Potential HIV latency-reversing agents with STAT1-activating activity from the leaves of Wikstroemia chamaedaphne.

Developing highly effective HIV latency-reversing agent is an inportmant approach for the treatment of AIDS via the "shock and kill" of latent HIV. In this study, two unreported modified daphnane-type diterpenes (chamaedaphnelide A and epi-chamaedaphnelide A) and one unreported tigliane-type diterpene (chamaedaphnelide B), along with four known daphnane-type diterpenes and one known tigliane-type diterpene were obtained from the leaves of Wikstroemia chamaedaphne. Chamaedaphnelide A and epi-chamaedaphnelide A represents the first A ring cleavage daphnane-type backbone. Chamaedaphnelide A, epi-chamaedaphnelide A, chamaedaphnelide B, and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate showed HIV latency-reversing activity, especially chamaedaphnelide B and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate displayed equally potential to positive drugs prostratin with reversing latent HIV on more than 100-fold compared to unstimulated cells. Furthermore, the activation of STAT1 was involved in the HIV latency-reversing activity of these diterpenes, firstly demonstrating that daphnane- and tigliane-type diterpenes can rapidly activate STAT1 activity. Indeed, these results also supported that activating STAT1 activity is a pathway for reversing latent HIV.

Schisandrin B promotes TH1 cell differentiation by targeting STAT1.

Schisandrin B (Sch B) is the major active ingredient of the traditional Chinese medicine Schisandra chinensis and has antitumor activity, anti-inflammatory activity. CD4+ Th subsets orchestrate immune responses to plenty of pathogen infections and participate in the pathogenesis of many immune-related diseases. However, little is known about the relationship between Sch B and T cell differentiation. Here, we showed that Sch B might participate in T cell receptor signaling pathway by using the TCMIO database. Importantly, Sch B promoted TH1 cell differentiation. Furthermore, Sch B did not affect TH2 cell and Treg differentiation. Mechanismly, Sch B increased the level of IFN-γ of CD4+ T cells by upregulating the phosphorylation of STAT1 protein. Then, STAT1 promoted T-bet expression in CD4+ T cells. In conclusion, Sch B modulates the differentiation of naïve CD4+ T cells into TH1 subset by STAT1/T-bet signaling, which may have the potential for the treatment of T cell-mediated-immune diseases.

Elevated STAT1 expression but not phosphorylation in lupus B cells correlates with disease activity and increased plasmablast susceptibility.

OBJECTIVES: SLE is characterized by two pathogenic key signatures, type I IFN and B-cell abnormalities. How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) - signal transducer and activator of transcription (STAT). JAK-STAT inhibition is an attractive therapeutic possibility for SLE. We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared with other autoimmune diseases and healthy controls (HD) and related it to disease activity. METHODS: Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T cells of 21 HD, 10 rheumatoid arthritis (RA), seven primary Sjögren's (pSS) and 22 SLE patients was analysed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs (peripheral blood mononuclear cells) of SLE patients and HD after IFNα and IFNγ incubation were further investigated. RESULTS: SLE patients showed substantially higher STAT1 but not pSTAT1 in B- and T-cell subsets. Increased STAT1 expression in B-cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker. STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ. CONCLUSION: Enhanced expression of STAT1 by B-cell candidates as a key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold a promise to block STAT1 expression and control plasmablast induction in SLE.

Rilpivirine attenuates liver fibrosis through selective STAT1-mediated apoptosis in hepatic stellate cells.

