The Modulation of Interferon Regulatory Factor-1 via Caspase-1-Mediated Alveolar Macrophage Pyroptosis in Ventilator-Induced Lung Injury.

Background: To examine the role of interferon regulatory factor-1 (IRF-1) and to explore the potential molecular mechanism in ventilator-induced lung injury. Methods: Wild-type C57BL/6 mice and IRF-1 gene knockout mice/caspase-1 knockout mice were mechanically ventilated with a high tidal volume to establish a ventilator-related lung injury model. The supernatant of the alveolar lavage solution and the lung tissues of these mice were collected. The degree of lung injury was examined by hematoxylin and eosin staining. The protein and mRNA expression levels of IRF-1, caspase-1 (p10), and interleukin (IL)-1ß (p17) in lung tissues were measured by western blot and quantitative real-time polymerase chain reaction, respectively. Pyroptosis of alveolar macrophages was detected by flow cytometry and western blotting for active caspase-1 and cleaved GSDMD. An enzyme-linked immunosorbent assay was used to measure the levels of IL-1ß, IL-18, IL-6, TNF-α, and high mobility group box protein 1 (HMGB-1) in alveolar lavage fluid. Results: IRF-1 expression and caspase-1-dependent pyroptosis in lung tissues of wild-type mice were significantly upregulated after mechanical ventilation with a high tidal volume. The degree of ventilator-related lung injury in IRF-1 gene knockout mice and caspase-1 knockout mice was significantly improved compared to that in wild-type mice, and the levels of GSDMD, IL-1ß, IL-18, IL-6, and HMGB-1 in alveolar lavage solution were significantly reduced (P < 0.05). The expression levels of caspase-1 (p10), cleaved GSDMD, and IL-1ß (p17) proteins in lung tissues of IRF-1 knockout mice with ventilator-related lung injury were significantly lower than those of wild-type mice, and the level of pyroptosis of macrophages in alveolar lavage solution was significantly reduced. Conclusions: IRF-1 may aggravate ventilator-induced lung injury by regulating the activation of caspase-1 and the focal death of alveolar macrophages.

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

TLR4/IL-6/IRF1 signaling regulates androgen receptor expression: A potential therapeutic target to overcome taxol resistance in ovarian cancer.

Ovarian cancer is poorly treatable due, at least in part, to induced drug resistance to taxol- and cisplatin-based chemotherapy. Recent studies showed that ectopic overexpression of toll-like receptor 4 (TLR4) in ovarian cancer cells leads to upregulation of the androgen receptor (AR) and transactivation of taxol resistance genes, thereby causing chemoresistance. In the present study, we examined the signaling pathways involving TLR4 and interleukin 6 (IL-6) that enhance AR expression. Based on transcriptomic analysis, we show that IL-6 functions as a hub gene among the upregulated genes in taxol-treated TLR4-overexpressing ovarian cancer cells. Both the TLR4 activator taxol and IL-6 can induce AKT phosphorylation, whereas TLR4 knockdown or inhibition of the IL-6 signal transducer GP130 abrogates AKT activation. Furthermore, expression of AR and IL-6 is downregulated in TLR4-knockdown, taxol-resistant cells. In addition, TLR4 knockdown inhibits GP130 and IL-6 receptor alpha (IL6Rα) activities, indicating that TLR4 plays a critical role in IL-6 signaling. On the other hand, nuclear translocation of AR is induced by IL-6 treatment, whereas knockdown of endogenous IL-6 reduces AR and TLR4 expression in taxol-resistant ovarian cancer cells. These results indicate that TLR4 and IL-6 play a crucial role in AR gene regulation and function. We also identify interferon regulatory factor 1 (IRF1) as a downstream target of IL-6 signaling and as a regulator of AR expression. Moreover, analysis of clinical samples indicates that high IL-6 expression correlates with poor progression-free survival in ovarian cancer patients treated with taxol. Overall, our findings indicate that the TLR4/IL-6/IRF1 signaling axis represents a potential therapeutic target to overcome AR-based taxol resistance in ovarian cancer.

A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.

An epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFß-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer.

Analysis of the relationship between microRNA-31 and interferon regulatory factor-1 in hepatocellular carcinoma cells.

