NFAT Pathway Research Articles

Regorafenib Attenuates Osteoclasts Differentiation by Inhibiting the NF-κB, NFAT, ERK, and p38 Signaling Pathways.

Osteolytic diseases such as osteoporosis and neoplastic bone metastases are caused by the excessive activation of osteoclasts. Inhibiting the excessive activation of osteoclasts is a crucial strategy for treating osteolytic diseases. This study investigated the roles and mechanisms of regorafenib, a tyrosine kinase inhibitor, on osteoclasts and osteolytic diseases. We first identified the potential targets and mechanisms of regorafenib on osteoclast-related osteolytic diseases using network pharmacological analysis and molecular docking techniques. Then, we verified its role and mechanism on osteoclasts via cellular and animal experiments. Network pharmacology analysis identified 89 common targets shared by regorafenib and osteoclast-related osteolytic diseases. Enrichment analysis suggested that regorafenib may act on osteoclast-related osteolytic diseases by modulating targets such as AKT1, CASP3, MMP9, and MAPK3, regulating biological processes such as cell proliferation, apoptosis, and phosphorylation regulation, and influencing signaling pathways such as MAPK, PI3K/AKT, and osteoclast differentiation. The molecular docking results indicated that regorafenib and AKT1, CASP3, MMP9, MAPK3, and MAPK14 were stably docked. Cell experiments demonstrated that regorafenib significantly inhibited osteoclast differentiation and bone resorption in RAW 264.7 cells and bone marrow macrophages in a dose-dependent manner, with up to 50% reduction at 800 nM concentration without exhibiting cytotoxic effects. Furthermore, Western blot and RT-qPCR results demonstrated that regorafenib inhibited osteoclast differentiation by blocking the transduction of RANKL-induced NF-κB, p38, ERK, and NFAT signaling pathways. In vivo studies using an ovariectomized mouse model showed that regorafenib significantly improved bone volume fraction (BV/TV), bone surface to total volume (BS/TV), and number of trabeculae (TB.N), as well as reduced trabecular separation (Tb.Sp) compared to the OVX groups (P < 0.05). TRAcP staining results revealed a reduction in the number of osteoclasts with regorafenib treatment (P < 0.01). These results indicate that regorafenib exerts its protective effects against osteoclast-related osteolytic disease by inhibiting the RANKL-induced NF-κB, NFAT, ERK, and p38 signaling pathways. This study proves that regorafenib may serve as a potential therapeutic agent for osteoclast-related osteolytic diseases.

Calcineurin-NFAT signaling controls neutrophils' ability of chemoattraction upon fungal infection.

Calcineurin-nuclear factor of activated T cells (CN-NFAT) inhibitors are widely clinically used drugs for immunosuppression, but besides their required T cell response inhibition, they also undesirably affect innate immune cells. Disruption of innate immune cell function can explain the observed susceptibility of CN-NFAT inhibitor-treated patients to opportunistic fungal infections. Neutrophils play an essential role in innate immunity as a defense against pathogens; however, the effect of CN-NFAT inhibitors on neutrophil function was poorly described. Thus, we tested the response of human neutrophils to opportunistic fungal pathogens, namely Candida albicans and Aspergillus fumigatus, in the presence of CN-NFAT inhibitors. Here, we report that the NFAT pathway members were expressed in neutrophils and mediated part of the neutrophil response to pathogens. Upon pathogen exposure, neutrophils underwent profound transcriptomic changes with subsequent production of effector molecules. Importantly, genes and proteins involved in the regulation of the immune response and chemotaxis, including the chemokines CCL2, CCL3, and CCL4 were significantly upregulated. The presence of CN-NFAT inhibitors attenuated the expression of these chemokines and impaired the ability of neutrophils to chemoattract other immune cells. Our results amend knowledge about the impact of CN-NFAT inhibition in human neutrophils.