OBJECTIVE: Liver fibrosis constitutes a major health problem worldwide due to its rapidly increasing prevalence and the lack of specific and effective treatments. Growing evidence suggests that signalling through cytokine-activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways regulates liver fibrosis and regeneration. Rilpivirine (RPV) is a widely used anti-HIV drug not reported to produce hepatotoxicity. We aimed to describe the potential hepatoprotective effects of RPV in different models of chronic liver injury, focusing on JAK-STAT signalling regulation. DESIGN: The effects of RPV on hepatic steatosis, inflammation and fibrogenesis were studied in a nutritional mouse model of non-alcoholic fatty liver disease, carbon tetrachloride-induced fibrosis and bile duct ligation-induced fibrosis. Primary human hepatic stellate cells (hHSC) and human cell lines LX-2 and Hep3B were used to investigate the underlying molecular mechanisms. RESULTS: RPV exerted a clear anti-inflammatory and antifibrotic effect in all the in vivo models of liver injury employed, and enhanced STAT3-dependent proliferation in hepatocytes and apoptosis in HSC through selective STAT1 activation. These results were reproduced in vitro; RPV undermined STAT3 activation and triggered STAT1-mediated pathways and apoptosis in HSC. Interestingly, this selective pro-apoptotic effect completely disappeared when STAT1 was silenced. Conditioned medium experiments showed that HSC apoptosis activated STAT3 in hepatocytes in an interleukin-6-dependent mechanism. CONCLUSION: RPV ameliorates liver fibrosis through selective STAT1-dependent induction of apoptosis in HSC, which exert paracrinal effects in hepatocytes, thus promoting liver regeneration. RPV's actions may represent an effective strategy to treat chronic liver diseases of different aetiologies and help identify novel therapeutic targets.

Differential Responsiveness of Monocyte and Macrophage Subsets to Interferon.

OBJECTIVE: Peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients exhibit a gene expression program (interferon [IFN] signature) that is attributed to overproduction of type I IFNs by plasmacytoid dendritic cells. Type I IFNs have been thought to play a role in the pathogenesis of SLE. This study was undertaken to examine an unexpected influence of monocyte/macrophages on the IFN signature. METHODS: Proinflammatory (classic) and antiinflammatory (nonclassic) monocyte/macrophages were sorted from mice and analyzed by RNA sequencing and quantitative polymerase chain reaction (qPCR). Type I IFN-α/ß/ω receptor (IFNAR-1) expression was determined by qPCR and flow cytometry. Macrophages were stimulated in vitro with IFNα, and pSTAT1was measured. RESULTS: Transcriptional profiling of peritoneal macrophages from mice with pristane-induced SLE unexpectedly indicated a strong IFN signature in classic, but not nonclassic, monocyte/macrophages exposed to the same type I IFN concentrations. Ifnar1 messenger RNA and IFNAR surface staining were higher in classic monocyte/macrophages versus nonclassic monocyte/macrophages (P < 0.0001 and P < 0.05, respectively, by Student's t-test). Nonclassic monocyte/macrophages were also relatively insensitive to IFNα-driven STAT1 phosphorylation. Humans exhibited a similar pattern: higher IFNAR expression (P < 0.0001 by Student's t-test) and IFNα-stimulated gene expression (P < 0.01 by paired Wilcoxon's rank sum test) in classic monocyte/macrophages and lower levels in nonclassic monocyte/macrophages. CONCLUSION: This study revealed that the relative abundance of different monocyte/macrophage subsets helps determine the magnitude of the IFN signature. Responsiveness to IFNα signaling reflects differences in IFNAR expression in classic (high IFNAR) compared to nonclassic (low IFNAR) monocyte/macrophages. Thus, the IFN signature depends on both type I IFN production and the responsiveness of monocyte/macrophages to IFNAR signaling.

The N6-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of STAT1 mRNA.

Compelling evidence indicates that epigenetic regulations orchestrate dynamic macrophage polarization. N6-methyladenosine (m6A) methylation is the most abundant epigenetic modification of mammalian mRNA, but its role in macrophage polarization is still completely unknown. Here, we show that the m6A-catalytic enzyme methyltransferase like 3 (METTL3) is specifically upregulated following the M1 polarization of mouse macrophages. Furthermore, METTL3 knockdown through siRNA transfection markedly inhibited M1, but enhanced M2, macrophage polarization. Conversely, its overexpression via plasmid transfection greatly facilitated M1, but attenuated M2, macrophage polarization. Further methylated RNA immunoprecipitation and in vitro m6A methylation assays suggested that METTL3 directly methylates mRNA encoding signal transducer and activator of transcription 1 (STAT1), a master transcription factor controlling M1 macrophage polarization, at its coding sequence and 3'-untranslated regions. In addition, METTL3-mediated STAT1 mRNA methylation significantly increased mRNA stability and subsequently upregulated STAT1 expression. In conclusion, METTL3 drives M1 macrophage polarization by directly methylating STAT1 mRNA, potentially serving as an anti-inflammatory target.

Met inhibition revokes IFNγ-induction of PD-1 ligands in MET-amplified tumours.

