Deletion Of Suppressor Of Cytokine Signaling 3 Research Articles

Abstract 14924: SM22-Cre-Mediated SOCS3 Deficient Mice Exhibit Pericardial Fibrosis, Diastolic Dysfunction, and Increased Mortality Rates Due to Augmented Activation of STAT3 in Fibroblasts

Background: Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible potent negative regulator of STAT3 signaling pathway. Here, we aimed to determine whether SOCS3 in non-cardiomyocytes would play a role in cardiovascular pathophysiology. Methods and Results: To investigate the role of SOCS3 in non-cardiomyocytes, we generated conditional knockout mice by crossing SOCS3-flox mice with SM22-α-Cre mice (SM22-SOCS3-KO mice). Mortality rates in the SM22-SOCS3-KO mice was significantly higher than that in the wild-type mice (WT). Heart weight to body weight ratio was significantly increased in SM22-SOCS3-KO mice compared with WT at 12 months of age. Echocardiographic analyses revealed that SM22-SOCS3-KO mice showed significantly increased left ventricular diastolic dysfunction compared with WT from 12 months of age. Sirius-red staining revealed that thickness of pericardium and cardiac interstitial fibrosis in SM22-SOCS3-KO mice were markedly greater compared with WT mice at 12 months of age. Western blot analyses showed that phosphorylated STAT3 was significantly increased in SM22-SOCS3-KO hearts compared with WT mice at 12 months of age. PCR array analysis revealed that the expression of pro-fibrotic ctgf , pdgf , and TGF-beta family genes including tgf-beta1 , tgf-beta2 , and tgf-beta3 , were significantly higher in SM22-SOCS3-KO hearts than those in WT at 6 months of age. We also found that circulating interleukin-6 concentration was significantly higher in SM22-SOCS3-KO mice compared with WT mice at 12 months of age. After interleukin-6 administration, in the heart tissue from SM22-SOCS3-KO mice, coimmunostaining of phosphorylated STAT3 and ER-TR7 revealed that STAT3 activation occurred in fibroblasts in the coronary arterial adventitia and pericardium. Conclusion: Thus, SM22-Cre-mediated SOCS3 deletion induces higher mortality rates, increased pericardial fibrosis, cardiac interstitial fibrosis, and increased diastolic dysfunction in aging mice, possibly through the increased activation of STAT3 in fibroblasts.

SOCS3 deletion in effector T cells confers an anti-tumorigenic role of IL-6 to the pro-tumorigenic cytokine

Interleukin (IL)-6 is abundantly expressed in the tumor microenvironment and is associated with poor patient outcomes. Here, we demonstrate that the deletion of the suppressor of cytokine signaling 3 (SOCS3) in Tcells potentiates anti-tumor immune responses by conferring the anti-tumorigenic function of IL-6 in mouse and human models. In Socs3-deficient CD8+ Tcells, IL-6 upregulates the expression of type I interferon (IFN)-regulated genes and enhances the anti-tumor effector function of Tcells, while also modifying mitochondrial fitness to increase mitochondrial membrane potential and reactive oxygen species (ROS) levels and to promote metabolic glycolysis in the energy state. Furthermore, Socs3 deficiency reduces regulatory Tcells and increases T helper 1 (Th1) cells. SOCS3 knockdown in human chimeric antigen receptor T (CAR-T) cells exhibits a strong anti-tumor response in humanized mice. Thus, genetic disruption of SOCS3 offers an avenue to improve the therapeutic efficacy of adoptive Tcell therapy.

Loss of aryl hydrocarbon receptor suppresses the response of colonic epithelial cells to IL22 signaling by upregulating SOCS3.

IL22 signaling plays an important role in maintaining gastrointestinal epithelial barrier function, cell proliferation, and protection of intestinal stem cells from genotoxicants. Emerging studies indicate that the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, promotes production of IL22 in gut immune cells. However, it remains to be determined if AhR signaling can also affect the responsiveness of colonic epithelial cells to IL22. Here, we show that IL22 treatment induces the phosphorylation of STAT3, inhibits colonic organoid growth, and promotes colonic cell proliferation in vivo. Notably, intestinal cell-specific AhR knockout (KO) reduces responsiveness to IL22 and compromises DNA damage response after exposure to carcinogen, in part due to the enhancement of suppressor of cytokine signaling 3 (SOCS3) expression. Deletion of SOCS3 increases levels of pSTAT3 in AhR KO organoids, and phenocopies the effects of IL22 treatment on wild-type (WT) organoid growth. In addition, pSTAT3 levels are inversely associated with increased azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumorigenesis in AhR KO mice. These findings indicate that AhR function is required for optimal IL22 signaling in colonic epithelial cells and provide rationale for targeting AhR as a means of reducing colon cancer risk.NEW & NOTEWORTHY AhR is a key transcription factor controlling expression of IL22 in gut immune cells. In this study, we show for the first time that AhR signaling also regulates IL22 response in colonic epithelial cells by modulating SOCS3 expression.