Role of TRPV1 and TRPA1 in TSLP production in nasal epithelial cells

BackgroundTRP protein is sensitive to external temperature changes, but its pathogenic mechanism in the upper airway mucosa is still unclear. ObjectiveTo investigate the mechanism of TRPV1and TRPA1 in regulating the secretion of inflammatory factors in nasal epithelial cells. MethodsThe expression of TRPV1 and TRPA1 in nasal mucosal epithelial cells was investigated using immunofluorescence assays. Epithelial cells were stimulated with TRPV1 and TRPA1 agonists and antagonists, and changes in Ca2+ release and inflammatory factor secretion in epithelial cells were detected. TSLP secretion stimulated with the calcium chelating agent EGTA was evaluated. The transcription factor NFAT was observed by immunofluorescence staining. ResultsTRPV1 and TRPA1 expression was detected in nasal epithelial cells, and Ca2+ influx was increased after stimulation with agonists. After the activation of TRPV1 and TRPA1, the gene expression of TSLP, IL-25, and IL-33 and the protein expression levels of TSLP and IL-33 were increased, and only TSLP could be inhibited by antagonists and siRNAs. After administration of EGTA, the secretion of TSLP was inhibited significantly, and the expression of the transcription factor NFAT in the nucleus was observed after activation of the TRPV1 and TRPA1 proteins in epithelial cells. ConclusionActivation of TRPV1 and TRPA1 on nasal epithelial cells stimulates the generation of TSLP through the Ca2+/NFAT pathway. It also induces upregulation of IL-25 and IL-33 gene expression levels and increased levels of IL-33 protein, leading to the development of airway inflammation.

Abstract 3979: PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3979.

Modulation of Dendritic Cell Function via Nanoparticle-Induced Cytosolic Calcium Changes.

Calcium nanoparticles have been investigated for applications, such as drug and gene delivery. Additionally, Ca2+ serves as a crucial second messenger in the activation of immune cells. However, few studies have systematically studied the effects of calcium nanoparticles on the calcium levels and functions within immune cells. In this study, we explore the potential of calcium nanoparticles as a vehicle to deliver calcium into the cytosol of dendritic cells (DCs) and influence their functions. We synthesized calcium hydroxide nanoparticles, coated them with a layer of silica to prevent rapid degradation, and further conjugated them with anti-CD205 antibodies to achieve targeted delivery to DCs. Our results indicate that these nanoparticles can efficiently enter DCs and release calcium ions in a controlled manner. This elevation in cytosolic calcium activates both the NFAT and NF-κB pathways, in turn promoting the expression of costimulatory molecules, antigen-presenting molecules, and pro-inflammatory cytokines. In mouse tumor models, the calcium nanoparticles enhanced the antitumor immune response and augmented the efficacy of both radiotherapy and chemotherapy without introducing additional toxicity. Our study introduces a safe nanoparticle immunomodulator with potential widespread applications in cancer therapy.

Inhibition of DYRK1A attenuates vascular remodeling in pulmonary arterial hypertension via suppressing STAT3/Pim-1/NFAT pathway

ABSTRACT Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling caused by the excessive proliferation and survival of pulmonary artery smooth muscle cells (PASMCs). Dual-specificity tyrosine regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in the regulation of multiple biological functions, including cell proliferation and survival. However, the role and underlying mechanisms of DYRK1A in PAH pathogenesis remain unclear. We found that DYRK1A was upregulated in PASMCs in response to hypoxia, both in vivo and in vitro. Inhibition of DYRK1A by harmine significantly attenuated hypoxia-induced pulmonary hypertension and pulmonary artery remodeling. Mechanistically, we found that DYRK1A promoted pulmonary arterial remodeling by enhancing the proliferation and survival of PASMCs through activating the STAT3/Pim-1/NFAT pathway, because STAT3 gain-of-function via adeno-associated virus serotype 2 (AAV2) carrying the constitutively active form of STAT3 (STAT3C) nearly abolished the protective effect of harmine on PAH. Collectively, our results reveal a significant role for DYRK1A in pulmonary arterial remodeling and suggest it as a drug target with translational potential for the treatment of PAH.

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts.

