Interferon establishes an antiviral state in numerous cell types through the induction of a set of immediate-early response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the alpha/beta-interferon-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced interferon-stimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified interferon regulatory factor 3 (IRF3), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of interferon-stimulated gene factor 3. IRF3, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p300, to form CIF. Activating interferon-stimulated genes without the need for prior synthesis of interferons might provide the host cell with a potential shortcut in the activation of its antiviral defense.

The hyperinduction of proinflammatory cytokines and chemokines such as TNF-alpha, IFN-beta, and CCL2/MCP-1 in primary human macrophages and respiratory epithelial cells by the highly pathogenic avian influenza H5N1 is believed to contribute to the unusual severity of human H5N1 disease. Here we show that TNF-alpha, IFN-beta, and IFN-lambda1 are the key mediators directly induced by the H5N1 virus in primary human macrophages. In comparison with human influenza (H1N1), the H5N1 virus more strongly activated IFN regulatory factor 3 (IRF3). IRF3 knockdown and p38 kinase inhibition separately and in combination led to a substantial reduction of IFN-beta, IFN-lambda1, and MCP-1 but only to a partial reduction of TNF-alpha. IRF3 translocation was independent of p38 kinase activity, indicating that IRF3 and p38 kinase are distinct pathways leading to cytokine production by H5N1 virus. We conclude that IRF3 and p38 kinase separately and predominantly contribute to H5N1-mediated induction of IFN-beta, IFN-lambda1, and MCP-1 but only partly control TNF-alpha induction. A more precise identification of the differences in the regulation of TNF-alpha and IFN-beta could provide novel targets for the design of therapeutic strategies for severe human H5N1 influenza and also for treating other causes of acute respiratory distress syndrome.

IFN-stimulated gene 56 (ISG56) is one of the first identified proteins induced by viruses and type I IFNs. In this study, we identified ISG56 as a virus-induced protein associated with MITA, an adapter protein involved in virus-triggered induction of type I IFNs. Overexpression of ISG56 inhibited Sendai virus-triggered activation of IRF3, NF-kappaB, and the IFN-beta promoter, whereas knockdown of ISG56 had opposite effects. Consistently, overexpression of ISG56 reversed cytoplasmic poly(I:C)-induced inhibition of vesicular stomatitis virus (VSV) replication, whereas knockdown of ISG56 inhibited VSV replication. Competitive coimmunoprecipitation experiments indicated that ISG56 disrupted the interactions between MITA and VISA or TBK1, two components in the virus-triggered IFN signaling pathways. These results suggest that ISG56 is a mediator of negative-feedback regulation of virus-triggered induction of type I IFNs and cellular antiviral responses.

Heme oxygenase-1 (HO-1) is a key cytoprotective, antioxidant, and antiinflammatory molecule. The pathophysiological functions of HO-1 have been associated with its enzymatic activities in heme catabolism. We have examined the immune functions of HO-1 by its conditional ablation in myeloid cells (HO-1(M-KO) mice). We demonstrate that myeloid HO-1 is required for the activation of interferon (IFN) regulatory factor (IRF) 3 after Toll-like receptor 3 or 4 stimulation, or viral infection. HO-1-deficient macrophages show reduced expression of IFN-beta and of primary IRF3 target genes encoding RANTES, IP-10 and MCP-1. In the presence of polyI:C, myeloid HO-1 knockout mice infected with Listeria monocytogenes, a model dependent on IFN-beta production, showed enhanced bacterial clearance and survival, whereas control mice succumbed to infection. Moreover, after induction of experimental autoimmune encephalomyelitis, mice with myeloid-specific HO-1 deficiency developed a higher incidence and an exacerbated, nonremitting clinical disease correlating with persistent activation of antigen-presenting cells, enhanced infiltration of Th17 cells, and a nonregressing myelin-specific T cell reactivity. Notably, these defects were rectified by exogenous administration of IFN-beta, confirming that HO-1 functions directly upstream of this critical immune pathway. These results uncover a novel direct function for myeloid HO-1 in the regulation of IFN-beta production, establishing HO-1 as a critical early mediator of the innate immune response.

