Type I IFN (IFN-I) increase the sensitivity of cells and mice to lethal infection with Listeria monocytogenes. Therefore the amount of IFN-I produced during infection might be an important factor determining Listeria virulence. Two commonly used strains of L. monocytogenes, EGD and LO28, were identified as, respectively, low and high inducers of IFN-I synthesis in infected macrophages. Increased IFN-I production resulted from the stronger ability of the LO28 strain to trigger the IRF3 signalling pathway and correlated with an increased sensitization of macrophages to lethal infection. In contrast, stimulation of NFkappaB, MAPK, or inflammasome signalling by the LO28 and EGD strains did not differ significantly. The LO28 strain was more virulent in wild-type (wt) C57/BL6 mice than the EGD strain whereas both strains were similarly virulent in IFN-I receptor-deficient C57/BL6 mice. Together our data suggest that isolates of wt L. monocytogenes differ in their ability to trigger the IRF3 signalling pathway and IFN-I production, and that the amount of IFN-I produced during infection is an important determinant of Listeria virulence.

Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor-like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN-β production. Co-immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand-induced IFN-β induction. Interestingly, Mal does not affect the induction of IL-6 and TNF-α upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN-β induction through the positive regulatory domains I-III enhancer element of the IFN-β gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)-induced IRF7 activation and concomitant IFN-β production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN-β production.

Chlamydophila pneumoniae infection of the vascular wall as well as activation of the transcription factor IFN regulatory factor (IRF)3 have been linked to development of chronic vascular lesions and atherosclerosis. The innate immune system detects invading pathogens by use of pattern recognition receptors, some of which are able to stimulate IRF3/7 activation and subsequent type I IFN production (e. g., IFN-beta). In this study, we show that infection of human endothelial cells with C. pneumoniae-induced production of IFN-beta, a cytokine that so far has been mainly associated with antiviral immunity. Moreover, C. pneumoniae infection led to IRF3 and IRF7 nuclear translocation in HUVECs and RNA interference experiments showed that IRF3 and IRF7 as well as the mitochondrial antiviral signaling (MAVS) were essential for IFN-beta induction. Finally, C. pneumoniae replication was enhanced in endothelial cells in which IRF3, IRF7, or MAVS expression was inhibited by small interfering RNA and attenuated by IFN-beta treatment. In conclusion, C. pneumoniae infection of endothelial cells activates an MAVS-, IRF3-, and IRF7-dependent signaling, which controls bacterial growth and might modulate development of vascular lesions.

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression.

For almost 10 years, Nod2 has been known as a cytosolic innate receptor able to sense peptidoglycan from Gram-positive and -negative bacteria and to trigger RIP2- and NF-κB-mediated pro-inflammatory and antibacterial response. Mutations in the gene encoding Nod2 in humans have been associated with Crohn's disease (CD). Mechanisms by which Nod2 variants can lead to CD development are still under investigation. The most admitted hypothesis suggests that the impaired function of Nod2 variants in intestinal epithelial and phagocytic cells results in deficiencies in epithelial-barrier function which subsequently lead to increased bacterial invasion and inflammation at intestinal sites. Very recent results have just reinforced this hypothesis by demonstrating that Nod2 wild-type (unlike Nod2 variants) could mediate autophagy, allowing an efficient bacterial clearance and adaptative immune response. Other recent data have attributed new roles to Nod2. Indeed, Nod2 has been shown to activate antiviral innate immune responses involving IRF3-dependent IFN-β production after viral ssRNA recognition through a RIP2-independent mechanism requiring the mitochondrial adaptor protein MAVS. Recently, Nod2 has been also shown to be exquisitely tuned to detect mycobacterial infections and mount a protective immunity against these pathogens.

The innate immune system senses cytosolic dsDNA and bacterial cyclic dinucleotides and initiates signaling via the adaptor STING to induce type 1 interferon (IFN) response. We demonstrate here that BTK-deficient cells have impaired IFN-β production and TBK1/IRF3 activation when stimulated with agonists or infected with pathogens that activate STING signaling. BTK interacts with STING and DDX41 helicase. The kinase and SH3/SH2 interaction domains of BTK bind, respectively, the DEAD-box domain of DDX41 and transmembrane region of STING. BTK phosphorylates DDX41, and its kinase activities are critical for STING-mediated IFN-β production. We show that Tyr364 and Tyr414 of DDX41 are critical for its recognition of AT-rich DNA and binding to STING, and tandem mass spectrometry identifies Tyr414 as the BTK phosphorylation site. Modeling studies further indicate that phospho-Tyr414 strengthens DDX41's interaction with STING. Hence, BTK plays a critical role in the activation of DDX41 helicase and STING signaling.

Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia DeltaE3L mutant virus triggers the production of beta interferon (IFN-beta), interleukin-6 (IL-6), CCL4, and CCL5. None of these immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding domain of E3 suffices to prevent activation of the innate immune response. DeltaE3L induction of IFN-beta, IL-6, CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is induced in keratinocytes infected with DeltaE3L, an event that depends on MAVS. The response of keratinocytes to DeltaE3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. In this study, human brain astrocytes were found to constitutively express TLR3, and this expression was increased by interferon-gamma (IFN-gamma) or double-stranded RNA (dsRNA). Treatment employing dsRNA in astrocytes induced IFN regulatory factor 3 (IRF3) phosphorylation, dimer formation and nuclear translocation followed by STAT1 activation. This treatment also activated nuclear factor-kappaB, p38 and c-Jun N-terminal kinase significantly, while activating extracellular signal-regulated kinase to a lesser extent. Treatment with anti-TLR3 antibody inhibited dsRNA-mediated interleukin-6 (IL-6) production. In the presence of mitogen-activated protein kinase inhibitors, astrocytes failed to secrete IL-6 in response to dsRNA treatment. Therefore, dsRNA-induced IL-6 production is dependent on mitogen-activated protein kinases and type I IFN production is dependent on IRF3 in brain astrocytes. These results suggest that brain inflammation, which produces inflammatory cytokines and type I IFNs, may enhance TLR3 expression in astrocytes. Additionally, upregulated TLR3 might modulate inflammatory processes by producing proinflammatory cytokines.

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

In mammals, Toll-like receptors (TLR) recognize ligands, including pathogen-associated molecular patterns (PAMPs), and respond with ligand-specific induction of genes. In this study, we establish evolutionary conservation in teleost fish of key components of the TLR-signaling pathway that act as switches for differential gene induction, including MYD88, TIRAP, TRIF, TRAF6, IRF3, and IRF7. We further explore this conservation with a molecular phylogenetic analysis of MYD88. To the extent that current genomic analysis can establish, each vertebrate has one ortholog to each of these genes. For molecular tree construction and phylogeny inference, we demonstrate a methodology for including genes with only partial primary sequences without disrupting the topology provided by the high-confidence full-length sequences. Conservation of the TLR-signaling molecules suggests that the basic program of gene regulation by the TLR-signaling pathway is conserved across vertebrates. To test this hypothesis, leukocytes from a model fish, rainbow trout (Oncorhynchus mykiss), were stimulated with known mammalian TLR agonists including: diacylated and triacylated forms of lipoprotein, flagellin, two forms of LPS, synthetic double-stranded RNA, and two imidazoquinoline compounds (loxoribine and R848). Trout leukocytes responded in vitro to a number of these agonists with distinct patterns of cytokine expression that correspond to mammalian responses. Our results support the key prediction from our phylogenetic analyses that strong selective pressure of pathogenic microbes has preserved both TLR recognition and signaling functions during vertebrate evolution.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3(-/-) and TLR4(-/-) mice, i.e. TLR3(-/-) mice were highly susceptible to JE, whereas TLR4(-/-) mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b(+)Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4(-/-) mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3(-/-) myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4(+) and CD8(+) T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b(+)Ly-6C(high) monocytes) and CD4(+)Foxp3(+) Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

The chronic inflammatory state that accompanies obesity is a major contributor to insulin resistance and other dysfunctional adaptations in adipose tissue. Cellular and secreted factors promote the inflammatory milieu of obesity, but the transcriptional pathways that drive these processes are not well described. Although the canonical inflammatory transcription factor NF-κB is considered to be the major driver of adipocyte inflammation, members of the interferon regulatory factor (IRF) family may also play a role in this process. Here, we determined that IRF3 expression is upregulated in the adipocytes of obese mice and humans. Signaling through TLR3 and TLR4, which lie upstream of IRF3, induced insulin resistance in murine adipocytes, while IRF3 knockdown prevented insulin resistance. Furthermore, improved insulin sensitivity in IRF3-deficient mice was associated with reductions in intra-adipose and systemic inflammation in the high fat-fed state, enhanced browning of subcutaneous fat, and increased adipose expression of GLUT4. Taken together, the data indicate that IRF3 is a major transcriptional regulator of adipose inflammation and is involved in maintaining systemic glucose and energy homeostasis.