Hyaloid Vasculature as a Major Source of STAT3+ (Signal Transducer and Activator of Transcription 3) Myeloid Cells for Pathogenic Retinal Neovascularization in Oxygen-Induced Retinopathy.

Myeloid cells are critically involved in inflammation-induced angiogenesis, although their pathogenic role in the ischemic retina remains controversial. We hypothesize that myeloid cells contribute to pathogenic neovascularization in retinopathy of prematurity through STAT3 (signal transducer and activator of transcription 3) activation. Approach and Results: Using the mouse model of oxygen-induced retinopathy, we show that myeloid cells (CD45+IsolectinB4 [IB4]+) and particularly M2-type macrophages (CD45+ Arg1+), comprise a major source of STAT3 activation (pSTAT3) in the immature ischemic retina. Most of the pSTAT3-expressing myeloid cells concentrated at the hyaloid vasculature and their numbers were strongly correlated with the severity of pathogenic neovascular tuft formation. Pharmacological inhibition of STAT3 reduced the load of IB4+ cells in the hyaloid vasculature and significantly reduced the formation of pathogenic neovascular tufts during oxygen-induced retinopathy, leading to improved long-term visual outcomes (ie, increased retinal thickness and scotopic b-wave electroretinogram responses). Genetic deletion of SOCS3 (suppressor of cytokine signaling 3), an endogenous inhibitor of STAT3, in myeloid cells, enhanced pathological and physiological neovascularization in oxygen-induced retinopathy, indicating that myeloid-STAT3 signaling is crucial for retinal angiogenesis. Circulating myeloid cells may migrate to the immature ischemic retina through the hyaloid vasculature and contribute to retinal neovascularization via activation of STAT3. Understanding how STAT3 modulates myeloid cells for vascular repair/pathology may provide novel therapeutic options in pathogenic angiogenesis.

SOCS3-deficient lung epithelial cells uptaking neutrophil-derived SOCS3 worsens lung influenza infection

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of TBK1 and interferon pathway and the expression of SOCS3 is closely correlated with symptoms of influenza patients. However, whether deletion of Socs3 in the lung epithelial cells would affect influenza lung replication and inflammation in vivo is unknown. To test this, we approached the influenza infected Socs3f/f and SpcCre.Socs3f/f mice. We first found that knockdown of Socs3 in lung epithelial cells reduced influenza replication. However, in the in vivo study, there was a reduction of SOCS3 in the influenza-infected neutrophils coincided with an increase of SOCS3 in the CD45−CD326+ lung epithelial cells in PR8-infected SpcCre.Socs3f/f mice. SOCS3-deficient neutrophils expressed higher levels of IL-17 that enhanced chemokine expression in the lung epithelial cells. Lung SOCS3-dificient epithelial cells increased expression of GM-CSF and PGE2 which promoted SpcCre.Socs3f/f neutrophils to yield SOCS3. SpcCre.Socs3f/f lung epithelial cells internalized SOCS3 released from GM-CSF + PGE2-stimulated SpcCre.Socs3f/f neutrophils, which could boost influenza replication in the lung epithelial cells. Thus, in the in vivo study, deletion of SOCS3 from lung epithelium could be nullified by the uptake from SOCS3 from infiltrated neutrophils. In addition, deletion of Socs3 from myeloid cells reduced lung influenza infection, but increased lung inflammation. Taken together, deletion of SOCS3 could suppress influenza replication, but intracellular SOCS3 communication between neutrophils and lung epithelial cells confounds this effect.

Deletion of suppressor of cytokine signaling 3 (SOCS3) in muscle stem cells does not alter muscle regeneration in mice after injury.

