Regulation Of IFN-γ Research Articles

Selective regulation of IFN-γ and IL-4 co-producing unconventional T cells by purinergic signaling.

Unconventional T cells, including mucosal-associated invariant T (MAIT), natural killer T (NKT), and gamma-delta T (γδT) cells, comprise distinct T-bet+, IFN-γ+ and RORγt+, IL-17+ subsets which play differential roles in health and disease. NKT1 cells are susceptible to ARTC2-mediated P2X7 receptor (P2RX7) activation, but the effects on other unconventional T-cell types are unknown. Here, we show that MAIT, γδT, and NKT cells express P2RX7 and are sensitive to P2RX7-mediated cell death. Mouse peripheral T-bet+ MAIT1, γδT1, and NKT1 cells, especially in liver, co-express ARTC2 and P2RX7. These markers could be further upregulated upon exposure to retinoic acid. Blocking ARTC2 or inhibiting P2RX7 protected MAIT1, γδT1, and NKT1 cells from cell death, enhanced their survival in vivo, and increased the number of IFN-γ-secreting cells without affecting IL-17 production. Importantly, this revealed the existence of IFN-γ and IL-4 co-producing unconventional T-cell populations normally lost upon isolation due to ARTC2/P2RX7-induced death. Administering extracellular NAD in vivo activated this pathway, depleting P2RX7-sensitive unconventional T cells. Our study reveals ARTC2/P2RX7 as a common regulatory axis modulating the unconventional T-cell compartment, affecting the viability of IFN-γ- and IL-4-producing T cells, offering important insights to facilitate future studies into how these cells can be regulated in health and disease.

Maternal separation alters peripheral immune responses associated with IFN-γ and OT in mice

The co-evolution of social behavior and the immune system plays a critical role in individuals' adaptation to their environment. However, also need for further research on the key molecules that co-regulate social behavior and immunity. This study focused on neonatal mice that were separated from their mothers for 4 hours per day between the 6th and 16th day after birth. The results showed that these mice had lower plasma levels of IFN-γ and oxytocin, but higher levels of plasma glucocorticoids (GC), then impacting their social abilities. Additionally, maternal separation led to decreased levels of BDNF, IGF2, and CREB mRNAs in the hippocampus, while levels in the prefrontal cortex (PFC) remained unaffected. Maternal separation also resulted in increased levels of oxytocin and CRH mRNA in the hypothalamus, as well as an increase in CD45+ lymphocyte subsets in the meninges and choroid plexus (CP), with CD8+ lymphocytes in meninges and CD4+ lymphocytes in CP showing an increase. In IFN-γ-/- mice, a decrease in social preference was observed alongside lower plasma oxytocin levels. Moreover, IFN-γ-/- mice exhibited reduced numbers of oxytocin neurons in the paraventricular nucleus of the paraventricular nucleus of hypothalamus (PVN), decreased BDNF levels in the PFC and hippocampus, and alterations in CD45+ lymphocytes in CP and meninges, with an increase in CD8+ lymphocytes in meninges and CD4+ lymphocytes in CP. These findings highlight the immunological impact of social stress on IFN-γ regulation, suggesting that the immunomodulatory molecule IFN-γ may influence social behavior by affecting synaptic efficiency in brain regions such as the hippocampus and PFC, which are linked to oxytocin in the PVN.

Th1 cells reduce the osteoblast-like phenotype in valvular interstitial cells by inhibiting NLRP3 inflammasome activation in macrophages

