigen-processing machinery (APM). Developing new approaches to reverse these defects could boost the efforts to restore antitumor immunity. Recent studies have shown that the expression of MHC-I and antigen-processing molecules is transcriptionally regulated by NOD-like receptor CARD domain containing 5 (NLRC5). To investigate whether NLRC5 could be used to improve tumor immunogenicity, we established stable lines of B16-F10 melanoma cells expressing NLRC5 (B16-5), the T cell co-stimulatory molecule CD80 (B16-CD80) or both (B16-5/80). Cells harboring NLRC5 constitutively expressed MHC-I and LMP2, LMP7 and TAP1 genes of the APM. The B16-5 cells efficiently presented the melanoma antigenic peptide gp10025-33 to Pmel-1 TCR transgenic CD8(+) T cells and induced their proliferation. In the presence of CD80, B16-5 cells stimulated Pmel-1 cells even without the addition of gp100 peptide, indicating that NLRC5 facilitated the processing and presentation of endogenous tumor antigen. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that the NLRC5-expressing tumor cells elicited antitumor immunity. Following intravenous injection, B16-5 and B16-5/80 cells formed fewer lung tumor foci compared to control cells. In mice depleted of CD8(+) T cells, B16-5 cells formed large subcutaneous and lung tumors. Finally, immunization with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Collectively, our findings indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity.

Interleukin-21-dependent modulation of T cell antigen receptor reactivity towards low affinity peptide ligands in autoreactive CD8(+) T lymphocytes.

IL-21 promotes autoimmune type-1 diabetes (T1D) in NOD mice by facilitating CD4(+) T cell help to CD8(+) T cells. IL-21 also enables autoreactive CD8(+) T cells to respond to weak TCR ligands and induce T1D. Here, we assessed whether IL-21 is essential for T1D induction in a mouse model where the disease can occur independently of CD4 help. In this model, which expresses lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) antigen under the rat insulin promoter (RIP-GP), LCMV infection activates CD8(+) T cells reactive to the GP-derived GP33 peptide that attack pancreatic islets and cause T1D. We show that IL-21 deficiency in RIP-GP mice did not impair T1D induction by LCMV expressing the wildtype GP33 peptide. Surprisingly, LCMV-L6F, expressing a weak peptide mimic of GP33, induced T1D more efficiently in Il21(-/-)RIP-GP mice than in controls. However, LCMV-C4Y expressing a very weak peptide mimic of GP33 did not induce T1D in Il21(-/-) mice, but T cells from the infected mice caused disease in lymphopenic RIP-GP mice upon adoptive transfer. Using Nur77(GFP) reporter mice, we show that CD8(+) T cells from Il21(-/-) mice expressing the GP33-specific transgenic P14 TCR showed increased reactivity towards low affinity TCR ligands. Collectively, our findings show that IL-21 is not always required for T1D induction by autoreactive CD8(+) T cells, and suggest that IL-21 may play an important role in regulating CD8(+) T cell reactivity towards low affinity TCR ligands.

The hepatocyte growth factor (HGF)-MET receptor tyrosine kinase signaling pathway: Diverse roles in modulating immune cell functions.

Hepatocyte growth factor (HGF) signaling via the MET receptor is essential for embryonic development and tissue repair. On the other hand, deregulated MET signaling promotes tumor progression in diverse types of cancers. Even though oncogenic MET signaling remains the major research focus, the HGF-MET axis has also been implicated in diverse aspects of immune cell development and functions. In the presence of other hematopoietic growth factors, HGF promotes the development of erythroid, myeloid and lymphoid lineage cells and thrombocytes. In monocytes and macrophages responding to inflammatory stimuli, induction of autocrine HGF-MET signaling can contribute to tissue repair via stimulating anti-inflammatory cytokine production. HGF-MET signaling can also modulate adaptive immune response by facilitating the migration of Langerhans cells and dendritic cells to draining lymph nodes. However, MET signaling has also been shown to induce tolerogenic dendritic cells in mouse models of graft-versus-host disease and experimental autoimmune encephalomyelitis. HGF-MET axis is also implicated in promoting thymopoiesis and the survival and migration of B lymphocytes. Recent studies have shown that MET signaling induces cardiotropism in activated T lymphocytes. Further understanding of the HGF-MET axis in the immune system would allow its therapeutic manipulation to improve immune cell reconstitution, restore immune homeostasis and to treat immuno-inflammatory diseases.