Nonalcoholic fatty liver disease (NAFLD) is a common result of obesity and metabolic syndrome. Hepatocyte injury and metabolic disorders are hallmarks of NAFLD. Stimulator of interferon genes (STING) and its downstream factor interferon regulatory factor 3 (IRF3) trigger inflammatory reaction in response to the presence of cytosolic DNA. STING has recently been shown to play an important role in early alcoholic liver disease. However, little is known about the role of STING-IRF3 pathway in hepatocyte injury. Here, we aimed to examine the effect of STING-IRF3 pathway on hepatocyte metabolism, inflammation and apoptosis.

We examined the activation of the STING-IRF3 pathway, a high-fat diet (HFD)-induced obese mouse model, and determined the role of this pathway in a free fatty acid (FFA)-induced hepatocyte inflammatory response, injury, and dysfunction in L-O2 human liver cells.

STING and IRF3 were upregulated in livers of HFD-fed mice and in FFA-induced L-O2 cells. Knocking down either STING or IRF3 led to a significant reduction in FFA-induced hepatic inflammation and apoptosis, as evidenced by modulation of the nuclear factor κB (NF-κB) signaling pathway, inflammatory cytokines, and apoptotic signaling. Additionally, STING/IRF3 knockdown enhanced glycogen storage and alleviated lipid accumulation, which were found to be associated with increased expression of hepatic enzymes in glycolysis and lipid catabolism, and attenuated expression of hepatic enzymes in gluconeogenesis and lipid synthesis.

Our results suggest that the STING-IRF3 pathway promotes hepatocyte injury and dysfunction by inducing inflammation and apoptosis and by disturbing glucose and lipid metabolism. This pathway may be a novel therapeutic target for preventing NAFLD development and progression.

The interferon (IFN) system is an early innate anti-virus host defense mechanism that takes place shortly after entry of the pathogen and long before the onset of adaptive immunity. Thus, African swine fever virus (ASFV), as an acute and persistent virus in pigs, is predicted to have evolved multiple genes for the manipulation and evasion of interferon. Although, ASFV is known to interfere with signaling pathways controlling the transcription of cytokines, surprisingly no individual virus gene manipulating the induction or impact of IFN has been described. Since an initial bioinformatics search of the ASFV genome failed to identify potential antagonists of the IFN response, our strategy was to functionally screen early expressed, "unassigned" ASFV genes without existing homologies, particularly from MGFs 360 and 530, in luciferase reporter assays for their inhibition of the induction and impact of IFN. Specifically, we used reporter plasmids containing the luciferase gene under the control of: (1) the IFN-β promoter, to screen for inhibition of induction of type I IFN stimulated by the addition of Poly(I:C); (2) the ISRE DNA elements, to screen for the inhibition of the impact of type I IFN; and (3) the GAS DNA elements to screen for the inhibition of the impact of type II IFN. Our initial experiments revealed six ASFV genes inhibiting one or more of the three luciferase assays. From these, we have selected a total of 3 genes for presentation. The ASFV A276R gene from MGF360 inhibited the induction of IFN-β via both the TLR3 and the cytosolic pathways, targeting IRF3, but not IRF7 or NF-κB. The ASFV A528R inhibited the induction of both NF-κB and IRF3 branches of the type I IFN induction signaling pathway and the impact of IFN response via both IFN type I and type II stimulation. The ASFV I329L gene is a functional viral TLR3 homologue inhibiting the induction of IFN at the level of TRIF. Thus, these genes reduce the IFN response by targeting different intracellular signaling intermediates. Their deletion from wild type virus may strengthen the host interferon response and so provide an attenuated form with more restricted virus spread after the initial infection, perhaps "buying" sufficient time to allow the development of a protective adaptive immune response. The demonstration of multiple ASFV genes for the evasion of IFN responses will demand technology to construct viruses with multiple gene deletions. An alternative would be a multigene DNA vaccine. Finally, our work clearly demonstrates that unassigned viral genes may be viewed as a repository of host evasion strategies, only identifiable through functional assays. These may be considered to be "ready-made tools" for the experimental manipulation of cell biology and immune responses in health and disease and, as proof of concept, we have constructed a T-cell restricted transgenic mouse expressing the ASFV gene A238L, a dual inhibitor of NF-κB and NFAT activation. The resulting T cell restricted A238L transgenic mice developed a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas. In contrast, transgenic mice similarly expressing a mutant A238L solely inhibiting transcription mediated by NF-κB were indistinguishable from wild type mice, suggesting a transgene-NFAT-dependent transformation. Elucidation of the molecular events associated with the development of this virus host evasion molecule induced tumor may clarify some mechanisms of tumorigenesis in general, and in the development of T cell acute lymphoblastic leukemia in particular.

Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses.

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage^1,2. Cytosolic DNA triggers immune responses by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway³. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)^4-7. This cyclic dinucleotide (CDN) activates STING⁸, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells⁹ and other CDNs, including those produced by bacteria^10-12 and synthetic CDNs used in cancer immunotherapy^13,14, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer¹³, host responsiveness to CDN-producing pathogenic microorganisms¹¹ and-potentially-for some inflammatory diseases.

During infection with an RNA virus, the DExD/H-box RNA helicases RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) activate the interferon regulatory factor 3 (IRF3), nuclear factor κB (NF-κB), c-Jun amino-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways through an unknown mechanism involving the adaptor protein MAVS (mitochondrial antiviral signaling). We used a Drosophila misexpression screen to identify DEAH-box polypeptide 15 (DHX15) as an activator of the p38 MAPK pathway. Human DHX15 contributed to the activation of the NF-κB, JNK, and p38 MAPK pathways, but not the IRF3 pathway, in response to the synthetic double-stranded RNA analog poly(I:C) (polyinosinic-polycytidylic acid), and DHX15 was required for optimal cytokine production in response to poly(I:C) and infection with RNA virus. DHX15 physically interacted with MAVS and mediated the MAVS-dependent activation of the NF-κB and MAPK pathways. Furthermore, DHX15 was required for poly(I:C)- and RNA virus-dependent, MAVS-mediated apoptosis. Thus, our findings indicate that, in RIG-I-like receptor signaling, DHX15 specifically stimulates the NF-κB and MAPK pathways downstream of MAVS and contributes to MAVS-mediated cytokine production and apoptosis.

Pestiviruses prevent alpha/beta interferon (IFN-alpha/beta) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral N(pro) nonstructural protein. N(pro) is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire N(pro) gene resulted in attenuation in pigs. In order to elaborate on the role of the N(pro)-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in N(pro) that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of N(pro). We describe two mutations in N(pro) that eliminate N(pro)-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for N(pro) to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-alpha/beta induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-alpha/beta induction is not a prerequisite for CSFV virulence.

The innate immune system forms our first line of defense against invading pathogens and relies for a major part on the activation of two transcription factors, NF-κB and IRF3. Signaling pathways that activate these transcription factors are intertwined at the level of the canonical IκB kinases (IKKα, IKKβ) and non-canonical IKK-related kinases (IKKε, TBK1). Recently, significant progress has been made in understanding the function and mechanism of action of IKKε in immune signaling. In addition, IKKε impacts on cell proliferation and transformation, and is thereby also classified as an oncogene. Studies with IKKε knockout mice have illustrated a key role for IKKε in inflammatory and metabolic diseases. In this review we will highlight the mechanisms by which IKKε impacts on signaling pathways involved in disease development and discuss its potential as a novel therapeutic target.