Muscles of older animals are more susceptible to injury and regenerate poorly, in part due to a persistent inflammatory response. The janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway mediates inflammatory signaling and is tightly regulated by the suppressor of cytokine signaling (SOCS) proteins, especially SOCS3. SOCS3 expression is altered in the muscle of aged animals and may contribute to the persistent inflammation and impaired regeneration. To test this hypothesis, we performed myotoxic injuries on mice with a tamoxifen-inducible deletion of SOCS3 specifically within the muscle stem cell compartment. Muscle stem cell-specific SOCS3 deletion reduced muscle mass at 14 days post-injury (-14%, P < 0.01), altered the myogenic transcriptional program, and reduced myogenic fusion based on the number of centrally-located nuclei per muscle fiber. Despite the delay in myogenesis, muscles with a muscle stem cell-specific deletion of SOCS3 were still able to regenerate after a single bout or multiple bouts of myotoxic injury. A reduction in SOCS3 expression in muscle stem cells is unlikely to be responsible for the incomplete muscle repair in aged animals.

Role of Suppressor of Cytokine Signaling 3 (SOCS3) in POMC Neurons in Metabolic and Cardiovascular Regulation during Chronic Leptin Infusion

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling and is expressed in proopiomelanocortin (POMC) neurons. We previously showed that activation of leptin receptors in POMC neurons mediates the chronic effects of leptin on blood pressure (BP). In this study, we examined whether deletion of SOCS3 specifically in POMC neurons amplifies the chronic metabolic and cardiovascular responses to leptin. 28‐week old male (n=5) and female (n=7) SOCS3flox/flox‐POMC/cre mice in which SOCS3 was selectively deleted in POMC neurons and control SOCS3flox/flox male (n=5) and female (n=7) mice were implanted with BP telemetry probes. After recovery, 24‐hr mean arterial pressure (MAP), heart rate (HR), body weight, and daily food intake were recorded for 5 consecutive days at baseline. Then leptin was infused (2 mg/kg/day, i.p) via osmotic minipumps for 14 days followed by 6‐days of recovery.Compared to SOCS3flox/flox controls, SOCS3flox/flox‐POMC/cre mice had similar body weight (36.5 ± 2.4 vs 38.5 ± 5.0 g in males and 24.7 ± 0.7 vs 25.2 ± 0.8 g in females) during baseline and consumed similar amounts of food (3.2 ± 0.3 vs 3.3 ± 0.4 g/day in males and 2.6 ± 0.2 vs 2.7 ± 0.1 g/day in females). MAP and HR were similar in SOCS3flox/flox‐POMC/cre and control mice in males (111 ± 5 vs 113 ± 2 mmHg; 546 ± 24 vs 543 ± 26 bpm) and females (105 ± 3 vs 106 ± 2 mmHg; 562 ± 20 vs 573 ± 25 bpm). Leptin infusion reduced body weight by 7–8% in male and female SOCS3flox/flox‐POMC/cre and control mice. Reductions of food intake were similar in SOCS3flox/flox‐POMC/cre and control mice in males (2.5 ± 0.4 vs 2.5 ± 0.7 g/day) and females (2.3 ± 0.3 vs 2.2 ± 0.4 g/day) when calculated as average daily food intake during 10 days of leptin infusion. Leptin infusion transiently increased MAP in male and female SOCS3flox/flox‐POMC/cre and control mice despite decreasing body weight and food intake. Increases of MAP and HR during leptin infusion were similar in male and female SOCS3flox/flox‐POMC/cre mice compared to control mice. Chronic leptin infusion produced similar reductions in fasting blood glucose concentration in SOCS3flox/flox‐POMC/cre and control mice.These results provide no evidence that SOCS3 deletion specifically in POMC neurons amplified the chronic effects of leptin to reduce food intake, body weight, and fasting glucose concentration in male or female mice. Deletion of SOCS3 in POMC neurons also did not substantially amplify the chronic effects of leptin on MAP and HR.Support or Funding InformationFunded by NHLBI PO1HL51971, NIGMS P20GM104357This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 021: Role of Central Nervous System Suppressor of Cytokine Signaling 3 (socs3) in Regulating Metabolic and Cardiovascular Function in Obesity

Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of leptin signaling, is upregulated in obesity and may contribute to development of leptin resistance. We determined whether deletion of SOCS3 in the entire central nervous system (CNS) would protect mice from adverse metabolic and cardiovascular effects of a high fat diet (HFD). SOCS3 flox/flox /Nestin-cre mice were generated by breeding SOCS3 flox/flox with Nestin-Cre mice. Male and female mice with CNS deletion of SOCS3 (SOCS3 flox/flox /Nestin-Cre, n=5-10) or control (SOCS3 flox/flox , n=6-10) were fed a HFD plus sucrose from 6 until 22 weeks of age. Mean arterial pressure (MAP) and heart rate (HR) were recorded by telemetry and oxygen consumption (VO 2 ) was monitored by indirect calorimetry in 22-week-old mice. Compared to control mice, SOCS3 flox/flox /Nestin-Cre mice were lighter (male: 34±3 vs. 45±3 and female: 27±1 vs. 37±2 g) and had elevated VO 2 (94±12 vs. 69±6 ml/kg/min) but there were no significant differences in food intake (male: 3.3±0.7 vs. 3.8±0.5 and female: 3.0±0.6 vs. 3.0±0.5 g/day) or plasma glucose (male: 148±8 vs. 198±31 and female 149±12 vs. 164±12 mg/dl). Male SOCS3 flox/flox /Nestin-Cre mice had similar MAP (115±2 vs. 116±1 mmHg) but higher HR (657±3 vs. 592±3 bpm) compared to control mice. However, female SOCS3 flox/flox /Nestin-cre mice had higher MAP (121±1 vs. 108±1 mmHg) and HR (655±2 vs. 606±5 bpm) compared to control mice. No significant differences were observed in glucose tolerance in SOCS3 flox/flox /Nestin-Cre vs. control mice (AUC: 391±36 vs. 429±54 mg/dL x 120 min in males and 459±70 vs. 372±51 mg/dL x 120 min in females). These results indicate that CNS SOCS3 deletion reduced body weight and increased energy expenditure and HR but did not improve glucose tolerance in male or female mice fed a HFD. However, HFD significantly increased BP in female SOCS3 flox/flox /Nestin-Cre mice compared to control mice fed a HFD, suggesting a sex difference in the role of CNS SOCS3 signaling in BP regulation in obesity. (NHLBI PO1HL51971and NIGMS P20GM104357)

Muscle-specific deletion of SOCS3 does not reduce the anabolic response to leucine in a mouse model of acute inflammation

Excessive inflammation reduces skeletal muscle protein synthesis leading to wasting and weakness. The janus kinase/signal transducers and activators of transcription-3 (JAK/STAT3) pathway is important for the regulation of inflammatory signaling. As such, suppressor of cytokine signaling-3 (SOCS3), the negative regulator of JAK/STAT signaling, is thought to be important in the control of muscle homeostasis. We hypothesized that muscle-specific deletion of SOCS3 would impair the anabolic response to leucine during an inflammatory insult. Twelve week old (n=8 per group) SOCS3 muscle-specific knockout mice (SOCS3-MKO) and littermate controls (WT) were injected with lipopolysaccharide (LPS, 1mg/kg) or saline and were studied during fasted conditions or after receiving 0.5g/kg leucine 3h after the injection of LPS. Markers of inflammation, anabolic signaling, and protein synthesis were measured 4h after LPS injection. LPS injection robustly increased mRNA expression of inflammatory molecules (Socs3, Socs1, Il-6, Ccl2, Tnfα and Cd68). In muscles from SOCS3-MKO mice, the Socs3 mRNA response to LPS was significantly blunted (∼6-fold) while STAT3 Tyr705 phosphorylation was exacerbated (18-fold). Leucine administration increased protein synthesis in both WT (∼1.6-fold) and SOCS3-MKO mice (∼1.5-fold) compared to basal levels. LPS administration blunted this effect, but there were no differences between WT and SOCS3-MKO mice. Muscle-specific SOCS3 deletion did not alter the response of AKT, mTOR, S6 or 4EBP1 under any treatment conditions. Therefore, SOCS3 does not appear to mediate the early inflammatory or leucine-induced changes in protein synthesis in skeletal muscle.

Changes in Leptin Signaling by SOCS3 Modulate Fasting-Induced Hyperphagia and Weight Regain in Mice

Weight regain frequently follows interventions that reduce body weight, leading to a failure in long-term obesity treatment. Inhibitory proteins of the leptin signaling pathway, such as the suppressor of cytokine signaling 3 (SOCS3), have been studied in conditions that predispose animals to obesity. However, whether SOCS3 modulates postrestriction hyperphagia and weight regain remains unknown. Mice lacking SOCS3 protein specifically in leptin receptor (LepR)-expressing cells (LepR SOCS3 knockout [KO]) were generated and studied in fasting and refeeding conditions. LepR SOCS3 KO mice exhibited increased leptin sensitivity in the hypothalamus. Notably, LepR SOCS3 KO males and females showed attenuated food intake and weight regain after 48 hours of fasting. Postrestriction hyperleptinemia was also prevented in LepR SOCS3 KO mice. Next, we studied possible mechanisms and neural circuits involved in the SOCS3 effects. SOCS3 deletion did not prevent fasting- or refeeding-induced c-Fos expression in the arcuate nucleus of the hypothalamus (ARH) nor fasting-induced increased excitability of ARH LepR-expressing cells. On the other hand, SOCS3 ablation reduced the mRNA levels of hypothalamic orexigenic neuropeptides during fasting (neuropeptide Y, agouti-related protein, orexin, and melanin-concentrating hormone). In summary, our findings suggest that increased leptin sensitivity contributes to the maintenance of a reduced body weight after food deprivation. In addition, the attenuated postrestriction food intake observed in mutant mice was not explained by fasting-induced changes in the activity of ARH neurons but exclusively by a lower transcription of orexigenic neuropeptides during fasting. These results indicate a partial dissociation between the regulation of neuronal activity and gene expression in ARH LepR-expressing cells.

