SOCS3-deficient Mice Research Articles

Fibroblast-specific SOCS3 deficient mice exhibits decreased myocardial fibrosis and increased cardiomyocyte survival after acute myocardial infarction

Abstract Background Left ventricular (LV) remodeling after acute myocardial infarction (AMI) results in poor cardiac performance leading to heart failure. LV remodeling after AMI is characterized by infarct expansion, LV dilation, and fibrosis of viable myocardium. Suppressor of cytokine signaling-3 (SOCS3) is an endogenous negative-feedback regulator of the STAT3 signaling pathway. We previously reported that myocardial SOCS3 plays a detrimental role in LV remodeling after AMI; however, the role of STAT3 and SOCS3 within fibroblast in LV remodeling after AMI remains elusive. Objective The aim of this study is to clarify the role of STAT3 and SOCS3 within fibroblasts in LV remodeling during AMI in mice. Methods To investigate the role of STAT3 signaling and SOCS3 within fibroblast, we generated tamoxifen-induced fibroblast-specific SOCS3-deficient (fib-SOCS3 KO) mice. AMI was induced by permanent ligation of the left anterior descending artery in mice. After 2 weeks of induction with tamoxifen, histological evaluation by Hematoxylin Eosin staining and Picro-Sirius Red staining, cardiac function evaluation by echocardiography using a Vevo 3100 Echo Imaging System (Visual Sonics), and protein expression using Western blot analysis were performed. Results There was no significant difference in 2-week survival rate between fib-SOCS3 KO group and wild type (WT). Two weeks after AMI, ventricular weight was less in fib-SOCS3 KO mice compared to WT mice (p<0.05). In terms of cardiac function, LV ejection fraction was predominantly increased in fib-SOCS3 KO mice compared with WT mice, as measured by the Area method (p<0.05). There was no difference in LV end-diastolic area, but LV end-systolic area was significantly less in fib-SOCS3 KO mice than in WT mice. Histologically, residual viable myocardium in the infarcted area was greater in fib-SOCS3 KO mice compared with WT mice. The percentage of fibrotic area in the LV short-axis plane was significantly less in fib-SOCS3 KO mice than in WT mice. Western blot analysis revealed that phosphorylated-STAT3 tended to be elevated in fib-SOCS3 KO mice than in WT mice. Conclusions Our data suggest that STAT3 and SOCS3 within fibroblast may play an important role in the prevention of LV remodeling after AMI.

Socs3 expression in myeloid cells modulates the pathogenesis of dextran sulfate sodium (DSS)-induced colitis.

Myeloid cells play a critical role in the pathogenesis of Inflammatory Bowel Diseases (IBDs), including Ulcerative Colitis (UC) and Crohn's Disease (CD). Dysregulation of the JAK/STAT pathway is associated with many pathological conditions, including IBD. Suppressors Of Cytokine Signaling (SOCS) are a family of proteins that negatively regulate the JAK/STAT pathway. Our previous studies identified that mice lacking Socs3 in myeloid cells developed a hyper-activated phenotype of macrophages and neutrophils in a pre-clinical model of Multiple Sclerosis. To better understand the function of myeloid cell Socs3 in the pathogenesis of colitis, mice with Socs3 deletion in myeloid cells (Socs3 ΔLysM) were utilized in a DSS-induced colitis model. Our results indicate that Socs3 deficiency in myeloid cells leads to more severe colitis induced by DSS, which correlates with increased infiltration of monocytes and neutrophils in the colon and increased numbers of monocytes and neutrophils in the spleen. Furthermore, our results demonstrate that the expression of genes related to the pathogenesis and diagnosis of colitis such as Il1β, Lcn2, S100a8 and S100a9 were specifically enhanced in Socs3-deficient neutrophils localized to the colon and spleen. Conversely, there were no observable differences in gene expression in Ly6C+ monocytes. Depletion of neutrophils using a neutralizing antibody to Ly6G significantly improved the disease severity of DSS-induced colitis in Socs3-deficient mice. Thus, our results suggest that deficiency of Socs3 in myeloid cells exacerbates DSS-induced colitis and that Socs3 prevents overt activation of the immune system in IBD. This study may provide novel therapeutic strategies to IBD patients with hyperactivated neutrophils.

Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

ObjectiveRecently, podocytes have been recognised not only as a physical barrier to prevent urinary protein loss but also as producers of proinflammatory cytokines. However, the roles of podocytes in the...

Prognostic significance of SOCS1 and SOCS3 tumor suppressors and oncogenic signaling pathway genes in hepatocellular carcinoma

BackgroundSOCS1 and SOCS3 genes are considered tumor suppressors in hepatocellular carcinoma (HCC) due to frequent epigenetic repression. Consistent with this notion, mice lacking SOCS1 or SOCS3 show increased susceptibility to diethylnitrosamine (DEN)-induced HCC. As SOCS1 and SOCS3 are important regulators of cytokine and growth factor signaling, their loss could activate oncogenic signaling pathways. Therefore, we examined the correlation between SOCS1/SOCS3 and key oncogenic signaling pathway genes as well as their prognostic significance in HCC.MethodsThe Cancer Genome Atlas dataset on HCC comprising clinical and transcriptomic data was retrieved from the cBioportal platform. The correlation between the expression of SOCS1 or SOCS3 and oncogenic pathway genes was evaluated using the GraphPad PRISM software. The inversely correlated genes were assessed for their impact on patient survival using the UALCAN platform and their expression quantified in the regenerating livers and DEN-induced HCC tissues of mice lacking Socs1 or Socs3. Finally, the Cox proportional hazards model was used to evaluate the predictive potential of SOCS1 and SOCS3 when combined with the genes of select oncogenic signaling pathways.ResultsSOCS1 expression was comparable between HCC and adjacent normal tissues, yet higher SOCS1 expression predicted favorable prognosis. In contrast, SOCS3 expression was significantly low in HCC, yet it lacked predictive potential. The correlation between SOCS1 or SOCS3 expression and key genes of the cell cycle, receptor tyrosine kinase, growth factor and MAPK signaling pathways were mostly positive than negative. Among the negatively correlated genes, only a few showed elevated expression in HCC and predicted survival. Many PI3K pathway genes showed mutual exclusivity with SOCS1 and/or SOCS3 and displayed independent predictive ability. Among genes that negatively correlated with SOCS1 and/or SOCS3, only CDK2 and AURKA showed corresponding modulations in the regenerating livers and DEN-induced tumors of hepatocyte-specific Socs1 or Socs3 deficient mice and predicted patient survival. The Cox proportional hazards model identified the combinations of SOCS1 or SOCS3 with CXCL8 and DAB2 as highly predictive.ConclusionsSOCS1 expression in HCC has an independent prognostic value whereas SOCS3 expression does not. The predictive potential of SOCS1 expression is increased when combined with other oncogenic signaling pathway genes.

P1627Increased pericardial fibrosis and cardiac dysfunction in smooth muscle cell-specific SOCS3 deficient mice

Abstract Background Suppressor of cytokine signaling-3 (SOCS3) is an intrinsic negative-feedback regulator of signal transducer and activator of transcription-3 (STAT3) signaling pathway. We have previously shown that myocardial SOCS3 plays an important role in cardiac hypertrophy and survival; however, the role of SOCS3 in smooth muscle cells in cardiovascular pathophysiology remains elusive. In this study, we determined whether STAT3 and SOCS3 in smooth muscle cells would play a role in cardiovascular pathophysiology. Methods and results To target inactivation of the SOCS3 gene to smooth muscle cells, SOCS3-flox mice were bred with transgenic mice expressing Cre recombinase under control of the mouse SM22-α promoter (sm-SOCS3-KO mice). Left ventricular weight to body weight ratio was significantly increased in sm-SOCS3-KO mice compared with wild-type mice at 52 weeks of age (p<0.05). Echocardiographic analyses of sm-SOCS3-KOmice showed significantly decreased cardiac function compared with wild-typefrom 52 weeks of age (p<0.05). Interestingly, Sirius-red staining revealed that thickness of pericardium in sm-SOCS3-KOmice was markedly greater compared with wild-typemice at 52 weeks of age (p<0.05). Cardiac interstitial fibrosis in sm-SOCS3-KOmice was also greater compared with wild-typemice (p<0.05). Western blot analyses showed that phosphorylated STAT3 was significantly increased in sm-SOCS3-KOhearts compared with wild-typemice at 52 weeks of age (p<0.05), whereas no significant differences were observed at 7 weeks of age. Immunostaining revealed that phosphorylated STAT3 positive cells were in thickened pericardial area in sm-SOCS3-KOhearts. Some infiltrated inflammatory cells were also found in thickened pericardial area in sm-SOCS3-KOmice hearts. Conclusion These results suggest that STAT3 and its negative-feedback regulator SOCS3 in smooth muscle cells play an important role in the pathogenesis of pericardial fibrosis and cardiac dysfunction.

