IRF-1 mRNA Research Articles

Innate Immune Response to Viral Infections in Primary Bronchial Epithelial Cells is Modified by the Atopic Status of Asthmatic Patients.

PurposeIn order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls.MethodsHBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-β and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR.ResultsPIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-β mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-β, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-β mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics.ConclusionsThe immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status.

Interferon regulatory factor 4/5 signaling impacts on microglial activation after ischemic stroke in mice.

Microglial activation is a key element in initiating and perpetuating inflammatory responses to stroke. Interferon regulatory factor 5 (IRF5) and IRF4 signaling have been found critical in mediating macrophage pro‐inflammatory (M1) and anti‐inflammatory (M2) phenotypes, respectively, in peripheral inflammation. We hypothesize that the IRF5/4 regulatory axis also mediates microglial activation after stroke. C57BL6 mice of 8–12 weeks were subject to a 90‐min middle cerebral artery occlusion, and the brains evaluated at 24 h, 3, 10 and 30 days after reperfusion. Flow cytometry was utilized to examine microglial activation and cytokine expression. RT‐PCR was performed for mRNA levels of IRF5/4 in sorted microglia. Microglial expression of IRF5/4 was examined by immunohistochemistry, and brain cytokine levels were determined by ELISA. Our results revealed that the IRF5 mRNA level in sorted microglia increased at 3 days of stroke; whereas IRF4 mRNA level exhibited biphasic increases, with a transient rise at 24 h and a peak at 10 days. The same pattern was seen in IRF5/4 protein colocalization with Iba‐1+ cells by IHC. Intracellular levels of TNF‐α and IL‐1β in microglia peaked at 3 days of stroke, and IL‐4+ IL‐10+ double‐positive microglia significantly increased at day 10. Brain levels of these cytokines were consistent with microglial cytokine changes. Worse behavior test results were seen at 3 days vs. 10 days of stroke. We conclude that microglia phenotypes are dynamic to ischemic stroke, and IRF5/4 signaling may regulate microglial M1/M2 activation and impact on stroke outcomes.

CD150 AND CD180 ARE INVOLVED IN REGULATION OF TRANSCRIPTION FACTORS EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150cell surface expression. From other side we attempted to regulate TFs expression via CD150and CD180cell surface receptors. Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia(CLL) cells isolated from peripheral blood of 67primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Median of PAX5and EBF1mRNA expression was higher in cell surface CD150positive (csCD150+) compared to csCD150- CLL cases or normal CD19+ and CD19+CD5+ B-cell subsets. Differences in mRNA expression of IRF8, IRF4and BLIMP1between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1and BCL6mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6was uniformly distributed between csCD150- and csCD150+ CLL cases. PU.1protein and CD20that is direct PU.1target gene positively correlated with CD150cell surface expression on CLL cells. Ligation of CD150and CD180alone or in combination upregulated IRF8and PU.1while downregulated the IRF4mRNA expression. Signaling via CD150or CD180alone elevated the level of BCL6mRNA. Strong downregulation of IRF4mRNA was observed after CD150, CD180or CD150 andCD180 coligation on CLL cells. We found that in CLL cells CD150is a negative regulator of SPIB while CD180is involved in upregulation of EBF1expression level. Moreover, CD180ligation on CLL cells caused increase of CD150mRNA level that is a one of the EBF1 target genes. Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 inCLL cells. CD150and CD180receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels.

