Human genetic and immunological determinants of critical COVID-19 pneumonia.

SARS-CoV-2 infection is benign in most individuals but, in around 10% of cases, it triggers hypoxaemic COVID-19 pneumonia, which leads to critical illness in around 3% of cases. The ensuing risk of death (approximately 1% across age and gender) doubles every five years from childhood onwards and is around 1.5 times greater in men than in women. Here we review the molecular and cellular determinants of critical COVID-19 pneumonia. Inborn errors of type I interferons (IFNs), including autosomal TLR3 and X-chromosome-linked TLR7 deficiencies, are found in around 1-5% of patients with critical pneumonia under 60 years old, and a lower proportion in older patients. Pre-existing auto-antibodies neutralizing IFNα, IFNß and/or IFNω, which are more common in men than in women, are found in approximately 15-20% of patients with critical pneumonia over 70 years old, and a lower proportion in younger patients. Thus, at least 15% of cases of critical COVID-19 pneumonia can be explained. The TLR3- and TLR7-dependent production of type I IFNs by respiratory epithelial cells and plasmacytoid dendritic cells, respectively, is essential for host defence against SARS-CoV-2. In ways that can depend on age and sex, insufficient type I IFN immunity in the respiratory tract during the first few days of infection may account for the spread of the virus, leading to pulmonary and systemic inflammation.

Brain-derived neurotrophic factor is down regulated after bovine alpha-herpesvirus 5 infection in both wild-type and TLR3/7/9 deficient mice.

Neurotrophic factors have been implicated in the control of neuronal survival and plasticity in different brain diseases. Meningoencephalitis caused by bovine alpha-herpesvirus 5 (BoHV-5) infection is a frequent neurological disease of young cattle, being the involvement of apoptosis in the development of neuropathological changes frequently discussed in the literature. It's well known that Toll-like receptors (TLRs) can activate neuroinflammatory response and consequently lead to neuronal loss. However, there are no studies evaluating the expression of neurotrophic factors and their association with brain pathology and TLRs during the infection by BoHV-5. The current study aimed to analyze brain levels of neurotrophic factors along with neuropathological changes during acute infection by BoHV-5 in wild-type (WT) and TLR3/7/9 (TLR3/7/9-/-) deficiency mice. The infection was induced by intracranial inoculation of 1 × 104 TCID50 of BoHV-5. Infected animals presented similar degrees of clinical signs and neuropathological changes. Both infected groups had meningoencephalitis and neuronal damage in CA regions from hippocampus. BoHV-5 infection promoted the proliferation of Iba-1 positive cells throughout the neuropil, mainly located in the frontal cortex. Moreover, significant lower levels of brain-derived neurotrophic factor (BDNF) were detected in both BoHV-5 infected WT and TLR3/7/9 deficient mice, compared with non-infected animals. Our study showed that BDNF down regulation was associated with brain inflammation, reactive microgliosis and neuronal loss after bovine alpha-herpesvirus 5 infection in mice. Moreover, we demonstrated that combined TLR3/7/9 deficiency does not alter those parameters.

Tumoural activation of TLR3-SLIT2 axis in endothelium drives metastasis.

Blood vessels support tumours by providing nutrients and oxygen, while also acting as conduits for the dissemination of cancer1. Here we use mouse models of breast and lung cancer to investigate whether endothelial cells also have active 'instructive' roles in the dissemination of cancer. We purified genetically tagged endothelial ribosomes and their associated transcripts from highly and poorly metastatic tumours. Deep sequencing revealed that metastatic tumours induced expression of the axon-guidance gene Slit2 in endothelium, establishing differential expression between the endothelial (high Slit2 expression) and tumoural (low Slit2 expression) compartments. Endothelial-derived SLIT2 protein and its receptor ROBO1 promoted the migration of cancer cells towards endothelial cells and intravasation. Deleting endothelial Slit2 suppressed metastatic dissemination in mouse models of breast and lung cancer. Conversely, deletion of tumoural Slit2 enhanced metastatic progression. We identified double-stranded RNA derived from tumour cells as an upstream signal that induces expression of endothelial SLIT2 by acting on the RNA-sensing receptor TLR3. Accordingly, a set of endogenous retroviral element RNAs were upregulated in metastatic cells and detected extracellularly. Thus, cancer cells co-opt innate RNA sensing to induce a chemotactic signalling pathway in endothelium that drives intravasation and metastasis. These findings reveal that endothelial cells have a direct instructive role in driving metastatic dissemination, and demonstrate that a single gene (Slit2) can promote or suppress cancer progression depending on its cellular source.

Influenza A virus enhances ciliary activity and mucociliary clearance via TLR3 in airway epithelium.