OBJECTIVE: MicroRNAs (miRNAs) play a role in the pathogenesis of hepatocellular carcinoma (HCC). This study was designed to elucidate the role of microRNA-31 (miR-31) in HCC. MATERIALS AND METHODS: HuH7 cell lines were transfected with miR-31 mimic or miR-31 inhibitor to investigate the role of miR-31 in regulating interferon regulatory factor-1 (IRF-1). The mRNA and protein expression levels of IRF-1 were quantitatively detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Subsequently, Dual-Luciferase reporter assay was also performed. RESULTS: The expression level of miR-31 was significantly up-regulated in HuH7 cells when compared with that in primary human hepatocytes (hHC). Dual-Luciferase reporter assay indicated that IRF-1 was the direct target of miR-31. The expression levels of IRF-1 were decreased in HuH7 and HepG2 cell lines. IRF-1 was negatively correlated with miR-31 in HCC tissues and paired adjacent tissues. The expression level of miR-31 was inversely correlated with IRF-1. MiR-31 inhibitor up-regulated the expression levels of IRF-1 in HuH7 cells, whereas miR-31 mimic down-regulated the expression levels of IRF-1. Furthermore, the miR-31 mimic repressed IRF-1-3'UTR reporter activity, whereas the miR-31 inhibitor enhanced IRF-1-3'UTR reporter activity depending on the concentration of miR-31 mimic and miR-31 inhibitor. CONCLUSIONS: These results indicated that miR-31 can regulate the expression level of IRF-1 in HCC, which probably provided novel theoretical evidence for the application of target miR-31 treatment of HCC.

Translation of IRF-1 Restricts Hepatic Interleukin-7 Production to Types I and II Interferons: Implications for Hepatic Immunity.

Interleukin-7 (IL-7) is an important cytokine with pivotal pro-survival functions in the adaptive immune system. However, the role of IL-7 in innate immunity is not fully understood. In the present study, the impact of hepatic IL-7 on innate immune cells was assessed by functional experiments as well as in patients with different stages of liver cirrhosis or acute-on-chronic liver failure (ACLF). Human hepatocytes and liver sinusoidal endothelial cells secreted IL-7 in response to stimulation with interferons (IFNs) of type I and II, yet not type III. De novo translation of interferon-response factor-1 (IRF-1) restricted IL-7 production to stimulation with type I and II IFNs. LPS-primed human macrophages were identified as innate immune target cells responding to IL-7 signaling by inactivation of Glycogen synthase kinase-3 (GSK3). IL-7-mediated GSK3 inactivation augmented LPS-induced secretion of pro-inflammatory cytokines and blunted LPS tolerance of macrophages. The IFN-IRF-1-IL-7 axis was present in liver cirrhosis patients. However, liver cirrhosis patients with or without ACLF had significantly lower concentrations of IL-7 in serum compared to healthy controls, which might contribute to LPS-tolerance in these patients. In conclusion, we propose the presence of an inflammatory cascade where IFNs of type I/II induce hepatocellular IL-7 in an IRF-1-restriced way. Beyond its role in adaptive immune responses, IL-7 appears to augment the response of macrophages to LPS and to ameliorate LPS tolerance, which may improve innate immune responses against invading pathogens.

Analysis of coding and non-coding transcriptome of peripheral B cells reveals an altered interferon response factor (IRF)-1 pathway in multiple sclerosis patients.

Several evidences emphasize B-cell pathogenic roles in multiple sclerosis (MS). We performed transcriptome analyses on peripheral B cells from therapy-free patients and age/sex-matched controls. Down-regulation of two transcripts (interferon response factor 1-IRF1, and C-X-C motif chemokine 10-CXCL10), belonging to the same pathway, was validated by RT-PCR in 26 patients and 21 controls. IRF1 and CXCL10 transcripts share potential seeding sequences for hsa-miR-424, that resulted up-regulated in MS patients. We confirmed this interaction and its functional effect by transfection experiments. Consistent findings indicate down-regulation of IRF1/CXCL10 axis, that may plausibly contribute to a pro-survival status of B cells in MS.

Recombinant human interferon regulatory factor-1 (IRF-1) protein expression and solubilisation study in Escherichia coli.