BACKGROUND: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression. CONCLUSIONS: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

Dual inhibition of STAT1 and STAT3 activation downregulates expression of PD-L1 in human breast cancer cells.

OBJECTIVES: Breast cancer is the most commonly diagnosed cancer, and it is a leading cause of cancer-related deaths in females worldwide. Triple-negative breast cancer (TNBC) constitutes 15% of breast cancer and shows distinct metastasis profiles with poor prognosis. Strong PD-L1 expression has been observed in some tumors, supporting their escape from immune surveillance. Targeting PD-L1 could be a promising therapeutic approach in breast cancer patients. We investigated potential molecular mechanisms for constitutive expression of PD-L1 by inhibiting upstream STAT1 and STAT3 signals. METHODS: PD-L1 expression in three breast cancer cell lines was measured using quantitative PCR and western blotting. Activation of STAT1 and STAT3 was blocked using pharmacological inhibitors and siRNA. The mechanism underlying the constitutive expression of PD-L1 was investigated using ChIP and co-immunoprecipitation assays. RESULTS: We found that individual inhibition of STAT1 and STAT3 activation partially downregulated PD-L1, while combined inhibition completely downregulated PD-L1 expression. Moreover, our results suggest that pSTAT1-pSTAT3 dimerize in cytosol and translocate to the nucleus, where they bind to PD-L1 promoter and induce PD-L1 expression. CONCLUSION: These findings provide a rationale for combined targeting of STAT1 and STAT3 for the development of immune-based cancer therapies for down regulation of PD-L1 expression.

Grape seed proanthocyanidin extract ameliorates murine autoimmune arthritis through regulation of TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND/AIMS: Grape seed proanthocyanidin extract (GSPE) has been reported to have a beneficial effect on regulating inf lammation. However, the anti-inflammatory mechanism of GSPE remains unclear. The aim of this study was to verify the influence of GSPE on the Toll-like receptor 4 (TLR4)-mediated signaling pathway in the regulation of murine autoimmune arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in dilute brown non-agouti (DBA)/1J mice. The mice were treated with GSPE (0 or 100 mg/kg) intraperitoneally. The severity of arthritis was assessed clinically, biochemically, and histologically. Immunostaining for TLR4 was performed. The expressions of TLR4 and downstream signaling molecules were analyzed by Western blot. The effect of GSPE on lipopolysaccharide (LPS)-induced TLR4 activation was also evaluated using RAW264.7 cells and fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis and from those with osteoarthritis. RESULTS: GSPE attenuated the clinical severity of arthritis and decreased histological damage. GSPE treatment reduced the number of TLR4-stained cells in the synovium of mice with CIA. GSPE also downregulated the expression of TLR4, myeloid differentiation factor 88 (MyD88) and phosphorylated IκBα synovial protein in CIA mice. Concurrently, GSPE inhibited the nuclear translocation of nuclear factor-κB (NF-κB) subunits (p65 and p50). LPS-induced TLR4 activation was suppressed by GSPE in human FLS as well as in murine macrophages in vitro. CONCLUSIONS: Our results demonstrated that GSPE ameliorated CIA by regulating the TLR4-MyD88-NF-κB signaling pathway.

Interferon-Alpha Reduces Human Hippocampal Neurogenesis and Increases Apoptosis via Activation of Distinct STAT1-Dependent Mechanisms.

Background: In humans, interferon-α treatment for chronic viral hepatitis is a well-recognized clinical model for inflammation-induced depression, but the molecular mechanisms underlying these effects are not clear. Following peripheral administration in rodents, interferon-α induces signal transducer and activator of transcription-1 (STAT1) within the hippocampus and disrupts hippocampal neurogenesis. Methods: We used the human hippocampal progenitor cell line HPC0A07/03C to evaluate the effects of 2 concentrations of interferon-α, similar to those observed in human serum during its therapeutic use (500 pg/mL and 5000 pg/mL), on neurogenesis and apoptosis. Results: Both concentrations of interferon-α decreased hippocampal neurogenesis, with the high concentration also increasing apoptosis. Moreover, interferon-α increased the expression of interferon-stimulated gene 15 (ISG15), ubiquitin-specific peptidase 18 (USP18), and interleukin-6 (IL-6) via activation of STAT1. Like interferon-α, co-treatment with a combination of ISG15, USP18, and IL-6 was able to reduce neurogenesis and enhance apoptosis via further downstream activation of STAT1. Further experiments showed that ISG15 and USP18 mediated the interferon-α-induced reduction in neurogenesis (potentially through upregulation of the ISGylation-related proteins UBA7, UBE2L6, and HERC5), while IL-6 mediated the interferon-α-induced increase in apoptosis (potentially through downregulation of aquaporin 4). Using transcriptomic analyses, we showed that interferon-α regulated pathways involved in oxidative stress and immune response (e.g., Nuclear Factor (erythroid-derived 2)-like 2 [Nrf2] and interferon regulatory factor [IRF] signaling pathway), neuronal formation (e.g., CAMP response element-binding protein [CREB] signaling), and cell death regulation (e.g., tumor protein(p)53 signaling). Conclusions: We identify novel molecular mechanisms mediating the effects of interferon-α on the human hippocampus potentially involved in inflammation-induced neuropsychiatric symptoms.

