Transcription Factor IRF3 Research Articles

Abstract 1211: Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer

Abstract Lurbinectedin (PM1183) is a new synthetic compound from the tetrahydroisoquinoline family, which has demonstrated a strong antiproliferative activity against a panel of human tumor models in preclinical assays and is currently being evaluated in phase III clinical trials in platinum-resistant ovarian cancer and small cell lung cancer. Lurbinectedin binds to DNA, inhibits trans-activated transcription, induces the degradation of elongating RNA Pol II and fools nucleotide excision repair to produce dsDNA breaks that need to be repaired mainly by homologous recombination (HR)1,2. Nearly 70% of patients diagnosed with ovarian cancer are in advanced stage, and the vast majority of them will eventually relapse after a primary cytoreductive surgery and several cycles of standard adjuvant chemotherapy including a platinum drug and a taxane. After a period of treatment with platinum drugs, patients will finally develop resistance, usually mediated by mechanisms such as drug detoxification or efflux and enhanced DNA repair. IRF-1 transcription factor expression has been shown to be up-regulated by cisplatin (CDDP) in ovarian cancer cells and might be limiting the response to the drug, likely by inhibiting cell proliferation3. Here we took advantage of the A2780/A2780cis human ovarian cancer cell lines, the second being a cisplatin resistant derivative, to investigate the role of IRF1 in the response of human ovarian cancer cells to cisplatin and lurbinectedin. A2780cis cells are, indeed, more resistant to cisplatin that their parental cell line but they do not differ in their resistance to lurbinectedin. Basal IRF-1 protein levels were actually higher in A2780cis cells than in their parental cell line, contributing to their resistance to cisplatin. Furthermore, cisplatin treatment induced the overexpression and nuclear localization of IRF-1 both, in A2780 and A2780cis cell lines. Contrarily, lurbinectedin did not induce the overexpression of IRF-1 neither in A2780 nor in A2780cis, explaining why this latter cell line is not resistant to the compound. Furthermore, lurbinectedin co-treatment with cisplatin reduced the expression of IRF-1 in A2780 and, more importantly, in A2780cis cells, explaining the synergism the combination has on these tumor cell lines. Thus, lurbinectedin not only did not activate the same mechanisms of resistance as cisplatin in ovarian cancer cells, but even reversed the resistance of these resistant cells to platinum drugs. 1 Santamaría Nuñez et al, 2016. Mol Cancer Ther 15(10):2399-2412 2 Romano et al, 2013. Int J Cancer. 2013 Nov;133(9):2024-33 3 Pavan et al, 2013. Eur J Cancer 49(4):964-973 Citation Format: Gema Santamaria Nuñez, Maria Jose Guillén, Juan F. Martínez-Leal, Pablo Avilés, Carlos M. Galmarini. Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer [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 1211. doi:10.1158/1538-7445.AM2017-1211

Lineage specification of human dendritic cells is marked by IRF8 expression in hematopoietic stem cells and multipotent progenitors.

The origin and specification of human dendritic cells (DCs) have not been investigated at clonal level. Using clonal assays combined with statistical computation to quantify the yield of granulocytes, monocytes, lymphocytes and three subsets of DCs from single human CD34+ progenitor cells, we show DC lineage specification occurs in parallel with myeloid and lymphoid lineages in HSCs, starting as a lineage bias defined by specific transcriptional programs correlated with the relative IRF8/PU.1 ratios, which is transmitted to most progeny and reinforced by FLT3L-driven IRF8 upregulation over division. We propose a model in which DC lineage specification is driven by parallel and inheritable transcriptional programs in HSCs, and reinforced over cell division by recursive interaction between transcriptional programs and extrinsic signals.

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF–derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

RNA-binding proteins, the guardians of the marginal zone.

RNA-binding proteins (RBPs) take control of binary cell-fate 'decisions' and cellular identity in lymphoid organs, as the RBP ZFP36L1 is shown to negatively regulate the stability of the transcription factors KLF2 and IRF8 to control the maintenance, survival and localization of marginal zone B cells.