Loss of SOCS3 in myeloid cells prolongs survival in a syngeneic model of glioma.

In glioma, microglia and macrophages are the largest population of tumor-infiltrating cells, referred to as glioma associated macrophages (GAMs). Herein, we sought to determine the role of Suppressor of Cytokine Signaling 3 (SOCS3), a negative regulator of Signal Transducer and Activator of Transcription 3 (STAT3), in GAM functionality in glioma. We utilized a conditional model in which SOCS3 deletion is restricted to the myeloid cell population. We found that SOCS3-deficient bone marrow-derived macrophages display enhanced and prolonged expression of pro-inflammatory M1 cytokines when exposed to glioma tumor cell conditioned medium in vitro. Moreover, we found that deletion of SOCS3 in the myeloid cell population delays intracranial tumor growth and increases survival of mice bearing orthotopic glioma tumors in vivo. Although intracranial tumors from mice with SOCS3-deficient myeloid cells appear histologically similar to control mice, we observed that loss of SOCS3 in myeloid cells results in decreased M2 polarized macrophage infiltration in the tumors. Furthermore, loss of SOCS3 in myeloid cells results in increased CD8+ T-cell and decreased regulatory T-cell infiltration in the tumors. These findings demonstrate a beneficial effect of M1 polarized macrophages on suppressing glioma tumor growth, and highlight the importance of immune cells in the tumor microenvironment.

SOCS3 expression within leptin receptor-expressing cells regulates food intake and leptin sensitivity but does not affect weight gain in pregnant mice consuming a high-fat diet

Pregnancy induces transitory metabolic changes including increases in food intake and body fat deposition, as well as leptin and insulin resistance. Recent findings have suggested that increased hypothalamic expression of suppressor of cytokine signaling-3 (SOCS3) is a key mechanism responsible for triggering those metabolic adaptations. Because obesity is a risk factor for gestational metabolic imbalances, we aimed to study the role of SOCS3 during pregnancy in obese mice. Female mice carrying a deletion of SOCS3 in leptin receptor-expressing cells (SOCS3 KO mice) were exposed to a chronic high-fat diet (HFD), and we then studied their energy balance and glucose homeostasis during pregnancy. SOCS3 deletion did not prevent diet-induced obesity or changes in body weight and adiposity observed during pregnancy. However, the typical increase in food intake during mid- and late-pregnancy was blunted in SOCS3 KO females. We also observed a slight improvement in glucose homeostasis and increased leptin sensitivity in the arcuate nucleus of the hypothalamus in pregnant SOCS3 KO mice on HFD. Despite this, SOCS3 KO mice had an increased number of uterine reabsorptions and fewer fetuses compared to the controls. Compared to control animals, a reduction in proopiomelanocortin and an increase in oxytocin mRNA levels were observed in the hypothalamus of pregnant SOCS3 KO mice. In contrast to previous studies using lean animals, conditional SOCS3 ablation did not prevent major gestational metabolic changes in diet-induced obese mice. Our findings contribute to the understanding of the role of SOCS3 in mediating pregnancy-induced metabolic adaptations.

Abstract 085: Role of Suppressor of Cytokines Signaling 3 (Socs3) in Modulating Chronic Metabolic and Cardiovascular Effects of Leptin