Saponin Quil A up-regulates type I interferon-regulated gene and type I and II interferon expressions which are suppressed by porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses innate immune response following infection of myeloid antigen-presenting cells. Poor innate immune response results in weak and delayed PRRSV-specific adaptive immunity, and facilitates PRRSV replication, pathogenesis, and persistent infection. Numerous efforts have been made to enhance the effective innate and adaptive immune defenses to PRRSV, however, only a few attempts have so far elicited satisfactory results. The present study aims to evaluate in vitro the potential of saponin quil A to enhance the expression of type I interferon (IFN)-regulated gene, type I and II IFNs, and pro-inflammatory cytokines in PRRSV-inoculated peripheral blood mononuclear cells (PBMC). Naïve PBMC from four PRRSV-seronegative pigs were inoculated with PRRSV and subsequently stimulated with quil A in the absence or presence of either polyinosinic:polycytidylic acid (poly IC) or lipopolysaccharide (LPS). The mRNA expression levels of myxovirus resistance 1 (Mx1), interferon regulatory factor 3 (IRF3), IRF7, 2′-5′-oligoadenylatesynthetase 1 (OAS1), stimulator of interferon genes (STING), osteopontin (OPN), IFNα, IFNβ, IFNγ, interleukin-2 (IL-2), IL-10, IL-13, tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFβ) were evaluated by real-time PCR. Compared with uninoculated PBMC, PRRSV significantly suppressed expression of all immune parameters except IL-2, IL-10, IL-13, and TGFβ. When compared with PRRSV-inoculated PBMC, stimulation with quil A significantly enhanced Mx1, IRF3, IRF7, OAS1, STING, IFNβ, and IFNγ mRNA expressions, and significantly reduced TGFβ mRNA expression. Our findings thus suggest that quil A has a potential to up-regulate the expression of type I IFN-regulated gene and type I and II IFNs which are suppressed by PRRSV. Therefore, it may serve as an effective immunostimulator for potentiating the innate immune defense to PRRSV.

Investigation of sphingosine kinase 1 in interferon responses during dengue virus infection.

Dengue virus (DENV) regulates sphingosine kinase (SK)-1 activity and chemical inhibition of SK1 reduces DENV infection. In primary murine embryonic fibroblasts (pMEFs) lacking SK1 however, DENV infection is enhanced and this is associated with induction of normal levels of interferon beta (IFN-β) but reduced levels of IFN-stimulated genes (ISGs). We have further investigated this link between SK1 and type I IFN responses. DENV infection downregulates cell-surface IFN-alpha receptor (IFNAR)1 in both wild-type (WT) and SK1−/− pMEF, but, consistent with poor ISG responses, shows reduced induction of phosphorylated (p)-STAT1 and key IFN regulatory factors (IRF)1 and −7 in SK1−/− pMEF. Direct IFN stimulation induced ISGs (viperin, IFIT1), CXCL10, IRF1 and −7 and p-STAT1. Responses, however, were significantly reduced in SK1−/− pMEF, except for IFN-stimulated CXCL10 and IRF7. Poor IFN responses in SK1−/− pMEF were associated with a small reduction in basal cell-surface IFNAR1 and IRF1 mRNA in uninfected SK1−/− compared with WT pMEF. In contrast, treatment of cells with the SK1 inhibitor, SK1-I or expression of an inhibitory SK1 short hairpin RNA (shRNA), both of which reduce DENV infection, does not alter basal IRF1 mRNA or affect type I IFN stimulation of p-STAT1. Thus, cells genetically lacking SK1 can induce many responses normally following DENV infection, but have adaptive changes in IFNAR1 and IRF1 that compromise DENV-induced type I IFN responses. This suggests a biological link between SK1 and IFN-stimulated pathways. Other approaches to reduce SK1 activity, however, do not influence these important antiviral pathways but reduce infection and may be useful antiviral strategies.

Abstract 1013: Location matters: unique functional and transcriptional, but not flow cytometric, characteristics of intratumoral FOXP3+ Tregs vs. Tregs from other sites of patients with lung tumors