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene, which encodes a mammalian transcription factor that serves various vital functions in a cell, such as cell cycle regulation, immunomodulation, and antiviral response. We report full-length human IRF-1 cDNA cloning and expression in E. coli/BL21 cells with complete solubilisation of recombinant protein. We cloned the gene by the RT-PCR technique using ORF-specific primers followed by expression of recombinant IRF-1 in E. coli under GST fusion system. The profound expression of recombinant protein was observed after inducing with 0.5 mM IPTG for 3 h at 37 °C. We observed few degradation products of low molecular mass along with full-length fusion protein. We successfully minimized the formation of low molecular mass degradation products of GST-huIRF-1 protein at 16 °C. Simultaneously, we achieved the expression of recombinant protein in soluble fraction of E. coli/BL21 cells at 20 °C with higher yield, which is crucial to the study of the biological functions of any protein. We further confirmed it by the immunoblotting technique using anti-IRF-1 and anti-GST antibodies under the induction of E. coli cells harboring the IRF-1 recombinant plasmid after sonicated and fractioned fractions. This work will serve as a platform for characterizing the recombinant protein that may pave the way to understand molecular mechanism of tumour suppression caused by this molecule.

Central Role of the NF-κB Pathway in the Scgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-Induced Airway Inflammation.

Lower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin (Scgb1a1)-expressing bronchiolar epithelial cells in RSV infection. Mice expressing a tamoxifen (TMX)-inducible Cre recombinase-estrogen receptor fusion protein (CreERTM) knocked into the Scgb1a1 locus were crossed with mice that harbor a RelA conditional allele (RelAfl ), with loxP sites flanking exons 5 to 8 of the Rel homology domain. The Scgb1a1CreERTM/+ × RelAfl/fl mouse is a RelA conditional knockout (RelACKO) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelACKO mice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NF-κB-dependent cytokines in response to RSV. In addition, RelACKO mice had reduced expression levels of interferon (IFN) regulatory factor 1/7 (IRF1/7) and retinoic acid-inducible gene I (RIG-I), components of the mucosal IFN positive-feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA polymerase II (Pol II) phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser2 Pol II formation, histone H3K122 acetylation, and cytokine secretion in vitro and in vivo TMX-treated RelACKO mice have less weight loss and reduced airway obstruction/hyperreactivity yet similar levels of IFN-γ production despite higher levels of virus production. These data indicate that the nonciliated Scgb1a1-expressing epithelium is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation and chemokine and mucosal IFN production via the RelA-BRD4 pathway.IMPORTANCE RSV infection is the most common cause of infant hospitalizations in the United States, resulting in 2.1 million children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NF-κB/RelA transcription factor in this subset of epithelial cells. These mice show an impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores and airway hyperreactivity yet increased levels of virus replication. We conclude that RelA-BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection.

Specific microRNA signatures responsible for immune disturbance related to hip fracture in aged rats.

BACKGROUND: Hip fracture is commonly associated with an overwhelming inflammatory response, which may lead to high rates of morbidity and mortality in the elderly. MicroRNAs (miRNAs) play important roles in the functions of immune system. However, the association between miRNA dysregulation and immune disturbance (IMD) related to elderly hip fracture is largely unknown. METHODS: In this study, microarray profiling was carried out to evaluate the differential expression patterns of miRNAs in plasma of the aged hip fracture rats with IMD, those without IMD, and normal aged rats, followed by validation using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Genes and signaling pathways of the dysregulated miRNAs related to elderly hip fracture-induced IMD were investigated in silico using Gene Ontology and analysis of Kyoto Encyclopedia of Genes or Genomes. RESULTS: Dead or moribund rats with hip fracture exhibited significantly reduced TNF-α/IL-10 ratio compared with healthy controls and other hip fracture rats, which were therefore named as hip fracture rats with IMD. Seven serum miRNAs in hip fracture rats with IMD were significantly downregulated. qRT-PCR and in silico analysis revealed that miR-130a-3p likely participated in regulating the hip fracture-induced IMD. Furthermore, Western blot experiment demonstrated that in lung tissue, the reduction of miR-130a-3p was accompanied with the increase of the protein expression of interferon regulatory factor-1 (IRF1) and sphingosine-1-phosphate receptor 1 (SIPR1). CONCLUSIONS: miR-130a-3p desregulation may be associated with elderly hip fracture-induced IMD, which might act as a new potential biomarker for the diagnosis and prognosis of elderly hip fracture-induced IMD and a potential therapeutic target as well.

Sulforaphane inhibits the interferon-γ-induced expression of MIG, IP-10 and I-TAC in INS­1 pancreatic ß-cells through the downregulation of IRF-1, STAT-1 and PKB.