Total alkaloids of Fritillaria unibracteata var. wabuensis bulbus ameliorate chronic asthma via the TRPV1/Ca2+/NFAT pathway

BackgroundAsthma is a chronic inflammatory disease that is challenging to treat. Fritillaria unibracteata var. wabuensis (FUW) is the plant origin for the famous Chinese antitussive medicine Fritillaria Cirrhosae Bulbus. The total alkaloids of Fritillaria unibracteata var. wabuensis bulbus (TAs-FUW) have anti-inflammatory properties and may be used to treat asthma. PurposeTo explore whether TAs-FUW have bioactivity against airway inflammation and a therapeutic effect on chronic asthma. MethodsThe alkaloids were extracted via ultrasonication in a cryogenic chloroform-methanol solution after ammonium-hydroxide percolation of the bulbus. UPLC-Q-TOF/MS was used to characterize the composition of TAs-FUW. An ovalbumin (OVA)-induced asthmatic mouse model was established. We used whole-body plethysmography, ELISA, western blotting, RT-qPCR, and histological analyses to assess the pulmonary pathological changes in these mice after TAs-FUW treatment. Additionally, TNF-α/IL-4–induced inflammation in BEAS-2B cells was used as an in vitro model, whereby the effects of various doses of TAs-FUW on the TRPV1/Ca2+-dependent NFAT-induced expression of TSLP were assessed. Stimulation and inhibition of TRPV1 receptors by capsaicin (CAP) and capsazepine (CPZ), respectively, were used to validate the effect of TAs-FUW. ResultsThe UPLC-Q-TOF/MS analysis revealed that TAs-FUW mainly contain six compounds (peiminine, peimine, edpetiline, khasianine, peimisine, and sipeimine). TAs-FUW improved airway inflammation and obstruction, mucus secretion, collagen deposition, and leukocyte and macrophage infiltration, and downregulated TSLP by inhibiting the TRPV1/NFAT pathway in asthmatic mice. In vitro, the application of CPZ demonstrated that the TRPV1 channel is involved in TNF-α/IL-4–mediated regulation of TSLP. TAs-FUW suppressed TNF-α/IL-4–induced TSLP generation expression by regulating the TRPV1/Ca2+/NFAT pathway. Furthermore, TAs-FUW reduced CAP-induced TSLP release by inhibiting TRPV1 activation. Notably, sipeimine and edpetiline each were sufficient to block the TRPV1-mediated Ca2+ influx. ConclusionOur study is the first to demonstrate that TNF-α/IL-4 can activate the TRPV1 channel. TAs-FUW can alleviate asthmatic inflammation by suppressing the TRPV1 pathway and thereby preventing the increase in cellular Ca2+ influx and the subsequent NFAT activation. The alkaloids in FUW may be used for complementary or alternative therapies in asthma.

Transcriptomic Differentiation of Phenotypes in Chronic Rhinosinusitis and Its Implications for Understanding the Underlying Mechanisms

Chronic rhinosinusitis (CRS) is a multifaceted disease with variable clinical courses and outcomes. We aimed to determine CRS-associated nasal-tissue transcriptome in clinically well-characterized and phenotyped individuals, to gain a novel insight into the biological pathways of the disease. RNA-sequencing of tissue samples of patients with CRS with polyps (CRSwNP), without polyps (CRSsNP), and controls were performed. Characterization of differently expressed genes (DEGs) and functional and pathway analysis was undertaken. We identified 782 common CRS-associated nasal-tissue DEGs, while 375 and 328 DEGs were CRSwNP- and CRSsNP-specific, respectively. Common key DEGs were found to be involved in dendritic cell maturation, the neuroinflammation pathway, and the inhibition of the matrix metalloproteinases. Distinct CRSwNP-specific DEGs were involved in NF-kβ canonical pathways, Toll-like receptor signaling, HIF1α regulation, and the Th2 pathway. CRSsNP involved the NFAT pathway and changes in the calcium pathway. Our findings offer new insights into the common and distinct molecular mechanisms underlying CRSwNP and CRSsNP, providing further understanding of the complex pathophysiology of the CRS, with future research directions for novel treatment strategies.

Glutamine Metabolism Underlies the Functional Similarity of T Cells between Nile Tilapia and Tetrapod.