Background and aimsInflammation is initiates the propagation phase of aortic valve calcification. The activation of NLRP3 signaling in macrophages plays a crucial role in the progression of calcific aortic valve stenosis (CAVS). IFN-γ regulates NLRP3 activity in macrophages. This study aimed to explore the mechanism of IFN-γ regulation and its impact on CAVS progression and valve interstitial cell transdifferentiation.Methods and resultsThe number of Th1 cells and the expression of IFN-γ and STAT1 in the aortic valve, spleen and peripheral blood increased significantly as CAVS progressed. To explore the mechanisms underlying the roles of Th1 cells and IFN-γ, we treated CAVS mice with IFN-γ-AAV9 or an anti-IFN-γ neutralizing antibody. While IFN-γ promoted aortic valve calcification and dysfunction, it significantly decreased NLRP3 signaling in splenic macrophages and Ly6C+ monocytes. In vitro coculture showed that Th1 cells inhibited NLPR3 activation in ox-LDL-treated macrophages through the IFN-γR1/IFN-γR2-STAT1 pathway. Compared with untreated medium, conditioned medium from Th1-treated bone marrow–derived macrophages reduced the osteogenic calcification of valvular interstitial cells.ConclusionInhibition of the NLRP3 inflammasome by Th1 cells protects against valvular interstitial cell calcification as a negative feedback mechanism of adaptive immunity toward innate immunity. This study provides a precision medicine strategy for CAVS based on the targeting of anti-inflammatory mechanisms.Graphical

Multi-omics analysis reveals GAPDH posttranscriptional regulation of IFN-γ and PHGDH as a metabolic checkpoint of microglia polarization

A “switch” in the metabolic pattern of microglia is considered to be required to meet the metabolic demands of cell survival and functions. However, how metabolic switches regulate microglial function remains controversial. We found here that exposure to amyloid-β triggers microglial inflammation accompanied by increasing GAPDH levels. The increase of GAPDH, a glycolysis enzyme, leads to the reduced release of interferon-γ (IFN-γ) from inflammatory microglia. Such alternation is translational and is regulated by the binding of glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through influencing IFN-γ expression, regulates microglia functions, including phagocytosis and cytokine production. Phosphoglycerate dehydrogenase (PHGDH), screened from different state microglia by metabolomics combined with METARECON analysis, is a metabolic enzyme adjacent downstream of GAPDH and synthesizes serine on the collateral pathway derived from glycolysis. Polarization of microglial with PHGDH as a metabolic checkpoint can be bidirectionally regulated by adding IL-4 or giving PHGDH inhibitors. Therefore, regulation of metabolic enzymes not only reprograms metabolic patterns, but also manipulates microglia functions. Further study should be performed to explore the mechanism of metabolic checkpoints in human microglia or more in vivo animal experiments, and may expand to the effects of various metabolic substrates or enzyme, such as lipids and amino acids, on the functions of microglia.

IL-15 Priming Alters IFN-γ Regulation in Murine NK Cells.

NK effector functions can be triggered by inflammatory cytokines and engagement of activating receptors. NK cell production of IFN-γ, an important immunoregulatory cytokine, exhibits activation-specific IFN-γ regulation. Resting murine NK cells exhibit activation-specific metabolic requirements for IFN-γ production, which are reversed for activating receptor-mediated stimulation following IL-15 priming. Although both cytokine and activating receptor stimulation leads to similar IFN-γ protein production, only cytokine stimulation upregulates Ifng transcript, suggesting that protein production is translationally regulated after receptor stimulation. Based on these differences in IFN-γ regulation, we hypothesized that ex vivo IL-15 priming of murine NK cells allows a switch to IFN-γ transcription upon activating receptor engagement. Transcriptional analysis of primed NK cells compared with naive cells or cells cultured with low-dose IL-15 demonstrated that primed cells strongly upregulated Ifng transcript following activating receptor stimulation. This was not due to chromatin accessibility changes in the Ifng locus or changes in ITAM signaling, but was associated with a distinct transcriptional signature induced by ITAM stimulation of primed compared with naive NK cells. Transcriptional analyses identified a common signature of c-Myc (Myc) targets associated with Ifng transcription. Although Myc marked NK cells capable of Ifng transcription, Myc itself was not required for Ifng transcription using a genetic model of Myc deletion. This work highlights altered regulatory networks in IL-15-primed cells, resulting in distinct gene expression patterns and IFN-γ regulation in response to activating receptor stimulation.

