Fang-Hsuean Liao

DUSP4 deficiency enhances CD25 expression and CD4+ T-cell proliferation without impeding T-cell development

The differentiation and activation of T cells are critically modulated by MAP kinases, which are in turn feed‐back regulated by dual‐specificity phosphatases (DUSPs) to determine the duration and magnitude of MAP kinase activation. DUSP4 (also known as MKP2) is a MAP kinase‐induced DUSP member that is dynamically expressed during thymocyte differentiation. We generated DUSP4‐deficient mice to study the function of DUSP4 in T‐cell development and activation. Our results show that thymocyte differentiation and activation‐induced MAP kinase phosphorylation were comparable between DUSP4‐deficient and WT mice. Interestingly, activated DUSP4−/− CD4+ T cells were hyperproliferative while DUSP4−/− CD8+ T cells proliferated normally. Further mechanistic studies suggested that the hyperproliferation of DUSP4−/− CD4+ T cells resulted from enhanced CD25 expression and IL‐2 signaling through increased STAT5 phosphorylation. Immunization of DUSP4−/− mice recapitulated the T‐cell hyperproliferation phenotype in antigen recall responses, while the profile of Th1/Th2‐polarized antibody production was not altered. Overall, these results suggest that other DUSPs may compensate for DUSP4 deficiency in T‐cell development, MAP kinase regulation, and Th1/Th2‐mediated antibody responses. More importantly, our data indicate that DUSP4 suppresses CD4+ T‐cell proliferation through novel regulations in STAT5 phosphorylation and IL‐2 signaling.

Effects of different ratios of monounsaturated and polyunsaturated fatty acids to saturated fatty acids on regulating body fat deposition in hamsters.

OBJECTIVE Effects of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid consumption on regulating body fat accumulation and body weight gain are controversial between animal and human studies. METHODS We designed a 2 x 2 factorial study, with two levels of MUFAs (60% and 30%) and two levels of polyunsaturated-to-saturated fatty acid (P/S) ratio (5 and 3) to prepare four kinds of experimental oils consisting of 60% MUFAs with a high or low P/S ratio (HMHR or HMLR, respectively) or 30% MUFAs with a high or low P/S ratio (LMHR or LMLR, respectively). Thirty-two male golden Syrian hamsters were randomly divided into four groups and fed the experimental diets containing 15% (w/w) fat for 12 wk. RESULTS No difference was observed in the mean daily food intake. Hamsters fed the LMLR diet had increased weight gain, epididymal and retroperitoneal white adipose tissues, plasma non-esterified fatty acids, insulin, hepatic acetyl coenzyme A carboxylase and malic enzyme activities, and mRNA expressions of peroxisome proliferator-activated receptor-alpha and sterol regulatory element-binding protein-1c among all groups (P < 0.05). Hamsters fed the HMHR diet had lower plasma insulin levels and hepatic acetyl coenzyme A carboxylase activities among groups (P < 0.05) and elevated hepatic acyl coenzyme A oxidase and carnitine palmitoyltransferase-I activities compared with those fed the LMLR diet (P < 0.05). CONCLUSION Hamsters fed the LMLR diet had increased weight gain and body fat accumulation, whereas the HMHR diet appeared to be beneficial in preventing white adipose tissue accumulation by decreasing plasma insulin levels and increasing hepatic lipolytic enzyme activities involved in beta-oxidation.

Differential effects of high MUFA with high or low P/S ratio (polyunsaturated to saturated fatty acids) on improving hepatic lipolytic enzymes and mediating PPARγ related with lipoprotein lipase and hormone-sensitive lipase of white adipose tissue in diet-induced obese hamster.