As the lowest organisms possessing T cells, fish are instrumental for understanding T cell evolution and immune defense in early vertebrates. This study established in Nile tilapia models suggests that T cells play a critical role in resisting Edwardsiella piscicida infection via cytotoxicity and are essential for IgM+ B cell response. CD3 and CD28 monoclonal antibody crosslinking reveals that full activation of tilapia T cells requires the first and secondary signals, while Ca2+ -NFAT, MAPK/ERK, NF-κB, and mTORC1 pathways and IgM+ B cells collectively regulate T cell activation. Thus, despite the large evolutionary distance, tilapia and mammals such as mice and humans exhibit similar T cell functions. Furthermore, it is speculated that transcriptional networks and metabolic reprogramming, especially c-Myc-mediated glutamine metabolism triggered by mTORC1 and MAPK/ERK pathways, underlie the functional similarity of T cells between tilapia and mammals. Notably, tilapia, frogs, chickens, and mice utilize the same mechanisms to facilitate glutaminolysis-regulated T cell responses, and restoration of the glutaminolysis pathway using tilapia components rescues the immunodeficiency of human Jurkat T cells. Thus, this study provides a comprehensive picture of T cell immunity in tilapia, sheds novel perspectives for understanding T cell evolution, and offers potential avenues for intervening in human immunodeficiency.

Glial cell reactivity and oxidative stress prevention in Alzheimer’s disease mice model by an optimized NMDA receptor antagonist

In Alzheimer's disease pathology, several neuronal processes are dysregulated by excitotoxicity including neuroinflammation and oxidative stress (OS). New therapeutic agents capable of modulating such processes are needed to foster neuroprotection. Here, the effect of an optimised NMDA receptor antagonist, UB-ALT-EV and memantine, as a gold standard, have been evaluated in 5XFAD mice. Following treatment with UB-ALT-EV, nor memantine, changes in the calcineurin (CaN)/NFAT pathway were detected. UB-ALT-EV increased neurotropic factors (Bdnf, Vgf and Ngf) gene expression. Treatments reduced astrocytic and microglial reactivity as revealed by glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) quantification. Interestingly, only UB-ALT-EV was able to reduce gene expression of Trem2, a marker of microglial activation and NF-κB. Pro-inflammatory cytokines Il-1β, Ifn-γ, Ccl2 and Ccl3 were down-regulated in UB-ALT-EV-treated mice but not in memantine-treated mice. Interestingly, the anti-inflammatory markers of the M2-migroglial phenotype, chitinase-like 3 (Ym1) and Arginase-1(Arg1), were up-regulated after treatment with UB-ALT-EV. Since iNOS gene expression decreased after UB-ALT-EV treatment, a qPCR array containing 84 OS-related genes was performed. We found changes in Il-19, Il-22, Gpx6, Ncf1, Aox1 and Vim gene expression after UB-ALT-EV. Hence, our results reveal a robust effect on neuroinflammation and OS processes after UB-ALT-EV treatment, surpassing the memantine effect in 5XFAD.

Activation of the Ca2+/NFAT Pathway by Assembly of Hepatitis C Virus Core Protein into Nucleocapsid-like Particles

Hepatitis C virus (HCV) is the primary pathogen responsible for liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been linked to several aspects of HCV pathology, including oncogenesis, immune evasion and stress responses. We and others have previously shown that C expression in various cell lines activates Ca2+ signaling and alters Ca2+ homeostasis. In this study, we identified two distinct C protein regions that are required for the activation of Ca2+/NFAT signaling. In the basic N-terminal domain, which has been implicated in self-association of C, amino acids 1–68 were critical for NFAT activation. Sedimentation analysis of four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain was not required for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein to function. Our results indicate that targeting of HCV C to the ER is necessary but not sufficient for inducing Ca2+/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with an intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane.

Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

The CCCH-Type Zinc Finger Antiviral Protein Relieves Immunosuppression of T Cells Induced by Avian Leukosis Virus Subgroup J via the NLP-PKC-δ-NFAT Pathway.