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling. Hypothalamic SOCS3 is upregulated in obese animals fed a high-fat diet and has been suggested to contribute to development of resistance to leptin’s anorexic effects. In this study we determined whether deletion of SOCS3 in the entire central nervous system (CNS) amplifies the chronic anorexic and blood pressure (BP) effects of physiological increases in plasma leptin in mice fed a normal diet. SOCS3 flox/flox -Nestin-cre mice were generated by breeding SOCS3 flox/flox with Nestin-cre mice. BP and heart rate (HR) were recorded by telemetry, and oxygen consumption (VO 2 ) was monitored by indirect calorimetry in 22-week-old SOCS3 flox/flox -Nestin-cre (n=4) and control mice (SOCS3 flox/flox , n=4). Compared to controls SOCS3 flox/flox -Nestin-cre mice were lighter (30±1 vs 33±1 g) and normoglycemic (124±7 vs 146±10 mg/dl), consumed less food (3.0±0.4 vs 3.6±0.2 g/day) and had similar VO 2 (77±6 vs 73±3 ml/kg/min). SOCS3 flox/flox -Nestin-cre mice had similar MAP (103±3 vs 107±3 mmHg) but higher HR (666±15 vs 602±17 bpm) compared to control mice. Chronic leptin infusion greatly reduced food in SOCS3 flox/flox -Nestin-cre (46±3 vs 35±4%) and increased MAP (15±3 vs 7±2 mmHg) and VO2 (18±3 vs 14±2%) compared to control mice. No significant changes were observed in HR in either group. Leptin infusion significantly reduced blood glucose levels in both groups (124±7 to 97±7 vs 146±10 to 105±7 mg/dl). These results indicate that SOCS3 deletion in the entire CNS reduces body weight and food intake, and amplifies leptin’s effect on appetite and blood pressure and also suggest the SOCS3 signaling attenuates the chronic actions of leptin on blood pressure as well as appetite regulation even in non-obese mice fed a normal diet. (NHLBI-PO1HL51971, NIGMS P20GM104357 and AHA SDG5680016)

Abstract P076: Suppressor of Cytokine Signaling 3 (SOCS3) in POMC Neurons and Its Role in Regulating Blood Pressure, Body Weight and Glucose in Obesity

Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of leptin signaling, may be involved in development of obesity-induced leptin resistance. Although we previously showed that activation of proopiomelanocortin (POMC) neurons mediates the chronic effects of leptin on blood pressure (BP), the role of SOCS3 in modulating BP in obesity is still unclear. In this study, we investigated the role of SOCS3 specifically in POMC neurons in regulating body weight, glucose handling and BP in mice fed a normal or high fat (45%, HFD) chow. Male and female SOCS3flox/flox-POMC/cre mice in which SOCS3 was selectively deleted in POMC neurons and control SOCS3flox/flox mice were used. Food intake and body weight were measured from 8 to 16 weeks of age, and a glucose tolerance test (GTT) was performed at 20 wks of age. At 22 wks of age, mice were implanted with telemetry probe to measure BP and heart rate (HR) and fed a HFD for 6 weeks. Compared to control mice, both male and female SOCS3flox/flox-POMC/cre mice were lighter at 16 wks (29.1 ± 3.5 vs 31.9 ± 3.6 g in male and 21.5 ± 2.2 vs 26.1 ± 5.7 in female, n=9-11, p&lt;0.05) but food intake was similar in both groups. Only male SOCS3flox/flox-POMC/cre mice exhibited improved glucose handling (AUC: 1059 ± 52 vs 1283 ± 54 mg/dL x 120 min, n=7-10, p&lt;0.05 ) and no differences were observed in female mice. When fed normal chow, BP was similar in SOCS3flox/flox-POMC/cre and control mice (116 ± 7 vs 113 ± 5 mmHg) at 23 wks. After a HFD for 6 weeks, SOCS3flox/flox-POMC/cre mice had a greater BP increase compared to control mice (7.2 ± 1.9 vs 0.9 ± 1.8 mmHg, n=4-9, P&lt;0.05) but no significant differences were observed in food intake or body weight between two groups. These results suggest that SOCS3 deletion specifically in POMC neurons reduced body weight in male and female mice, and improved glucose handling only in male mice. HFD increased BP in SOCS3flox/flox-POMC/cre but not in control mice, suggesting that SOCS3 in POMC neurons may modulate BP response to HFD.

Preferential Recruitment of Neutrophils into the Cerebellum and Brainstem Contributes to the Atypical Experimental Autoimmune Encephalomyelitis Phenotype.