Fra-2 regulates B cell development by enhancing IRF4 and Foxo1 transcription.

The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7-stimulated pro-B cell cultures revealed a reduced differentiation from large pre-B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2-deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro-B cell proliferation and small pre-B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2-deficient pro-B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

IRF5 distinguishes severe asthma in humans and drives Th1 phenotype and airway hyperreactivity in mice.

Severe asthma (SA) is a significant problem both clinically and economically, given its poor response to corticosteroids (CS). We recently reported a complex type 1-dominated (IFN-γ-dominated) immune response in more than 50% of severe asthmatics despite high-dose CS treatment. Also, IFN-γ was found to be critical for increased airway hyperreactivity (AHR) in our model of SA. The transcription factor IRF5 expressed in M1 macrophages can induce a Th1/Th17 response in cocultured human T cells. Here we show markedly higher expression of IRF5 in bronchoalveolar lavage (BAL) cells of severe asthmatics as compared with that in cells from milder asthmatics or healthy controls. Using our SA mouse model, we demonstrate that lack of IRF5 in lymph node migratory DCs severely limits their ability to stimulate the generation of IFN-γ- and IL-17-producing CD4+ T cells and IRF5-/- mice subjected to the SA model displayed significantly lower IFN-γ and IL-17 responses, albeit showing a reciprocal increase in Th2 response. However, the absence of IRF5 rendered the mice responsive to CS with suppression of the heightened Th2 response. These data support the notion that IRF5 inhibition in combination with CS may be a viable approach to manage disease in a subset of severe asthmatics.

Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells.

Chemokine receptor type 6 (CCR6)+CD4+ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4+ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6+CD4+ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6+CD4+ T cells infected with HIV-1. The selective protection of CCR6+CD4+ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.

Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

The T helper 9 (Th9) cell transcriptional network is formed by an equilibrium of signals induced by cytokines and antigen presentation. Here we show that, within this network, two interferon regulatory factors (IRF), IRF1 and IRF4, display opposing effects on Th9 differentiation. IRF4 dose-dependently promotes, whereas IRF1 inhibits, IL-9 production. Likewise, IRF1 inhibits IL-9 production by human Th9 cells. IRF1 counteracts IRF4-driven Il9 promoter activity, and IRF1 and IRF4 have opposing function on activating histone modifications, thus modulating RNA polymerase II recruitment. IRF1 occupancy correlates with decreased IRF4 abundance, suggesting an IRF1-IRF4-binding competition at the Il9 locus. Furthermore, IRF1 shapes Th9 cells with an interferon/Th1 gene signature. Consistently, IRF1 restricts the IL-9-dependent pathogenicity of Th9 cells in a mouse model of allergic asthma. Thus our study reveals that the molecular ratio between IRF4 and IRF1 balances Th9 fate, thus providing new possibilities for manipulation of Th9 differentiation.

Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help.

To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. Peri-follicular GC precursors first expressed intermediate levels of BCL6 while co-expressing the transcription factors RelB and IRF4, the latter known to repress Bcl6 transcription. Transition of GC precursors to the BCL6hi follicular state was associated with cell division, although the number of required cell divisions was immunogen dose dependent. Potentiating T cell help or CD40 signaling in these GC precursors actively repressed GC B cell maturation and diverted their fate towards plasmablast differentiation, whereas depletion of CD4+ T cells promoted this initial transition. Thus while CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, transition to the BCL6hi follicular state is promoted by a regional and transient diminution of T cell help.

A method for the detection of virus infectivity in single cells and real time: Towards an automated fluorescence neutralization test

Virus neutralizing antibodies are critical correlates of protection in vaccine development and are discriminatory in the plaque reduction neutralization test when used for the diagnosis of viral infections. However, neutralization assays are time consuming, labor intensive and highly variable, thus limiting their use. Advances in automated live imaging of cells opened new possibilities for standard virus diagnostic techniques such as neutralization assays. To this end, a reporter cell line based on the translocation of the transcription factor IRF3 in response to infection is proposed. Image acquisition of signal in a microplate format allowed the setup of a rapid, semi-automated and high-throughput fluorescent neutralization test. The study is extended to the live imaging of IRF3 translocations that could potentially cut the time of analysis to few hours. The fluorescent neutralization test is suitable for high-throughput assays and expandable to other viruses of global importance such as Zika virus.

YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation.

The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKɛ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.

FCRL4 and FCRL5 expression distinguishes three human memory B cell subsets in tonsils.

Abstract FCRL4 and FCRL5 are two members of the human Fc receptor like family with the capacity to regulate antigen receptor signaling. FCRL4 was shown to bind IgA, whereas FCRL5 binds IgG. Although the expression of FCRL4 and FCRL5 on human B cell subsets has each been individually evaluated, no study thus far addressed their potential co-expression. Here we report that three distinct subpopulations of CD38lowIgD-memory B cells exist in human tonsils, one that co-expresses FCRL4 and FCRL5, another that is FCRL5 single-positive, and a third that lacks both proteins. We found that the FCRL5+FCRL4+ subset expresses the highest levels of surface FCRL5, CD11c, FAS, PD-1, CD32, CCR1, CCR5 and CCR6 protein, as well as the lowest levels of LAIR-1 and CD21 among the three memory B cell populations. Furthermore, microarray analysis demonstrated that the three memory B cells subsets each possess distinct gene expression profiles. The FCRL5+FCRL4− memory B subset overexpressed dozens of immunoglobulin variable genes compared to the other two subsets, in addition to the transcription factors XBP1, PRDM1 and IRF4 that drive plasmablast and plasma cell differentiation. These results suggest that the FCRL5+FCRL4− memory B subset is committed to become plasmablast. Using KREC assay we established that the FCRL5+FCRL4+ subset underwent more rounds of cell divisions than the other two subsets. The low number of cell divisions measured in the FCRL5+FCRL4− subset suggests that the cells bypassed the germinal center. The surprising association of FCRL4 and FCRL5 expression with distinct memory B cell subsets could point to the functional role of these FCRL proteins and their immunoglobulin ligands to drive B cell development.

PD-1 blockade alters fate commitment of myeloid progenitors during tumor-mediated “emergency” myelopoiesis

Abstract PD-1 is an immune inhibitory receptor for which blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of the how PD-1 compromises anti-tumor function and how such effect is reversed by PD-1 blockade remain poorly understood. The rapid change in hematopoietic output that occurs in response to immunologic stress is known as “emergency” myelopoiesis. This process is co-opted by tumors to enhance the generation of tumor-promoting myeloid cells, mostly undifferentiated myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We analyzed the myeloid compartment of tumor-bearing mice and determined that myeloid cells express PD-1 and PD-L1. Using PD-1 KO mice or WT mice treated with PD-1 blocking antibody in cancer-driven myelopoiesis experiments we determined a cell-switch fate from MDSCs to effector myeloid cells. PD-1 deletion or blockade, which impaired tumor growth and metastases, stimulated the differentiation of hematopoietic progenitor cells and resulted in increase of Ly6Chimonocytes and monocyte-derived DC (mo-DC). Abrogation of PD-1 signaling enhanced the maturation of MDSCs by inducing the lineage commitment transcription factors IRF8, IRF4 and RORC1. IRF8 and IRF4 promote monocytes/macrophage and hinder granulocyte differentiation, whereas RORC1 is required for expansion of Ly6Chi monocytes and resolution of inflammation. These results reveal a role of the PD-1:PD-L1 pathway in the differentiation of lineage-committed myeloid progenitors to promote undifferentiated MDSCs and suppress effector monocytes/DCs. Switch of myeloid progenitor fate commitment might be a key mechanism by which PD-1 blockade mediates anti-tumor function.