Lack of SOCS3 increases LPS-induced murine acute lung injury through modulation of Ly6C(+) macrophages

BackgroundSOCS3 (suppressor of cytokine signaling 3) is a negative regulator of JAK/STAT3 signaling pathway and participates in the regulation of lung inflammation in a mouse model with acute lung injury (ALI). However, it is not well understood how SOCS3 regulates lung inflammation in the ALI mouse model.MethodIn the present study, we investigated the effects of SOCS3 on modulation of Ly6C(+) monocyte phenotypes in a mouse model with lipopolysaccharide (LPS)-induced ALI. Conditional SOCS3(Lyz2cre) mice with myeloid cell-restricted depletion of SOCS3 gene were created by breeding transgenic Lyz2Cre mice with SOCS3(fl/fl) mice. Wilde-type (WT) and SOCS3(Lyz2cre) mice were intratracheal instilled with 5 mg/kg LPS for 2 days. Lung, bronchoalveolar lavage (BAL) and blood were collected for analysis by flow cytometry, ELISA, qRT-PCR and Western blot analysis.ResultsThe studies in the ALI mouse model revealed that myeloid cell-restricted SOCS3 deficiency exacerbated the severity of ALI as compared to the WT mice. The increased severity of ALI in SOCS3-deficient mice was associated with higher populations of neutrophils, T lymphocytes and Ly6C(+) monocytes in the inflamed lung tissues. In addition, CCR2 and CXCL15 were elevated, and accompanied by greater expression and activation of STAT3 in the lung of SOCS3-deficient mice. SOCS3-deficient bone marrow-derived macrophages (BMDMs) expressed a higher amount of TNF-alpha, and adoptive transfer of the SOCS3-deficient Ly6C(+) BMDMs into WT mice enhanced the severity of ALI than adoptive transfer of WT control BMDMs. However, depletion of Ly6C(+) circulating monocytes by anti-Ly6C(+) neutralizing antibody moderately attenuated neutrophil infiltration and resulted in lower prevalence of Ly6C(+) cells in the lung of treated mice.ConclusionMyeloid cell-restricted lack of SOCS3 induced more severe ALI through modulation of Ly6C(+) subtype macrophages. The results provide insight into a new role of SOCS3 in modulation of Ly6C(+) monocyte phenotypes and provide a novel therapeutic strategy for ALI by molecular intervention of macrophages subtypes.

Suppressor of Cytokine Signaling 3 in Macrophages Prevents Exacerbated Interleukin-6-Dependent Arginase-1 Activity and Early Permissiveness to Experimental Tuberculosis.

