Transcription Factor NFAT5 Research Articles

Activation of the transcription factor NFAT5 in the tumor microenvironment enforces CD8+ T cell exhaustion.

Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. Here we found that NFAT5, an NFAT family transcription factor that lacks an AP-1 docking site, was highly expressed in exhausted CD8+ T cells in the context of chronic infections and tumors but was selectively required in tumor-induced CD8+ T cell exhaustion. Overexpression of NFAT5 in CD8+ T cells reduced tumor control, while deletion of NFAT5 improved tumor control by promoting the accumulation of tumor-specific CD8+ T cells that had reduced expression of the exhaustion-associated proteins TOX and PD-1 and produced more cytokines, such as IFNɣ and TNF, than cells with wild-type levels of NFAT5, specifically in the precursor exhausted PD-1+TCF1+TIM-3-CD8+ T cell population. NFAT5 did not promote T cell exhaustion during chronic infection with clone 13 of lymphocytic choriomeningitis virus. Expression of NFAT5 was induced by TCR triggering, but its transcriptional activity was specific to the tumor microenvironment and required hyperosmolarity. Thus, NFAT5 promoted the exhaustion of CD8+ T cells in a tumor-selective fashion.

Serine protease 35 regulates the fibroblast matrisome in response to hyperosmotic stress.

Hyperosmotic stress occurs in several diseases, but its long-term effects are largely unknown. We used sorbitol-treated human fibroblasts in 3D culture to study the consequences of hyperosmotic stress in the skin. Sorbitol regulated many genes, which help cells cope with the stress condition. The most robustly regulated gene encodes serine protease 35 (PRSS35). Its regulation by hyperosmotic stress was dependent on the kinases p38 and JNK and the transcription factors NFAT5 and ATF2. We identified different collagens and collagen-associated proteins as putative PRSS35 binding partners. This is functionally important because PRSS35 affected the extracellular matrix proteome, which limited cell proliferation. The in vivo relevance of these findings is reflected by the coexpression of PRSS35 and its binding partners in human skin wounds, where hyperosmotic stress occurs as a consequence of excessive water loss. These results identify PRSS35 as a key regulator of the matrisome under hyperosmotic stress conditions.

The role of the osmosensitive transcription factor NFAT5 in corneal edema resorption after injury

The osmosensitive transcription factor nuclear factor of activated T cells 5 (NFAT5; or tonicity-responsive enhancer binding protein; TonEBP) plays a key role in macrophage-driven regulation of cutaneous salt and water balance. In the immune-privileged and transparent cornea, disturbances in fluid balance and pathological edema result in corneal transparency loss, which is one of the main causes of blindness worldwide. The role of NFAT5 in the cornea has not yet been investigated. We analyzed the expression and function of NFAT5 in naive corneas and in an established mouse model of perforating corneal injury (PCI), which causes acute corneal edema and transparency loss. In uninjured corneas, NFAT5 was mainly expressed in corneal fibroblasts. In contrast, after PCI, NFAT5 expression was highly upregulated in recruited corneal macrophages. NFAT5 deficiency did not alter corneal thickness in steady state; however, loss of NFAT5 led to accelerated resorption of corneal edema after PCI. Mechanistically, we found that myeloid cell-derived NFAT5 is crucial for controlling corneal edema, as edema resorption after PCI was significantly enhanced in mice with conditional loss of NFAT5 in the myeloid cell lineage, presumably due to increased pinocytosis of corneal macrophages. Collectively, we uncovered a suppressive role for NFAT5 in corneal edema resorption, thereby identifying a novel therapeutic target to combat edema-induced corneal blindness.

Epstein-Barr virus infection controls the concentration of the intracellular antioxidant glutathione by upregulation of the glutamate transporter EAAT3 in tumor cells.

Epstein-Barr virus or human herpesvirus 4 (EBV/HHV-4) is an omnipresent oncovirus etiologically associated with various B-cell lymphomas and epithelial cancers. The malignant transformation associated with the persistent expression of viral proteins often deregulates the host cellular machinery and EBV infection is coupled to elevated levels of reactive oxygen species. Here, we investigated the role that the glutamate transporter EAAT3 plays in regulating the antioxidant system as a protective mechanism of EBV-infected cells against the virus-induced oxidative stress. Our study demonstrated that the expression of EAAT3 was upregulated and localized to the plasma membrane in EBV latently infected and de novo EBV-infected cells. EAAT3 was regulated by the transcription factor NFAT5 in the infected cells. Membrane localized EAAT3 was found to be involved in the transportation of glutamate from the extracellular space into the cell, as EAAT3 and NFAT5 inhibitors markedly reduced the levels of intracellular glutamate levels in EBV latently infected cells. Additionally, our data demonstrated a notable decrease in the intracellular glutathione levels following treatment with an EAAT3 inhibitor. Collectively, our results suggest that upregulation of the glutamate transporter EAAT3 is an adaptation of EBV-infected cells to maintain cellular redox homeostasis against the virus-induced oxidative stress, and that this cellular balance could be therapeutically destroyed by targeting EAAT3 to impede EBV-associated cancers.