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1.

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1(-/-)Ifng(-/-) mice with dimethylnitrosamine or carbon tetrachloride. Ifng(-/-) and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1(-/-)Ifng(-/-) mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng(-/-) mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.

The nod-like receptor, Nlrp12, plays an anti-inflammatory role in experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is an organ-specific autoimmune disease resulting in demyelinating plaques throughout the central nervous system. In MS, the exact role of microglia remains unknown. On one hand, they can present antigens, skew T cell responses, and upregulate the expression of pro-inflammatory molecules. On the other hand, microglia may express anti-inflammatory molecules and inhibit inflammation. Microglia express a wide variety of immune receptors such as nod-like receptors (NLRs). NLRs are intracellular receptors capable of regulating both innate and adaptive immune responses. Among NLRs, Nlrp12 is largely expressed in cells of myeloid origins. It plays a role in immune inflammatory responses by negatively regulating the nuclear factor-kappa B (NF-κB) pathway. Thus, we hypothesize that Nlrp12 suppresses inflammation and ameliorates the course of MS. METHODS: We used experimental autoimmune encephalomyelitis (EAE), a well-characterized mouse model of MS. EAE was induced in wild-type (WT) and Nlrp12 (-/-) mice with myelin oligodendrocyte glycoprotein (MOG):complete Freud's adjuvant (CFA). The spinal cords of healthy and immunized mice were extracted for immunofluorescence and pro-inflammatory gene analysis. Primary murine cortical microglia cell cultures of WT and Nlrp12 (-/-) were prepared with cortices of 1-day-old pups. The cells were stimulated with lipopolysaccharide (LPS) and analyzed for the expression of pro-inflammatory genes as well as pro-inflammatory molecule secretions. RESULTS: Over the course of 9 weeks, the Nlrp12 (-/-) mice demonstrated increased severity in the disease state, where they developed the disease earlier and reached significantly higher clinical scores compared to the WT mice. The spinal cords of immunized WT mice relative to healthy WT mice revealed a significant increase in Nlrp12 messenger ribonucleic acid (mRNA) expression at 1, 3, and 5 weeks post injection. A significant increase in the expression of pro-inflammatory genes Ccr5, Cox2, and IL-1ß was found in the spinal cords of the Nlrp12 (-/-) mice relative to the WT mice (P < 0.05). A significant increase in the level of gliosis was observed in the spinal cords of the Nlrp12 (-/-) mice compared to the WT mice after 9 weeks of disease (P < 0.05). Primary Nlrp12 (-/-) microglia cells demonstrated a significant increase in inducible nitric oxide synthase (iNOS) expression (P < 0.05) and secreted significantly (P < 0.05) more tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and nitric oxide (NO). CONCLUSION: Nlrp12 plays a protective role by suppressing inflammation during the development of EAE. The absence of Nlrp12 results in an increased inflammatory response.

IL-15 trans-presentation regulates homeostasis of CD4(+) T lymphocytes.

Interleukin-15 (IL-15) is essential for the survival of memory CD8(+) and CD4(+) T cell subsets, and natural killer and natural killer T cells. Here, we describe a hitherto unreported role of IL-15 in regulating homoeostasis of naive CD4(+) T cells. Adoptive transfer of splenocytes from non-obese diabetic (NOD) mice results in increased homeostatic expansion of T cells in lymphopenic NOD.scid.Il15(-/-) mice when compared to NOD.scid recipients. The increased accumulation of CD4(+) T cells is also observed in NOD.Il15(-/-) mice, indicating that IL-15-dependent regulation also occurs in the absence of lymphopenia. NOD.scid mice lacking the IL-15Rα chain, but not those lacking the common gamma chain, also show increased accumulation of CD4(+) T cells. These findings indicate that the IL-15-mediated regulation occurs directly on CD4(+) T cells and requires trans-presentation of IL-15. CD4(+) T cells expanding in the absence of IL-15 signaling do not acquire the characteristics of classical regulatory T cells. Rather, CD4(+) T cells expanding in the absence of IL-15 show impaired antigen-induced activation and IFN-γ production. Based on these findings, we propose that the IL-15-dependent regulation of the naive CD4(+) T-cell compartment may represent an additional layer of control to thwart potentially autoreactive cells that escape central tolerance, while permitting the expansion of memory T cells.