Apolipoprotein L1 (APOL1) risk variants greatly elevate the risk of kidney disease in African Americans. Here we report a cohort of patients who developed collapsing focal segmental glomerulosclerosis while receiving therapeutic interferon, all of whom carried the APOL1 high-risk genotype. This finding raised the possibility that interferons and the molecular pattern recognition receptors that stimulate interferon production may contribute to APOL1-associated kidney disease. In cell culture, interferons and Toll-like receptor (TLR) agonists increased APOL1 expression by up to 200-fold, in some cases with the appearance of transcripts not detected under basal conditions. PolyI:C, a double-stranded RNA TLR3 agonist, increased APOL1 expression by upregulating interferons directly or through an interferon-independent, IFN-regulatory factor 3 (IRF3)-dependent pathway. Using pharmacological inhibitors, small hairpin RNA knockdown, and chromatin immunoprecipitation, we found that the interferon-independent TLR3 pathway relied on signaling through TBK1, NF-κB, and Jak kinases, and on binding of IRF1, IRF2, and STAT2 at the APOL1 transcription start site. We also demonstrate that overexpression of the APOL1 risk variants is more injurious to cells than overexpression of the wild-type APOL1 protein. Our study illustrates that antiviral pathways may be important inducers of kidney disease in individuals with the APOL1 high-risk genotype and identifies potential targets for prevention or treatment.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m⁶A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m⁶A modification abrogates immune gene activation and adjuvant activity. m⁶A reader YTHDF2 sequesters m⁶A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m⁶A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered type I interferon (IFN)(2) induction pathways. In this study, we identified two deubiquitinating enzymes, OTUB1 and OTUB2, as negative regulators of virus-triggered type I IFN induction. Overexpression of OTUB1 and OTUB2 inhibited virus-induced activation of IRF3 and NF-kappaB, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects. Coimmunoprecipitations indicated OTUB1 and -2 interacted with TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-kappaB activation, respectively. Furthermore, we found that OTUB1 and OTUB2 mediated virus-triggered deubiquitination of TRAF3 and -6. These findings suggest that OTUB1 and OTUB2 negatively regulate virus-triggered type I IFN induction and cellular antiviral response by deubiquitinating TRAF3 and -6.

Type I interferon (IFN) is an important host defense cytokine against intracellular pathogens, mainly viruses. In assessing IFN production in response to group B streptococcus (GBS), we find that IFN-beta was produced by macrophages upon stimulation with both heat-killed and live GBS. Exposure of macrophages to heat-killed GBS activated a Toll-like receptor (TLR)-dependent pathway, whereas live GBS activated a TLR/NOD/RIG-like receptor (RLR)-independent pathway. This latter pathway required bacterial phagocytosis, proteolytic bacterial degradation, and phagolysosomal membrane destruction by GBS pore-forming toxins, leading to the release of bacterial DNA into the cytosol. GBS DNA in the cytosol induced IFN-beta production via a pathway dependent on the activation of the serine-threonine kinase TBK1 and phosphorylation of the transcription factor IRF3. Thus, activation of IFN-alpha/-beta production during infection with GBS, commonly considered an extracellular pathogen, appears to result from the interaction of GBS DNA with a putative intracellular DNA sensor or receptor.

BACKGROUND

Toll-like receptor 4 (TLR4) is activated in response to cerebral ischemia leading to substantial brain damage. In contrast, mild activation of TLR4 by preconditioning with low dose exposure to lipopolysaccharide (LPS) prior to cerebral ischemia dramatically improves outcome by reprogramming the signaling response to injury. This suggests that TLR4 signaling can be altered to induce an endogenously neuroprotective phenotype. However, the TLR4 signaling events involved in this neuroprotective response are poorly understood. Here we define several molecular mediators of the primary signaling cascades induced by LPS preconditioning that give rise to the reprogrammed response to cerebral ischemia and confer the neuroprotective phenotype.

METHODS

C57BL6 mice were preconditioned with low dose LPS prior to transient middle cerebral artery occlusion (MCAO). Cortical tissue and blood were collected following MCAO. Microarray and qtPCR were performed to analyze gene expression associated with TLR4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity. Protein expression was determined using Western blot or ELISA. MyD88-/- and TRIF-/- mice were utilized to evaluate signaling in LPS preconditioning-induced neuroprotection.

RESULTS

Gene expression analyses revealed that LPS preconditioning resulted in a marked upregulation of anti-inflammatory/type I IFN-associated genes following ischemia while pro-inflammatory genes induced following ischemia were present but not differentially modulated by LPS. Interestingly, although expression of pro-inflammatory genes was observed, there was decreased activity of NFκB p65 and increased presence of NFκB inhibitors, including Ship1, Tollip, and p105, in LPS-preconditioned mice following stroke. In contrast, IRF3 activity was enhanced in LPS-preconditioned mice following stroke. TRIF and MyD88 deficient mice revealed that neuroprotection induced by LPS depends on TLR4 signaling via TRIF, which activates IRF3, but does not depend on MyD88 signaling.