Suppressor of cytokine signaling 3 (SOCS3) is a feedback inhibitor of interleukin (IL)-6 signaling in macrophages. In the absence of this molecule, macrophages become extremely prone to an IL-6-dependent expression of arginase-1 (Arg1) and nitric oxide synthase (NOS)2, the prototype markers for alternative or classical macrophage activation, respectively. Because both enzymes are antipodean macrophage effector molecules in Mycobacterium tuberculosis (Mtb) infection, we assessed the relevance of SOCS3 for macrophage activation during experimental tuberculosis using macrophage-specific SOCS3-deficient (LysMcreSOCS3loxP/loxP) mice. Aerosol infection of LysMcreSOCS3loxP/loxP mice resulted in remarkably higher bacterial loads in infected lungs and exacerbated pulmonary inflammation. This increased susceptibility to Mtb infection was accompanied by enhanced levels of both classical and alternative macrophage activation. However, high Arg1 expression preceded the increased induction of NOS2 and at early time points of infection mycobacteria were mostly found in cells positive for Arg1. This sequential activation of Arg1 and NOS2 expression in LysMcreSOCS3loxP/loxP mice appears to favor the initial replication of Mtb particularly in Arg1-positive cells. Neutralization of IL-6 in Mtb-infected LysMcreSOCS3loxP/loxP mice reduced arginase activity and restored control of mycobacterial replication in LysMcreSOCS3loxP/loxP mice. Our data reveal an unexpected role of SOCS3 during experimental TB: macrophage SOCS3 restrains early expression of Arg1 and helps limit Mtb replication in resident lung macrophages, thereby limiting the growth of mycobacteria. Together, SOCS3 keeps IL-6-dependent divergent macrophage responses such as Nos2 and Arg1 expression under control and safeguard protective macrophage effector mechanisms.

Context-dependent effects of SOCS3 in angiotensin II-induced vascular dysfunction and hypertension in mice: mechanisms and role of bone marrow-derived cells.

Carotid artery disease is a major contributor to stroke and cognitive deficits. Angiotensin II (Ang II) promotes vascular dysfunction and disease through mechanisms that include the IL-6/STAT3 pathway. Here, we investigated the importance of suppressor of cytokine signaling 3 (SOCS3) in models of Ang II-induced vascular dysfunction. We examined direct effects of Ang II on carotid arteries from SOCS3-deficient (SOCS3(+/-)) mice and wild-type (WT) littermates using organ culture and then tested endothelial function with acetylcholine (ACh). A low concentration of Ang II (1 nmol/l) did not affect ACh-induced vasodilation in WT but reduced that of SOCS3(+/-) mice by ∼50% (P < 0.05). In relation to mechanisms, effects of Ang II in SOCS3(+/-) mice were prevented by inhibitors of STAT3, IL-6, NF-κB, or superoxide. Systemic Ang II (1.4 mg/kg per day for 14 days) also reduced vasodilation to ACh in WT. Surprisingly, SOCS3 deficiency prevented most of the endothelial dysfunction. To examine potential underlying mechanisms, we performed bone marrow transplantation. WT mice reconstituted with SOCS3(+/-) bone marrow were protected from Ang II-induced endothelial dysfunction, whereas reconstitution of SOCS3(+/-) mice with WT bone marrow exacerbated Ang II-induced effects. The SOCS3 genotype of bone marrow-derived cells did not influence direct effects of Ang II on vascular function. These data provide new mechanistic insight into the influence of SOCS3 on the vasculature, including divergent effects depending on the source of Ang II. Bone marrow-derived cells deficient in SOCS3 protect against systemic Ang II-induced vascular dysfunction.

Abstract B51: SOCS3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cell leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributes of SOCS3. Citation Format: Hao Yu, Yudong Liu, Jessy Deshane, Etty (Tika) Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B51. doi:10.1158/1538-7445.CHTME14-B51

Abstract A25: SOCS3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3. Citation Format: Hao Yu, Yudong Liu, Braden C. McFarland, Etty N. Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A25. doi:10.1158/1538-7445.CHTME14-A25

Abstract 1068: Socs3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3. Citation Format: Hao Yu, Hongwei Qin. Socs3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2014-1068

AB0150 Regulation of SOCS1, SOCS2 and SOCS3 expression in patients with early arthritis

BackgroundMost cytokines involved in the pathogenesis of rheumatoid arthritis (RA) act through receptors that trigger distinct signalling pathways that are initiated through activation of the JAK/STAT system which up-regulates the...

Abstract A80: Role of myeloid SOCS3 in prostate cancer progression.