CREATINE KINASE EXPRESSION IS PROTECTIVE IN A MOUSE COLITIS MODEL

Abstract BACKGROUND We have previously identified a loss of expression of creatine pathway proteins in mucosal tissue samples from inflammatory bowel disease patients. This is important as creatine is vital to ATP buffering in energetically active cells. The goal of this study was to determine the impact of decreased creatine kinase (CK) expression on mucosal inflammation by utilizing a whole body CK Brain and CK Mitochondrial knockout mouse (CKB/MitKO) in the dextran sulfate sodium (DSS) model of acute colitis. METHODS CKB/MitKO and matched controls were administered 1.5% DSS in drinking water for 5 days and assessed by body weight loss, disease activity index, mucosal permeability, histology and inflammatory cytokine production by Mesoscale analysis. In subsequent experiments, interferon-γ (IFN-γ) was given by intraperitoneal injection during the DSS experiment. We also analyzed transcriptional differences between CKB/MitKO and controls using a transcription factor array and western blot. Finally, we evaluated the impact of CK on the regulation of IFN-γ and transcriptional regulation of IFN-γ production in immune cells by flow cytometry. RESULTS Loss of CKB/Mit did not result in detectable baseline inflammation or barrier dysfunction in CKB/MitKO mice as compared to controls. However, in the setting of the chemical irritant DSS, CKB/MitKO mice had significantly increased weight loss, disease activity, intestinal permeability, histologic inflammation and decreased colon length. While control mice treated with DSS had significant increases in mucosal IFN-γ, the CKB/MitKO mice had strikingly absent mucosal protein concentrations of IFN-γ. Although IFN-γ is commonly considered a pro-inflammatory cytokine, it has also been shown to help promote inflammatory resolution. Therefore, we proceeded to add back IFN-γ in DSS experiments and we identified significant protection from weight loss and histologic damage with the addition of IFN-γ. We went on to identify transcription factor differences between CKB/MitKO and control splenocytes that revealed a >40-fold increase in the hypoxia responsive factor, HIF-1a, in CKB/MitKO splenocytes. We confirmed a causative relationship between HIF-1 expression and IFN-γ by treating mouse splenocytes with a HIF stabilizer and identified that HIF stabilization results in a reduction of IFN-γ production by CD4+ and CD8+ T cells. CONCLUSIONS CK is an essential energetic regulator. Using CK knockout mice we have identified an important role for CK in regulating IFN-γ production, mediated at least in part by HIF stabilization. These findings support a novel role for CK in tissue protection by immune regulation during acute colitis. Better understanding the role of energetics and the creatine pathway in particular may provide novel therapeutic options in inflammatory bowel disease.

IFNγ enhances ferroptosis by increasing JAK‑STAT pathway activation to suppress SLCA711 expression in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare type of tumor with a poor prognosis. Ferroptosis is a relatively novel form of programmed cell death driven by iron-dependent lipid peroxidation accumulation. Recent evidence suggests that IFNγ facilitates erastin-induced ferroptosis, which contributed to anticancer therapy in various types of cancer. However, it has remained elusive whether the regulation of IFNγ on ferroptosis has a positive role in the treatment of ACC. Thus, the aim of the present study was to explore the effects of IFNγ on erastin-induced ferroptosis in the ACC cell line NCI-H295R and investigate the underlying mechanisms. Cell viability was assessed using a Cell Counting Kit-8 assay, an ethynyldioxyuridine proliferation assay and Live/Dead staining. The levels of iron, reactive oxygen species, lipid peroxidation and mitochondrial damage were also assessed. Western blot and reverse transcription-quantitative PCR analyses were used to determine the underlying molecular mechanisms involved in the erastin-induced ferroptosis of NCI-H295R cells. The results suggested that IFNγ promoted erastin-induced ferroptotic cell death. Furthermore, IFNγ enhanced erastin-induced ferroptosis, as evidenced by the accumulation of iron, as well as the increase in lipid peroxidation and promotion of mitochondrial damage. Further analysis suggested that IFNγ enhanced ferroptosis by suppressing the expression of solute carrier family 7 member 11, an important negative regulator of ferroptosis, and this was achieved via activation of the JAK/STAT pathway in NCI-H295R cells. The present study provided experimental evidence on the activity and mechanism of ferroptosis enhanced by IFNγ in ACC and may give critical insight into the immunotherapeutic management of ACC.

