A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition.

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes that influence Cryptosporidium parvum infection and/or host cell survival. Gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection and impact on the viability of host cells in the context of parasite infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFNλ reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that required STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFNλ production critical to early protection against this infection.

Extracellular Nucleotides and Histamine Suppress TLR3- and RIG-I-Mediated Release of Antiviral IFNs from Human Airway Epithelial Cells.

Respiratory viruses stimulate the release of antiviral IFNs from the airway epithelium. Previous studies have shown that asthmatic patients show diminished release of type I and type III IFNs from bronchial epithelia. However, the mechanism of this suppression is not understood. In this study, we report that extracellular nucleotides and histamine, which are elevated in asthmatic airways, strongly inhibit release of type I and type III IFNs from human bronchial airway epithelial cells (AECs). Specifically, ATP, UTP, and histamine all inhibited the release of type I and type III IFNs from AECs induced by activation of TLR3, retinoic acid-inducible gene I (RIG-I), or cyclic GMP-AMP synthase-STING. This inhibition was at least partly mediated by Gq signaling through purinergic P2Y2 and H1 receptors, but it did not involve store-operated calcium entry. Pharmacological blockade of protein kinase C partially reversed inhibition of IFN production. Conversely, direct activation of protein kinase C with phorbol esters strongly inhibited TLR3- and RIG-I-mediated IFN production. Inhibition of type I and type III IFNs by ATP, UTP, histamine, and the proteinase-activated receptor 2 (PAR2) receptor agonist SLIGKV also occurred in differentiated AECs grown at an air-liquid interface, indicating that the suppression is conserved following mucociliary differentiation. Importantly, histamine and, more strikingly, ATP inhibited type I IFN release from human airway cells infected with live influenza A virus or rhinovirus 1B. These results reveal an important role for extracellular nucleotides and histamine in attenuating the induction of type I and III IFNs from AECs and help explain the molecular basis of the suppression of IFN responses in asthmatic patients.

Induction of Innate Immune Response by TLR3 Agonist Protects Mice against SARS-CoV-2 Infection.

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.

Antiviral Toll-like Receptor Signaling in Non-Parenchymal Liver Cells Is Restricted to TLR3.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). METHODS: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-ß, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. RESULTS: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-ß or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. CONCLUSION: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.

Distinct trans-placental effects of maternal immune activation by TLR3 and TLR7 agonists: implications for schizophrenia risk.

Exposure to infection in utero predisposes towards psychiatric diseases such as autism, depression and schizophrenia in later life. The mechanisms involved are typically studied by administering mimetics of double-stranded (ds) virus or bacterial infection to pregnant rats or mice. The effect of single-stranded (ss) virus mimetics has been largely ignored, despite evidence linking prenatal ss virus exposure with psychiatric disease. Understanding the effects of gestational ss virus exposure has become even more important with recent events. In this study, in pregnant mice, we compare directly the effects, on the maternal blood, placenta and the embryonic brain, of maternal administration of ds-virus mimetic poly I:C (to activate Toll-like receptor 3, TLR3) and ss-virus mimetic resiquimod (to activate TLR7/8). We find that, 4 h after the administration, both poly I:C and resiquimod elevated the levels of IL-6, TNFα, and chemokines including CCL2 and CCL5, in maternal plasma. Both agents also increased placental mRNA levels of IL-6 and IL-10, but only resiquimod increased placental TNFα mRNA. In foetal brain, poly I:C produced no detectable immune-response-related increases, whereas pronounced increases in cytokine (e.g. Il-6, Tnfα) and chemokine (e.g. Ccl2, Ccl5) expression were observed with maternal resiquimod administration. The data show substantial differences between the effect of maternal exposure to a TLR7/8 activator as compared to a TLR3 activator. There are significant implications for future modelling of diseases where maternal ss virus exposure contributes to environmental disease risk in offspring.

Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

HIF-1α Regulation of Cytokine Production following TLR3 Engagement in Murine Bone Marrow-Derived Macrophages Is Dependent on Viral Nucleic Acid Length and Glucose Availability.

