Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity.

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1ß for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1ß was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1ß, resulting in severe inflammation.

Carbazole to indolazepinone scaffold morphing leads to potent cell-active dengue antivirals.

According to WHO, dengue virus is classed among major threats for future pandemics and remains at large an unmet medical need as there are currently no relevant antiviral drugs whereas vaccine developments have met with safety concerns, mostly due to secondary infections caused by antibody-dependant-enhancement in cross infections among the four dengue serotypes. This adds extra complexity in dengue antiviral research and has impeded the progress in this field. Following through our previous effort which born the allosteric, dual-mode inhibitor SP-471P (a carbazole derivative, EC50 1.1 µM, CC50 100 µM) we performed further optimisation while preserving the two arylamidoxime arms and the bromoaryl domain present in SP-471P. Examination of the relative positions of these functionalities within this three-point pharmacophore ultimately led us to an indolazepinone scaffold and our lead compound SP-1769B. SP-1769B is among the most cell-efficacious against all serotypes (DENV2/3 EC50 100 nM, DENV1/4 EC50 0.95-1.25 µM) and safest (CC50 > 100 µM) anti-dengue compounds in the literature that also completely inhibits a secondary ADE-driven infection.

Cutaneous anaphylactoid reaction to polyoxyethylene hydrogenated castor oil in dogs.

BACKGROUND: Polyoxyethylene hydrogenated castor oil (HCO ethoxylates) is a nonionic surfactant used as an excipient for ointments and injections in human and veterinary drugs. Several polyethylene glycol (PEG) derivatives can be obtained depending on the number of moles of ethylene oxide (EO). HCO ethoxylates have the potential to cause anaphylactoid reactions. There is little published information about these types of reactions in dogs. OBJECTIVE: To determine the potential for HCO-ethoxylate-containing drugs to cause anaphylactoid reactions in dogs, employing intradermal testing (IDT) with various concentrations of HCO ethoxylates (HCO-25, -40, -60 and -80). ANIMALS: Four healthy male laboratory dogs. MATERIALS AND METHODS: We performed IDT with drugs containing HCO ethoxylates and HCO ethoxylates alone to determine threshold concentrations. The IDT scores and threshold concentrations were compared. Analysis of skin biopsies from IDT sites was used to measure the percentage of degranulated mast cells. The effect of histamine at IDT sites was investigated by pre-treatment with an antihistamine. RESULTS: All HCO-ethoxylate-containing drugs caused a wheal-and-flare reaction. The threshold concentrations (0.001% and 0.00001%) of each HCO-ethoxylate depended on the number of moles of EO (p < 0.05). Mast cell degranulation was enhanced by all HCO ethoxylates. The HCO-60-induced reaction was suppressed by an oral antihistamine. CONCLUSIONS AND CLINICAL RELEVANCE: The threshold concentration can serve as a consideration for developing safe new drug formulations and for clinical decision-making around using drugs containing PEG derivatives. IDT is useful to predict the risk of adverse effects. Antihistamines could demonstrate a prophylactic effect.

A humanized mouse model to study mast cells mediated cutaneous adverse drug reactions.

Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.

MRGPR-mediated activation of local mast cells clears cutaneous bacterial infection and protects against reinfection.

Mast cells (MCs) are strategically distributed at barrier sites and prestore various immunocyte-recruiting cytokines, making them ideal targets for selective activation to treat peripheral infections. Here, we report that topical treatment with mastoparan, a peptide MC activator (MCA), enhances clearance of Staphylococcus aureus from infected mouse skins and accelerates healing of dermonecrotic lesions. Mastoparan functions by activating connective tissue MCs (CTMCs) via the MRGPRX2 (Mas-related G protein-coupled receptor member X2) receptor. Peripheral CTMC activation, in turn, enhances recruitment of bacteria-clearing neutrophils and wound-healing CD301b+ dendritic cells. Consistent with MCs playing a master coordinating role, MC activation also augmented migration of various antigen-presenting dendritic cells to draining lymph nodes, leading to stronger protection against a second infection challenge. MCAs therefore orchestrate both the innate and adaptive immune arms, which could potentially be applied to combat peripheral infections by a broad range of pathogens.

Collaboration between Distinct Rab Small GTPase Trafficking Circuits Mediates Bacterial Clearance from the Bladder Epithelium.