Pathogen-associated T follicular helper cell plasticity is critical in anti-viral immunity.

T follicular helper (Tfh) cells are critical in providing help for B cells in the germinal center reaction. Tfh cell plasticity, especially with regard to their expression of effector Th cytokines, has been described, but lacks in-depth analysis with genetic approaches. In this study, we systemically compared transcriptomic profiles of Tfh cells derived from various types of immune responses and found gene clusters corresponding to effector Th cells were differentially induced in response to pathogens or immune responses. Of special interest, a subset of Tfh cells producing IFN-γ was generated in an influenza virus infection, partially dependent on the innate cytokine IL-12. Lineage-tracing mouse model revealed unique developmental regulation of IFN-γ+ Tfh cells, while selective ablation of these cells impaired the induction of IgG2c+ germinal center B cells and the control of influenza infection. These results indicate that pathogen-associated Tfh cell plasticity is necessary for host immunity, which has implications in vaccine design.

Interferon-γ activated T-cell IRGM–autophagy axis in oral lichen planus

Oral lichen planus (OLP) is an inflammatory immune disease featured by dense T-cell infiltrate and basal keratinocytes degeneration. Immunity related GTPase M (IRGM) is vital for the induction of autophagy. Our previous studies have demonstrated aberrant autophagy in OLP, however, the involvement of IRGM–autophagy axis in OLP has not yet been revealed. The expression of IRGM and autophagy activity were evaluated in oral mucosal tissues and peripheral T cells of OLP patients and healthy controls, respectively. We found significant upregulation of IRGM and LC3B in lesions of patients with OLP as compared with healthy donors. IRGM, LC3B and NOD2 levels were also elevated in the peripheral T cells of OLP. Then, knockdown of IRGM after electrotransfection with siRNA resulted in attenuated autophagy, growth inhibition, and apoptosis of T cells. In addition, preincubation with IFN-γ promoted the expression of IRGM mRNA and induced autophagy in T cells. Furthermore, IFN-γ decreased the proliferation and apoptosis of T cells, whereas facilitated the viability of keratinocytes in a co-culture system of activated T cells and keratinocytes. Taken together, activated IRGM–autophagy axis under IFN-γ regulation in T cells might participate in the immunoregulatory mechanism of OLP.

Identification of Novel Genetic Regulatory Region for Proprotein Convertase FURIN and Interferon Gamma in T Cells.

The proprotein convertase enzyme FURIN promotes the proteolytic maturation of pro-proteins and thereby it serves as an important factor for maintaining cellular homeostasis. In T cells, FURIN is critical for maintaining the T regulatory cell dependent peripheral immune tolerance and intact T helper cell polarization. The enzymatic activity of FURIN is directly associated with its expression levels, but genetic determinants for cell-type specific Furin gene regulation have remained elusive. By exploring the histone acetyltransferase p300 binding patterns in T helper cells, a putative regulatory region at ca. 20kB upstream of Furin gene was identified. When this region was deleted with CRISPR/Cas9 the production of Furin mRNA was significantly reduced in activated mouse T cells. Genome-wide RNA profiling by sequencing revealed that the novel Furin regulator region also impacted the expression of several genes that have previously been associated with the Th1 type hall mark cytokine IFNγ regulation or function. Finally, Furin genetic regulatory region was found to specifically promote the secretion of IFNγ by activated T cells. In sum, our data unravels the presence of Furin expression regulatory region in T cells that has characteristics of a super-enhancer for Th1 cell fate.

