The Interferon-Inducible Human PLSCR1 Protein Is a Restriction Factor of Human Cytomegalovirus.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and it has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the levels of human cytomegalovirus (HCMV) plaque formation in OUMS-36T-3 (36T-3) cells with high basal expression of PLSCR1 were significantly lower than those in human embryonic lung (HEL) cells with low basal expression of PLSCR1. In addition, the levels of HCMV plaque formation and replication in PLSCR1-knockout (KO) 36T-3 cells were significantly higher than those in parental 36T-3 cells and were comparable to those in HEL cells. Furthermore, compared to that in PLSCR1-KO cells, the expression of HCMV major immediate early (MIE) proteins was repressed and/or delayed in parental 36T-3 cells after HCMV infection. We also showed that PLSCR1 expression decreased the levels of the cAMP-responsive element (CRE)-binding protein (CREB)•HCMV immediate early protein 2 (IE2) and CREB-binding protein (CBP)•IE2 complexes, which have been suggested to play important roles in the IE2-mediated transactivation of the viral early promoter through interactions with CREB, CBP, and IE2. Interestingly, PLSCR1 expression repressed CRE- and HCMV MIE promoter-regulated reporter gene activities. These observations reveal, for the first time, that PLSCR1 negatively regulates HCMV replication by repressing the transcription from viral MIE and early promoters, and that PLSCR1 expression may contribute to the IFN-mediated suppression of HCMV infection. IMPORTANCE Because several IFN-stimulated genes (ISGs) have been reported to suppress HCMV replication, HCMV replication is thought to be regulated by an IFN-mediated host defense mechanism, but the mechanism remains unclear. PLSCR1 expression is induced in response to viral infection and IFN treatment, and PLSCR1 has been reported to play an important role in IFN-dependent antiviral responses. Here, we demonstrate that HCMV plaque formation and major immediate early (MIE) gene expression are significantly increased in PLSCR1-KO human fibroblast cells. PLSCR1 reduces levels of the CREB•IE2 and CBP•IE2 complexes, which have been suggested to play important roles in HCMV replication through its interactions with CREB, CBP, and IE2. In addition, PLSCR1 expression represses transcription from the HCMV MIE promoter. Our results indicate that PLSCR1 plays important roles in the suppression of HCMV replication in the IFN-mediated host defense system.

CREBBP/EP300 mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-associated macrophage polarization via FBXW7-NOTCH-CCL2/CSF1 axis.

Epigenetic alterations play an important role in tumor progression of diffuse large B-cell lymphoma (DLBCL). However, the biological relevance of epigenetic gene mutations on tumor microenvironment remains to be determined. The core set of genes relating to histone methylation (KMT2D, KMT2C, EZH2), histone acetylation (CREBBP, EP300), DNA methylation (TET2), and chromatin remodeling (ARID1A) were detected in the training cohort of 316 patients by whole-genome/exome sequencing (WGS/WES) and in the validation cohort of 303 patients with newly diagnosed DLBCL by targeted sequencing. Their correlation with peripheral blood immune cells and clinical outcomes were assessed. Underlying mechanisms on tumor microenvironment were investigated both in vitro and in vivo. Among all 619 DLBCL patients, somatic mutations in KMT2D (19.5%) were most frequently observed, followed by mutations in ARID1A (8.7%), CREBBP (8.4%), KMT2C (8.2%), TET2 (7.8%), EP300 (6.8%), and EZH2 (2.9%). Among them, CREBBP/EP300 mutations were significantly associated with decreased peripheral blood absolute lymphocyte-to-monocyte ratios, as well as inferior progression-free and overall survival. In B-lymphoma cells, the mutation or knockdown of CREBBP or EP300 inhibited H3K27 acetylation, downregulated FBXW7 expression, activated the NOTCH pathway, and downstream CCL2/CSF1 expression, resulting in tumor-associated macrophage polarization to M2 phenotype and tumor cell proliferation. In B-lymphoma murine models, xenografted tumors bearing CREBBP/EP300 mutation presented lower H3K27 acetylation, higher M2 macrophage recruitment, and more rapid tumor growth than those with CREBBP/EP300 wild-type control via FBXW7-NOTCH-CCL2/CSF1 axis. Our work thus contributed to the understanding of aberrant histone acetylation regulation on tumor microenvironment as an alternative mechanism of tumor progression in DLBCL.

CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses.

CREB binding protein (CBP), a transcriptional coactivator and acetyltransferase, is involved in the pathogenesis of inflammation-related diseases. High mobility group box-1 protein (HMGB1) is a critical mediator of lethal sepsis, which has prompted investigation for the development of new treatment for inflammation. Here, we report that the potent and selective inhibition of CBP bromodomain by SGC-CBP30 blocks HMGB1-mediated inflammatory responses in vitro and in vivo. Our data suggest that CBP bromodomain inhibition suppresses LPS-induced expression and release of HMGB1, when the inhibitor was given 8 h post LPS stimulation; moreover, CBP bromodomain inhibition attenuated pro-inflammatory activity of HMGB1. Furthermore, our findings provide evidence that SGC-CBP30 down-regulated rhHMGB1-induced activation of MAPKs and NF-κB signaling by triggering the reactivation of protein phosphatase 2A (PP2A) and the stabilization of MAPK phosphatase 1 (MKP-1). Collectively, these results suggest that CBP bromodomain could serve as a candidate therapeutic target for the treatment of lethal sepsis via inhibiting LPS-induced expression and release of HMGB1 and suppressing the pro-inflammatory activity of HMGB1.

Pharmacological Modulation of the Wnt/ß-Catenin Pathway Inhibits Proliferation and Promotes Differentiation of Long-Lived Memory CD4+ T Cells in Antiretroviral Therapy-Suppressed Simian Immunodeficiency Virus-Infected Macaques.

The major obstacle to human immunodeficiency type 1 virus (HIV-1) eradication is a reservoir of latently infected cells that persists despite long-term antiretroviral therapy (ART) and is maintained through cellular proliferation. Long-lived memory CD4+ T cells with high self-renewal capacity, such as central memory (CM) T cells and stem cell memory (SCM) T cells, are major contributors to the viral reservoir in HIV-infected individuals on ART. The Wnt/ß-catenin signaling pathway regulates the balance between self-renewal and differentiation of SCM and CM T cells, and pharmacological manipulation of this pathway offers an opportunity to interfere with the proliferation of latently infected cells. Here, we evaluated in vivo a novel approach to inhibit self-renewal of SCM and CM CD4+ T cells in the rhesus macaque (RM) model of simian immunodeficiency (SIV) infection. We used an inhibitor of the Wnt/ß-catenin pathway, PRI-724, that blocks the interaction between the coactivator CREB-binding protein (CBP) and ß-catenin, resulting in the cell fate decision to differentiate rather than proliferate. Our study shows that PRI-724 treatment of ART-suppressed SIVmac251-infected RMs resulted in decreased proliferation of SCM and CM T cells and modified the SCM and CM CD4+ T cell transcriptome toward a profile of more differentiated memory T cells. However, short-term treatment with PRI-724 alone did not significantly reduce the size of the viral reservoir. This work demonstrates for the first time that stemness pathways of long-lived memory CD4+ T cells can be pharmacologically modulated in vivo, thus establishing a novel strategy to target HIV persistence.IMPORTANCE Long-lasting CD4+ T cell subsets, such as central memory and stem cell memory CD4+ T cells, represent critical reservoirs for human immunodeficiency virus (HIV) persistence despite suppressive antiretroviral therapy. These cells possess stem cell-like properties of enhanced self-renewal/proliferation, and proliferation of latently infected memory CD4+ T cells plays a key role in maintaining the reservoir over time. Here, we evaluated an innovative strategy targeting the proliferation of long-lived memory CD4+ T cells to reduce viral reservoir stability. Using the rhesus macaque model, we tested a pharmacological inhibitor of the Wnt/ß-catenin signaling pathway that regulates T cell proliferation. Our study shows that administration of the inhibitor PRI-724 decreased the proliferation of SCM and CM CD4+ T cells and promoted a transcriptome enriched in differentiation genes. Although the viral reservoir size was not significantly reduced by PRI-724 treatment alone, we demonstrate the potential to pharmacologically modulate the proliferation of memory CD4+ T cells as a strategy to limit HIV persistence.

Branched mannans from the mushroom Cantharellus cibarius enhance the anticancer activity of natural killer cells against human cancers of lung and colon.