Abstract FOXP3+ Tregs are considered important to limiting antitumor immunity but are rarely characterized clinically. We studied 227 samples from 66 lung cancer patients using blood, lymph node (LN), tumor and distant lung samples, with a mean age of 67.8±1.1 years, 67% males, 62% adenocarcinoma, 31% squamous cell carcinoma, 7% miscellaneous tumors, mean tumor size of 3.4±0.3 cm, and 21% rate of metastases. In addition to quantitating FOXP3+CD4+ Tregs, we evaluated their expression of 35 markers by flow cytometry: CD15s, CD25, CD26, CD27, CD39, CD40L, CD45RA/RO, CD62L, CD69, CD101, CD120b, CD161, CCR4, CTLA4, GARP, GITR, Helios, HLA-DR, ICOS, LAP, neuropilin, PD-1, TIGIT, Tim3, CCR4, CCR5, CCR7, CXCR3, CXCR4 and CCR8, and tested Treg suppressive function. We used PrimeFlow to evaluate mRNA expression of target genes in 100% pure human Treg cells, gated on CD4+FOXP3+ cells. FOXP3+ Tregs (%) in the CD4 cels of tumors and LN were significantly increased (p<0.0001 Kruskal-Wallis) compared to other sites; tumors 19.2±8.5%, LN 14.7±8.5%, healthy PBMC donors 7.2±2.5%, PBMC lung cancer 7.2±2.7%, and lungs 6.6±3.2%. Tregs of lung tumors were remarkably suppressive vs. all other sites (p<0.0001 vs. PBMC & LN, p=0.0106 vs. lung Tregs). None of the 35 markers evaluated by flow cytometry were statistically significantly different for tumor Tregs vs. other sites. However, PrimeFlow showed tumor Tregs, but not FOXP3- T cells or Tregs from other sites, had upregulated mRNA expression of 4 transcription factors (TF): Eos, Irf4, Satb1 and Gata1. These 4 TFs plus LEF1 were recently described as a quintet of Treg self-locking signature TF; expression of any 2 TF plus FOXP3 promoted full Treg gene expression and function. Tumor Tregs also showed significant upregulation of FOXP3 mRNA and protein. Tumor and lung Tregs expressed more Eos, Irf4, Satb1 and Gata1 mRNA, and much higher levels of FOXP3 protein per cell, while in PBMC, LNs and healthy donor Tregs upregulation of TFs did not correspond with increase of FOXP3 protein, indicating the “Treg self-locking signature” is subject to regulation by local factors. Indeed, cultured tumor Tregs downregulated FOXP3 protein, mRNA and TF expression, while PBMC and LN Treg incubated in tumor-conditioned media upregulated FOXP3 mRNA and protein, and Treg TF expression, moving toward the tumor Treg-like phenotype. We found significantly increased numbers and suppressive function of FOXP3+ Tregs within lung tumors vs. other sites in the same patients. In addition, while large-scale flow cytometric studies were not useful in identifying key features of tumor Tregs vs. Tregs at other sites, PrimeFlow showed that tumor Treg have a unique phenotype with upregulated expression of FOXP3 mRNA and protein, as well as Eos, Irf4, Satb1 and Gata1 mRNAs. These features appear to be malleable and associated with features of the local tumor microenvironment. Citation Format: Wayne W. Hancock, Tatiana Akimova, Tianyi Zhang, Evgeniy Eruslanov, Sunil Singhal, Steven M. Albelda. Location matters: unique functional and transcriptional, but not flow cytometric, characteristics of intratumoral FOXP3+ Tregs vs. Tregs from other sites of patients with lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1013. doi:10.1158/1538-7445.AM2017-1013

Hypermethylation of Interferon Regulatory Factor 8 (IRF8) Confers Risk to Vogt-Koyanagi-Harada Disease

Aberrant methylation change of IRF8 confers risk to various tumors, and abnormal expression of IRF8 is involved in many autoimmune diseases, including ocular Behcet’s disease. However, whether the methylation change of IRF8 is associated with Vogt-Koyanagi-Harada (VKH) disease remains unknown. In the present study, we found a decreased IRF8 mRNA expression in association with a higher methylation level in monocyte-derived dendritic cells (DCs) from active VKH patients compared with the normal and inactive subjects. DCs incubated with cyclosporin a (CsA) or dexamethasone (DEX) showed a lower methylation and higher mRNA expression of IRF8 in active VKH patients. A demethylation reagent, 5-Aza-2′-deoxycytidine (DAC) showed a notable demethylation effect as evidenced by increasing the mRNA expression and reducing the methylation level of IRF8. It also suppressed the Th1 and Th17 responses through down-regulating the expression of co-stimulatory molecules (CD86, CD80, CD40), and reducing the production of pro-inflammatory cytokines (IL-6, IL-1β, IL-23, IL-12) produced by DCs. These findings shows that hypermethylation of IRF8 in DCs confers risk to VKH disease. Demethylation of IRF8 may offer a novel therapeutic strategy protect against VKH disease.

Neutralization of interleukin-9 ameliorates symptoms of allergic rhinitis by reducing Th2, Th9, and Th17 responses and increasing the Treg response in a murine model.