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins, negative regulators of the JAK/STAT pathway, have been implicated in having a key protective role in tumor initiation and progression. However, the role of SOCS3 in regulating the differentiation and functions of myeloid-derived suppressor cells (MDSC) is poorly understood. Recent studies show the important role of MDSCs in promoting tumor progression as well as metastasis by regulating immune surveillance. To determine the role of myeloid SOCS3 in tumor progression, Conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3fl/fl) and SOCS3fl/fl C57BL/6 mice were used as recipients for subcutaneous transplantation of TRAMP-derived mouse prostate tumor cells. Our results indicate that tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Gr1+/CD11b+ cells in both spleen and tumor, and less CD8+ T-cell infiltration in tumors. Tumors from myeloid-specific SOCS3-deficient mice also express high levels of Arginase-1 and have elevated Akt signaling. In vitro, SOCS3-deficient bone-marrow-derived mononuclear cells (BDMC) exhibit heightened STAT3 activation and are subject to differentiation towards the MDSC phenotype. These findings suggest that myeloid cell SOCS3 provides protection from tumor progression by regulating MDSC differentiation. Citation Format: Hao Yu, Yudong Liu, Etty N. Benveniste, Hongwei Qin. Role of myeloid SOCS3 in prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A80.

Inactivation ofSocs3in the Hypothalamus Enhances the Hindbrain Response to Endogenous Satiety Signals via Oxytocin Signaling

Leptin is an adipocyte-derived hormone that controls energy balance by acting primarily in the CNS, but its action is lost in common forms of obesity due to central leptin resistance. One potential mechanism for such leptin resistance is an increased hypothalamic expression of Suppressor of cytokine signaling 3 (Socs3), a feedback inhibitor of the Jak-Stat pathway that prevents Stat3 activation. Ample studies have confirmed the important role of Socs3 in leptin resistance and obesity. However, the degree to which Socs3 participates in the regulation of energy homeostasis in nonobese conditions remains largely undetermined. In this study, using adult mice maintained under standard diet, we demonstrate that Socs3 deficiency in the mediobasal hypothalamus (MBH) reduces food intake, protects against body weight gain, and limits adiposity, suggesting that Socs3 is necessary for normal body weight maintenance. Mechanistically, MBH Socs3-deficient mice display increased hindbrain sensitivity to endogenous, meal-related satiety signals, mediated by oxytocin signaling. Thus, oxytocin signaling likely mediates the effect of hypothalamic leptin on satiety circuits of the caudal brainstem. This provides an anatomical substrate for the effect of leptin on meal size, and more generally, a mechanism for how the brain controls short-term food intake as a function of the energetic stores available in the organism to maintain energy homeostasis. Any dysfunction in this pathway could potentially lead to overeating and obesity.

O016 Inhibition of the JAK/STAT pathway protects against neuroinflammation

Introduction Suppressor Of Cytokine Signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT activation, cytokine signaling and inflammatory gene expression in macrophages/microglia. Methods To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3fl/fl) were tested for experimental autoimmune encephalomyelitis (EAE). Results The myeloid specific SOCS3-deficient mice are vulnerable to MOG-induced EAE, with a severe, non-resolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid specific SOCS3-deficient mice, as is enhanced STAT3/4 signaling and expression of inflammatory cytokines/chemokines, and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation, and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of anti-inflammatory M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses. LysMCre-SOCS3fl/fl mice are also protected from atypical EAE by the administration of a JAK/STAT3 inhibitor, AZD1480. Severity of disease is attenuated, which is associated with a reduction in STAT1/3 activation, less Th1/Th17 cell infiltration in the cerebellum, reductions in inflitrating neutrophils and macrophages, and decreased M1 proinflammatory gene expression. Mice with classical EAE are also protected against disease severity by treatment with AZD1480. Conclusion Collectively, these findings indicate the JAK/STAT3 signaling pathway may serve as a therapeutic target for neuroinflammatory diseases.

Signal transducer and activator of transcription-3/suppressor of cytokine signaling-3 (STAT3/SOCS3) axis in myeloid cells regulates neuroinflammation

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3(fl/fl)) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses.

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30-120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity.