Control of type I interferon responses by the AIM2 inflammasome

Abstract The immune system considers the presence of DNA in the cytosol as a sign of potential pathogen invasion and mounts inflammatory responses. Cytosolic DNA can be sensed by cyclic GMP-AMP synthase (c-GAS) leading to the production of type I Interferons (IFN). It can also be recognized by the PYHIN DNA sensor absent in melanoma (AIM2) resulting in the activation of inflammasome responses i.e. production of pro-inflammatory cytokines IL-1β and IL-18 as well as pyroptosis. Our results show that AIM2 inflammasome activation limits the production of type I IFNs in immune cells such as macrophages and dendritic cells. Further analysis showed that the recognition of cytosolic DNA and the assembly of inflammasome complex appear to be required for AIM2 regulation of IFN-β. Biochemical evidence showed an augmented phosphorylation of the transcription factor IRF-3 resulting in increased IFN-β production in AIM2-deficient macrophages. These findings may have implications in the treatment of autoimmune disorders such as systemic lupus erythematosus (SLE) associated with unregulated type I IFN signaling. (Supported by NIH R01AI119015 and Charles Hood Child Health Research Award)

ITK signaling via the Ras/IRF4 pathway regulates the development and function of type 1 regulatory T cells

Abstract Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging T cell receptor (TCR), express high levels of the immunosuppressive cytokine IL-10 but not Foxp3, and can suppress inflammation and promote immune tolerance. Here, using IL-10GFP/Foxp3RFP dual reporter transgenic mouse system, we show that ITK, a key modulator of TCR signaling, is required for the development of Tr1 cells in various organs following TCR activation, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell fate programming and regulates the balance between IL-10 and IFN-γ production during Tr1 cell differentiation. The requirement for ITK function during Tr1 cell development and suppressive function can be restored by the expression of the transcription factor IRF4. Downstream of ITK, Ras, and MAP kinases ERK1/2, p38 and JNK, are required for Tr1 cell differentiation, but not Th17 cell differentiation. We have previously shown that Th17 differentiation is also dependent on ITK signaling. However, in the absence of ITK, expression of the constitutively active HRasG12V rescued IRF4 expression and Tr1 cell differentiation, but failed to restore Th17 cell differentiation. We conclude that the TCR/ITK signaling through the Ras/MAPK/IRF4 pathway is specifically required for functional development of Tr1 cells, and that targeting these signaling components may be of therapeutic benefit.

IRF4-dependent DCs promote the establishment of CD8+ T cell memory during influenza infection.

Abstract The transcription factor IRF4 governs the development and function of CD11b+ dendritic cells (DCs). To determine the role of IRF4-dependent DCs in influenza infection, we compared responses of wild-type and CD11c-cre-Irf4f/f(KO) mice. The KO mice lack one lung-resident CD11b+ DC subset, and the other DC subsets lack IRF4. Upon infection with A/PuertoRico/8/34 virus, KO lung DCs contained increased IL-12 and reduced IL-10 RNA, which correlated with elevated numbers of CD103+ lung migratory DCs in the mLN. Consistent with this, KO mice showed greater numbers of T-bet+ and IFNg+ influenza antigen-specific CD8+ and CD4+ T cells, reduced numbers of Foxp3+ T regulatory cells, and decreased numbers of CD8+ T memory precursor cells. These altered effector T cell populations in KO mice impacted the memory T cell response to influenza virus. Numbers of virus-specific lung resident memory CD8+ T cells were significantly reduced in KO mice. Upon challenge with heterosubtypic virus, KO mice showed deficient expansion of virus-specific IFNγ+ CD8+ T cells. Taken together, these data show that KO mice mount a stronger antiviral T cell response upon primary infection but then have a weaker CD8+ T cell memory response to heterosubtypic virus. This supports the hypothesis that during influenza infection, the functional responses of IRF4-dependent DCs lead to increased differentiation of CD8+ memory precursor T cells and numbers of lung resident CD8+memory T cells.

Gene-enhancer variants reveal diverse TCR-mediated differentiation.

The transcription factor IRF4 acts as a 'rheostat' for TCR signaling. Discrete levels of IRF4 can activate distinct transcriptional programs in T cells due to binding sites of variable affinity in groups of target genes.