CONCLUSIONS

Our results characterize several critical mediators of the TLR4 signaling events associated with neuroprotection. LPS preconditioning redirects TLR4 signaling in response to stroke through suppression of NFκB activity, enhanced IRF3 activity, and increased anti-inflammatory/type I IFN gene expression. Interestingly, this protective phenotype does not require the suppression of pro-inflammatory mediators. Furthermore, our results highlight a critical role for TRIF-IRF3 signaling as the governing mechanism in the neuroprotective response to stroke.

RIG-I-like receptors (RLRs) MDA5 and RIG-I are key players in the innate antiviral response. Upon recognition of viral RNA, they interact with MAVS, eventually inducing type I interferon production. The interferon induction pathway is commonly targeted by viruses. How enteroviruses suppress interferon production is incompletely understood. MDA5 has been suggested to undergo caspase- and proteasome-mediated degradation during poliovirus infection. Additionally, MAVS is reported to be cleaved during infection with coxsackievirus B3 (CVB3) by the CVB3 proteinase 3C(pro), whereas MAVS cleavage by enterovirus 71 has been attributed to 2A(pro). As yet, a detailed examination of the RLR pathway as a whole during any enterovirus infection is lacking. We performed a comprehensive analysis of crucial factors of the RLR pathway, including MDA5, RIG-I, LGP2, MAVS, TBK1, and IRF3, during infection of CVB3, a human enterovirus B (HEV-B) species member. We show that CVB3 inhibits the RLR pathway upstream of TBK1 activation, as demonstrated by limited phosphorylation of TBK1 and a lack of IRF3 phosphorylation. Furthermore, we show that MDA5, MAVS, and RIG-I all undergo proteolytic degradation in CVB3-infected cells through a caspase- and proteasome-independent manner. We convincingly show that MDA5 and MAVS cleavages are both mediated by CVB3 2A(pro), while RIG-I is cleaved by 3C(pro). Moreover, we show that proteinases 2A(pro) and 3C(pro) of poliovirus (HEV-C) and enterovirus 71 (HEV-A) exert the same functions. This study identifies a critical role of 2A(pro) by cleaving MDA5 and MAVS and shows that enteroviruses use a common strategy to counteract the interferon response in infected cells.

OBJECTIVE

Human enteroviruses (HEVs) are important pathogens that cause a variety of diseases in humans, including poliomyelitis, hand, foot, and mouth disease, viral meningitis, cardiomyopathy, and more. Like many other viruses, enteroviruses target the host immune pathways to gain replication advantage. The MDA5/MAVS pathway is responsible for recognizing enterovirus infections in the host cell and leads to expression of type I interferons (IFN-I), crucial antiviral signaling molecules. Here we show that three species of HEVs all employ the viral proteinase 2A (2A(pro)) to proteolytically target MDA5 and MAVS, leading to an efficient blockade upstream of IFN-I transcription. These observations suggest that MDA5/MAVS antagonization is an evolutionarily conserved and beneficial mechanism of enteroviruses. Understanding the molecular mechanisms of enterovirus immune evasion strategies will help to develop countermeasures to control infections with these viruses in the future.

The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-kappaB essential modulator (NEMO, also called IKKgamma) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-kappaB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-kappaB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFalpha-mediated NF-kappaB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity.

OBJECTIVE

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear, and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity, including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing. This work not only provides a new understanding of the abilities of MERS-CoV and closely related bat viruses to subvert virus sensing but also might prove useful in revealing new strategies for the development of vaccines and antivirals.

Hepatitis B virus (HBV) infection remains one of the most serious health problems worldwide. Whilst studies have shown that HBV impairs interferon (IFN) production from dendritic cells in chronic hepatitis B patients, it remains unknown whether HBV inhibits IFN production in human hepatocytes. Using transient transfection assays in a primary human hepatocyte cell line (PH5CH8), this study demonstrated that HBV polymerase inhibits IFN-beta promoter activity induced by Newcastle disease virus, Sendai virus or poly(I : C) in a dose-dependent manner, whilst ectopic expression of the HBV core and X proteins had no effect on IFN-beta promoter activity. In addition, HBV polymerase blocked cellular IFN-beta expression and consequent antiviral immunity revealed by an infection protection assay. Furthermore, overexpression of key molecules on the IFN-beta induction axis, together with HBV polymerase, resulted in a block of IFN-beta promoter activity triggered by RIG-I, IPS-1, TRIF, TBK1 and IKKepsilon, but not by an IFN regulatory factor 3 dominant-positive mutant (IRF3-5D), suggesting that HBV polymerase prevents IFN-beta expression at the TBK1/IKKepsilon level. Further studies showed that HBV polymerase inhibited phosphorylation, dimerization and nuclear translocation of IRF3, in response to Sendai virus infection. Finally, it was shown that HBV polymerase-mediated dampening of the interaction between TBK1/IKKepsilon and DDX3 may be involved in the inhibitory effect on IFN-beta induction. Taken together, these findings reveal a novel role of HBV polymerase in HBV counteraction of IFN-beta production in human hepatocytes.