Mycobacterial Cord Factor Reprograms the Macrophage Response to IFN-γ towards Enhanced Inflammation yet Impaired Antigen Presentation and Expression of GBP1.

Mycobacteria survive in macrophages despite triggering pattern recognition receptors and T cell-derived IFN-γ production. Mycobacterial cord factor trehalose-6,6-dimycolate (TDM) binds the C-type lectin receptor MINCLE and induces inflammatory gene expression. However, the impact of TDM on IFN-γ-induced macrophage activation is not known. In this study, we have investigated the cross-regulation of the mouse macrophage transcriptome by IFN-γ and by TDM or its synthetic analogue trehalose-6,6-dibehenate (TDB). As expected, IFN-γ induced genes involved in Ag presentation and antimicrobial defense. Transcriptional programs induced by TDM and TDB were highly similar but clearly distinct from the response to IFN-γ. The glycolipids enhanced expression of a subset of IFN-γ-induced genes associated with inflammation. In contrast, TDM/TDB exerted delayed inhibition of IFN-γ-induced genes, including pattern recognition receptors, MHC class II genes, and IFN-γ-induced GTPases, with antimicrobial function. TDM downregulated MHC class II cell surface expression and impaired T cell activation by peptide-pulsed macrophages. Inhibition of the IFN-γ-induced GTPase GBP1 occurred at the level of transcription by a partially MINCLE-dependent mechanism that may target IRF1 activity. Although activation of STAT1 was unaltered, deletion of Socs1 relieved inhibition of GBP1 expression by TDM. Nonnuclear Socs1 was sufficient for inhibition, suggesting a noncanonical, cytoplasmic mechanism. Taken together, unbiased analysis of transcriptional reprogramming revealed a significant degree of negative regulation of IFN-γ-induced Ag presentation and antimicrobial gene expression by the mycobacterial cord factor that may contribute to mycobacterial persistence.

Eomesodermin promotes interaction of RelA and NFATc2 with the Ifng promoter and multiple conserved noncoding sequences across the Ifng locus in mouse lymphoma BW5147 cells

The T-box transcription factor Eomesodermin (Eomes) regulates the lineage-dependent expression of interferon γ (IFN-γ). We previously showed that Eomes promotes IFN-γ production and interacts with multiple conserved noncoding sequences (CNS) across the Ifng locus in mouse lymphoma BW5147 cells. In the present study, we investigated the transcriptional regulation of IFN-γ by the nuclear factor κB (NF-κB) subunit RelA and nuclear factor of activated T cells c2 (NFATc2, also known as NFAT1) in Eomes-transfected BW5147 cells. Eomes promoted the interaction of RelA and NFATc2 with the Ifng promoter and five CNS, including CNS-22 and CNS+30 upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). The dual NF-κB and STAT3 inhibitor TPCA-1 moderately reduced the PMA- and IM-induced IFN-γ transcription in Eomes-transfected BW5147 cells. TPCA-1 interfered with RelA binding to the Ifng promoter, CNS-22 and CNS+30. Moreover, TPCA-1 reduced the interaction of Eomes or NFATc2 with the Ifng promoter and CNS+30. The present results indicate that Eomes promotes the interaction of RelA and NFATc2 with the Ifng promoter and multiple CNS across the Ifng locus in BW5147 cells.