Hypoxia-inducible factor-1α (HIF-1α) is an important regulator of glucose metabolism and inflammatory cytokine production in innate immune responses. Viruses modulate HIF-1α to support viral replication and the survival of infected cells, but it is unclear if this transcription factor also plays an important role in regulating antiviral immune responses. In this study, we found that short and long dsRNA differentially engage TLR3, inducing distinct levels of proinflammatory cytokine production (TNF-α and IL-6) in bone marrow-derived macrophages from C57BL/6 mice. These responses are associated with differential accumulation of HIF-1α, which augments NF-κB activation. Unlike TLR4 responses, increased HIF-1α following TLR3 engagement is not associated with significant alterations in glycolytic activity and was more pronounced in low glucose conditions. We also show that the mechanisms supporting HIF-1α stabilization may differ following stimulation with short versus long dsRNA and that pyruvate kinase M2 and mitochondrial reactive oxygen species play a central role in these processes. Collectively, this work suggests that HIF-1α may fine-tune proinflammatory cytokine production during early antiviral immune responses, particularly when there is limited glucose availability or under other conditions of stress. Our findings also suggest we may be able to regulate the magnitude of proinflammatory cytokine production during antiviral responses by targeting proteins or molecules that contribute to HIF-1α stabilization.

Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes.

The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes.

Changes in MDA5 and TLR3 Sensing of the Same Diabetogenic Virus Result in Different Autoimmune Disease Outcomes.

Seemingly redundant in function, melanoma differentiation-associated protein 5 (MDA5) and toll-like receptor- 3 (TLR3) both sense RNA viruses and induce type I interferon (IFN-I). Herein, we demonstrate that changes in sensing of the same virus by MDA5 and TLR3 can lead to distinct signatures of IFN-α and IFN-ß resulting in different disease outcomes. Specifically, infection with a diabetogenic islet ß cell-tropic strain of coxsackievirus (CB4) results in diabetes protection under reduced MDA5 signaling conditions while reduced TLR3 function retains diabetes susceptibility. Regulating the induction of IFN-I at the site of virus infection creates a local site of interferonopathy leading to loss of T cell regulation and induction of autoimmune diabetes. We have not demonstrated another way to prevent T1D in the NOD mouse, rather we believe this work has provided compounding evidence for a specific control of IFN-I to drive a myriad of responses ranging from virus clearance to onset of autoimmune diabetes.

Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice.

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2-/- mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3-/- mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2-/- and TLR3-/- mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2-/- and TLR3-/- mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2-/-, and TLR3-/- mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses.

Toll-like receptor 3 (TLR3) is a member of the TLR family, mediating the transcriptional induction of type I interferons (IFNs), proinflammatory cytokines, and chemokines, thereby collectively establishing an antiviral host response. Studies have shown that unlike other TLR family members, TLR3 is the only RNA sensor that is utterly dependent on the Toll-interleukin-1 receptor (TIR)|-domain-containing adaptor-inducing IFN-|ß (TRIF). However, the details of how the TLR3-TRIF signaling pathway works in an antiviral response and how it is regulated are unclear. In this review, we focus on recent advances in understanding the antiviral mechanism of the TRIF pathway and describe the essential characteristics of TLR3 and its antiviral effects. Advancing our understanding of TLR3 may contribute to disease diagnosis and could foster the development of novel treatments for viral diseases.

Emodin from Aloe Inhibits Porcine Reproductive and Respiratory Syndrome Virus via Toll-Like Receptor 3 Activation.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows and respiratory diseases in growing and finishing pigs and results in great economic losses to the swine industry. Although vaccines are available, PRRSV remains a major threat to the pig farms. Thus, there is an urgent need to develop antiviral drugs to compensate for vaccines. In this study, we report that Aloe extract (Ae) can strongly inhibit PRRSV in Marc-145 cells and porcine alveolar macrophages lines (iPAMs) in vitro. Furthermore, we identified a novel anti-PRRSV molecule, Emodin, from Ae by high-performance liquid chromatography (HPLC). Emodin exerted its inhibitory effect through targeting the whole stages of PRRSV infectious cycle. Moreover, we also found that Emodin can inactivate PRRSV particles directly. Notably, we confirmed that Emodin was able to significantly induce Toll-like receptor 3 (TLR3) (p < 0.01), IFN-α (p < 0.05) and IFN-ß expression in iPAMs, indicating that induction of antiviral agents via TLR3 activation by Emodin might contribute to its anti-PRRSV effect. These findings imply that the Emodin from Aloe could hamper the proliferation of PRRSV in vitro and might constitute a new approach for treating PRRSV infection.

Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor.

Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO's in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.

PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy.

With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic-co-glycolic acid) (PLGA) particles containing antigens and toll-like receptor ligands induces vigorous antitumor immune responses in vivo. Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8+ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.

A computational approach for detecting physiological homogeneity in the midst of genetic heterogeneity.

The human genetic dissection of clinical phenotypes is complicated by genetic heterogeneity. Gene burden approaches that detect genetic signals in case-control studies are underpowered in genetically heterogeneous cohorts. We therefore developed a genome-wide computational method, network-based heterogeneity clustering (NHC), to detect physiological homogeneity in the midst of genetic heterogeneity. Simulation studies showed our method to be capable of systematically converging genes in biological proximity on the background biological interaction network, and capturing gene clusters harboring presumably deleterious variants, in an efficient and unbiased manner. We applied NHC to whole-exome sequencing data from a cohort of 122 individuals with herpes simplex encephalitis (HSE), including 13 individuals with previously published monogenic inborn errors of TLR3-dependent IFN-α/ß immunity. The top gene cluster identified by our approach successfully detected and prioritized all causal variants of five TLR3 pathway genes in the 13 previously reported individuals. This approach also suggested candidate variants of three reported genes and four candidate genes from the same pathway in another ten previously unstudied individuals. TLR3 responsiveness was impaired in dermal fibroblasts from four of the five individuals tested, suggesting that the variants detected were causal for HSE. NHC is, therefore, an effective and unbiased approach for unraveling genetic heterogeneity by detecting physiological homogeneity.

SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.

IRF5 regulates airway macrophage metabolic responses.

Interferon regulatory factor 5 (IRF5) is a master regulator of macrophage phenotype and a key transcription factor involved in expression of proinflammatory cytokine responses to microbial and viral infection. Here, we show that IRF5 controls cellular and metabolic responses. By integrating ChIP sequencing (ChIP-Seq) and assay for transposase-accessible chromatin using sequencing (ATAC)-seq data sets, we found that IRF5 directly regulates metabolic genes such as hexokinase-2 (Hk2). The interaction of IRF5 and metabolic genes had a functional consequence, as Irf5-/- airway macrophages but not bone marrow-derived macrophages (BMDMs) were characterized by a quiescent metabolic phenotype at baseline and had reduced ability to utilize oxidative phosphorylation after Toll-like receptor (TLR)-3 activation, in comparison to controls, ex vivo. In a murine model of influenza infection, IRF5 deficiency had no effect on viral load in comparison to wild-type controls but controlled metabolic responses to viral infection, as IRF5 deficiency led to reduced expression of Sirt6 and Hk2. Together, our data indicate that IRF5 is a key component of AM metabolic responses following influenza infection and TLR-3 activation.

Therapeutic vaccination targeting CD40 and TLR3 controls melanoma growth through existing intratumoral CD8 T cells without new T cell infiltration.

Dendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently priming T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human patients. While the efficacy of vaccines based on CD40 and toll like receptor stimulation has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells. In this study we vaccinated mice bearing established murine melanoma tumors with agonistic anti-CD40, polyI:C, and tumor antigen. Vaccination led to increased intratumoral T cell numbers and delayed tumor growth, yet did not require trafficking of T cells from the periphery. Pre-existing intratumoral T cells exhibited an acute burst in proliferation but became less functional in response to vaccination. However, the increased intratumoral T cell numbers yielded increased numbers of effector T cells per tumor. Together, our data indicate that the existing T cell response and intratumoral dendritic cells are critical for vaccination efficacy. It also suggests that circulating T cells responding to vaccination may not be an appropriate biomarker for vaccine efficacy.

TLR3 controls constitutive IFN-ß antiviral immunity in human fibroblasts and cortical neurons.

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/ß induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-ß protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-ß secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-ß immunity.