Sulforaphane (SFN) is a dietary isothiocyanate abundantly available in cruciferous vegetables and has been shown to possess anti-inflammatory and immunomodulatory activities. Chemokines are important mediators of inflammation and immune responses due to their ability to recruit and activate macrophages and leukocytes. To date, little is known about the SFN-mediated regulation of chemokine expression in pancreatic ß-cells. In this study, we investigated the inhibitory effects and mechanisms of SFN on the interferon-γ (IFN-γ)-induced expression of a subset of chemokines, including monokine induced by IFN-γ (MIG), IFN-inducible protein of 10 kDa (IP-10) and IFN-inducible T­cell alpha chemoattractant (I-TAC), in INS­1 cells, a rat pancreatic ß-cell line. Notably, IFN-γ treatment led to an increase in the mRNA expression levels of MIG, IP-10 and I-TAC in the INS­1 cells. However, SFN strongly blocked the mRNA expressions of MIG, IP-10 and I-TAC induced by IFN-γ in INS­1 cells. On the mechanistic level, SFN significanlty decreased not only the mRNA expression levels of interferon regulatory factor-1 (IRF-1), but also the phosphorylation levels of signal transducer and activator of transcription-1 (STAT-1) and protein kinase B (PKB) which were induced by IFN-γ in the INS­1 cells. Pharmacological inhibition experiments further revealed that treatment with JAK inhibitor I weakly inhibited the IFN-γ-induced expression of IP-10, whereas it strongly suppressed the IFN-γ-induced expression of MIG and I-TAC in the INS­1 cells. Moreover, treatment with LY294002, a PI3K/PKB inhibitor, was able to slightly repress IFN­Î³­induced expressions of MIG and I-TAC, but not IP-10, in INS­1 cells. Importantly, the IFN-γ-induced increase in the expression levels of MIG, IP-10 and I-TAC in the INS-1 cells was strongly inhibited by SFN, but not by other natural substances, such as curcumin, sanguinarine, resveratrol, triptolide and epigallocatechin gallate (EGCG), suggesting the specificity of SFN in downregulating the levels of these chemokines. To the best of our knowledge, these results collectively demonstrate for the first time that SFN strongly inhibits the IFN-γ-induced expression of MIG, IP-10 and I-TAC in INS­1 cells and this inhibition is, at least in part, mediated through the reduced expression and phosphorylation levels of IRF-1, STAT-1 and PKB.

Epigenetic silencing of IRF1 dysregulates type III interferon responses to respiratory virus infection in epithelial to mesenchymal transition.

Chronic oxidative injury produced by airway disease triggers a transforming growth factor-ß (TGF-ß)-mediated epigenetic reprogramming known as the epithelial-mesenchymal transition (EMT). We observe that EMT silences protective mucosal interferon (IFN)-I and III production associated with enhanced rhinovirus (RV) and respiratory syncytial virus (RSV) replication. Mesenchymal transitioned cells are defective in inducible interferon regulatory factor 1 (IRF1) expression by occluding RelA and IRF3 access to the promoter. IRF1 is necessary for the expression of type III IFNs (IFNLs 1 and 2/3). Induced by the EMT, zinc finger E-box binding homeobox 1 (ZEB1) binds and silences IRF1. Ectopic ZEB1 is sufficient for IRF1 silencing, whereas ZEB1 knockdown partially restores IRF1-IFNL upregulation. ZEB1 silences IRF1 through the catalytic activity of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), forming repressive H3K27(me3) marks. We observe that IRF1 expression is mediated by ZEB1 de-repression, and our study demonstrates how airway remodelling/fibrosis is associated with a defective mucosal antiviral response through ZEB1-initiated epigenetic silencing.

Increase of IRF-1 gene expression and impairment of T regulatory cells suppression activity on patients with myelodysplastic syndrome: A longitudinal one-year study.

