Cover Picture: Eur. J. Immunol. 7'13

The cover features human skin explants that had been pre‐incubated with control peptide, a peptide mimetic of SOCS1 or KIR peptide respectively, prior to stimulation with IFN‐γ. The level of STAT1 phosphorylation was then assessed by immunohistochemistry on cryosections (red staining). The images are taken from the article by Madonna et al. (pp. 1883–1895) in which the authors show that a peptide mimicking the SOCS1 kinase inhibitory region inhibits expression of STAT1‐dependents genes in IFN‐γ‐treated human skin explants. Madonna et al. further show that the SOCS1 mimetic reduces the migration of T cells towards supernatants from mimetic‐treated keratinocytes, indicating therapeutic potential for SOCS1 mimetics in the treatment of type 1 immune‐mediated skin diseases.     

SOCS和IFN对JAK/STAT活化的影响和意义

信号抑制因子(SOCS)是在细胞因子诱导下依赖JAK/STAT通路的信号传导而产生的,继而抑制细胞因子的信号传导,因此SOCS蛋白质被认为可以形成局部经典负反馈回路.IFN是由辅助性T细胞Ⅰ类亚群和自然杀伤细胞等分泌的一种细胞因子,具有多种免疫调节功能.在炎症反应中IFN首先激活JAK,然后再活化与之相结合的STATI和STAT3,从而引起iNOS诱生型一氧化氮表达增强而诱发炎症反应,而IFN在诱导细胞的抗病毒和抗生长反应中也起关键作用.IFN可正向调节SOCS的表达,但是SOCS的过度表达又可以抑制IFN受体的磷酸化及STAT1、STAT3的活化.     

IFN‐γ directly inhibits murine B‐cell precursor leukemia‐initiating cell proliferation early in life

The early‐life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL‐10 and IFN‐γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eμ‐ret transgenic mouse model of B‐cell precursor ALL to assess the influence of IFN‐γ on the early‐life burden of leukemia‐initiating cells. The absence of IFN‐γ activity resulted in greater numbers of leukemia‐initiating cells early in life and was associated with accelerated leukemia onset. The leukemia‐initiating cells from IFN‐γ‐knockout mice had reduced suppressor of cytokine signaling (SOCS‐1) expression, were significantly more sensitive to IFN‐γ, and exhibited more rapid expansion in vivo than their wild‐type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN‐γ‐knockout leukemia cells. These results demonstrate that the influence of IFN‐γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS‐mediated reduction in B‐cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection‐independent cytokine production on leukemogenesis.     

STS‐1 promotes IFN‐α induced autophagy by activating the JAK1‐STAT1 signaling pathway in B cells

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN‐α. IFN‐α induces autophagy via the JAK1‐STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS‐1 (suppressor of T‐cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS‐1 promoted IFN‐α‐induced autophagy in B cells by enhancing the JAK1‐STAT1 signaling activation. STS‐1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c‐cbl, and subsequently promoted IFN‐α‐induced phosphorylation of tyrosine kinase 2, leading to JAK1‐STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN‐α‐induced autophagy promoted by STS‐1, indicating that STS‐1 promotes IFN‐α‐induced autophagy via the JAK1‐STAT1 signaling. Our results demonstrate the importance of STS‐1 in regulating IFN‐α‐induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.     

Decidual NK cell-derived conditioned medium (dNK-CM) mediates VEGF-C secretion in extravillous cytotrophoblasts

Citation Eastabrook GDM, Hu Y, Tan R, Dutz JP, MacCalman CD, von Dadelszen P. Decidual NK cell‐derived conditioned medium (dNK‐CM) mediates VEGF‐C secretion in extravillous cytotrophoblasts. Am J Reprod Immunol 2012; 67: 101–111 Problem The regulatory mechanisms involved in VEGF‐C secretion by trophoblasts during placentation are poorly understood. We investigated whether or not decidual natural killer cell conditioned medium (dNK‐CM) stimulated VEGF‐C secretion in the extravillous cytotrophoblast (EVT) cell line HTR8/SVneo. Method of Study The effects of dNK‐CM and recombinant IFN‐γ on VEGF‐C induction by HTR8/SVneo were studied in the absence or presence of IFN‐γ or its receptor blocking antibodies, p38 inhibitor (SB202190), JAK inhibitor (JAK inhibitor‐1, JI‐1), and on STAT1 knockdown HTR8/SVneo. VEGF‐C was quantified by ELISA. FACS was used to investigate the phosphorylations of Tyr701 or Ser727 of STAT1 on stimulated HTR8/SVneo. Results dNK‐CM facilitated VEGF‐C secretion by HTR8/SVneo. IFN‐γ and IFN‐γR1 or IFN‐γR2 blocking antibodies reduced both dNK‐CM‐ and IFN‐γ‐induced VEGF‐C secretion. Phosphorylations on Tyr701 or Ser727 of STAT1 were elevated upon stimulation. Secretion of VEGF‐C was reduced by treatment with SB202190, JI‐1, or STAT1 knockdown by siRNA. Conclusion VEGF‐C production by trophoblasts is regulated by soluble factors secreted by dNK through p38 and JAK‐STAT1 pathways.     