IL-7-dependent STAT1 activation limits homeostatic CD4+ T cell expansion.

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7-dependent STAT1 and STAT5 activation. Consequently, the IL-7-induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, "switching on" an alternate IL-7-dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients.

The effect of leptin and resveratrol on JAK/STAT pathways and Sirt-1 gene expression in the renal tissue of ischemia/reperfusion induced rats.

OBJECTIVE: Our study aimed to investigate the possible modifying effects of leptin and combined use of resveratrol on rat renal I/R injury and their relationship on signal pathways and apoptosis-related mechanisms. BACKGROUND: Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure. METHODS: Male Sprague Dawley rats were divided into 5 groups: Control, I/R, I/R+leptin, I/R+resveratrol and I/R+leptin+resveratrol. Leptin (10 µg/kg BW) was administered (i.p.) 30 min prior to I/R. Resveratrol was administered by gavage at 20 mg/kg BW per d for 12 d prior to I/R. The left renal artery was exposed to 1 h of ischemia and 1 h of reperfusion. RESULTS: Resveratrol treatment alone increased TNF-α, TNF-α R1, NF-κB, SIRT-1, STAT1 and STAT3 mRNA levels and decreased caspase 3 protein levels. Leptin treatment alone significantly decreased the caspase 3 protein levels. The combined use of resveratrol and leptin significantly increased STAT3, and caspase 3 mRNA levels, and decreased the caspase 3 protein levels. Apoptosis was significantly decreased especially in the leptin and leptin+resveratrol groups. CONCLUSION: The present study suggest that a combined use of resveratrol and leptin has preventive and regulatory effects on renal I/R injury; the mechanism involves decreasing apoptosis, likely by altering the JAK/STAT pathway and SIRT1 expression (Fig. 8, Ref. 24).

Ethyl Caffeate Ameliorates Collagen-Induced Arthritis by Suppressing Th1 Immune Response.

The present study was designed to assess the antiarthritic potential of ECF in collagen-induced arthritis (CIA) and explore its underlying mechanism. Methods. In vitro, lymphocyte proliferation assay was measured by CCK-8 kit. In vivo, the therapeutic potential of ECF on CIA was investigated; surface marker, Treg cell, and intracellular cytokines (IL-17A and IFN-γ) were detected by flow cytometry. Th1 cell differentiation assay was performed, and mRNA expression in interferon-γ-related signaling was examined by q-PCR analysis. Results. In vitro, ECF markedly inhibited the proliferation of splenocytes in response to ConA and anti-CD3. In vivo, ECF treatment reduced the severity of CIA, inhibited IFN-γ and IL-6 secretion, and decreased the proportion of CD11b+Gr-1+ splenic neutrophil. Meanwhile, ECF treatment significantly inhibited the IFN-γ expression in CD4+T cell without obviously influencing the development of Th17 cells and T regulatory cells. In vitro, ECF suppressed the differentiation of naive CD4+ T cells into Th1. Furthermore, ECF intensely blocked the transcriptional expression in interferon-γ-related signaling, including IFN-γ, T-bet, STAT1, and STAT4. Conclusion. Our results indicated that ECF exerted antiarthritic potential in collagen-induced arthritis by suppressing Th1 immune response and interferon-γ-related signaling.

INHIBITORY EFFECT OF EMODIN ON RAW 264.7 ACTIVATED WITH DOUBLE STRANDED RNA ANALOGUE POLY I:C.