A novel independent Th-cell subset, characterized by high expression of interleukin (IL)-9, has been recognized as the Th9 subset. Although Th9 cells are important in many diseases, their contribution to allergic rhinitis (AR) remains unclear. We therefore first determined whether Th9 cells were present in a mouse model of AR. We then investigated the their involvement in the distribution of CD4+ T-cell subsets and the symptoms of AR by treating mice with anti-IL-9 antibodies (Abs). Anti-IL-9 Abs were administered intranasally during rechallenge of ovalbumin (OVA)-induced AR in BALB/c mice. We measured nasal rubbing motion, sneezing and eosinophils, as well as the Th1 (Th1 cell percentage, Ifn-? mRNA/protein, T-bet mRNA), Th2 (Th2 cell percentage, Il-4 mRNA/protein, Gata3 mRNA), Th9 (Th9 cell percentages Il-9 mRNA/protein, PU.1 and Irf4 mRNA), Th17 (Th17 cell percentage, Il-17 mRNA/protein, Ror?t mRNA), and Treg (Treg cell percentage, Foxp3 mRNA) responses in the nasal mucosa. Treatment with anti-IL-9 Abs markedly reduced nasal rubbing, sneezing, eosinophil infiltration, and Th2, Th9, and Th17 responses, and increased the Treg response. Our findings emphasize the importance of IL-9/Th9 in the pathogenesis of AR, and suggest that anti-IL-9 Ab treatment may be an effective therapeutic strategy for AR.

Abstract 45: IRF5/IRF4 Regulatory Axis Mediated Microglial M1/M2 Polarization After Ischemic Stroke

Introduction: Microglia have a prominent role in initiating, sustaining and resolving post-stroke inflammation. The pathology of stroke largely depends on the spectrum of microglial activation with the classically (M1; pro-inflammatory) and/or alternatively activated (M2; anti-inflammatory) phenotype. It has been reported that interferon regulatory factor 5 (IRF5) and IRF4 regulate the M1 and M2 phenotype of macrophages respectively in systemic inflammation; however, it is unknown if microglial activation is regulated by the same mechanism after stroke. We hypothesize that IRF5/IRF4 directs M1/M2 microglial polarization respectively after stroke and the balance of the IRF5/IRF4 regulatory axis influences stroke outcomes. Methods: C57BL6 mice (8-12 weeks) were subjected to a 90-minute middle cerebral artery occlusion (MCAO). Stroke outcomes were evaluated at 12h, 3d, 10d of MCAO. Protein and mRNA levels of IRF4/5 at each time point were examined by IHC, Western blot and RT-PCR. Microglial phenotypes were evaluated by flow cytometry (FC) and determined by the ratio of MHC-II + (M1) or CD206 + (M2) microglia number over the total microglia. IRF4/IRF5 conditional knockout (CKO) mice will also be utilized to examine the effect of gene deletion on stroke outcomes. Results: IRF4 protein level increased at 10d in the ipsilateral brain lysates, but IRF5 level decreased at day 10 compared to that of 3d. M2 microglia number was higher at 10d vs. 3d by FC analysis; while M1 microglia ratio was lower at 10d vs. 3d. The Iba1 + IRF4 + (M2) and Iba1 + IRF5 + (M1) cell number by IHC showed the same pattern as that in FC data. In addition, IRF4 mRNA in FC-sorted microglia was up-regulated at 3d of stroke and peaked at 10d, but IRF5 mRNA significantly increased at 3d vs. sham and declined to baseline at 10d. IRF5-CKO mice had significantly smaller infarct volume and improved behavior deficits than IRF5 flox/flox mice while IRF4-CKO mice had worse outcomes than that of IRF4 flox/flox mice. Conclusion: IRF5-mediated pro-inflammatory phenotype (M1) is predominate in the acute stage of stroke that switches to IRF4-mediated anti-inflammatory phenotype (M2) in the chronic stage. IRF5/IRF4 regulatory axis regulates the microglial M1/M2 activation and impacts stroke outcomes.

Estradiol Suppresses TLR4-triggered Apoptosis of Decidual Stromal Cells and Drives an Anti-inflammatory TH2 Shift by Activating SGK1.