Regulation of lipid metabolism in pancreatic beta cells by interferon gamma: A link to anti-viral function

Interferons (IFN) have been shown to alter lipid metabolism in immune and some non-hematopoietic cells and this affects host cell response to pathogens. In type 1 diabetes, IFNγ acts as a proinflammatory cytokine that, along with other cytokines, is released during pancreatic beta cell autoinflammation and contributes to immune response and beta cell dysfunction. The hypothesis tested herein is that IFN modifies beta cell lipid metabolism and this is associated with enhanced anti-viral response and beta cell stress. Treatment of INS-1 cells with IFNγ for 6 to 24 h led to a dynamic change in TAG and lipid droplet (LD) levels, with a decrease at 6 h and an increase at 24 h. The later accumulation of TAG was associated with increased de novo lipogenesis (DNL), and impaired mitochondrial fatty acid oxidation (FAO). Gene expression results suggested that IFNγ regulates lipolytic, lipogenic, LD and FAO genes in a temporal manner. The changes in lipid gene expression are dependent on the classical Janus kinase (JAK) pathway. Pretreatment with IFNγ robustly enhanced anti-viral gene expression induced by the viral mimetic polyinosinic: polycytidylic acid (PIC), and this potentiating effect of IFNγ was markedly attenuated by inhibitors of DNL. The IFNγ-induced accumulation of lipid, however, was insufficient to cause endoplasmic reticulum (ER) stress. These studies demonstrated a non-canonical effect of IFNγ in regulation of pancreatic beta cell lipid metabolism that is intimately linked with host cell defense and might alter cellular function early in the progression to type 1 diabetes.

Th1-Th2 Cross-Regulation Controls Early Leishmania Infection in the Skin by Modulating the Size of the Permissive Monocytic Host Cell Reservoir

The impact of T helper (Th) 1 versus Th2 immunity onintracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility. However, observations in multiple infectious settings demonstrate deficiencies in mediators of Th1-Th2 immunity, which have paradoxical or no impact. We report that prior to influencing activation, Th1/Th2 immunity first controls the size of the permissive host cell reservoir. During early Leishmania infection of the skin, IFN-γ- or STAT6-mediated changes in phagocyte activation were counteracted by changes in IFN-γ-mediated recruitment of permissive CCR2+ monocytes. Monocytes were required for early parasite expansion andacquired an alternatively activated phenotype despite the Th1 dermal environment required fortheir recruitment. Surprisingly, STAT6 did not enhance intracellular parasite proliferation, but rather modulated the size and permissiveness of the monocytic host cell reservoir via regulation of IFN-γ and IL-10. These observations expand our understanding of the Th1-Th2 paradigm during infection.

Contributions of Creatine Kinase to Mucosal Immune Responses in Colitis

Intestinal epithelial cells facilitate nutrient transport as well as protect against invasion of luminal bacteria and microbial products. Disruption of the intestinal epithelial barrier is a hallmark of IBD. The regulation of intestinal epithelial barrier is made possible by junctional complexes including tight junctions and adherens junctions that are supported by the actin cytoskeleton. Maintenance of the actin cytoskeleton is an energy demanding process and intestinal epithelial cells shuttle energy to the locations of the cellular junctions through the creatine‐ATP energy circuit. Within the cell, creatine and its associated enzyme(s) creatine kinase(s) (CK) facilitate the shuttling of high energy phosphates in the form of phosphocreatine between sites of ATP generation. Work in our lab has shown that CK contributes to apical junction assembly in vitro and that supplementation with creatine in murine colitis models is protective. Further studies have shown that mice lacking the brain and mitochondrial isoforms of CK (CK dKO) are significantly more susceptible to DSS‐induced colitis as measured by multiple disease parameters, including disease activity, weight loss, intestinal permeability and by histological assessment. In addition to impaired barrier function, mice lacking CK showed defective inflammatory responses underscored by nearly non‐existent levels of colonic IFNγ. Flow cytometry from splenocytes and MLNs of CK dKO mice showed that T cells produced less IFNγ upon stimulation in vitro, and this finding was confirmed in human PBMC treated with the CK inhibitor cyclocreatine. These results implicate a CK‐dependent regulation of IFNγ in protective innate immune responses during mucosal inflammation, and ongoing studies are focused on elucidating the mechanism of CK activity on T cell signaling.Support or Funding InformationCrohn's and Colitis Foundation RFA #546183