The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

Avian leukosis virus subgroup J (ALV-J) can cause neoplastic diseases in poultry and is still widely prevalent in China. Chicken telomerase reverse transcriptase (chTERT) is the core component of telomerase, which is closely related to the occurrence and development of tumors. Our previous studies showed that chTERT is overexpressed in ALV-J tumors, but the mechanism is still not completely clear. Therefore, this study aims to analyze the possible molecular mechanism of chTERT overexpression in ALV-J tumors from the perspective of DNA methylation and promoter mutation. Methylation sequencing of the chTERT amplicon showed that ALV-J replication promoted the methylation level of the chTERT promoter. And the methylation level of the chTERT promoter in ALV-J tumors was significantly higher than that in tumor-adjacent and normal tissues. Compared with the tumor-adjacent and normal tissues, the chTERT promoter in each ALV-J tumors tested had a mutation of −183 bp C > T, and 36.0% (9/25) of the tumors also had mutations of −184 bp T > C, −73 bp::GGCCC and −56 bp A > T in the chTERT promoter, which formed the binding sites for the transcription factors NFAT5, TFAP2A and ZEB1, respectively. The results of RT–qPCR and Western blotting showed that the occurrence of these mutations significantly increased the expression level of chTERT. In conclusion, this study demonstrated that the high expression of chTERT in ALV-J tumors is positively correlated with the level of hypermethylation and mutation in its promoter, which provides a new perspective for further research on the molecular mechanism of chTERT in ALV-J tumorigenesis.

Low-Salt Diet Reduces Anti-CTLA4 Mediated Systemic Immune-Related Adverse Events while Retaining Therapeutic Efficacy against Breast Cancer.

Simple SummaryImmunotherapy has transformed breast cancer treatment. However, ICI-induced systemic inflammatory immune-related adverse events (irAE) remain a major clinical challenge. In our current communication, using breast tumor models, we demonstrated that a low salt diet could reduce irAE development following ICI therapy. Importantly, a low salt diet did not change the anti-tumor efficiency. Our current study provides a basis for future clinical trials to verify the role of a low salt diet in long-term immunotherapeutic efficiency in breast cancer patients.Immune checkpoint inhibitor (ICI) therapy has revolutionized the breast cancer treatment landscape. However, ICI-induced systemic inflammatory immune-related adverse events (irAE) remain a major clinical challenge. Previous studies in our laboratory and others have demonstrated that a high-salt (HS) diet induces inflammatory activation of CD4+T cells leading to anti-tumor responses. In our current communication, we analyzed the impact of dietary salt modification on therapeutic and systemic outcomes in breast-tumor-bearing mice following anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) monoclonal antibody (mAb) based ICI therapy. As HS diet and anti-CTLA4 mAb both exert pro-inflammatory activation of CD4+T cells, we hypothesized that a combination of these would lead to enhanced irAE response, while low-salt (LS) diet through blunting peripheral inflammatory action of CD4+T cells would reduce irAE response. We utilized an orthotopic murine breast tumor model by injecting Py230 murine breast cancer cells into syngeneic C57Bl/6 mice. In an LS diet cohort, anti-CTLA4 mAb treatment significantly reduced tumor progression (day 35, 339 ± 121 mm3), as compared to isotype mAb (639 ± 163 mm3, p < 0.05). In an HS diet cohort, treatment with anti-CTLA4 reduced the survival rate (day 80, 2/15) compared to respective normal/regular salt (NS) diet cohort (8/15, p < 0.05). Further, HS plus anti-CTLA4 mAb caused an increased expression of inflammatory cytokines (IFNγ and IL-1β) in lung infiltrating and peripheral circulating CD4+T cells. This inflammatory activation of CD4+T cells in the HS plus anti-CTLA4 cohort was associated with the upregulation of inflammasome complex activity. However, an LS diet did not induce any significant irAE response in breast-tumor-bearing mice upon treatment with anti-CTLA4 mAb, thus suggesting the role of high-salt diet in irAE response. Importantly, CD4-specific knock out of osmosensitive transcription factor NFAT5 using CD4cre/creNFAT5flox/flox transgenic mice caused a downregulation of high-salt-mediated inflammatory activation of CD4+T cells and irAE response. Taken together, our data suggest that LS diet inhibits the anti-CTLA4 mAb-induced irAE response while retaining its anti-tumor efficacy.