A pro-inflammatory cytokine profile at the feto-maternal interface may predispose immune maladaptation notably in early miscarriages. We investigated the involvement of estradiol (E2)-activated serum-glucocorticoid regulated kinase 1 (SGK1) in preserving the tolerogenic and pro-survival intrauterine microenvironment beneficial to gestation maintenance. Decidual SGK1 was down-regulated in early miscarriage, consistent with the lower serum E2 concentration seen in pregnancy loss. Lipopolysaccharide (LPS)/Toll-like receptors 4 (TLR4) signaling induced apoptosis and the pro-inflammatory T helper type (TH) 1 response of decidual stromal cells (DSCs) were associated with miscarriage. SGK1 activation was suppressed by LPS/TLR4 signaling and would be rescued by E2 administration via the PI3K signaling pathway in DSCs. SGK1 activation attenuated TLR4-mediated cell apoptosis, while promoting cell viability of DSCs by up-regulating the pro-survival genes BCL2 and XIAP, and enhancing the phosphorylation of FOXO1. Furthermore, E2-induced SGK1 activation reduced the secretion of pro-inflammatory TH1 cytokines, and promoted the generation of TH2 cytokines and elevated IRF4 mRNA and protein levels in LPS-incubated DSCs. Pharmacologic inhibition of SGK1 or suppression by small interfering (si) RNA increased the phosphorylation and nuclear translocation of NF-κB to reverse the pro-TH2 and anti-inflammatory effects of E2 pretreatment, leading to compromised pregnancy. These findings suggest that the E2-mediated SGK1 activation suppressed LPS-mediated apoptosis and promoted the anti-inflammatory TH2 responses in DSCs, ultimately contributing to a successful pregnancy.

Characterization of non-lethal West Nile Virus (WNV) infection in horses: Subclinical pathology and innate immune response.

Most natural West Nile virus (WNV) infections in humans and horses are subclinical or sub-lethal and non-encephalitic. Yet, the main focus of WNV research remains on the pathogenesis of encephalitic disease, mainly conducted in mouse models. We characterized host responses during subclinical WNV infection in horses and compared outcomes with those obtained in a novel rabbit model of subclinical WNV infection (Suen etal. 2015. Pathogens, 4: 529). Experimental infection of 10 horses with the newly emerging WNV-strain, WNVNSW2011, did not result in neurological disease in any animal but transcriptional upregulation of both type I and II interferon (IFN) was seen in peripheral blood leukocytes prior to or at the time of viremia. Likewise, transcript upregulation for IFNs, TNFα, IL1β, CXCL10, TLRs, and MyD88 was detected in lymphoid tissues, while IFNα, CXCL10, TLR3, ISG15 and IRF7 mRNA was upregulated in brains with histopathological evidence of mild encephalitis, but absence of detectable viral RNA or antigen. These responses were reproduced in the New Zealand White rabbits (Oryctolagus cuniculus) experimentally infected with WNVNSW2011, by intradermal footpad inoculation. Kinetics of the anti-WNV antibody response was similar in horses and rabbits, which for both species may be explained by the early IFN and cytokine responses evident in circulating leukocytes and lymphoid organs. Given the similarities to the majority of equine infection outcomes, immunocompetent rabbits appear to represent a valuable small-animal model for investigating aspects of non-lethal WNV infections, notably mechanisms involved in abrogating morbidity.

Immunomodulatory Drugs (IMiDs), Lenalidomide and Pomalidomide, Suppress Th1-Inducing Capacity of Dendritic Cells but Enhance Th2-Mediated Allergic Response