PDTM-20. THE HISTONE MUTATION H3.3-G34R ENCOUNTERED IN PEDIATRIC HIGH GRADE GLIOMA MODIFIES THE TUMOR IMMUNE MICROENVIRONMENT RENDERING IT MORE PERMISSIVE FOR IMMUNE MEDIATED THERAPIES

Abstract Pediatric high grade gliomas (pHGGs) have a median survival (MS) of 9–15 months and are the most common malignant brain tumors in children. No significant improvement in the MS of these patients has been registered in decades. Thus, a representative in vivo model to study these tumors is critical. Recurrent mutations in genes encoding histones H3.3 and H3.1 have been described, which are distinctive of pHGGs. To stablish a mouse model for pHGG harboring the mutation H3.3-G34R, which co-occurs with inactivating mutations in ATRX and TP53, we used the Sleeping Beauty transposase system (H3.3-G34R group). The MS of mice harboring H3.3-G34R mutation was significantly longer than the MS of the WT-H3.3 control group (p< 0.0001). We performed RNA-Sequencing of these tumors which yielded 233 differentially expressed genes between these two groups (p< 0.05). Gene set enrichment analysis of the upregulated genes in H3.3-G34R group showed significant GOs related to immune response activation and response to IFNγ. Thus, we next studied the tumor microenvironment (TME) to determine the distinctive immune cell populations, at the phenotypic and functional level. We observed that the H3.3-G34R TME exhibited less frequency of Gr-1+/CD11b+ cells (myeloid-derived suppressor cells: MDSC). MDSC are immature myeloid cells that impair T cell functions and the adaptive immune response. Moreover, the H3.3-G34R TME had increased numbers of total macrophages (p< 0.05), a higher proportion of M1 anti-tumor macrophages, and higher levels of CD8+ T cells (p< 0.01). Also, the H3.3-G34R renders glioma cells more sensitive to IFNγ regulation as it induced a higher pSTAT1/STAT1 ratio in H3.3-G34R cells. These results shed light on the interaction between the immune system and the H3.3-G34R glioma cells and how this could be harnessed to develop immune mediated therapies specific for pHGG harboring the H3.3-G34R mutation.

Exosomes Derived From T Regulatory Cells Suppress CD8+ Cytotoxic T Lymphocyte Proliferation and Prolong Liver Allograft Survival.

BackgroundCD8+ cytotoxic T lymphocytes (CTLs) have been proved to exert crucial roles in immunological rejection. Exosomes (EXOs) secreted by CD4+CD25+ regulatory T (Treg) cells is believed to be deeply involved in immune regulation. Nevertheless, whether immunomodulatory effect of CD4+CD25+ Treg cells on CD8+ CTL depends on EXOs remains unknown and needs to be explored.Material/MethodsWe purified CD4+CD25+ Treg cells followed by in vitro amplification. EXOs in culture supernatants of Treg cells was separated and identified. The effect of CD4+CD25+ Treg cells and CD4+CD25+ Treg cells-derived EXOs on CD8+ CTL viability, proliferation, cell cycle mRNA, intracellular cytokines, and protein expression were investigated.ResultsWe successfully obtained EXOs from CD4+CD25+ Treg cells. The inhibition effect of EXOs on CD8+ CTL was concentration-dependent. In addition, the inhibition effect of CD4+CD25+ Treg cells could be reversed by GW4869, an EXOs inhibitor. The inhibition effect was associated with the regulation of IFN-γ and perforin. Our in vivo experiments proved that natural CD4+CD25+ Treg cells-released EXOs can prolong liver allograft survival.ConclusionsCD4+CD25+ Treg cells-derived EXOs could become an alternative tool for manipulating the immune system to discover novel underlying immunomodulatory mechanisms.