Aberrant IL-4 production by SOCS3-over-expressing T cells during infection with Leishmania major exacerbates disease manifestations

Suppressor of cytokine signaling (SOCS) 3 is a major negative feedback regulator of signal transducer and activator of transcription 3-activating cytokines. Studies using T-cell-specific SOCS3-deficient mice indicate that the absence of SOCS3 in T cells results in exacerbation of disease progression after infection by Leishmania major due to skewing of the T(h)3 cell phenotype accompanied by hyper-production of IL-10 and transforming growth factor β (TGF-β). Here we show that transgenic mice over-expressing the SOCS3 gene in T cells (Lck-SOCS3 Tg mice) are also susceptible to infection by L. major. Forced expression of SOCS3 in T cells did not affect the production of the anti-inflammatory cytokines IL-10 and TGF-β or that of the protective T(h)1 type cytokine IFN-γ, which is required for parasite clearance. CD4(+) T cells isolated from infected-Lck-SOCS3 Tg mice produced much higher levels of IL-4 when they were re-stimulated with L. major antigen in vitro. Exacerbation of disease progression in Lck-SOCS3 Tg mice was completely reversed by administration of a neutralizing antibody against IL-4. These data suggest that tight regulation of SOCS3 expression in T(h) cells is crucial for disease control during infection by L. major.

Leukemia Inhibitory Factor Regulates Trophoblast Giant Cell Differentiation via Janus Kinase 1-Signal Transducer and Activator of Transcription 3-Suppressor of Cytokine Signaling 3 Pathway

Suppressor of cytokine signaling 3 (SOCS3) inhibits leukemia-inhibitory factor (LIF) signaling and acts as a negative regulator. Deletion of SOCS3 causes embryonic lethality because of placental failure, and genetic reduction of LIF or the LIF receptor (LIFR) in SOCS3-deficient mice rescues placental defects and embryonic lethality; this indicates that SOCS3 is an essential inhibitor of LIFR signaling. However, the downstream signaling molecule that acts as a link between the LIFR and SOCS3 has not been identified. In this study we explored the downstream signaling of LIFR. The administration of LIF to SOCS3-heterozygous pregnant mice promotes trophoblast giant cell differentiation and accelerates placental failure in SOCS3-deficient mice. SOCS3-deficient trophoblast stem cells show enhanced and prolonged signal transducer and activator of transcription 3 (Stat3) activation by LIF stimulation. Further, in the trophoblasts of SOCS3-deficient placenta and differentiating cells from the choriocarcinoma-derived cell line Rcho-1 cells, constitutive activation of Stat3 is observed. The forced expression of SOCS3, dominant-negative Stat3, and dominant-negative Janus kinase 1 (JAK1) in Rcho-1 cells significantly suppressed the trophoblast giant cell differentiation of these cells. In addition, the number of trophoblast giant cells is significantly reduced concomitant with an increased number of precursor trophoblasts in JAK1-deficient placentas. Finally, JAK1 deficiency rescues placental defects and embryonic lethality in SOCS3-deficient mice. These results indicate that the LIFR signaling is finely coordinated by JAK1, Stat3, and SOCS3 and regulates trophoblast giant cell differentiation. In addition, these data establish that LIFR-JAK1-Stat3-SOCS3 signaling is an essential pathway for the regulation of trophoblast giant cell differentiation.

Suppression of SOCS3 expression in the pancreatic β-cell leads to resistance to type 1 diabetes

Type 1 diabetes results from the selective destruction of insulin-producing pancreatic β-cells during islet inflammation, which involves inflammatory cytokines and free radicals. However, mechanisms for protecting β-cells from destruction have not been clarified. In this study, we define the role of SOCS3 on β-cell destruction using β-cell-specific SOCS3-conditional knockout (cKO) mice. The β-cell-specific SOCS3-deficient mice were resistant to the development of diabetes caused by streptozotocin (STZ), a genotoxic methylating agent, which has been used to trigger β-cell destruction. The islets from cKO mice demonstrated hyperactivation of STAT3 and higher induction of Bcl-xL than did islets from WT mice, and SOCS3-deficient β-cells were more resistant to apoptosis induced by STZ in vitro than were WT β-cells. These results suggest that enhanced STAT3 signaling protects β-cells from destruction induced by a genotoxic stress and that STAT3/SOCS3 can be a potential therapeutic target for the treatment of type 1 diabetes.