Rab small GTPases control membrane trafficking through effectors that recruit downstream mediators such as motor proteins. Subcellular trafficking typically involves multiple Rabs, with each specific step mediated by a distinct Rab protein. We describe a collaboration between two distinct Rab-protein-orchestrated trafficking circuits in bladder epithelial cells (BECs) that expels intracellular uropathogenic Escherichia coli (UPEC) from their intracellular niche. RAB11a and RAB27b and their trafficking circuitry are simultaneously involved in UPEC expulsion. While RAB11a recruits its effector RAB11FIP3 and cytoskeletal motor Dynein, RAB27b mobilizes the effector MyRIP and motor Myosin VIIa to mediate bacterial expulsion. This collaboration is coordinated by deposition of the exocyst complex on bacteria-containing vesicles, an event triggered by the innate receptor Toll-like receptor 4. Both RAB11a and RAB27b are recruited and activated by the exocyst complex components SEC6/SEC15. Thus, the cell autonomous defense system can mobilize and coalesce multiple subcellular trafficking circuitries to combat infections.

Erratum: E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP.

This corrects the article DOI: 10.1038/ncomms15865.

Dengue virus activates cGAS through the release of mitochondrial DNA.

Cyclic GMP-AMP synthetase (cGAS) is a DNA-specific cytosolic sensor, which detects and initiates host defense responses against microbial DNA. It is thus curious that a recent study identified cGAS as playing important roles in inhibiting positive-sense single-stranded RNA (+ssRNA) viral infection, especially since RNA is not known to activate cGAS. Using a dengue virus serotype 2 (DENV-2) vaccine strain (PDK53), we show that infection creates an endogenous source of cytosolic DNA in infected cells through the release of mitochondrial DNA (mtDNA) to drive the production of cGAMP by cGAS. Innate immune responses triggered by cGAMP contribute to limiting the spread of DENV to adjacent uninfected cells through contact dependent gap junctions. Our result thus supports the notion that RNA virus indirectly activates a DNA-specific innate immune signaling pathway and highlights the breadth of the cGAS-induced antiviral response.

E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP.

Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.

ArfGAP Domain-Containing Protein 2 (ADAP2) Integrates Upstream and Downstream Modules of RIG-I Signaling and Facilitates Type I Interferon Production.

Transcription of type I interferon genes during RNA virus infection requires signal communication between several pattern recognition receptor (PRR)-adaptor complexes located at distinct subcellular membranous compartments and a central cytoplasmic TBK1-interferon regulatory factor 3 (IRF3) kinase-transcription factor module. However, how the cell integrates signal transduction through spatially distinct modules of antiviral signaling pathways is less defined. RIG-I is a major cytosolic PRR involved in the control of several RNA viruses. Here we identify ArfGAP domain-containing protein 2 (ADAP2) as a key novel scaffolding protein that integrates different modules of the RIG-I pathway, located at distinct subcellular locations, and mediates cellular antiviral type I interferon production. ADAP2 served to bridge the mitochondrial membrane-bound upstream RIG-I adaptor MAVS and the downstream cytosolic complex of NEMO (regulatory subunit of TBK1), TBK1, and IRF3, leading to IRF3 phosphorylation. Furthermore, independently, ADAP2 also functioned as a major orchestrator of the interaction of TBK1 with NEMO and IRF3. Mutational and in vitro cell-free reconstituted RIG-I signaling assay-based analyses identified that the ArfGAP domain of ADAP2 mediates the interferon response. TRAF3 acted as a trigger for ADAP2 to recruit RIG-I pathway component proteins into a single macromolecular complex. This study provides important novel insights into the assembly and integration of different modules of antiviral signaling cascades.

A novel 4,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivative inhibits tumor cell invasion and potentiates the apoptotic effect of TNFα by abrogating NF-κB activation cascade.