IFN-γ inhibits 5-HT-induced melanin biosynthesis via downregulation of 5-HT receptors in vivo/in vitro

Hypopigmentation disorders, such as vitiligo, are difficult for treatment due to complicated pathogenesis, resulting from multiple factors including neural and immune elements. 5-HT and IFN-γ both play crucial roles in these skin diseases. However, the interactions between 5-HT and IFN-γ in regulation of melanogenesis is still unknown. Our study aimed at exploring whether IFN-γ affects 5-HT-induced melanogenesis and searching the mechanism. In our study, IFN-γ attenuated 5-HT-induced pigmentation and green fluorescent protein (GFP) expression in zebrafishes. In addition, we found that IFN-γ decreased serum serotonin levels as well as the cutaneous expression of tryptophan hydroxylase 1 (TPH1), 5-HT1A receptor (5-HT1AR) and 5-HT1B receptor (5-HT1BR) in C57BL/6 mice. Moreover, IFN-γ attenuated 5-HT-induced melanin biosynthesis as well as the expression of 5-HT1AR, 5-HT1BR and 5-HT2A receptor (5-HT2AR) in B16F10 cells, which blocked by interferon-γ receptor 1 and interferon-γ receptor 2 (IFNGR1/IFNGR2) antibodies. In summary, IFN-γ not only affects melanogenesis alone, but also inhibits 5-HT response on melanin biosynthesis. Mediated by IFNGR1/IFNGR2, IFN-γ downregulated 5-HT receptors expression, which directly affect 5-HT-induced melanin biosynthesis. Our work may give insights into the drug development of hypopigmentation disorders with neuro-immune derangement.

Sirt1 in the Regulation of Interferon Gamma in Severe Aplastic Anemia

Recent studies have indicated that Sirt1 plays critical roles in the suppression of inflammation, T cell activation, and differentiation of hematopoietic progenitor cells. Severe aplastic anemia (SAA) is an immune-mediated disease that is characterized by elevated cytotoxic lymphocytes and type 1 cytokines. As a negative effector cytokine, interferon gamma (IFNγ) takes part in aplastic anemia through its inhibitory effect on hematopoiesis. In this study, we investigated the role of Sirt1 in the regulation of IFNγ in patients with SAA. A significant decrease in relative SIRT1 (p< 0.05) and increase in IFNG (p< 0.05) expression levels was observed in the sorted CD8+T cells of SAA patients compared to the controls. There was a significant negative correlation (r = –0.53, p < 0.05) between SIRT1 and IFNG expression in SAA patients. SRT3025, a Sirt1 activator, was shown to significantly reduce IFNγ (p < 0.01) and elevate Sirt1 (p < 0.05) expression in the CD8+T cells of SAA patients, and also showed a therapeutic role in an aplastic anemia mouse model. In conclusion, the defective Sirt1 may be correlated to the abnormal IFNγ expression in SAA patients, and activation of Sirt1 signaling may help improve the inflammatory status of SAA.

Activated β-catenin in Foxp3+ regulatory T cells links inflammatory environments to autoimmunity

Foxp3+ regulatory T cells (Treg cells) are the central component of peripheral immune tolerance. While dysregulated Treg cytokine signature has been observed in autoimmune diseases, the regulatory mechanisms underlying pro- and anti-inflammatory cytokine production are elusive. Here, we identify imbalance between IFN-γ and IL-10 as a shared Treg signature, present in patients with multiple sclerosis (MS) and under high salt conditions. RNA-sequencing analysis on human Treg subpopulations reveals β-catenin as a key regulator of IFN-γ and IL-10 expression. The activated β-catenin signature is enriched in human IFN-γ+ Treg cells, which is confirmed in vivo with Treg specific β-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional Treg phenotype. Moreover, we identify prostaglandin E receptor 2 (PTGER2) as a regulator for IFN-γ and IL-10 production under high salt environment, with skewed activation of the β-catenin-SGK1-Foxo axis. Our findings reveal a novel PTGER2-β-catenin loop in Treg cells linking environmental high salt conditions to autoimmunity.