Activation of AMPK inhibits inflammation in MRL/lpr mouse mesangial cells

Recent reports show that 5‐amino‐4‐imidazole carboxamide riboside (AICAR), a pharmacological activator of AMP‐activated protein kinase (AMPK), inhibits the lipopolysaccharide (LPS)‐induced production of proinflammatory cytokines. MRL/MPJ‐Fas^lpr (MRL/lpr) mice show an intrinsic decreased threshold for the production of inflammatory mediators when stimulated. In our current studies, we sought to determine if AMPK activation would inhibit inflammatory mediator production in stimulated kidney mesangial cells. Cultured mesangial cells from MRL/lpr mice were treated with AICAR and stimulated with LPS/interferon (IFN)‐γ. AICAR decreased dose‐dependently inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 and interleukin‐6 production in LPS/IFN‐γ‐stimulated mesangial cells. Mechanistically, AICAR inhibited the LPS/IFN‐γ‐stimulated PI3K/Akt signalling inflammatory cascade but did not affect LPS/IFN‐γ‐mediated inhibitory kappa B phosphorylation or nuclear factor (NF)‐κB (p65) nuclear translocation. Treatment with the adenosine kinase inhibitor 5′‐iodotubercidin blocked the ability of AICAR to activate AMPK and prevented AICAR from inhibiting the LPS/IFN‐γ‐stimulated PI3K/Akt pathway and attenuating iNOS expression. Taken together, these observations suggest that AICAR inhibits LPS/IFN‐γ‐induced Akt phosphorylation through AMPK activation and may serve as a potential therapeutic target in inflammatory diseases.     

N-acetyl cysteine regulates the phosphorylation of JAK proteins following CD40-activation of human memory B cells

During their development, human B lymphocytes migrate into various environments, each presenting important variations in their redox balance depending on oxygen availability. The modulation of the cells surroundings redox balance leads to the regulation of reactive oxygen species produced by the cell. These molecules are involved in the state of oxidation of the cytosol and affect many pathways involved in cell development, differentiation and protein secretion. B lymphocytes cultured in presence of interleukin (IL)-2, IL-4, IL-10 and under CD154 stimulation, present increases in their intracellular levels of ROS. However, when N-acetyl cysteine (NAC), an antioxidant, is added, STAT3 phosphorylation is decreased. In this study, we show that in activated human memory B cells, NAC inhibited STAT3 phosphorylation on tyrosine 705 but not on Serine 727. Moreover, higher concentrations of NAC decreased STAT3 synthesis. Two other antioxidants, α-tocopherol and Trolox, did not affect STAT3 phosphorylation. Furthermore, two kinases involved in STAT3 activation, known as JAK2 and JAK3, appeared down-regulated in presence of NAC. In parallel, 3 h after antioxidants incubation, we have observed a decrease in SOCS1 and SOCS3 protein levels, which seems time-related to antioxidant treatment. The decrease in the phosphorylation of JAK2 and JAK3, earlier in the process, could explain the downregulation of STAT3 and offer a hypothesis on the mechanism of action of NAC antioxidant properties which were confirmed by a decrease in the level of S-glutathionylation of proteins. The reduced expression of SOCS1 and SOCS3 appears directly linked to the inhibition of this STAT3-regulated pathway. In summary, NAC appears as a potential regulator of the STAT3 pathway.     