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-β and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.

Viral infection activates Toll-like receptor and RIG-I (retinoic acid-inducible gene I) signaling pathways, leading to phosphorylation of IRF3 (interferon regulatory factor 3) and IRF7 and stimulation of type I interferon (IFN) transcription, a process important for innate immunity. We show that upon vesicular stomatitis virus infection, IRF3 and IRF7 are modified not only by phosphorylation but by the small ubiquitin-related modifiers SUMO1, SUMO2, and SUMO3. SUMOylation of IRF3 and IRF7 was dependent on the activation of Toll-like receptor and RIG-I pathways but not on the IFN-stimulated pathway. However, SUMOylation of IRF3 and IRF7 was not dependent on their phosphorylation, and vice versa. We identified Lys(152) of IRF3 and Lys(406) of IRF7 to be their sole small ubiquitin-related modifier (SUMO) conjugation site. IRF3 and IRF7 mutants defective in SUMOylation led to higher levels of IFN mRNA induction after viral infection, relative to the wild type IRFs, indicating a negative role for SUMOylation in IFN transcription. Together, SUMO modification is an integral part of IRF3 and IRF7 activity that contributes to postactivation attenuation of IFN production.

The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

Type I interferon (IFN) signaling is the principal response mediating antiviral innate immunity. IFN transcription is dependent upon the activation of transcription factors IRF3/IRF7 and NF-κB. Many viral proteins have been shown as being capable of interfering with IFN signaling to facilitate evasion from the host innate immune response. Here, we report that a viral miRNA, miR-K12-11, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for the modulation of IFN signaling and acts through targeting I-kappa-B kinase epsilon (IKKɛ). Ectopic expression of miR-K12-11 resulted in decreased IKKɛ expression, while inhibition of miR-K12-11 was found to restore IKKɛ expression in KSHV-infected cells. Importantly, expression of miR-K12-11 attenuated IFN signaling by decreasing IKKɛ-mediated IRF3/IRF7 phosphorylation and by inhibiting the activation of IKKɛ-dependent IFN stimulating genes (ISGs), allowing miR-K12-11 suppression of antiviral immunity. Our data suggest that IKKɛ targeting by miR-K12-11 is an important strategy utilized by KSHV to modulate IFN signaling during the KSHV lifecycle, especially in latency. We also demonstrated that IKKɛ was able to enhance KSHV reactivation synergistically with the treatment of 12-O-tetradecanoylphorbol 13-acetate. Moreover, inhibition of miR-K12-11 enhanced KSHV reactivation induced by vesicular stomatitis virus infection. Taken together, our findings also suggest that miR-K12-11 can contribute to maintenance of KSHV latency by targeting IKKɛ.

Calcium and its major downstream effector, calcium/calmodulin-dependent protein kinase II (CaMKII), are found to be important for the functions of immune cells. Lipopolysaccharide (LPS) has been shown to induce intracellular calcium release in macrophages; however, whether and how CaMKII is required for Toll-like receptor (TLR) signaling remain unknown. Here we demonstrate that TLR 4, 9, and 3 ligands markedly induce intracellular calcium fluxes and activate CaMKII-alpha in macrophages. Selective inhibition or RNA interference of CaMKII significantly suppresses TLR4, 9, 3-triggered production of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferon-alpha/beta (IFN-alpha/beta) in macrophages. Coincidently, overexpression of constitutively active CaMKII-alpha significantly enhances production of the above cytokines. In addition to the activation of mitogen-activated protein kinase and nuclear factor kappaB pathways, CaMKII-alpha can directly bind and phosphorylate transforming growth factor beta-activated kinase 1 (TAK1) and IFN regulatory factor 3 (IRF3; serine on 386) via the N-terminal part of its regulatory domain. Therefore, CaMKII can be activated by TLR ligands, and in turn promotes both myeloid differentiating factor 88 and Toll/IL-1 receptor domain-containing adaptor protein-inducing IFN-beta-dependent inflammatory responses by directly activating TAK1 and IRF3. The cross-talk with the calcium/CaMKII pathway is needed for full activation of TLR signaling in macrophages.