The JAK/STAT pathway is critical for development, regulation, and termination of immune responses, and dysregulation of the JAK/STAT pathway, that is, hyperactivation, has pathological implications in autoimmune and neuroinflammatory diseases. Suppressor of cytokine signaling 3 (SOCS3) regulates STAT3 activation in response to cytokines that play important roles in the pathogenesis of neuroinflammatory diseases, including IL-6 and IL-23. We previously demonstrated that myeloid lineage-specific deletion of SOCS3 resulted in a severe, nonresolving atypical form of experimental autoimmune encephalomyelitis (EAE), characterized by lesions, inflammatory infiltrates, elevated STAT activation, and elevated cytokine and chemokine expression in the cerebellum. Clinically, these mice exhibit ataxia and tremors. In this study, we provide a detailed analysis of this model, demonstrating that the atypical EAE observed in LysMCre-SOCS3(fl/fl) mice is characterized by extensive neutrophil infiltration into the cerebellum and brainstem, increased inducible NO synthase levels in the cerebellum and brainstem, and prominent axonal damage. Importantly, infiltrating SOCS3-deficient neutrophils produce high levels of CXCL2, CCL2, CXCL10, NO, TNF-α, and IL-1β. Kinetic studies demonstrate that neutrophil infiltration into the cerebellum and brainstem of LysMCre-SOCS3(fl/fl) mice closely correlates with atypical EAE clinical symptoms. Ab-mediated depletion of neutrophils converts the atypical phenotype to the classical EAE phenotype and, in some cases, a mixed atypical/classical phenotype. Blocking CXCR2 signaling ameliorates atypical EAE development by reducing neutrophil infiltration into the cerebellum/brainstem. Thus, neutrophils lacking SOCS3 display elevated STAT3 activation and expression of proinflammatory mediators and play a critical role in the development of atypical EAE.

RAFting the rapids of axon regeneration signaling.

The functional regeneration of damaged axons and severed connections in the mature central nervous system (CNS) remains a challenging goal of neurological research. Mature CNS neurons are refractory to axon regeneration for two major reasons, one, because the activity of cell-intrinsic mechanisms that drive axon growth during development is low – and often further suppressed after an injury – and two, because certain molecules that are part of mature extracellular matrix and myelin act as strong inhibitors of axon growth. Genetic removal of growth inhibitory molecules can increase axon sprouting, but is not sufficient to enable long-range axon growth. Since axon growth is robust during early developmental stages, it has long been hypothesized that mature injured neurons may be “reprogrammed” to the earlier growth state by re-activation of the intracellular growth signaling cascades that drive axon elongation in the developing fetus.

SOCS3 deficiency in leptin receptor-expressing cells mitigates the development of pregnancy-induced metabolic changes.

ObjectiveDuring pregnancy, women normally increase their food intake and body fat mass, and exhibit insulin resistance. However, an increasing number of women are developing metabolic imbalances during pregnancy, including excessive gestational weight gain and gestational diabetes mellitus. Despite the negative health impacts of pregnancy-induced metabolic imbalances, their molecular causes remain unclear. Therefore, the present study investigated the molecular mechanisms responsible for orchestrating the metabolic changes observed during pregnancy.MethodsInitially, we investigated the hypothalamic expression of key genes that could influence the energy balance and glucose homeostasis during pregnancy. Based on these results, we generated a conditional knockout mouse that lacks the suppressor of cytokine signaling-3 (SOCS3) only in leptin receptor-expressing cells and studied these animals during pregnancy.ResultsAmong several genes involved in leptin resistance, only SOCS3 was increased in the hypothalamus of pregnant mice. Remarkably, SOCS3 deletion from leptin receptor-expressing cells prevented pregnancy-induced hyperphagia, body fat accumulation as well as leptin and insulin resistance without affecting the ability of the females to carry their gestation to term. Additionally, we found that SOCS3 conditional deletion protected females against long-term postpartum fat retention and streptozotocin-induced gestational diabetes.ConclusionsOur study identified the increased hypothalamic expression of SOCS3 as a key mechanism responsible for triggering pregnancy-induced leptin resistance and metabolic adaptations. These findings not only help to explain a common phenomenon of the mammalian physiology, but it may also aid in the development of approaches to prevent and treat gestational metabolic imbalances.