Several studies have shown that mushroom polysaccharides enhance the ability of natural killer (NK) cells to recognize cancer cells as foreign and thereby enhance the effectiveness of host immune defence mechanisms. Nevertheless, the use of NK cells in cancer treatment requires finding selective stimulators of their cytotoxicity without disturbing organism homeostasis. Our studies revealed that Cantharellus cibarius polysaccharides present in the CC2a fraction, mainly composed of an O-2 and O-3 branched (1→6)-linked mannan, not only beneficially influenced the viability and proliferation of the human natural killer cells NK92 but also enhanced their anticancer properties against the human lung and colon cancer cells A549 and LS180, and at the same time did not affect the human lung and colon epithelial cells NL20 and CCD841 CoN. Furthermore, the CC2a fraction used alone was also nontoxic to the normal epithelium, while it inhibited the viability of these cancer cells. Nevertheless, the therapeutic potential of NK92 cells was greatly enhanced after coincubation with these polysaccharides and the observed effect was dependent on the CC2a concentrations. The beneficial effect of CC2a on NK92 cells was associated with stimulation of p38 and Erk expression as well as induction of the transcription factor CREB. The discovered beneficial impact of the CC2a fraction on NK92 cells suggested the therapeutic use of the investigated compound especially as an adjuvant. Furthermore, taking into account the abundance of these water soluble mannans in C. cibarius, the results also suggest that an increase in the intake of C. cibarius may promote innate immunity response against cancer through the enhancement of NK cell activity.

CBP/p300 Drives the Differentiation of Regulatory T Cells through Transcriptional and Non-Transcriptional Mechanisms.

Regulatory T cells (Treg) are immunosuppressive and negatively impact response to cancer immunotherapies. CREB-binding protein (CBP) and p300 are closely related acetyltransferases and transcriptional coactivators. Here, we evaluate the mechanisms by which CBP/p300 regulate Treg differentiation and the consequences of CBP/p300 loss-of-function mutations in follicular lymphoma. Transcriptional and epigenetic profiling identified a cascade of transcription factors essential for Treg differentiation. Mass spectrometry analysis showed that CBP/p300 acetylates prostacyclin synthase, which regulates Treg differentiation by altering proinflammatory cytokine secretion by T and B cells. Reduced Treg presence in tissues harboring CBP/p300 loss-of-function mutations was observed in follicular lymphoma. Our findings provide novel insights into the regulation of Treg differentiation by CBP/p300, with potential clinical implications on alteration of the immune landscape. SIGNIFICANCE: This study provides insights into the dynamic role of CBP/p300 in the differentiation of Tregs, with potential clinical implications in the alteration of the immune landscape in follicular lymphoma.

Methamphetamine exacerbates neuroinflammatory response to lipopolysaccharide by activating dopamine D1-like receptors.

Methamphetamine (METH) is a highly addictive and widely abused drug worldwide. Although much research is on the drug's direct effects, METH may also alter host immunity. The mechanism by which METH influences immunity remains elusive. Here, C57BL6/J mice were intraperitoneally injected with 5 mg/kg METH four times at two-hour intervals. The microglial inhibitor minocycline or dopamine D1-like receptor antagonist SCH-23390 was also applied prior to METH injection. Twenty-four hours following the first METH injection, mice were challenged by lipopolysaccharide (LPS) at a dose of 330 µg/kg, and the hippocampus (Hip), caudate putamen (CPU), nucleus accumbens (NAc) and prefrontal cortex (PFC) were collected 4 h after LPS administration. IL-6 and TNF-α levels were detected by ELISA. The activation of D1-like receptors and microglial marker Iba1 were examined by immunohistochemical staining and Western blot. Finally, we examined the phosphorylation of ERK1/2 and CREB. We found that METH exposure increased LPS-induced IL-6 and TNF-α production in the Hip, CPU and NAc regions. METH also augmented microglia activation and D1/5DR expression in response to LPS. Moreover, administering SCH-23390 significantly reduced IL-6 and TNF-α production and Iba1 expression following LPS challenge. Similar inhibitory effects were also observed by minocycline administration. Moreover, phosphorylation of ERK1/2 and CREB was increased after METH and LPS exposure but decreased by SCH-23390. These data illustrate that METH exacerbates neuroinflammation response in LPS-stimulated mouse brains through dopamine D1-like receptors, microglia, and relevant signaling proteins, which may have therapeutic implications.