The CCCH-type zinc finger antiviral protein (ZAP) can recognize and induce the degradation of mRNAs and proteins of certain viruses, as well as exerting its antiviral activity by activating T cells. However, the mechanism of ZAP that mediates T cell activation during virus infection remains unclear. Here, we found a potential function of ZAP that relieves immunosuppression of T cell induced by avian leukosis virus subgroup J (ALV-J) via a novel signaling pathway that involves norbin-like protein (NLP), protein kinase C delta (PKC-δ), and nuclear factor of activated T cell (NFAT). Specifically, ZAP expression activated T cells by promoting the dephosphorylation and nuclear translocation of NFAT. Furthermore, knockdown of ZAP weakened the reactivity and antiviral response of T cells. Mechanistically, ZAP reduced PKC-δ activity by upregulating and reactivating NLP by competitively binding with viral protein. Knockdown of NLP decreased the dephosphorylation of PKC-δ by ZAP expression. Moreover, we show that knockdown of PKC-δ reduced the phosphorylation levels of NFAT and enhanced its nuclear translocation. Taken together, these data revealed that ZAP relieves immunosuppression caused by ALV-J and mediates T cell activation through the NLP-PKC-δ-NFAT pathway. IMPORTANCE The evolution of the host defense system is driven synchronously in the process of resisting virus invasion. Accordingly, host innate defense factors effectively work to suppress virus replication. However, it remains unclear whether the host innate defense factors are involved in antiviral immune responses against the invasion of immunosuppressive viruses. Here, we found that CCCH-type zinc finger antiviral protein (ZAP) effectively worked in resistance to immunosuppression caused by avian leukosis virus subgroup J (ALV-J), a classic immunosuppressive virus. Evidence showed that ZAP released the phosphatase activity of NLP inhibited by ALV-J and further activated NFAT by inactivating PKC-δ. This novel molecular mechanism, i.e., ZAP regulation of the antiviral immune response by mediating the NLP-PKC-δ-NFAT pathway, has greatly enriched the understanding of the functions of host innate defense factors and provided important scientific ideas and a theoretical basis for research on immunosuppressive viruses and antiviral immunity.

The Genetic Landscape of Ocular Adnexa MALT Lymphoma Reveals Frequent Aberrations in NFAT and MEF2B Signaling Pathways.

A comprehensive constellation of somatic non-silent mutations and copy number (CN) variations in ocular adnexa marginal zone lymphoma (OAMZL) is unknown. By utilizing whole-exome sequencing in 69 tumors we define the genetic landscape of OAMZL. Mutations and CN changes in CABIN1 (30%), RHOA (26%), TBL1XR1 (22%), and CREBBP (17%) and inactivation of TNFAIP3 (26%) were among the most common aberrations. Candidate cancer driver genes cluster in the B-cell receptor (BCR), NFkB, NOTCH and NFAT signaling pathways. One of the most commonly altered genes is CABIN1, a calcineurin inhibitor acting as a negative regulator of the NFAT and MEF2B transcriptional activity. CABIN1 deletions enhance BCR-stimulated NFAT and MEF2B transcriptional activity, while CABIN1 mutations enhance only MEF2B transcriptional activity by impairing binding of mSin3a to CABIN1. Our data provide an unbiased identification of genetically altered genes that may play a role in the molecular pathogenesis of OAMZL and serve as therapeutic targets.

Role of Nuclear Factor of Activated T Cells (NFAT) Pathway in Regulating Autophagy and Inflammation in Retinal Pigment Epithelial Cells.

Nuclear factor of activated T cells (NFAT) family of transcription factors are substrates of calcineurin and play an important role in integrating Ca2+ signaling with a variety of cellular functions. Of the five NFAT proteins (NFAT1-5), NFAT1-4 are subject to dephosphorylation and activation by calcineurin, a Ca2+-calmodulin-dependent phosphatase. Increased levels of intracellular Ca2+ activates calcineurin, which in turn dephosphorylates and promotes nuclear translocation of NFAT. We investigated the functions of NFAT proteins in the retinal pigment epithelial cells (RPE). Our results show that NFAT-mediated luciferase activity was induced upon treatment with the bacterial endotoxin, lipopolysaccharide (LPS) and treatment with the NFAT peptide inhibitor, MAGPHPVIVITGPHEE (VIVIT) decreased LPS-induced NFAT luciferase activity. LPS-induced activation of NFAT-regulated cytokines (IL-6 and IL-8) is inhibited by treatment of cells with VIVIT. We also investigated the effects of NFAT signaling on the autophagy pathway. Our results show that inhibition of NFAT with VIVIT in cells deprived of nutrients resulted in cytosolic retention of transcription Factor EB (TFEB), decreased expression of TFEB-regulated coordinated Lysosomal Expression and Regulation CLEAR network genes and decreased starvation-induced autophagy flux in the RPE cells. In summary, these studies suggest that the NFAT pathway plays an important role in the regulation of autophagy and inflammation in the RPE.