CC group of chemokines and associated gene expression of transcription factors: Deciphering immuno-pathogenetic aspect of oral submucous fibrosis

BackgroundOral submucous fibrosis (OSMF) is a chronic disease with significantly increasing malignant transformation rate. To date the pathogenesis of OSMF has been considered to be associated with areca nut constituents and their action on fibroblasts. However, fibrosis is also associated with immunological factors such as chemokines. In-depth analysis of such factors is the need of the hour in OSMF to better understand the pathogenesis so that effective therapeutic strategies can be developed in the future. Materials and methodClinically diagnosed cases of OSMF (n=21) and healthy individuals (n=10) were enrolled in the present study. Chemokines such as CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL28, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11 were assessed using the chemokine bead array in conjunction with the flow cytometry, along with real-time PCR (RT-PCR). The transcription factors CREB, NF-κB and NFAT5 were also studied for their expressions. The analysis of pg/ml (picogram/milliliter) values was done by using LEGENDplex™ Data Analysis Software. ResultsThe results obtained demonstrated early phase transient increase in CXCL-11, CCL20, CXCL9, CCL3, CCL2, CXCL10 and CXCL8. However, the expression of CCL3, CXCL10 and CXCL8 was higher in the late stage as compared to the early stage. The relative gene expression of CREB, NF-κB, NFAT5 were upregulated in the late stage of OSMF when compared to normal. ConclusionDistinctive sets of chemokine expression during the early and late stages of OSMF suggest a unique pattern of disease progression playing an important role in the pathogenesis.

Computational System Level Approaches for Discerning Reciprocal Regulation of IL10 and IL12 in Leishmaniasis.

IL12 and IL10 are two of the major cytokines which control the fate of Leishmaniasis. This paper presents two models healthy state and diseased state which shows how secretion of IL12 is responsible for parasite elimination and IL10 can jeopardize the parasite elimination and promote its survival. Epigenetic modification in the host IL12 and IL10 promoter can decide the fate of parasites. It was observed that reciprocal relationship exists between IL12 and IL10 and that is majorly controlled by a transcription factor NFAT5 from Rel family of transcription factors. By targeting this transcription factor at the cellular level, it might be possible to modulate the release of powerful pro-inflammatory cytokines, thereby reducing parasite survival. The mathematical models developed here serves as a step towards finding a key component that can pave a way for therapeutic investigation.

NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

Effect of MgCl2 and GdCl3 on ORAI1 Expression and Store-Operated Ca2+ Entry in Megakaryocytes.

In chronic kidney disease, hyperphosphatemia upregulates the Ca2+ channel ORAI and its activating Ca2+ sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca2+ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+. The present study explored whether sustained exposure to Mg2+ or Gd3+ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl2 or 50 µM GdCl3. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and SOCE from the increase in [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, Mg2+ and Gd3+ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg2+ and the CaSR agonist Gd3+ interfere with phosphate-induced dysregulation of [Ca2+]i in megakaryocytes.

Photoperiod-dependent developmental reprogramming of the transcriptional response to seawater entry in Atlantic salmon (Salmo salar).

The developmental transition of juvenile salmon from a freshwater resident morph (parr) to a seawater (SW) migratory morph (smolt), known as smoltification, entails a reorganization of gill function to cope with the altered water environment. Recently, we used RNAseq to characterize the breadth of transcriptional change which takes place in the gill in the FW phase of smoltification. This highlighted the importance of extended exposure to short, winter-like photoperiods (SP) followed by a subsequent increase in photoperiod for completion of transcriptional reprogramming in FW and efficient growth following transfer to SW. Here, we extend this analysis to examine the consequences of this photoperiodic history-dependent reprogramming for subsequent gill responses upon exposure to SW. We use RNAseq to analyze gill samples taken from fish raised on the photoperiod regimes we used previously and then challenged by SW exposure for 24 hours. While fish held on constant light (LL) throughout were able to hypo-osmoregulate during a 24 hours SW challenge, the associated gill transcriptional response was highly distinctive from that in fish which had experienced a 7-week period of exposure to SP followed by a return to LL (SPLL) and had consequently acquired the characteristics of fully developed smolts. Fish transferred from LL to SP, and then held on SP for the remainder of the study was unable to hypo-osmoregulate, and the associated gill transcriptional response to SW exposure featured many transcripts apparently regulated by the glucocorticoid stress axis and by the osmo-sensing transcription factor NFAT5. The importance of these pathways for the gill transcriptional response to SW exposure appears to diminish as a consequence of photoperiod mediated induction of the smolt phenotype, presumably reflecting preparatory developmental changes taking place during this process.

Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.

Co-expression of glycosylated aquaporin-1 and transcription factor NFAT5 contributes to aortic stiffness in diabetic and atherosclerosis-prone mice.

Increased stiffness characterizes the early change in the arterial wall with subclinical atherosclerosis. Proteins inducing arterial stiffness in diabetes and hypercholesterolaemia are largely unknown. This study aimed at determining the pattern of protein expression in stiffening aorta of diabetic and hypercholesterolaemic mice. Male Ins2+/Akita mice were crossbred with ApoE−/− (Ins2+/Akita: ApoE−/−) mice. Relative aortic distension (relD) values were determined by ultrasound analysis and arterial stiffness modulators by immunoblotting. Compared with age‐ and sex‐matched C57/BL6 control mice, the aortas of Ins2+/Akita, ApoE−/− and Ins2+/Akita:ApoE−/− mice showed increased aortic stiffness. The aortas of Ins2+/Akita, ApoE−/− and Ins2+/Akita:ApoE−/− mice showed greater expression of VCAM‐1, collagen type III, NADPH oxidase and iNOS, as well as reduced elastin, with increased collagen type III‐to‐elastin ratio. The aorta of Ins2+/Akita and Ins2+/Akita:ApoE−/− mice showed higher expression of eNOS and cytoskeletal remodelling proteins, such as F‐actin and α‐smooth muscle actin, in addition to increased glycosylated aquaporin (AQP)‐1 and transcription factor NFAT5, which control the expression of genes activated by high glucose‐induced hyperosmotic stress. Diabetic and hypercholesterolaemic mice have increased aortic stiffness. The association of AQP1 and NFAT5 co‐expression with aortic stiffness in diabetes and hypercholesterolaemia may represent a novel molecular pathway or therapeutic target.

The transcription factor NFAT5 limits infection-induced type I interferon responses.

Type I interferon (IFN-I) provides effective antiviral immunity but can exacerbate harmful inflammatory reactions and cause hematopoietic stem cell (HSC) exhaustion; therefore, IFN-I expression must be tightly controlled. While signaling mechanisms that limit IFN-I induction and function have been extensively studied, less is known about transcriptional repressors acting directly on IFN-I regulatory regions. We show that NFAT5, an activator of macrophage pro-inflammatory responses, represses Toll-like receptor 3 and virus-induced expression of IFN-I in macrophages and dendritic cells. Mice lacking NFAT5 exhibit increased IFN-I production and better control of viral burden upon LCMV infection but show exacerbated HSC activation under systemic poly(I:C)-induced inflammation. We identify IFNβ as a primary target repressed by NFAT5, which opposes the master IFN-I inducer IRF3 by binding to an evolutionarily conserved sequence in the IFNB1 enhanceosome that overlaps a key IRF site. These findings illustrate how IFN-I responses are balanced by simultaneously opposing transcription factors.

Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance.

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50–100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400–500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

Uric Acid Activates Aldose Reductase and the Polyol Pathway for Endogenous Fructose Production and Fat Accumulation in the Development of Fatty Liver

Dietary sugars containing fructose have been strongly associated with the development of fatty liver, but recent studies also suggest fructose can be produced in the liver via the polyol pathway where it may also induce hepatic fat accumulation. Here we investigate whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats, and whether this stimulation was associated with endogenous fructose production and fat accumulation. Uric acid dose stimulated the expression of aldose reductase in HepG2 cells in association with endogenous fructose production and triglyceride accumulation. The mechanism was mediated by uric acid‐induced oxidative stress with the stimulation of the transcription factor NFAT5. Uric acid could amplify the effects of elevated glucose levels to stimulate hepatocyte triglyceride accumulation. Hyperuricemic rats also showed elevated hepatic aldose reductase expression, endogenous fructose accumulation and fat accumulation that was significantly reduced by co‐administration of the xanthine oxidase inhibitor, allopurinol. These studies suggest that uric acid, which is generated during fructose metabolism, may act as a positive feedback to stimulate endogenous fructose production by stimulating aldose reductase in the polyol pathway. These studies provide an amplifying mechanism by which soft drinks rich in glucose and fructose can induce non‐alcoholic fatty liver disease.Support or Funding InformationSupported by NIH Grant R01 DK108859This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Inflammatory Functions of Macrophages and T Lymphocytes by NFAT5.