AMPKα1-sensitivity of Orai1 and Ca(2+) entry in T - lymphocytes.

BACKGROUND/AIMS: T-lymphocyte activation and function critically depends on Ca(2+) signaling, which is regulated by store operated Ca(2+) entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) by treatment of the cells with Ca(2+) ionophore or following inhibition of endosomal Ca(2+) ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca(2+) entry and Ca(2+)-sensitive regulation of T-lymphocyte function. METHODS: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk(-/-)) mice and from their wildtype (ampk(+/+)) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca(2+)]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca(2+)]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. RESULTS: Expression of surface markers in CD4(+) and CD8(+) T-cells were similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. SOCE and increase of [Ca(2+)]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. The difference of Ca(2+) entry between ampk(-/-) and ampk(+/+) T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk(-/-) lymphocytes was higher than proliferation of ampk(+/+) T-lymphocytes, a difference reversed by Orai1 silencing. CONCLUSIONS: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca(2+) activity.

SOCS1 prevents potentially skin-reactive cytotoxic T lymphocytes from gaining the ability to cause inflammatory lesions.

Suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of T lymphocyte homeostasis. SOCS1-deficient mice accumulate CD8(+) T cells, which display a memory-like phenotype and proliferate strongly to IL-15. Socs1(-/-) mice develop inflammatory skin lesions, however, the underlying mechanisms are not well understood. In order to investigate the role of SOCS1 in regulating CD8(+) T cells potentially reactive to tissue antigens (Ags) of the skin, we generated Socs1(-/-) mice expressing MHC-I-restricted Pmel-1 transgenic TCR specific to the melanoma-derived gp100 Ag, which is also expressed by normal melanocytes. Socs1(-/-) Pmel-1 cells express increased levels of memory markers CD44, Ly6C, CD122, and CD62L, and show downregulation of TCR and upregulation of CD5, suggesting in vivo TCR stimulation. However, stimulation of Socs1(-/-)Pmel-1 cells with gp100-derived peptide induced only marginal proliferation in vitro despite eliciting strong effector functions, which was associated with elevated Blimp-1 induction. Following adoptive transfer to Rag1(-/-) mice, Socs1(-/-)Pmel-1 cells underwent lymphopenia-induced proliferation and caused severe skin pathology characterized by inflammatory lesions in ears, muzzle, extremities, and eyes. These findings underscore the importance of SOCS1 in regulating potentially skin-reactive cytotoxic T lymphocytes, which could get activated under conditions that promote Ag-nonspecific, cytokine-driven proliferation.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes.

Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3α,ß. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3α,ß inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3(KI)). T cells from gsk3(KI) mice were compared to T cells from corresponding wild type mice (gsk3(WT)). As a result, in gsk3(KI) CD4(+) cells surface CD62L (L-selectin) was significantly less abundant than in gsk3(WT) CD4(+) cells. Upon activation in vitro T cells from gsk3(KI) mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3(KI) T cells, suggesting that GSK3 induces effector function in CD8(+) T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3α,ß is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells.

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 µg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 µM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.

Rapamycin sensitive ROS formation and Na(+)/H(+) exchanger activity in dendritic cells.

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 µM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 µM) and partially reversed by additional application of tert-butylhydroperoxide (10 µM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.

Effect of thymoquinone on cytosolic pH and Na+/H+ exchanger activity in mouse dendritic cells.

The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na(+)/H(+) exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 µM). Cytosolic pH (pH(i)) was determined from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 µg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 µM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 µM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.