A Direct Comparison of the In Vivo Efficacy and in Vitro Signaling of Chimeric Antigen Receptors and Endogenous T Cell Receptors

The adoptive transfer of chimeric antigen receptor (CAR) expressing T cells targeting the CD19 antigen induces remission in 70-90% of pediatric patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL). Despite initial enthusiasm, emerging data indicate the durability of remission is limited in a substantial number of patients, therefore, addressing relapse will be critical for increasing the clinical efficacy of CAR T cell therapy. CARs are synthetic receptors designed to redirect the antigen specificity of T cells while recapitulating the T cell activating properties of the endogenous T cell receptor (TCR). Clinical and pre-clinical observations have demonstrated that CAR T cells have inferior sensitivity to low levels of antigen and have diminished persistence in vivo compared to T cells responding to TCR activation. These observations suggest the CAR does not fully replicate the signaling of a TCR which subsequently leads to suboptimal T cell function which may underlie the inability of CAR T cell therapy to induce durable remissions in a significant number of patients.In these studies, we directly compared the in vivo and in vitro consequences of CAR versus TCR activation, as well as the signal transduction of each of these receptors. To accomplish this, we created a novel, syngeneic model system in which a 2nd generation CAR is introduced into OTI TCR transgenic T cells to generate a population of T cells (CAR-OTI cells) in which each cell recognizes the CD19 antigen via its CAR as well as the ovalbumin (OVA) antigen via its TCR, thus providing a model in which the function and signaling of each receptor could be studied within a uniform population of T cells. To stimulate either the CAR or the TCR, we generated antigen-expressing cell lines from the pre-B ALL cell line, E2aPbx, that expressed either the cognate antigen for the CAR (CD19), the TCR (OVA) or both. These cell lines allowed us to stimulate either receptor in a model which preserved the properties of the cell-to-cell interactions expected in vivo . We validated the activity of each receptor in this model, by demonstrating similar in vitro degranulation, proliferation and target cell lysis in CAR-OTI cells upon co-incubation with CAR-antigen or TCR-antigen positive E2aPbx cells. Despite the similarities seen in vitro, CAR-OTI cells adoptively transferred into mice with established E2aPbx leukemia demonstrated enhanced expansion at 4 (p<0.001) and 6 days (p<0.01) post-infusion in TCR-activated CAR-OTI cells, relative to CAR-activated CAR-OTI cells. Likewise, clearance of leukemia from the bone marrow (p<0.001) and survival (p<0.01) were each superior in mice in which CAR-OTI cells were activated through the TCR, relative to the CAR. These results demonstrate the significant difference in in vivo behavior and efficacy of CAR- versus TCR-mediated T cell activation, and suggest these differences may result from differences in the signals generated by each receptor.Using our model of CAR-OTI cells and E2aPbx cells expressing either the CAR- or TCR-antigen, we interrogated proteins in the canonical TCR signal transduction cascade after CAR or TCR activation. We demonstrated that CAR activation resulted in increased and prolonged phosphorylation of Zap70, a kinase central to canonical TCR signal transduction. Despite the increased activation of Zap70, the CAR signal demonstrated diminished activation of the MAPK family members, Erk (p<0.001) and p38 (p<0.05), which correlated with the diminished in vivo efficacy seen in this model. Furthermore, this model was used to define the global phosphoproteome of CAR- and TCR-activated CAR-OTI cells by a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) methodology in conjunction with mass spectrometry (MS). Our SILAC-MS analysis demonstrated multiple differences in the signal transduction cascade of CARs relative to TCRs, including proteins of the signalosome as well as the NFAT and NF-kB pathways. These data demonstrate that a CAR generates an “altered” signal, relative to that of a TCR, correlating with diminished in vivo expansion and efficacy in the treatment of established leukemia. These findings underscore the need to better understand the biology of chimeric antigen receptors as a means of rationally altering their design to improve their clinical effectiveness in mediating durable remissions in patients with relapsed/refractory ALL. DisclosuresNo relevant conflicts of interest to declare.