Objectives:The aim of this study was to assess the relationship between high monounsaturated fatty acids (MUFAs) with different levels of polyunsaturated-to-saturated fatty acid (P/S) ratios and body fat loss in diet-induced obesity (DIO) models.Design:Male Golden Syrian hamsters were randomly assigned to the control group (n=12) and obesity group (n=24) for 4 weeks of the high-fat DIO period; afterward, six hamsters from each group were killed. The remaining control hamsters were still fed a low-fat diet. For an additional 8 weeks, the remaining obesity hamsters were switched to a low-fat diet and subdivided into three subgroups (n=6/group): the obesity-control (ObC) group, high MUFA with high P/S ratio oil (HMHR) group and olive oil (OO) group. Serum insulin and leptin concentrations were measured, and hepatic fatty acid metabolic enzymes and adipose differentiation markers were determined using enzyme activities analysis, western blot and semiquantification reverse-transcription PCR.Results:No difference was observed in the mean energy intake through all study periods. After the DIO period, the obesity group increased in weight gain and epididymal fat weight compared with the control group. DIO hamsters in the HMLR group had significant reductions in white adipose tissue deposition and plasma leptin levels, suppression in adipose peroxisome proliferator-activated receptor-γ (PPARγ) and lipoprotein lipase (LPL) mRNA expressions and increases in hepatic acyl-CoA oxidase and carnitine palmitoyltransferase-I activities and mRNA levels compared with those in the ObC group. The HMHR group had upregulated phosphorylation of hormone-sensitive lipase (HSL) relative to total HSL protein levels compared with the OO group. However, the OO group had significantly elevated hepatic de novo lipogenesis compared with the HMHR group.Conclusions:HMHR seemed to be beneficial in depleting white adipose tissue accumulation by decreasing adipose PPARγ and LPL mRNA expressions and mediating phosphorylation of HSL, and by improving hepatic lipolytic enzyme activities and mRNA expressions involved in β-oxidation in DIO hamsters.

Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization*

Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability.

Protein phosphatase 4 is an essential positive regulator for Treg development, function, and protective gut immunity

BackgroundProtein phosphates 4 (PP4), encoded by the ppp4c gene, is a ubiquitously expressed phosphatase that has been implicated in the regulation of cytokine signaling and lymphocyte survival; recent reports suggest that PP4 may be involved in pre-TCR signaling and B cell development. However, whether PP4 also modulates the functions of peripheral T cells has not been investigated due to the lack of a suitable in vivo model. Treg cells are a specialized subset of CD4 helper T cells that can suppress the proliferation of activated effector T cells. In the absence of this negative regulation, autoimmune syndromes and inflammatory diseases, such as human Crohn’s disease, will arise.ResultsIn this report, we generated mice with T cell-specific ablation of the ppp4c gene (CD4cre:PP4f/f) and a Foxp3-GFP reporter gene to examine the roles of PP4 in Treg development and function. Characterizations of the CD4cre:PP4f/f mice showed that PP4 deficiency induced partial αβ T lymphopenia and T cell hypo-proliferation. Further analyses revealed significant reductions in the numbers of thymic and peripheral Treg cells, as well as in the efficiency of in vitro Treg polarization. In addition, PP4-deficient Treg cells exhibited reduced suppressor functions that were associated with decreased IL-10, CTLA4, GITR and CD103 expression. More interestingly, the CD4cre:PP4f/f mice developed spontaneous rectal prolapse and colitis with symptoms similar to human Crohn’s disease. The pathogenesis of colitis required the presence of commensal bacteria, and was correlated with reduced Treg cells in the gut. Nevertheless, PP4-deficient Treg cells were still capable of suppressing experimental colitis, suggesting that multiple factors contributed to the onset of the spontaneous colitis.ConclusionsWhile the molecular mechanisms remain to be investigated, our results clearly show that PP4 plays a non-redundant role for the differentiation, suppressor activity and gut homeostasis of Treg cells. The onset of spontaneous colitis in the CD4cre:PP4f/f mice further suggests that PP4 is essential for the maintenance of protective gut immunity. The CD4cre:PP4f/f mice thus may serve as a good model for studying the interactions between Treg cells and gut commensal bacteria for the regulation of mucosal immunity.

T cell proliferation and adaptive immune responses are critically regulated by protein phosphatase 4

ABSTRACT The clonal expansion of activated T cells is pivotal for the induction of protective immunity. Protein phosphatase 4 (PP4) is a ubiquitously expressed serine/threonine phosphatase with reported functions in thymocyte development and DNA damage responses. However, the role of PP4 in T cell immunity has not been thoroughly investigated. In this report, our data showed that T cell-specific ablation of PP4 resulted in defective adaptive immunity, impaired T cell homeostatic expansion, and inefficient T cell proliferation. This hypo-proliferation was associated with a partial G1-S cell cycle arrest, enhanced transcriptions of CDK inhibitors and elevated activation of AMPK. In addition, resveratrol, a known AMPK activator, induced similar G1-S arrests, while lentivirally-transduced WT or constitutively-active AMPKα1 retarded the proliferation of WT T cells. Further investigations showed that PP4 co-immunoprecipitated with AMPKα1, and the over-expression of PP4 inhibited AMPK phosphorylation, thereby implicating PP4 for the negative regulation of AMPK. In summary, our results indicate that PP4 is an essential modulator for T cell proliferation and immune responses; they further suggest a potential link between PP4 functions, AMPK activation and G1-S arrest in activated T cells.