Studies have demonstrated that abnormalities in interferon regulatory factor-1 (IRF-1) expression might develop myelodysplastic syndromes (MDS). IRF-1 was described as modulator of T regulatory (Treg) cells by suppressing Foxp3 on mice. We aimed to determine the role of Treg and IRF-1 in MDS. Thirty-eight MDS patients fulfilling WHO criteria and classified according to risk scores were evaluated at time 0 (T0) and after 12 months (T12) for: Treg suppression activity in coculture with T effector (Teff) cells; IRF-1 and Foxp3 genetic expression by qRT-PCR; IL-2, -4, -6, -10, -17, TNFα and IFNγ production by Cytometric Bead Array. No differences in Foxp3 expression (T0=0.06±0.06 vs T12=0.06±0.12, p=0.5), Treg number (T0=5.62±2.84×105 vs T12=4.87±2.62×105; p=0.3) and Teff percentage (T0=16.8±9.56% vs T12=13.1±6.3%; p=0.06) were observed on T12. Low risk MDS patients showed a higher number of Treg (5.2±2.6×105) versus high risk group (2.6±1.2×105, p=0.03). Treg suppression activity was impaired on T0 and T12.Cytokine production and IRF-1 expression were increased on T12. The correlation between IRF-1 and FoxP3 was negative (r2=0.317, p=0.045) on T0. These results suggest a hyper activity of the immune system, probably secondary to Treg suppression activity impairment. This state may induce the loss of tolerance culminating in the proliferation of MDS clones.

DUSP1 Maintains IRF1 and Leads to Increased Expression of IRF1-dependent Genes: A MECHANISM PROMOTING GLUCOCORTICOID INSENSITIVITY.

Although the mitogen-activated protein kinase (MAPK) phosphatase, DUSP1, mediates dexamethasone-induced repression of MAPKs, 14 of 46 interleukin-1ß (IL1B)-induced mRNAs were significantly enhanced by DUSP1 overexpression in pulmonary A549 cells. These include the interferon regulatory factor, IRF1, and the chemokine, CXCL10. Of these, DUSP1-enhanced mRNAs, 10 including CXCL10, were IRF1-dependent. MAPK inhibitors and DUSP1 overexpression prolonged IRF1 expression by elevating transcription and increasing IRF1 mRNA and protein stability. Conversely, DUSP1 silencing increased IL1B-induced MAPK phosphorylation while significantly reducing IRF1 protein expression at 4 h. This confirms a regulatory network whereby DUSP1 switches off MAPKs to maintain IRF1 expression. There was no repression of IRF1 expression by dexamethasone in primary human bronchial epithelial cells, and in A549 cells IL1B-induced IRF1 protein was only modestly and transiently repressed. Although dexamethasone did not repress IL1B-induced IRF1 protein expression at 4-6 h, silencing of IL1B plus dexamethasone-induced DUSP1 significantly reduced IRF1 expression. IL1B-induced expression of CXCL10 was largely insensitive to dexamethasone, whereas other DUSP1-enhanced, IRF1-dependent mRNAs showed various degrees of repression. With IL1B plus dexamethasone, CXCL10 expression was also IRF1-dependent, and expression was reduced by DUSP1 silencing. Thus, IL1B plus dexamethasone-induced DUSP1 maintains expression of IRF1 and the IRF1-dependent gene, CXCL10. This is supported by chromatin immunoprecipitation showing IRF1 recruitment to be essentially unaffected by dexamethasone at the CXCL10 promoter or at the promoters of more highly repressed IRF1-dependent genes. Since IRF1-dependent genes, such as CXCL10, are central to host defense, these data may help explain the reduced effectiveness of glucocorticoids during asthma exacerbations.

Decreased expression of pSTAT, IRF-1 and DAP10 signalling molecules in peripheral blood lymphocytes of patients with metastatic melanoma.

AIMS: As numerous signalling molecules regulate effector functions of peripheral blood lymphocytes (PBLs) that have an important anti-tumour activity, the aim of this study was to analyse their level in patients with metastatic melanoma (MM) compared with healthy controls (HCs). METHODS: Peripheral blood mononuclear cells (PBMCs) of 36 MMs and 28 HCs were analysed for the level of perforin, interferon-regulating transcription factor-1 (IRF-1), DAP10 and Src homology 2 domain-containing tyrosine phosphatase-1 by reverse transcriptase PCR, level of phosphorylated signal transducers and activators of transcription (pSTAT)-1, pSTAT-4, pSTAT-5 by western blot and interferon (IFN)-γ production by ELISA. The expression of activating NKG2D and inhibitory killer immunoglobulin-like receptors (KIR), CD158a and CD158b, on PBL, CD3-CD56+ natural killer (NK) cells and CD3+CD8+ cytotoxic T lymphocytes (CTLs), as well as the percentage of CD14+HLA-DR- cells in PBMC were estimated by flow cytometry. RESULTS: Patients with MM, compared with HCs, had significantly lower level of cytotoxic molecule perforin and decreased IFN-γ production, as well as lower level of pSTAT-1, pSTAT-4, pSTAT-5 and IRF-1 signalling molecules in PBMC. Furthermore, MM had decreased expression of activating NKG2D receptor on PBL and NK cells and low level of its DAP10 signalling molecule contrary to no changes in KIR expression on all investigated cells. These results could be associated with increased percentage of immunosuppressive CD14+HLA-DR- myeloid-derived suppressor cells detected in patients with MM. CONCLUSIONS: The altered signalling molecules of PBL could represent biomarkers of impaired cytotoxic and immunoregulatory function of these cells, indicating melanoma-associated immunosuppression that facilitates tumour progression.