Background: Analogs of thalidomide, lenalidomide (LEN) and pomalidomide (POM), are novel anti-multiple myeloma (MM) drugs (so-called Immunomodulatory Drugs; IMiDs). LEN has been reported to enhance function of effector immune cells such as T cells and NK cells and to induce selective reduction of regulatory T cells. Meanwhile, LEN and POM treatments lead to relatively high rate onset of itchy skin with rash like allergic reaction. However, cellular and molecular mechanisms underlying their immunomodulatory effects still remain largely unclear. Although there is evidence indicating the immunomodulatory actions of LEN on mouse conventional dendritic cells (DCs), there are few reports showing their effects on human DC subsets. DCs are pivotal in orchestrating both innate and acquire immunity as the center of the immune regulatory system, and series of analyses have clarified a functional plasticity of DCs to induce Th1 or Th2 response. Therefore, we focused on the effects of LEN and POM on the function of human myeloid DCs (mDCs), which are the major regulators to induce Th1 or Th2 responses and play a pathogenic role in allergy by their dysregulated Th2-inducing function (Ito T. J Exp Med 2005; 202: 1213).Methods: Using cell sorting, flow cytometry, real-time PCR, and ELISA methods, function and signaling were analyzed in blood human CD11+ mDCs from healthy adult volunteers. Sera were obtained from 24 MM patients with before and after LEN therapy. Written informed consent was obtained for all patients. This study was approved by the Institutional Review Board of Kansai Medical University.Results: We found that both agents at clinical in vivo plasma concentration of 0.1 µM to 1 µM did not affect the mDC survival and their CD86 and OX40-ligand expression in response to toll-like receptor (TLR)-ligands (LPS, poly IC, or R848) and Th2-inducing cytokine, thymic stromal lymphopoietin (TSLP) for 24 h culture. Either LEN or POM inhibited dose-dependently mDC-derived Th1-associated cytokines, IL-12 in response to R848 (1 µM LEN; 5.2 % of cont. and 0.3 µM POM; 12 % of cont.), IFN-λ in response to poly IC (1 µM LEN; 5.4 % of cont. and 0.3 µM POM; 10 % of cont.), and TNFα in response to R848 (1 µM LEN; 36 % of cont. and 0.3 µM POM; 17 % of cont.), but enhanced IL-10 production in response to R848 (1 µM LEN; 4.8-fold increase and 0.3 µM POM; 7.5-fold increase). When mDCs were stimulated with TSLP, both agents significantly enhanced the production of Th2-recruiting chemokine CCL17/TARC (1 µM LEN; 2.4 fold increase and 0.3 µM POM; 3.9 fold increase), which functions as chemoattractant for memory Th2 cells and contribute to aggravation of allergic inflammation. These capacities of POM were stronger than those of LEN. In addition, we revealed that, in response to LPS or R848, both LEN and POM downregulated not only IRF4 mRNA in human mDCs as in LEN-treated myeloma cells, but also STAT4 mRNA, which is important for DC-mediated Th1 response and IL-12 production. In mDCs treated with TSLP, both agents were found to upregulate STAT6 mRNA, which is responsible for TSLP-mediated CCL17 secretion (Arima K. Sci Signal2010;3:ra4). Clinically, serum CCL17/TARC levels are associated with the disease activity of atopic dermatitis and sensitively reflect short-term changes in skin conditions. We also found, in MM patients, serum CCL17 levels at the onset of LEN-associated rash during 4 weeks (n=9; 1271 ± 376 pg/ml) were significantly higher (p <0.001) compared to those without rash in LEN treatment (n=15; 476 ± 236 pg/ml) and those before treatment (296 ± 172 pg/ml).Conclusion: Our data suggest that the IMiDs, while suppress the Th1-inducing capacity of DCs, rather lead to enhance allergic response at DC phase through modulation of signaling pathways in this stream of action. Thus, IMiDs could have the potential to shift DC-mediated response from Th1 to Th2, and our findings provide a plausible explanation for pathogenesis of IMiD-associated rash in the treatment for MM. DisclosuresIto:Celgene corporation: Honoraria; GSK: Patents & Royalties.

FOXO3, IRF4, and xIAP Are Correlated with Immune Activation in HIV-1-Infected Men Who Have Sex with Men During Early HIV Infection.

Forkhead box O (FOXO)1, FOXO3, interferon regulatory factor (IRF)4, X-linked inhibitor of apoptosis protein (xIAP), and E74-like factor (ELF)4 have been described as important regulators of T cell functions and differentiation. However, whether these molecules are associated with HIV-1 disease progression is still unknown. In this study, we showed that the levels of FOXO3, IRF4, and xIAP mRNA in rapid progressors (RPs) were significantly higher than in HIV-negative healthy controls (HCs). Moreover, FOXO3 expression was positively correlated with HIV-1 viral load and CD4+ T cell activation. Remarkably, increased CD4+ and CD8+ T cell activation was apparent in RPs compared with typical progressors and HCs. In addition, a profile of higher apoptosis, more CD8+ TEM cells, and fewer CD4+ and CD8+ Naive T cells were observed in early HIV infection patients with low CD4+ T cell counts. Furthermore, in vitro, IRF4 and xIAP expression was enhanced in peripheral blood mononuclear cells from healthy people following T cell receptor stimulation. T cell activation was decreased by treatment with siRNA inhibiting FOXO3, IRF4, and xIAP. Our results show that significantly increased levels of FOXO3, IRF4, and xIAP mRNA in Chinese HIV-1-infected patients were related to T cell immune activation, implicating them as potential targets for developing new therapeutic avenues to slow down HIV-1 disease progression.