Functional conservation of suppressors of cytokine signaling proteins between teleosts and mammals: Atlantic salmon SOCS1 binds to JAK/STAT family members and suppresses type I and II IFN signaling

Suppressor of cytokine signaling (SOCS) proteins are crucially involved in the control of inflammatory responses through their impact on various signaling pathways including the JAK/STAT pathway. Although all SOCS protein family members are identified in teleost fish, their functional properties in non-mammalian vertebrates have not been extensively studied. To gain further insight into SOCS functions in bony fish, we have identified and characterized the Atlantic salmon (Salmo salar) SOCS1, SOCS2 and CISH genes. These genes exhibited sequence conservation with their mammalian counterparts and they were ubiquitously expressed. SOCS1 in mammalian species has been recognized as a key negative regulator of interferon (IFN) signaling and recent data for the two model fish Tetraodon (Tetraodon nigroviridis) and zebrafish (Danio rerio) suggest that these functions are conserved from teleost to mammals. In agreement with this we here demonstrate a strong negative regulatory activity of salmon SOCS1 on type I and type II IFN signaling, while SOCS2a and b and CISH only moderately affected IFN responses. SOCS1 also inhibited IFNγ-induced nuclear localization of STAT1 and a direct interaction between SOCS1 and STAT1 and between SOCS1 and the Tyk2 kinase was found. Using SOCS1 mutants lacking either the KIR domain or the ESS, SH2 and SOCS box domains showed that all domains affected the ability of SOCS1 to inhibit IFN-mediated signaling. These results are the first to demonstrate that SOCS1 is a potent inhibitor of IFN-mediated JAK-STAT signaling in teleost fish.     

BJ‐2266 ameliorates experimental autoimmune encephalomyelitis through down‐regulation of the JAK/STAT signaling pathway

CD4⁺ T cells differentiate into distinct effector subsets upon antigenic stimulation. Cytokines, and micro‐environmental factors present during T‐cell priming, direct differentiation of naïve CD4⁺ T cells into pro‐inflammatory Th1 and Th17 cells. From extensive screening of 2,4,5‐trimethylpyridin‐3‐ol derivatives with various functional groups at C(6)‐position, BJ‐2266, a 6‐thioureido‐derivative, showed potent inhibitory activity on in vitro T helper (Th)‐cell differentiation. This compound inhibited IFN‐γ and IL‐17 production from polyclonal CD4⁺ T cells and ovalbumin (OVA)‐specific CD4⁺ T cells that were activated by T‐cell receptor (TCR) engagement. We assessed the inhibitory effect of BJ‐2266 in experimental autoimmune encephalomyelitis (EAE). Our results suggest that BJ‐2266 treatment significantly suppresses EAE disease progression with reduced generation of Th1 and Th17 cells. Notably, Th‐cell differentiation was significantly suppressed by BJ‐2266 treatment with no effect on apoptosis, activation and proliferation of activated T cells. Furthermore, adoptive transfer of BJ‐2266 treated MOG‐reactive Th1 and Th17 cells led to a lower EAE disease score and better clinical recovery from EAE. The underlying mechanism of BJ‐2266 effect involved the inhibition of JAK/STAT phosphorylation that is critical for Th‐cell differentiation. We conclude that BJ‐2266 regulates the JAK/STAT pathway in response to cytokine signals and subsequently suppresses the differentiation of Th‐cell responses.     

Counter-regulation mechanism of IL-4 and IFN-α signal transduction through cytosolic retention of the pY-STAT6:pY-STAT2:p48 complex

IFN‐α and IL‐4 induce Th1 and Th2 responses, respectively, and often display antagonistic actions against each other. To elucidate the molecular mechanism of counter‐regulation, we have investigated the signal interception by IFN‐α and IL‐4, employing a human B‐cell line Ramos, sensitive to both cytokines. In these cells, IFN‐α effectively inhibited IL‐4‐induced Fc epsilon receptor II (CD23) expression, whereas IL‐4 suppressed IFN‐α‐mediated IRF7 expression. The counter‐regulatory action by IL‐4 and IFN‐α proceeded with a delayed kinetics requiring 4 h. Notably, IFN‐α did not affect the IL‐4‐induced tyrosine phosphorylation of STAT6, but induced a time‐dependent cytoplasmic accumulation of phosphotyrosine(pY)‐STAT6 and a corresponding decrease in nuclear pY‐STAT6. By confocal analysis and co‐immunoprecipitation assays, we demonstrated the colocalization and molecular interaction of IL‐4‐induced pY‐STAT6 with IFN‐α‐induced pY‐STAT2:p48 in the cytosol. In addition, the over‐expression of STAT2 or STAT6 induced the concomitant cytosolic accumulation of pY‐STAT6 or pY‐STAT2, leading to the suppression of IL‐4‐induced CD23 or IFN‐α‐induced IRF7 gene expression, respectively. Our data suggest that the signals ensued by IFN‐α and IL‐4 induce cytoplasmic sequestration of IL‐4‐activated STAT6 and IFN‐α‐activated STAT2:p48 in B cells through the formation of pY‐STAT6:pY‐STAT2:p48 complex, which provides a novel mechanism by which IFN‐α and IL‐4 cross‐regulate their signaling into the nucleus.