5-Azacytidine modulates interferon regulatory factor 1 in macrophages to exert a cardioprotective effect.

Macrophages are actively involved in inflammatory responses during the progression of cardiac injury, including myocardial infarction (MI). A previous study showed that 5-azacytidine (5AZ), a DNA methylation inhibitor, can ameliorate cardiac injury by shifting macrophages toward an anti-inflammatory phenotype via iNOS inhibition. Here, we show that the beneficial effect of 5AZ is associated with sumoylation of interferon regulatory factor-1 (IRF1) in macrophages. IRF1 is a critical transcription factor for iNOS induction and is antagonized by IRF2. In the stimulated macrophages, IRF1 accumulated in the nucleus without degradation by 5AZ treatment. In animal study, 5AZ administration resulted in significant improvements in cardiac function and fibrosis. IRF1-expressing macrophages were more abundant in the 5AZ-treated MI group than in the PBS-treated MI group. Because sumoylated IRF1 is known to mimic IRF2, we examined the IRF1 sumoylation. Sumoylated IRF1 was resistant to degradation and significantly increased in the 5AZ-treated MI group. Collectively, 5AZ had a protective effect after MI by potentiation of IRF1 sumoylation and is suggested as a novel therapeutic intervention for cardiac repair.

Sphingosine-1-phosphate inhibits IL-1-induced expression of C-C motif ligand 5 via c-Fos-dependent suppression of IFN-ß amplification loop.

The neuroinflammation associated with multiple sclerosis involves activation of astrocytes that secrete and respond to inflammatory mediators such as IL-1. IL-1 stimulates expression of many chemokines, including C-C motif ligand (CCL) 5, that recruit immune cells, but it also stimulates sphingosine kinase-1, an enzyme that generates sphingosine-1-phosphate (S1P), a bioactive lipid mediator essential for inflammation. We found that whereas S1P promotes IL-1-induced expression of IL-6, it inhibits IL-1-induced CCL5 expression in astrocytes. This inhibition is mediated by the S1P receptor (S1PR)-2 via an inhibitory G-dependent mechanism. Consistent with this surprising finding, infiltration of macrophages into sites of inflammation increased significantly in S1PR2(-/-) animals. However, activation of NF-κB, IFN regulatory factor-1, and MAPKs, all of which regulate CCL5 expression in response to IL-1, was not diminished by the S1P in astrocytes. Instead, S1PR2 stimulated inositol 1,4,5-trisphosphate-dependent Ca(++) release and Elk-1 phosphorylation and enhanced c-Fos expression. In our study, IL-1 induced the IFNß production that supports CCL5 expression. An intriguing finding was that S1P induced c-Fos-inhibited CCL5 directly and also indirectly through inhibition of the IFN-ß amplification loop. We propose that in addition to S1PR1, which promotes inflammation, S1PR2 mediates opposing inhibitory functions that limit CCL5 expression and diminish the recruitment of immune cells.

STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.

The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells.

The TH9 subset of helper T cells was initially shown to contribute to the induction of autoimmune and allergic diseases, but subsequent evidence has suggested that these cells also exert antitumor activities. However, the molecular events that account for their effector properties are elusive. Here we found that the transcription factor IRF1 enhanced the effector function of TH9 cells and dictated their anticancer properties. Under TH9-skewing conditions, interleukin 1ß (IL-1ß) induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to the promoters of Il9 and Il21 and enhanced secretion of the cytokines IL-9 and IL-21 from TH9 cells. Furthermore, IL-1ß-induced TH9 cells exerted potent anticancer functions in an IRF1- and IL-21-dependent manner. Our findings thus identify IRF1 as a target for controlling the function of TH9 cells.

Fungal engagement of the C-type lectin mincle suppresses dectin-1-induced antifungal immunity.

Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target.