Transcription factor IRF8 governs enhancer landscape dynamics during mononuclear phagocyte development

Abstract Monocytes and dendritic cells (DCs) are mononuclear phagocytes essential for innate and adaptive immunity. These phagocytes develop from hematopoietic stem cells via intermediate progenitors, such as granulocyte-monocyte progenitors (GMPs), monocyte-DC progenitors (MDPs), and common monocyte progenitors (cMoPs) or common DC progenitors (CDPs). However, the molecular mechanism underlying their differentiation potential remains incompletely understood. Recent studies suggest that promoter-distal enhancers are key for cell fate decision. In this study, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of enhancer-related histone modifications in GMPs, MDPs, cMoPs, CDPs, monocytes, DCs, and neutrophils. We found that monocyte- and DC-specific enhancers were gradually established at progenitor stages before the expression of associated genes. Of the transcription factors predicted to bind to these enhancers, IRF8, essential for monocyte and DC development, was found to be required for the establishment of these enhancers, particularly those common to both monocyte and DC lineages. Somewhat unexpectedly, global gene expression patterns were comparable between the remaining Irf8−/− progenitors and their wild-type counterparts. Nevertheless, the enhancer landscapes of Irf8−/− progenitors were significantly distinct from those of wild-type cells, with the enhancer landscapes of Irf8−/− GMPs, MDPs, and cMoPs all remaining similar to that of wild-type GMPs. Our results illustrate the dynamic process by which key transcription factors regulate enhancer formation and thereby direct future gene expression to achieve mononuclear phagocyte development.

Dynamic programming and memory of monocytes by varying signal strengths of innate stimulants

Abstract In response to changing external signal strength, innate monocytes may be dynamically programmed into distinct functional states, which may bear significance relevance in host pathophysiology. However, molecular mechanisms that control innate memory dynamics are not well understood nor extensively studied. Using lipopolysaccharide (LPS), the model stimulant of Toll-Like-Receptor 4 (TLR4), we observed that the expressions of pro-inflammatory mediators are preferentially sustained in monocytes adapted by subclinical super-lower dose LPS, and suppressed/tolerized in monocytes programmed by higher dosages of LPS. These phenomena are well conserved in both mice and human monocytes. At the molecular level, we observed that monocytes programmed by super-low dose LPS maintain higher levels of transcription factor IRF5. We also observed that the inflammatory monocyte programming is dependent upon TRAM/TRIF but not MyD88. Similar to LPS, we also observed biphasic inflammatory adaptation and tolerance in monocytes challenged with varying dosages of TLR7 agonist. In sharp contrast, rising doses of TLR3 agonist preferentially caused inflammatory adaptation without inducing tolerance. At the functional level, we observed that in vivo programming of monocytes by subclinical dose LPS exacerbates the progression of atherosclerosis. Taken together, our study provides novel mechanisms for the dynamic programming/memory of innate monocytes and their pathological implications in atherosclerosis.

Transmissible gastroenteritis virus infection induces NF-κB activation through RLR-mediated signaling

Transmissible gastroenteritis virus (TGEV) is a porcine enteric coronavirus which causes lethal severe watery diarrhea in piglets. The pathogenesis of TGEV is strongly associated with inflammation. In this study, we found that TGEV infection activates transcription factors NF-κB, IRF3 and AP-1 in a time- and dose-dependent manner in porcine kidney cells. Treatment with the NF-κB-specific inhibitor BAY11-7082 significantly decreased TGEV-induced proinflammatory cytokine production, but did not affect virus replication. Phosphorylation of NF-κB subunit p65 and proinflammatory cytokine production were greatly decreased after knockdown of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) or its adaptors MAVS and STING, while only slight reduction was observed in cells following silencing of Toll-like receptor adaptors, MyD88 and TRIF. Furthermore, TGEV infection significantly upregulated mRNA expression of RIG-I and MDA5. Taken together, our results indicate that the RLR signaling pathway is involved in TGEV-induced inflammatory responses.