Our knowledge of the molecular mechanisms of urinary tract infection (UTI) pathogenesis has advanced greatly in recent years. In this review, we provide a general background of UTI pathogenesis, followed by an update on the mechanisms of UTI susceptibility, with a particular focus on genetic variation affecting innate immunity. The innate immune response of the host is critically important in the antibacterial defence mechanisms of the urinary tract, and bacterial clearance normally proceeds without sequelae. However, slight dysfunctions in these mechanisms may result in acute disease and tissue destruction. The symptoms of acute pyelonephritis are caused by the innate immune response, and inflammation in the urinary tract decreases renal tubular function and may give rise to renal scarring, especially in paediatric patients. In contrast, in children with asymptomatic bacteriuria (ABU), bacteria persist without causing symptoms or pathology. Pathogenic agents trigger a response determined by their virulence factors, mediating adherence to the urinary tract mucosa, signalling through Toll-like receptors (TLRs) and activating the defence mechanisms. In ABU strains, such virulence factors are mostly not expressed. However, the influence of the host on UTI severity cannot be overestimated, and rapid progress is being made in clarifying host susceptibility mechanisms. For example, genetic alterations that reduce TLR4 function are associated with ABU, while polymorphisms reducing IRF3 or CXCR1 expression are associated with acute pyelonephritis and an increased risk for renal scarring. It should be plausible to "individualize" diagnosis and therapy by combining information on bacterial virulence and the host response.

BACKGROUND

Degraded extracellular matrix can stimulate the innate immune system via the Toll-Like Receptor-4 (TLR4). In the pancreas, syndecan-anchored heparan sulphate (HS) on the ductal epithelium can be cleaved off its protein cores by the proteases (trypsin and elastase) and potentially activate TLR4 signalling.

METHODS

To investigate this signalling event, a low sulphated HS (500 μg/ml) was infused into the biliary-pancreatic duct of C57BL/6J wild-type mice. Phosphate buffered saline (PBS) and lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Mice were sacrificed after 1, 3, 6, 9, and 48 hours and tissues were analysed for neutrophil and cytokine contents. In order to study the TLR4 signalling pathway of HS in the pancreas, genetically engineered mice lacking TLR4, Myeloid Differentiation primary response gene (88) (MyD88) or Interferon Regulatory Factor 3 (IRF3) were subjected to pancreatic infusion of HS.

RESULTS

Neutrophil sequestration and corresponding myeloperoxidase (MPO) activity in the pancreas were increased 9 hours following HS challenge. In wild-type mice, the monocyte chemoattractant protein-1(MCP-1) increased at 3 hours after infusion, while RANTES increased after 9 hours.TLR4, MyD88, and IRF3 knockout mice showed an abrogated neutrophil recruitment and myeloperoxidase activity in the HS group, while the LPS response was only abolished in TLR4 and MyD88 knockouts.

CONCLUSIONS

The results of this study show that HS is capable of initiating a TLR4-dependent innate immune response in the pancreas which is distinctly different from that induced by LPS. This inflammatory response was mediated predominantly through IRF3- dependent pathway. Release of HS into the pancreatic duct may be one important mediator in the pancreatic ductal defence.

SOCS1 can regulate TLR-mediated signal transduction, yet mechanistic studies in murine macrophages have been confusing and contradictory. This study has used an adenoviral transfection system to determine the role of SOCS1 in the regulation of TNF-alpha production by activated human monocytes. Monocytes were infected with AdV-SOCS1 or with an empty vector control, AdV-GFP, for 24 h before activation with the TLR4 ligand, LPS. SOCS1 did not regulate TNF-alpha mRNA or protein production within the first two hours of TLR4 activation. However, SOCS1 suppressed the sustained production of TNF-alpha by primary human monocytes and synovial fluid macrophages ex vivo. In addition, SOCS1 regulated the production of IL-6, but not IL-10, by monocytes. Analysis of the early signaling pathway downstream of TLR4 demonstrated that SOCS1 had no regulatory effect on the activation or on the DNA binding capacity of NFkappaB. The late effects of LPS are mediated in part through the MyD88-independent pathway activating IRF3 and initiating the production of IFN-beta. In response to adenoviral infection and before LPS exposure, monocytes expressed enhanced levels of IFN-beta and Myxovirus A mRNA, an anti-viral molecule characterizing IFN-beta activity. These two genes were reduced in AdV-SOCS1-infected cells. Further, SOCS1 regulated IFN-dependent pathways in LPS-activated cells as evidenced by reduced IFN-beta production and STAT1 phosphorylation. Using AdV-infection to dissect SOCS1 control of IFN-dependent pathways, this study suggests that SOCS1-regulation of the IFN-dependent component of the LPS-induced TLR4 signaling pathway may contribute to the down-regulation of inflammatory cytokine production by AdV-SOCS1-infected human monocytes.