Condensed-bicyclic 4,6-substituted1,2,4-triazolo-1,3,4-thiadiazole derivatives (CBTT) have been shown to possess a wide spectrum of pharmacological activities. In this study, several novel CBTT derivatives were synthesized and investigated for their possible role as anti-neoplastic agents. The anti-proliferative effect of various CBTT derivatives was analyzed against tumor cell lines by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. One of the potential CBTT derivative, 5-(3-(2,3-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)flurobenzonitrile (DTTF) was found to be the most potent against cervical cancer SiHa cells and exhibited minimal effect against normal cells. Molecular docking analysis indicated that transcription factor NF-κB was one of the potential molecular targets modulated by DTTF. Specifically, the drug blocked the TNFα-induced phosphorylation of upstream IκBα kinase in a time-dependent manner leading to the suppression of NF-κB activation and nuclear translocation. DTTF also potentiated the apoptotic effect of TNFα, as well as significantly inhibited migration and invasion of tumor cells. Overall, these findings indicate a potential novel role and mechanism(s) of action of DTTF as an anticancer agent against diverse malignancies.

Global functional profiling of human ubiquitome identifies E3 ubiquitin ligase DCST1 as a novel negative regulator of Type-I interferon signaling.

Type I interferon (IFN-I) mediated innate immune response controls virus infections by inducing the expression of interferon stimulated genes (ISGs). Although ubiquitination plays key roles in immune signaling regulation, a human genome-wide understanding of the role of E3 ubiquitin ligases in interferon mediated ISG induction is lacking. Here, we report a genome-wide profiling of the effect of ectopic expression of 521 E3 ubiquitin ligases and substrate recognition subunits encoded in the human genome (which constitutes 84.4% of all ubiquitination related genes encoded in the human genome, hereafter termed Human Ubiquitome) on IFNß mediated induction of interferon stimulated DNA response element (ISRE) driven reporter activity. We identified 96 and 42 genes of the human ubiquitome as novel negative and positive regulators of interferon signaling respectively. Furthermore, we characterized DCST1 as a novel E3 ubiquitin ligase negatively regulating interferon response. Ectopic expression and gene silencing of DCST1 respectively attenuated and increased ISRE reporter activity. DCST1 regulated Type I interferon signaling by interacting with and promoting ubiquitination-mediated degradation of STAT2, an essential component of antiviral gene induction. In summary, this study provided a systems level view on the role of human ubiquitination associated genes in Type I interferon response.

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

Annexin-A1 controls an ERK-RhoA-NFκB activation loop in breast cancer cells.

Wound healing is critical for normal development and pathological processes including cancer cell metastasis. MAPK, Rho-GTPases and NFκB are important regulators of wound healing, but mechanisms for their integration are incompletely understood. Annexin-A1 (ANXA1) is upregulated in invasive breast cancer cells resulting in constitutive activation of NFκB. We show here that silencing ANXA1 increases the formation of stress fibers and focal adhesions, which may inhibit wound healing. ANXA1 regulated wound healing is dependent on the activation of ERK1/2. ANXA1 increases the activation of RhoA, which is dependent on ERK activation. Furthermore, active RhoA is important in NF-κB activation, where constitutively active RhoA potentiates NFκB activation, while dominant negative RhoA inhibits NFκB activation in response to CXCL12 stimulation and active MEKK plasmids. These findings establish a central role for ANXA1 in the cell migration through the activation of NFκB, ERK1/2 and RhoA.

Bruton's tyrosine kinase phosphorylates DDX41 and activates its binding of dsDNA and STING to initiate type 1 interferon response.

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-ß 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-ß 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.

Annexin-A1 regulates microRNA-26b* and microRNA-562 to directly target NF-κB and angiogenesis in breast cancer cells.

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3' UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer.

Human genome-wide RNAi screen identifies an essential role for inositol pyrophosphates in Type-I interferon response.

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

The Nod1, Nod2, and Rip2 axis contributes to host immune defense against intracellular Acinetobacter baumannii infection.

Acinetobacter baumannii is a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controlling A. baumannii are not well understood. Although viewed as an extracellular pathogen, A. baumannii can also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity against A. baumannii is not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance of A. baumannii by human airway epithelial cells in vitro. A. baumannii infection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival of A. baumannii in lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control of A. baumannii infection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed for A. baumannii infection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses to A. baumannii infection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control of A. baumannii infection through the production of ß-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control of A. baumannii and may contribute to the development of effective immune therapeutics and vaccines against A. baumannii.

Annexin-A1 regulates TLR-mediated IFN-ß production through an interaction with TANK-binding kinase 1.

TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-ß and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-ß and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-ß and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-ß and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.