BACKGROUND: Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) is a compound which can be found in Polygoni Multiflori Radix (PMR). PMR is the root of Polygonum multiflorum. PMR is used to treat dizziness, spermatorrhea, sores, and scrofula as well as chronic malaria traditionally in China and Korea. The anti-tumor property of emodin was already reported. However, anti-viral activity of emodin on macrophages are not fully reported. MATERIALS AND METHODS: Effects of emodin on RAW 264.7 mouse macrophages induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA, were evaluated. RESULTS: Emodin restored the cell viability in poly I: C-induced RAW 264.7 at concentrations of up to 50 µM. Emodin significantly inhibited the production of nitric oxide, IL-1α, IL-Ιß, IL-6, GM-CSF, G-CSF, M-CSF, MCP-1, MIP-1a, MIP-Ιß, MIP-2, RANTES, and IP-10 as well as calcium release and mRNA expression of signal transducer and activated transcription 1 (STAT1) in poly I:C-induced RAW 264.7 (P < 0.05). CONCLUSION: This study shows the inhibitory effect of emodin on poly I: C-induced RAW 264.7 via calcium-STAT pathway.

Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

OBJECTIVE: Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. METHODS: PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNß reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. RESULTS: Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNß promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. CONCLUSION: These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies.

GABAergic Neurons and Their Modulatory Effects on GnRH3 in Zebrafish.

γ-Aminobutyric acid (GABA) is a major amino acid neurotransmitter in the vertebrate brain. To provide detailed information on the distribution of the GABA in zebrafish (Danio rerio), neurons were labeled with mCherry driven by the glutamic acid decarboxylase 67 (gad67) promoter. In the transgenic line Tg(gad67:mCherry), mCherry-positive gad67 cell bodies were predominantly localized to the olfactory bulb, pallial zones, subpallium zones, parvocellular preoptic nucleus, periventricular gray zone of optic tectum, torus semicircularis, posterior tuberculum, medial longitudinal fascicle, caudal zone of periventricular hypothalamus, and oculomotor nucleus. mCherry-positive fibers were widely distributed in the olfactory bulbs, subpallium, thalamus, ventral hypothalamic zone, tectum opticum, mesencephalon, and rhombencephalon. mCherry-positive neurons were also observed in the retina and the spinal cord. The anatomical relationships between GABAergic and gonadotrophin-releasing hormone 3 (GnRH3) neurons were investigated by crossing Tg(gad67:mCherry) fish with the previously established Tg(gnrh3:EGFP) transgenic line. GnRH3 cell bodies and fibers were contacted by GABAergic fibers directly in the ventral telencephalon and anterior tuberal nucleus. A subpopulation of GnRH3 neurons in the ventral telencephalic area was also labeled with mCherry, so some GnRH3 neurons are also GABAergic. GABAB receptor agonist (baclofen) and antagonist (CGP55845) treatments indicated that GABAB receptor signaling inhibited gnrh3 expression in larval fish but was stimulatory in adult fish. The expression of pituitary lhß and fshß was stimulated by intraperitoneal injection of baclofen in adult fish. We conclude that GABA via GABAB receptors regulates GnRH3 neurons in a developmentally dependent manner in zebrafish.

In Vitro and In Vivo Anti-Allergic and Anti-Inflammatory Effects of eBV, a Newly Developed Derivative of Bee Venom, through Modulation of IRF3 Signaling Pathway in a Carrageenan-Induced Edema Model.

BACKGROUND: Bee venom (BV), a type of toxin extracted from honeybees (Apis mellifera), has been empirically and widely used to treat inflammatory diseases throughout Asia. Essential BV (eBV) was developed by removing phospholipase A2 (PLA2) and histamine to lower occurrence of allergic reaction. This study investigated the anti-allergic and anti-inflammatory activities of eBV in vitro and in vivo and its underlying mechanism of action. METHODS: The anti-inflammatory potential of eBV was assessed in vivo using a carrageenan-induced paw edema model. To further investigate the mechanism by which eBV exerts anti-allergic and anti-inflammatory effects, compound 48/80-stimulated RBL-2H3 cells and lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells were studied in vitro. RESULTS: Release of ß-hexosaminidase and histamine was increased by eBV in a dose-dependent manner, but these levels were lower in eBV compared to original BV at the same concentration. In addition, eBV suppressed compound 48/80-induced expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RBL-2H3 cells. eBV was also shown to suppress nitric oxide (NO) production by down-regulating mRNA expression and subsequent protein expression of inflammatory mediators in LPS-induced RAW 264.7 cells. Phosphorylation of activators and signal transducers of transcription 1/interferon regulatory factor 3 (STAT1/IRF3) was attenuated by eBV treatment. eBV significantly inhibited carrageenan-induced acute edema in vivo. Serum levels of prostaglandin E2 (PGE2), TNF-α, and IL-1ß were also down-regulated by eBV. CONCLUSIONS: These results demonstrate that eBV inhibits allergic and inflammatory response by reducing inflammatory mediator production via regulation of the STAT1/IRF3 signaling pathway, suggesting that eBV is a feasible candidate for regulation of allergic-inflammatory response in complementary and alternative medicine.