IFN-α/IFN-λ responses to respiratory viruses in paediatric asthma.

We analysed the influence of rhinovirus (RV) in nasopharyngeal fluid (NPF) on type I and III interferon (IFN) responses (e.g. IFN-α and IFN -: λ) and their signal transduction, at baseline and during disease exacerbation, in cohorts of pre-school children with and without asthma.At the time of recruitment into the Europe-wide study PreDicta, and during symptoms, NPF was collected and the local RV colonisation was analysed. Peripheral blood mononuclear cells (PBMCs) were challenged in vitro with RV or not. RNA was analysed by quantitative real-time PCR and gene arrays. Serum was analysed with ELISA for IFNs and C-reactive protein.We found that PBMCs from asthmatic children infected in vitro with the RV1b serotype upregulated MYD88, IRF1, STAT1 and STAT2 mRNA, whereas MYD88, IRF1, STAT1 and IRF9 were predominantly induced in control children. Moreover, during symptomatic visits because of disease exacerbation associated with RV detection in NPF, IFN-α production was found increased, while IFN-λ secretion was already induced by RV in asthmatic children at baseline.During asthma exacerbations associated with RV, asthmatic children can induce IFN-α secretion, indicating a hyperactive immune response to repeated respiratory virus infection.

Increased circulating IL-9-producing CD8+ T cells are associated with eosinophilia and high FeNO in allergic asthmatics.

Allergic asthma is a chronic airway disorder mediated by Th2 cells. It has been shown that IL-9-producing CD8+ cytotoxic T (Tc9) cells promote the subsequent onset of allergic airway inflammation in mice mediated by abnormal Th2 immunity. Whether Tc9 cells are associated with the immunopathogenesis of asthmatic patients remains unknown. In the present study, peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque gradient centrifugation from all subjects. The frequency of Tc9 cells was measured by flow cytometry. Serum IL-9 levels were assessed by enzyme-linked immunosorbent assay (ELISA). mRNA expression levels of IL-9, STAT6, and IRF4 in PBMCs from healthy controls and asthmatic patients were detected by reverse transcription-quantitative polymerase chain reaction. The results showed that the numbers of Tc9 cells in allergic asthmatics were significantly increased, compared with healthy controls (P<0.0001). Notably, IL-9 protein and mRNA levels were increased in allergic asthmatics and STAT6 and IRF4 mRNA levels were elevated, as compared with healthy controls. In addition, circulating numbers of Tc9 cells were positively correlated with blood eosinophil counts and fractioned exhaled nitric oxide (FeNO) levels in asthmatic patients. Moreover, the number of Tc9 cells and serum IL-9 levels in asthmatic patients were significantly decreased after treatment with glucocorticoids (P<0.05). These findings suggest that increased circulating Tc9 cells are associated with eosinophilia and high FeNO of allergic asthma, and that abnormal Tc9 immunity may contribute to the pathogenesis of allergic asthmatics.

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-1β (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.

IRF3 is involved in human acute myeloid leukemia through regulating the expression of miR-155

Acute myeloid leukemia (AML) is a serious disease of the hematopoietic system characterized by de-differentiation and uncontrolled proliferation of immature hematopoietic precursor cells in the bone marrow. However, the underlying mechanism of AML development remains largely unknown. Here in this study, we report the function of IRF3, a member of the interferon-regulatory factor (IRF) family, in human AML. We first show that IRF3 mRNA and protein levels are significantly up-regulated in human AML compared with healthy donors. IRF3 knockdown inhibits cellular proliferation and colony formation in OCI/AML-2 and OCI/AML-3 cells. In addition, IRF3 knockdown induces apoptosis of OCI/AML-2 and OCI/AML-3 cells, whereas IRF3 overexpression promotes cell survival. Further mechanism study shows that IRF3 is positively correlated with miR-155, which is considered as an oncogenic microRNA in AML. We show that IRF3 binds to the promoter of miR-155 and promotes the expression of miR-155 in OCI/AML-2 and OCI/AML-3 cells. In conclusion, our evidence show that IRF3 overexpression in AML promotes cell growth and survival, and miR-155 is involved, indicating that IRF3 may be a potential new biomarker and therapeutic target for AML.