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection.

Macrophages are key target cells for HIV-1. HIV-1(BaL) induced a subset of interferon-stimulated genes in monocyte-derived macrophages (MDMs), which differed from that in monocyte-derived dendritic cells and CD4 T cells, without inducing any interferons. Inhibition of type I interferon induction was mediated by HIV-1 inhibition of interferon-regulated factor (IRF3) nuclear translocation. In MDMs, viperin was the most up-regulated interferon-stimulated genes, and it significantly inhibited HIV-1 production. HIV-1 infection disrupted lipid rafts via viperin induction and redistributed viperin to CD81 compartments, the site of HIV-1 egress by budding in MDMs. Exogenous farnesol, which enhances membrane protein prenylation, reversed viperin-mediated inhibition of HIV-1 production. Mutagenesis analysis in transfected cell lines showed that the internal S-adenosyl methionine domains of viperin were essential for its antiviral activity. Thus viperin may contribute to persistent noncytopathic HIV-1 infection of macrophages and possibly to biologic differences with HIV-1-infected T cells.

A20 (also known as TNFAIP3) is a cytoplasmic protein that plays a key role in the negative regulation of inflammation and immunity. Polymorphisms in the A20 gene locus have been identified as risk alleles for multiple human autoimmune diseases, and A20 has also been proposed to function as a tumor suppressor in several human B-cell lymphomas. A20 expression is strongly induced by multiple stimuli, including the proinflammatory cytokines TNF and IL-1, and microbial products that trigger pathogen recognition receptors, such as Toll-like receptors. A20 functions in a negative feedback loop, which mediates its inhibitory functions by downregulating key proinflammatory signaling pathways, including those controlling NF-κB- and IRF3-dependent gene expression. Activation of these transcription factors is controlled by both K48- and K63- polyubiquitination of upstream signaling proteins, respectively triggering proteasome-mediated degradation or interaction with other signaling proteins. A20 turns off NF-κB and IRF3 activation by modulating both types of ubiquitination. Induction of K48-polyubiquitination by A20 involves its C-terminal zinc-finger ubiquitin-binding domain, which may promote interaction with E3 ligases, such as Itch and RNF11 that are involved in mediating A20 inhibitory functions. A20 is thought to promote de-ubiquitination of K63-polyubiquitin chains either directly, due to its N-terminal deubiquitinase domain, or by disrupting the interaction between E3 and E2 enzymes that catalyze K63-polyubiquitination. A20 is subject to different mechanisms of regulation, including phosphorylation, proteolytic processing, and association with ubiquitin binding proteins. Here we review the expression and biological activities of A20, as well as the underlying molecular mechanisms.

BACKGROUND

Sepsis syndrome is characterized by inappropriate amplified systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates a pleiotropic cytoprotective defense program including antioxidants and protects against several inflammatory disorders by inhibiting oxidative tissue injuries. However, the role of enhanced Nrf2 activity in modulating innate immune responses to microbial infection and pathogenesis of sepsis is unclear.

OBJECTIVE

To determine whether Nrf2 in myeloid leukocytes alters inflammatory response and protects against sepsis.

METHODS

Mice with deletion of Nrf2 or kelch-like ECH-associated protein (Keap1) in myeloid leukocyte cells and respective floxed controls were subjected to cecal ligation and puncture-induced sepsis and were assessed for survival, organ injury, systemic inflammation, and bacteremia. Using LPS-stimulated peritoneal macrophages, Toll-like receptor (TLR) 4 surface trafficking and downstream signaling events were analyzed.

RESULTS

Mortality, organ injury, circulating levels of inflammatory mediators, and bacteremia were markedly reduced in LysM-Keap1(-/-) compared with respective floxed controls (Keap1(f/f) or Nrf2(f/f)) and significantly elevated in LysM-Nrf2(-/-) mice after cecal ligation and puncture. Peritoneal macrophages from septic LysM-Keap1(-/-) mice showed a greater bacterial phagocytic activity compared with LysM-Nrf2(-/-) and floxed controls. LPS stimulation resulted in greater reactive oxygen species-induced cell surface transport of TLR4 from trans-Golgi network and subsequent TLR4 downstream signaling (recruitment of MYD88 and TRIF, phosphorylation of IkB and IRF3, and cytokine expression) in macrophages of LysM-Nrf2(-/-) compared with LysM-Keap1(-/-) mice and floxed controls.

CONCLUSIONS

Our study shows that Nrf2 acts as a critical immunomodulator in leukocytes, controls host inflammatory response to bacterial infection, and protects against sepsis.