A prebiotic formula improves the gastrointestinal bacterial flora in toddlers

We aimed to investigate the effect of enriched 3-prebiotic formula (including inulin, fructooligosaccharides, and galactooligosaccharides) on toddler gut health by measuring fecal microbiota. Our results revealed that the consumption of 3-prebiotic formula three times per day giving total intake of 1.8 g prebiotic ingredients significantly showed the increased number of probiotic Bifidobacterium spp. colonies and the reduced populations of both C. perfringens and total anaerobic bacteria on the fecal bacterial flora in toddlers at 18~36 months. In addition, total organic acids in the fecal samples significantly increased which improves the utilization of bifidus under acidic conditions after consumption of the 3-prebiotic formula. Therefore, using the formula enriched with prebiotic may maintain gut health in toddlers.

Medium-Chain Triglycerides Lower Blood Lipids and Body Weight in Streptozotocin-Induced Type 2 Diabetes Rats

Medium-chain triglycerides (MCTs) are distinguished from other triglycerides in that each fat molecule consists of 6 to 12 carbons in length. MCTs and long-chain triglycerides (LCTs) are absorbed and utilized in different ways. The aim of this study was to assess the effects of replacing soybean oil with MCT oil, in a low- or high-fat diet, on lipid metabolism in rats with streptozotocin-induced type 2 diabetes mellitus (T2DM). There were, thirty-two T2DM Sprague-Dawley rats divided into low-fat-soybean oil (LS), low-fat-MCT oil (LM), high-fat-soybean oil (HS), and high-fat-MCT oil (HM) groups. After 8 weeks, blood sugar, serum lipids, liver lipids, and enzyme activities related to lipid metabolism were measured. Under a high-fat diet condition, replacement of soybean oil with MCT oil lowered serum low-density lipoprotein cholesterol (LDL-C), non-esterified fatty acids, and liver total cholesterol; whilst it increased serum high-density lipoprotein cholesterol (HDL-C) and the HDL-C/LDL-C ratio. A low-fat diet with MCT oil resulted in lower body weight and reproductive white adipose tissues compared to the HS groups, and higher hepatic acyl-CoA oxidase activities (the key enzyme in the peroxisomal beta-oxidation) compared to the LS group in T2DM rats. In conclusion, MCTs showed more protective effects on cardiovascular health in T2DM rats fed a high-fat diet, by improving serum lipid profiles and reducing hepatic total cholesterol.

Subnanometer Gold Clusters Adhere to Lipid A for Protection against Endotoxin-Induced Sepsis

Endotoxicity originating from a dangerous debris (i.e., lipopolysaccharide, LPS) of Gram-negative bacteria is a challenging clinical problem, but no drugs or therapeutic strategies that can successfully address this issue have been identified yet. In this study, we report a subnanometer gold cluster that can efficiently block endotoxin activity to protect against sepsis. The endotoxin blocker consists of a gold nanocluster that serves as a flakelike substrate and a coating of short alkyl motifs that act as an adhesive to dock with LPS by compacting the intramolecular hydrocarbon chain-chain distance ( d-spacing) of lipid A, an endotoxicity active site that can cause overwhelming cytokine induction resulting in sepsis progression. Direct evidence showed the d-spacing values of lipid A to be decreased from 4.19 Å to either 3.85 or 3.54 Å, indicating more dense packing densities in the presence of subnanometer gold clusters. In terms of biological relevance, the concentrations of key pro-inflammatory NF-κB-dependent cytokines, including plasma TNF-α, IL-6, and IL-1β, and CXC chemokines, in LPS-challenged mice showed a noticeable decrease. More importantly, we demonstrated that the treatment of antiendotoxin gold nanoclusters significantly prolonged the survival time in LPS-induced septic mice. The ultrasmall gold nanoclusters could target lipid A of LPS to deactivate endotoxicity by compacting its packing density, which might constitute a potential therapeutic strategy for the early prevention of sepsis caused by Gram-negative bacterial infection.