The transcription factor NFAT5, also known as TonEBP, belongs to the family of Rel homology domain-containing factors, which comprises the NF-κB proteins and the calcineurin-dependent NFAT1 to NFAT4. NFAT5 shares several structural and functional features with other Rel-family factors, for instance it recognizes DNA elements with the same core sequence as those bound by NFAT1 to 4, and like NF-κB it responds to Toll-like receptors (TLR) and activates macrophage responses to microbial products. On the other hand, NFAT5 is quite unique among Rel-family factors as it can be activated by hyperosmotic stress caused by elevated concentrations of extracellular sodium ions. NFAT5 regulates specific genes but also others that are inducible by NF-κB and NFAT1 to 4. The ability of NFAT5 to do so in response to hypertonicity, microbial products, and inflammatory stimuli may extend the capabilities of immune cells to mount effective anti-pathogen responses in diverse microenvironment and signaling conditions. Recent studies identifying osmostress-dependent and -independent functions of NFAT5 have broadened our understanding of how NFAT5 may modulate immune function. In this review we focus on the role of NFAT5 in macrophages and T cells in different contexts, discussing findings from in vivo mouse models of NFAT5 deficiency and reviewing current knowledge on its mechanisms of regulation. Finally, we propose several questions for future research.

Sodium chloride is an ionic checkpoint for human TH2 cells and shapes the atopic skin microenvironment.

The incidence of allergic diseases has increased over the past 50 years, likely due to environmental factors. However, the nature of these factors and the mode of action by which they induce the type 2 immune deviation characteristic of atopic diseases remain unclear. It has previously been reported that dietary sodium chloride promotes the polarization of T helper 17 (TH17) cells with implications for autoimmune diseases such as multiple sclerosis. Here, we demonstrate that sodium chloride also potently promotes TH2 cell responses on multiple regulatory levels. Sodium chloride enhanced interleukin-4 (IL-4) and IL-13 production while suppressing interferon-γ (IFN-γ) production in memory T cells. It diverted alternative T cell fates into the TH2 cell phenotype and also induced de novo TH2 cell polarization from naïve T cell precursors. Mechanistically, sodium chloride exerted its effects via the osmosensitive transcription factor NFAT5 and the kinase SGK-1, which regulated TH2 signature cytokines and master transcription factors in hyperosmolar salt conditions. The skin of patients suffering from atopic dermatitis contained elevated sodium compared to nonlesional atopic and healthy skin. These results suggest that sodium chloride represents a so far overlooked cutaneous microenvironmental checkpoint in atopic dermatitis that can induce TH2 cell responses, the orchestrators of atopic diseases.

The role of chondroitin sulfate in regulating hypertrophy during MSC chondrogenesis in a cartilage mimetic hydrogel under dynamic loading

Mesenchymal stem cells (MSCs) are promising for cartilage regeneration, but readily undergo terminal differentiation. The aim of this study was two-fold: a) investigate physiochemical cues from a cartilage-mimetic hydrogel under dynamic compressive loading on MSC chondrogenesis and hypertrophy and b) identify whether Smad signaling and p38 MAPK signaling mediate hypertrophy during MSC chondrogenesis. Human MSCs were encapsulated in photoclickable poly(ethylene glycol) hydrogels containing chondroitin sulfate and RGD, cultured under dynamic compressive loading or free swelling for three weeks, and evaluated by qPCR and immunohistochemistry. Loading inhibited hypertrophy in the cartilage-mimetic hydrogel indicated by a reduction in pSmad 1/5/8, Runx2, and collagen X proteins, while maintaining chondrogenesis by pSmad 2/3 and collagen II proteins. Inhibiting pSmad 1/5/8 under free swelling culture significantly reduced collagen X protein, similar to the loading condition. Chondroitin sulfate was necessary for load-inhibited hypertrophy and correlated with enhanced S100A4 expression, which is downstream of the osmotic responsive transcription factor NFAT5. Inhibiting p38 MAPK under loading reduced S100A4 expression, and upregulated Runx2 and collagen X protein. Findings from this study indicate that chondroitin sulfate with dynamic loading create physiochemical cues that support MSC chondrogenesis and attenuate hypertrophy through Smad 1/5/8 inhibition and p38 MAPK upregulation.

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5.

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.