Camu-Camu Fruit Extract Inhibits Oxidative Stress and Inflammatory Responses by Regulating NFAT and Nrf2 Signaling Pathways in High Glucose-Induced Human Keratinocytes.

Myrciaria dubia (HBK) McVaugh (camu-camu) belongs to the family Myrtaceae. Although camu-camu has received a great deal of attention for its potential pharmacological activities, there is little information on the anti-oxidative stress and anti-inflammatory effects of camu-camu fruit in skin diseases. In the present study, we investigated the preventative effect of 70% ethanol camu-camu fruit extract against high glucose-induced human keratinocytes. High glucose-induced overproduction of reactive oxygen species (ROS) was inhibited by camu-camu fruit treatment. In response to ROS reduction, camu-camu fruit modulated the mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NFAT) signaling pathways related to inflammation by downregulating the expression of proinflammatory cytokines and chemokines. Furthermore, camu-camu fruit treatment activated the expression of nuclear factor E2-related factor 2 (Nrf2) and subsequently increased the NAD(P)H:quinone oxidoreductase1 (NQO1) expression to protect keratinocytes against high-glucose-induced oxidative stress. These results indicate that camu-camu fruit is a promising material for preventing oxidative stress and skin inflammation induced by high glucose level.

The inhibitory receptor Tim-3 fails to suppress IFN-\u03b3 production via the NFAT pathway in NK-cell, unlike that in CD4+ T cells

BackgroundT cell immunoglobulin and mucin domain-containing-3 (Tim-3) is a negative regulator expressed on T cells, and is also expressed on natural killer (NK) cells. The function of Tim-3 chiefly restricts IFNγ-production in T cells, however, the impact of Tim-3 on NK cell function has not been clearly elucidated.ResultsIn this study, we demonstrated down-regulation of Tim-3 expression on NK cells while Tim-3 is upregulated on CD4+ T cells during HIV infection. Functional assays indicated that Tim-3 mediates suppression of CD107a degranulation in NK cells and CD4+ T cells, while it fails to inhibit the production of IFN-γ by NK cells. Analyses of downstream pathways using an antibody to block Tim-3 function demonstrated that Tim-3 can inhibit ERK and NFκB p65 signaling; however, it failed to suppress the NFAT pathway. Further, we found that the NFAT activity in NK cells was much higher than that in CD4+ T cells, indicating that NFAT pathway is important for promotion of IFN-γ production by NK cells.ConclusionsThus, our data show that the expression of Tim-3 on NK cells is insufficient to inhibit IFN-γ production. Collectively, our findings demonstrate a potential mechanism of Tim-3 regulation of NK cells and a target for HIV infection immunotherapy.

Role of NFAT in the Progression of Diabetic Atherosclerosis.

Nuclear factor of activated T cells (NFAT) is a transcription factor with a multidirectional regulatory function, that is widely expressed in immune cells, including cells in the cardiovascular system, and non-immune cells. A large number of studies have confirmed that calcineurin/NFAT signal transduction is very important in the development of vascular system and cardiovascular system during embryonic development, and plays some role in the occurrence of vascular diseases such as atherosclerosis, vascular calcification, and hypertension. Recent in vitro and in vivo studies have shown that NFAT proteins and their activation in the nucleus and binding to DNA-related sites can easily ɨnduce the expression of downstream target genes that participate in the proliferation, migration, angiogenesis, and vascular inflammation of vascular wall related cells in various pathophysiological states. NFAT expression is regulated by various signaling pathways, including CD137-CD137L, and OX40-OX40L pathways. As a functionally diverse transcription factor, NFAT interacts with a large number of signaling molecules to modulate intracellular and extracellular signaling pathways. These NFAT-centered signaling pathways play important regulatory roles in the progression of atherosclerosis, such as in vascular smooth muscle cell phenotypic transition and migration, endothelial cell injury, macrophage-derived foam cell formation, and plaque calcification. NFAT and related signaling pathways provide new therapeutic targets for vascular diseases such as atherosclerosis. Hence, further studies of the mechanism of NFAT in the occurrence and evolution of atherosclerosis remain crucial.