The effect of the TLR9 ligand CpG-oligodeoxynucleotide on the protective immune response to radiation-induced lung fibrosis in mice.

CpG-oligodeoxynucleotide (CpG-ODN) is not only reported to protect against airway hyper responsiveness but is also known as a potent vaccine adjuvant for anti-tumor therapy. Little is known about the effect of CpG-ODN in mice with radiation-induced lung fibrosis (RILF), a common late stage form of tissue damage that occurs after thorax radiotherapy (RT). Here, we evaluated the immunomodulatory effects of CpG-ODN on the development of RILF. Mice were divided into four groups: (1) RT, single dose of 12Gy to the whole thorax; (2) CpG, only intraperitoneal injection of CpG-ODN for total 5 weeks; (3) RT+CpG, irradiation plus CpG-ODN treatment before and after irradiation for total 5 weeks; and (4) control (CTL): No RT or CpG-ODN treatment. In this study, we found that CpG-ODN treatment attenuated lung fibrosis and collagen deposition by increasing the number of M1 macrophagocytes, levels of Type-2 cytokines and TGF-ß. CpG-ODN administration up-regulated the expression of TLR9 and STAT1 phosphorylation and reversed the expression of Type-2 immune response key transcription factor GATA-3. Activation of the JAK-STAT1 signaling pathway further enhanced M1 macrophage differentiation and Type-1 cytokine production. This study reveals the mitigating effect of early exposure to CpG-ODN on lung injury caused by irradiation in mice. The potential mechanism of action may be related to enhancement of Type-1 immunity. In conclusion, CpG-ODN may be a potential therapeutic target to treat RILF.

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea.

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser(727) (but not Tyr(701)) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways.R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT: Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity.

Autoantibody-Positive Healthy Individuals Display Unique Immune Profiles That May Regulate Autoimmunity.

OBJECTIVE: Antinuclear antibodies (ANAs) are detected in ∼18% of females, yet autoimmune disease develops in only 5-8%. Immunologic differences between ANA-positive healthy individuals and patients with systemic lupus erythematosus (SLE) may elucidate the regulatory mechanisms by which ANA-positive individuals avoid transition to clinical autoimmune disease. METHODS: Healthy individuals (n = 790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sjögren's syndrome. From this screening, 31 European American ANA-positive healthy individuals were selected and demographically matched to ANA-negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry. RESULTS: Of 790 individuals screened, 57 (7%) were ANA-positive. The majority of proinflammatory cytokines, including interferon-γ (IFNγ), tumor necrosis factor, interleukin-17 (IL-17), and granulocyte colony-stimulating factor, exhibited a stepwise increase in serum levels from ANA-negative controls to ANA-positive healthy individuals to SLE patients (P < 0.0001). IFNα, IFNß, IL-12p40, and stem cell factor/c-Kit ligand were increased in SLE patients only (P < 0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA-positive individuals (P < 0.001). Further, IL-1 receptor antagonist (IL-1Ra) was down-regulated in SLE patients only (P < 0.0001). ANA-positive individuals had increased frequencies of monocytes, memory B cells, and plasmablasts and increased levels of pSTAT-1 and pSTAT-3 following IFNα stimulation compared with ANA-negative controls (P < 0.05). CONCLUSION: ANA-positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL-12p40, and stem cell factor/c-Kit ligand. Further, severely decreased levels of IL-1Ra in SLE patients compared with ANA-positive individuals may contribute to disease development. These results highlight the importance of IFN-related pathways and regulatory elements in SLE pathogenesis.