Histone acetyltransferase CBP is critical for conventional effector and memory T-cell differentiation in mice.

Compared with naïve T cells, memory CD8+ T cells have a transcriptional landscape and proteome that are optimized to generate a more rapid and robust response to secondary infection. Additionally, rewired kinase signal transduction pathways likely contribute to the superior recall response of memory CD8+ T cells, but this idea has not been experimentally confirmed. Herein, we utilized an MS approach to identify proteins that are phosphorylated on tyrosine residues in response to Listeria-induced T-cell receptor (TCR) stimulation in both naïve and memory CD8+ T cells from mice and separated by fluorescence- and flow cytometry-based cell sorting. This analysis identified substantial differences in tyrosine kinase signaling networks between naïve and memory CD8+ T cells. We also observed that an important axis in memory CD8+ T cells couples Janus kinase 2 (JAK2) hyperactivation to the phosphorylation of CREB-binding protein (CBP). Functionally, JAK2-catalyzed phosphorylation enabled CBP to bind with higher affinity to acetylated histone peptides, indicating a potential epigenetic mechanism that could contribute to rapid initiation of transcriptional programs in memory CD8+ T cells. Moreover, we found that CBP itself is essential for conventional effector and memory CD8+ T-cell formation. These results indicate how signaling pathways are altered to promote CD8+ memory cell formation and rapid responses to and protection from repeat infections.

Challenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.

The structural and functional characterization of large multidomain signaling proteins containing long disordered linker regions represents special methodological and conceptual challenges. These proteins show extreme structural heterogeneity and have complex posttranslational modification patterns, due to which traditional structural biology techniques provide results that are often difficult to interpret. As demonstrated through the example of two such multidomain proteins, CREB-binding protein (CBP) and its paralogue, p300, even the expression and purification of such proteins are compromised by their extreme proteolytic sensitivity and structural heterogeneity. In this chapter, we describe the effective expression of CBP and p300 in a eukaryotic host, Sf9 insect cells, followed by their tandem affinity purification based on two terminal tags to ensure their structural integrity. The major focus of this chapter is on the development of novel accessory tools, single-domain camelid antibodies (nanobodies), for structural-functional characterization. Specific nanobodies against full-length CBP and p300 can specifically target their different regions and can be used for their marking, labeling, and structural stabilization in a broad range of in vitro and in vivo studies. Here, we describe four high-affinity nanobodies binding to the KIX and the HAT domains, either mimicking known interacting partners or revealing new functionally relevant conformations. As immunization of llamas results in nanobody libraries with a great sequence variation, deep sequencing and interaction analysis with different regions of the proteins provide a novel approach toward developing a panel of specific nanobodies.

Oligomeric proanthocyanidins attenuate airway inflammation in asthma by inhibiting dendritic cells maturation.

To date, although a promising anti-inflammatory activity of oligomeric proanthocyanidins (OPCs) has been observed in asthma, the mechanism responsible for these immunomodulatory properties remains obscure. Dendritic cells (DCs) that reside in the airway have been widely perceived as an important contributor to asthma. Our study was to demonstrate OPCs' effects on maturation and immunoregulation of pulmonary CD11c+ dendritic cells (DCs). BALB/c mice were exposed to ovalbumin (OVA) to induce murine model of asthma. In addition, pulmonary DCs and bone marrow-derived DCs (BMDCs) cultures were used to evaluate impacts of OPCs on DCs function. The results obtained here indicated that OPCs treatment dramatically reduced airway inflammation, such as the infiltration of inflammatory cells and the levels of allergen-specific serum IgE and Th2 cytokines. The expression of co-stimulatory molecules especially CD86 distributed on pulmonary DCs and bone marrow-derived DCs (BMDCs) also markedly declined. The phosphorylation of cAMP responsive element-binding protein (CREB) was significantly inhibited while no changes were observed in the expression of cAMP responsive element modulator (CREM). By transferring BMDCs into the airways of naïve mice, we found that OPCs-treated DCs (DC+OVA+OPC) were much less potent in promoting CD4+ T cells proliferation than OVA-pulsed DCs (DC+OVA), followed by the ameliorated eosinophilic inflammation in airway. Our findings tailor a novel profile of OPCs in the regulation of DCs function, shedding new light on the therapeutic potential of OPCs in asthma management.

Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense.

The activation of interferon (IFN)-regulatory factor-3 (IRF3), characterized by phosphorylation and nuclear translocation of the latent transcription factor, is central to initiating innate antiviral responses. Whereas much has been learned about the upstream pathways and signaling mechanisms leading to IRF3 activation, how activated IRF3 operates in the nucleus to control transcription of IFNs remains obscure. Here we identify EAP30 (a.k.a, SNF8/VPS22), an endosomal sorting complex required for transport (ESCRT)-II subunit, as an essential factor controlling IRF3-dependent antiviral defense. Depletion of EAP30, but not other ESCRT-II subunits, compromised IRF3-dependent induction of type I and III IFNs, IFN-stimulated genes (ISGs) and chemokines by double-stranded RNA or viruses. EAP30, however, was dispensable for the induction of inflammatory mediators of strict NF-κB target. Significantly, knockdown of EAP30 also impaired the establishment of an antiviral state against vesicular stomatitis virus and hepatitis C virus, which are of distinct viral families. Mechanistically, EAP30 was not required for IRF3 activation but rather acted at a downstream step. Specifically, a fraction of EAP30 localized within the nucleus, where it formed a complex with IRF3 and its transcriptional co-activator, CREB-binding protein (CBP), in a virus-inducible manner. These interactions promoted IRF3 binding to target gene promoters such as IFN-ß, IFN-λ1 and ISG56. Together, our data describe an unappreciated role for EAP30 in IRF3-dependent innate antiviral response in the nucleus.

Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth.

The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4+ T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPß regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 ß (IL-1ß) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPß signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPßsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

Acetylation Modulates IL-2 Receptor Signaling in T Cells.

Ligand binding to the cognate cytokine receptors activates intracellular signaling by recruiting protein tyrosine kinases and other protein modification enzymes. However, the roles of protein modifications other than phosphorylation remain unclear. In this study, we examine a novel regulatory mechanism of Stat5, based on its acetylation. As for phosphorylation, IL-2 induces the acetylation of signaling molecules, including Stat5, in the murine T cell line CTLL-2. Stat5 is acetylated in the cytoplasm by CREB-binding protein (CBP). Acetylated Lys696 and Lys700 on Stat5 are critical indicators for limited proteolysis, which leads to the generation of a truncated form of Stat5. In turn, the truncated form of Stat5 prevents transcription of the full-length form of Stat5. We also demonstrate that CBP physically associates with the IL-2 receptor ß-chain. CBP, found in the nucleus in resting CTLL-2 cells, relocates to the cytoplasm after IL-2 stimulation in an MEK/ERK pathway-dependent manner. Thus, IL-2-mediated acetylation plays an important role in the modulation of cytokine signaling and T cell fate.

Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Modulates Interferon-ß Expression Mainly Through Attenuating Interferon-Regulatory Factor 3 Phosphorylation.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that emerged from classic PRRSV causes more severe damage to the swine industry. The earlier reports indicating inhibition of interferon-ß (IFN-ß) expression by PRRSV through total blockage of IFN-regulatory factor 3 (IRF3) nuclear translocation made us investigate the mechanism of IFN-ß expression in HP-PRRSV infection. For this purpose, the IRF3 nuclear translocation in the control group [Poly (I:C)] and test group [Poly (I:C)+HP-PRRSV] was detected by immunofluorescence, and the results showed that IRF3 nuclear translocation in cells with PRRSV was weaker than cells without PRRSV, which was different from the previous study. In addition, the IFN-ß mRNA and protein expression was observed to be inhibited by HP-PRRSV along with decreased IRF3 mRNA and total protein, and IRF3 nuclear translocation of test group was suppressed in MARC-145 and porcine alveolar macrophage cells in comparison with the control group. The quantity of phosphorylated IRF3 protein was also reduced after HP-PRRSV infection. However, CREB-binding protein (CBP) expression did not change between the control and test group. These results indicate that the inhibition of IFN-ß expression is mainly due to the quantitative change in the amount of phosphorylated IRF3 in the cytoplasm, but not dependent on the complete blockage of IRF3 nuclear translocation or the restraining of CBP expression in the nucleus by HP-PRRSV.

Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-ß) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

Inverse Correlation between IL-10 and HIF-1α in Macrophages Infected with Histoplasma capsulatum.

Hypoxia-inducible factor (HIF)-1α is a transcription factor that regulates metabolic and immune response genes in the setting of low oxygen tension and inflammation. We investigated the function of HIF-1α in the host response to Histoplasma capsulatum because granulomas induced by this pathogenic fungus develop hypoxic microenvironments during the early adaptive immune response. In this study, we demonstrated that myeloid HIF-1α-deficient mice exhibited elevated fungal burden during the innate immune response (prior to 7 d postinfection) as well as decreased survival in response to a sublethal inoculum of H. capsulatum The absence of myeloid HIF-1α did not alter immune cell recruitment to the lungs of infected animals but was associated with an elevation of the anti-inflammatory cytokine IL-10. Treatment with mAb to IL-10 restored protective immunity to the mutant mice. Macrophages (Mϕs) constituted most IL-10-producing cells. Deletion of HIF-1α in neutrophils or dendritic cells did not alter fungal burden, thus implicating Mϕs as the pivotal cell in host resistance. HIF-1α was stabilized in Mϕs following infection. Increased activity of the transcription factor CREB in HIF-1α-deficient Mϕs drove IL-10 production in response to H. capsulatum IL-10 inhibited Mϕ control of fungal growth in response to the activating cytokine IFN-γ. Thus, we identified a critical function for Mϕ HIF-1α in tempering IL-10 production following infection. We established that transcriptional regulation of IL-10 by HIF-1α and CREB is critical for activation of Mϕs by IFN-γ and effective handling of H. capsulatum.

A Rhesus Rhadinovirus Viral Interferon (IFN) Regulatory Factor Is Virion Associated and Inhibits the Early IFN Antiviral Response.

UNLABELLED: The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with a transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in beta interferon (IFN-ß) transcription, fails to effectively bind to the IFN-ß promoter, thus inhibiting the activation of IFN-ß genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and, furthermore, that virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFN-ß promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only viruses known to encode viral homologues to cellular interferon regulatory factors (IRFs), known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind immunomodulation by the RRV vIRFs during infection.

Cloning and characterization of SCIRR69: a novel transcriptional factor belonging to the CREB/ATF family.

The complete cDNA sequence of a novel gene, SCIRR69 (spinal cord injury and regeneration related no. 69 gene), was obtained by RACE technique. It codes for a protein of 521 amino acid residues homologous to human CREB3l2 (also known as BBF2H7) and mouse CREB3l2. The protein contains a basic DNA binding and leucine zipper dimerization (B-ZIP) motif and a hydrophobic region representing a putative transmembrane domain, similar to the structure of other CREB/ATF transcription factors. Monoclonal antibody against SCIRR69 was developed and could recognize the SCIRR69 protein in both native and denatured forms. Constructing of SCIRR69 fusion proteins with the GAL4 DNA-binding domain disclosed that SCIRR69 functioned as a transcriptional activator and its N-terminal 60 amino acids accounted for the activation ability. SCIRR69 resides in the cytoplasm of primary neurons, whereas neuron damage by incision led to the cleavage and translocation from the cytoplasm to the nucleus. These results suggest that SCIRR69 is activated by proteolytic cleavage at the transmembrane domain in response to neuron damage and its amino-terminal cytoplasmic domain translocates into the nucleus to activate the transcription of target genes.

Adenosine augments IL-10 production by microglial cells through an A2B adenosine receptor-mediated process.

Microglia are activated by pathogen-associated molecular patterns and produce proinflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and a ligand of four G protein-coupled adenosine receptors (ARs), which are the A(1)AR, A(2A)AR, A(2B)AR, and A(3)AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. In this study, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of proinflammatory cytokines. Because the order of potency of selective AR agonists in inducing IL-10 production was NECA > IB-MECA > CCPA ≥ CGS21680, and the A(2B)AR antagonist MRS1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A(2B)AR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered short hairpin RNA blocked the enhancing effect of adenosine on IL-10 production, confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A(2B)ARs augment IL-10 production by activated murine microglia.