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro

Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8−/−) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8−/− mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8fl/fl;Lyz2cre/+) mice. We found that trabecular bone volume in the Irf8fl/fl;Lyz2cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8fl/fl;Lyz2cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8fl/fl;Lyz2cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.

Abstract 3226: Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models

Abstract PD-1 - PD-L1 pathway disruptors (e.g. nivolumab, pembrolizumab) are now being approved for treatment of patients with locally evolved or metastatic melanoma and advanced non-small cell lung cancer (NSCLC) who have progressed after first line platinum-based chemotherapy. These drugs have also antitumor efficacy in other tumor types (renal cell carcinoma, bladder, Hodgkin lymphoma, colorectal carcinoma with mismatch-repair deficiency ⋯). However, there is still needs to identify predictive biomarkers of response in order to select patients who will benefit from treatments. PD-L1 expression, mutation numbers, or mismatch repair (MMR) deficiencies were already demonstrated to influence response to PD-1 targeting therapies. However, they did not completely discriminate responders from non-responders or cut off levels are not yet precisely defined. Syngenic models are one of the only experimental models to evaluate immune system targeting therapies. Eight mouse models (4T1, A20, B16-F10, CT-26, EMT-6, MBT-2, LLC and Renca) were tested for response to PD-1 or PD-L1 targeting antibodies. From them, 3 models were characterized as non-responder (i.e. 4T1, LLC and RenCa) while other 7 models were showing to be sensitive to PD-1 inhibitors. PD-L1 expression (using RT-qPCR, WB and flow cytometry) was analyzed on cell lines at baseline and after 24h incubation with IFNg. At baseline, there is no significant difference between responders and non-responders whatever the level of analysis (mRNA, total PD-L1 protein expression or cell surface PD-L1 expression). However, after incubation with IFNg, there is a trend to segregate between responders and non-responders. Increased level of PD-L1 at the cell surface was evidenced for responders. IFNg signaling pathway used interferon regulatory factors (IRFs). IRF1 have been demonstrated to be responsible for both constitutive PD-L1 expression and early induction of PD-L1 after IFNg stimulation. mRNA expression of IRF1, IRF3, IRF7 and IRF9 were analyzed at baseline and after 24h incubation with IFNg. At baseline or after IFNg stimulation, there is no significant difference for IRF3, IRF7 and IRF9 mRNA expression between responders and non-responders. However, IRF1 mRNA is less expressed at baseline in responders as compared to non-responders. Moreover, increase in IRF1 expression after IFNg stimulation is higher in responders when compared to non-responders, which correlates well with changes in PD-L1 cell surface expression after IFNg stimulation. The total number of genomic variations was also analyzed using whole exome sequencing. Responder cell lines have highest number of variations in comparison to non-responder cell lines. In depth characterization of these syngeneic models will increase the understanding of how translatable are the results acquired from preclinical mouse studies. Citation Format: Anais Lagrange, Romain Boidot, Marc Hillairet de Boisferon, Olivier Duchamp, Jean-François Mirjolet, François Ghiringhelli. Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3226.

Two Genetic Variations in the IRF8 region are associated with Behçet's disease in Han Chinese.

Several modulatory factors in the TLR signaling pathway including IRF3, IRF7, IRF8, TRIM20, MYD88 and NF-κB1 have been associated with autoimmune disease. In this study, we investigated the association of 13 SNPs for these genes with Behçet’s disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. Haplotype and linkage disequilibrium (LD) analysis were performed by Haploview4.2. IRF8 mRNA expression and cytokine production was tested by real-time PCR and ELISA. Two SNPs near IRF8 were associated with BD (for rs17445836 GG genotype, Pc = 9.56 × 10−8, OR = 2.044; for rs11642873 AA genotype, Pc = 9.24 × 10−7, OR = 1.776). No significant association was found for the 13 SNPs tested with VKH syndrome. Haplotype analysis of the two positive SNPs revealed that the AG haplotype was significantly increased in BD patients (Pc = 2.60 × 10−8, OR = 1.646). Functional studies revealed an increased mRNA expression of IRF8 and IFN-γ production and a decreased production of IL-10 in rs17445836 carriers with the GG genotype. Increased expression of IRF8 as well as IFN-γ production and a decreased production of IL-10 were found in individuals carrying the rs11642873/AA genotype. In conclusion, this study indicates that IRF8 may contribute to the genetic susceptibility of BD by regulating IRF8 expression and cytokine production.