ME-13 * LOSS OF SOCS3 IN MYELOID CELLS DELAYS TUMOR GROWTH AND PROLONGS SURVIVAL IN A SYNGENEIC MODEL OF GLIOBLASTOMA

Glioblastoma (GBM), a particularly devastating type of brain tumor, remains a challenging and difficult disease to treat. Despite aggressive therapies, patients diagnosed with GBM have a median survival of 12-16 months. On a cellular level, GBM tumors are extremely heterogeneous, consisting of resident tumor cells, tumor initiating cells, infiltrating immune cells, endothelial cells, and other tumor associated stromal cells, which makes developing targeted therapies a challenge. In GBM, microglia (MG) and macrophages (MP) are the largest population of tumor-infiltrating cells. GBM tumor cells recruit MG/MP via the secretion of chemo-attractants; however, upon arrival at the tumor, the MG/MP adopt an anti-inflammatory phenotype, and in turn accidentally aide in tumor promotion through the secretion of various cytokines. We have previously determined that macrophages with deletion of Suppressor of Cytokine Signaling 3 (SOCS3), the negative regulator of Signal Transducer and Activator of Transcription 3 (STAT3), have an enhanced M1 (pro-inflammatory) response. Herein, we sought to determine the role of SOCS3 in MG/MP functionality in GBM. In this report, we utilized a conditional model in which SOCS3 deletion is restricted to the myeloid cell population. We found that deletion of SOCS3 in the myeloid cell population delays intracranial tumor growth and increases survival of mice bearing orthotopic glioma tumors. Intracranial tumors from mice with SOCS3 deficient myeloid cells have an increase in the number of infiltrating immune cells, including monocytes, neutrophils and macrophages. Lastly, SOCS3 deficient bone marrow derived macrophages display enhanced and prolonged expression of pro-inflammatory M1 cytokines (TNF-α, IL-6) when exposed to tumor cell conditioned medium in vitro. Therefore, loss of SOCS3 in MG/MP in the tumor promotes an inflammatory, immune response early and thus delays tumor growth. These preliminary findings highlight an important role of the STAT3/SOCS3 signaling axis in MG/MP in GBM tumors.

Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice

Leptin acts on its receptor (ObR) in the hypothalamus to inhibit food intake and energy expenditure. Leptin and ObR are also expressed in the gastrointestinal tract; however, the physiological significance of leptin signaling in the gut remains uncertain. Suppressor of cytokine signaling 3 (SOCS3) is a key negative feedback regulator of ObR-mediated signaling in the hypothalamus. We now show that gastrointestinal epithelial cell-specific SOCS3 conditional knockout (T3b-SOCS3 cKO) mice developed gastric tumors by enhancing leptin production and the ObRb/signal transducer and activator of transcription 3 (STAT3) signaling pathway. All T3b-SOCS3 cKO mice developed tumors in the stomach but not in the bowels by 2 months of age, even though the SOCS3 deletion occurred in both the epithelium of stomach and bowels. The tumors developed in the absence of the inflammatory response and all cKO mice died within 6 months. These tumors displayed pathology and molecular alterations, such as an increase in MUC2 (Mucin 2, oligomeric mucus/gel-forming) and TFF3 (trefoil factor 3), resembling human intestinal-type gastric tumors. Administration of antileptin antibody to T3b-SOCS3 cKO mice reduced hyperplasia of gastric mucosa, which is the step of the initiation of gastric tumor. These data suggest that SOCS3 is an antigastric tumor gene that suppresses leptin overexpression and ObRb/STAT3 hyperactivation, supporting the hypothesis that the leptin/ObRb/STAT3 axis accelerates tumorigenesis and that it may represent a new therapeutic target for the treatment of gastric cancer.

Cardiac-Specific Deletion of SOCS-3 Prevents Development of Left Ventricular Remodeling After Acute Myocardial Infarction

The study investigated the role of myocardial suppressor of cytokine signaling-3 (SOCS3), an intrinsic negative feedback regulator of the janus kinase and signal transducer and activator of transcription (JAK-STAT) signaling pathway, in the development of left ventricular (LV) remodeling after acute myocardial infarction (AMI). LV remodeling after AMI results in poor cardiac performance leading to heart failure. Although it has been shown that JAK-STAT-activating cytokines prevent LV remodeling after AMI in animals, little is known about the role of SOCS3 in this process. Cardiac-specific SOCS3 knockout mice (SOCS3-CKO) were generated and subjected to AMI induced by permanent ligation of the left anterior descending coronary artery. Although the initial infarct size after coronary occlusion measured by triphenyltetrazolium chloride staining was comparable between SOCS3-CKO and control mice, the infarct size 14 days after AMI was remarkably inhibited in SOCS3-CKO, indicating that progression of LV remodeling after AMI was prevented in SOCS3-CKO hearts. Prompt and marked up-regulations of multiple JAK-STAT-activating cytokines including leukemia inhibitory factor and granulocyte colony-stimulating factor (G-CSF) were observed within the heart following AMI. Cardiac-specific SOCS3 deletion enhanced multiple cardioprotective signaling pathways including STAT3, AKT, and extracellular signal-regulated kinase (ERK)-1/2, while inhibiting myocardial apoptosis and fibrosis as well as augmenting antioxidant expression. Enhanced activation of cardioprotective signaling pathways by inhibiting myocardial SOCS3 expression prevented LV remodeling after AMI. Our data suggest that myocardial SOCS3 may be a key molecule in the development of LV remodeling after AMI.