BACKGROUND: Viral respiratory tract infections, such as influenza A virus (IAV), are common and life-threatening illnesses worldwide. The mechanisms by which viruses are removed from the respiratory tract are indispensable for airway host defense. Mucociliary clearance is an airway defense mechanism that removes pathogens from the respiratory tract. The coordination and modulation of the ciliary beating of airway epithelial cells play key roles in maintaining effective mucociliary clearance. However, the impact of respiratory virus infection on ciliary activity and mucociliary clearance remains unclear. METHODS: Tracheal samples were taken from wild-type (WT) and Toll-like receptor 3 (TLR3)-knockout (KO) mice. Transient organ culture of murine trachea was performed in the presence or absence of IAV, polyI:C, a synthetic TLR3 ligand, and/or reagents. Subsequently, cilia-driven flow and ciliary motility were analyzed. To evaluate cilia-driven flow, red fluorescent beads were loaded into culture media and movements of the beads onto the tracheal surface were observed using a fluorescence microscope. To evaluate ciliary motility, cilia tips were labeled with Indian ink diluted with culture medium. The motility of ink-labeled cilia tips was recorded by high-speed cameras. RESULTS: Short-term IAV infection significantly increased cilia-driven flow and ciliary beat frequency (CBF) compared with the control level in WT culture. Whereas IAV infection did not elicit any increases of cilia-driven flow and CBF in TLR3-KO culture, indicating that TLR3 was essential to elicit an increase of cilia-driven flow and CBF in response to IAV infection. TLR3 activation by polyI:C readily induced adenosine triphosphate (ATP) release from the trachea and increases of cilia-driven flow and CBF in WT culture, but not in TLR3-KO culture. Moreover, blockade of purinergic P2 receptors (P2Rs) signaling using P2R antagonist, suramin, suppressed polyI:C-mediated increases of cilia-driven flow and CBF, indicating that TLR3-mediated ciliary activation depended on released extracellular ATP and the autocrine ATP-P2R loop. CONCLUSIONS: IAV infection readily increases ciliary activity and cilia-driven flow via TLR3 activation in the airway epithelium, thereby hastening mucociliary clearance and "sweeping" viruses from the airway as an initial host defense response. Mechanically, extracellular ATP release in response to TLR3 activation promotes ciliary activity through autocrine ATP-P2R loop.

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19.

Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.

Divergent Roles of Kupffer Cell TLR2/3 Signaling in Alcoholic Liver Disease and the Protective Role of EGCG.

BACKGROUND & AIMS: Toll-like receptor 2 (TLR2) and TLR3 regulate hepatic immunity under pathological conditions, but their functions and potential drug targets in alcoholic liver disease (ALD) remain poorly understood. METHODS: ALD-associated liver injury were induced in TLR2 knockout (TLR2-/-), TLR3-/-, TLR2-/- bone marrow transplanted (BMT), TLR3-/- BMT, IL-10-/- mice, and their wild-type littermates through ethanol challenge with or without co-administered epigallocatechin-3-gallate (EGCG). Moreover, Kupffer cells were depleted by GdCl3 injection to evaluate their pathogenic roles in ALD. RESULTS: We identified that deficiency of TLR2 and TLR3 significantly alleviated and aggravated ALD-induced liver injury, respectively. Mechanistically, Kupffer cell inactivation, M1 to M2 polarization, and IL-10 production via STAT3 activation contributed to hepatic protection mediated by concurrent TLR2 inhibition and TLR3 agonism. These findings were further confirmed in TLR2 and TLR3 BMT mice. We also identified a novel ALD-protective agent EGCG which directly interacted with Kupffer cell TLR2/3 to induce IL-10 production. Deficiency of IL-10 aggravated ALD injury and blunted EGCG-mediated hepatoprotection while depletion of Kupffer cells partially recovered liver injury but abolished EGCG's actions. CONCLUSIONS: Altogether, our results illustrate the divergent roles of Kupffer cells TLR2/3 in ALD progression via anti-inflammatory cytokine IL-10 production.

A Population of Radio-Resistant Macrophages in the Deep Myenteric Plexus Contributes to Postoperative Ileus Via Toll-Like Receptor 3 Signaling.

Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter's contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-ß reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.

Multiphasic disseminated encephalomyelitis associated with herpes virus infection in a patient with TLR3 deficiency.

We report a case of a 14-year-old girl that presented headache, amaurosis, drowsiness, fever, vomiting and diffuse reduction of muscle strength. She had been diagnosed with ADEM one year before and had a previous diagnosis of Toll-Like 3 receptor deficiency. Cerebrospinal fluid analysis revealed pleocytosis (28/mm3, 12/mm3 red blood cells, 70% lymphocytes cells, 2% monocytes cells, 28% neutrophils), normal total protein (38 pg/mL) and normal glucose level (53/mm3). Studies for CSF oligoclonal bands and serum anti-MOG were negative but polymerase chain reaction (PCR) testing was positive for herpes virus 1. In the first ADEM episode, PCR for herpes virus was also positive. Magnetic resonance imaging (MRI) of the brain revealed disseminated hyperintense lesions on T2-weighted and FLAIR images in the white matter of frontal, parietal and temporal lobes, corresponding to extensive asymmetric areas of demyelination that produced mass effect and gadolinium enhancement. Electroencephalography demonstrated irregular diffuse and generalized slow-wave activity with predominance in frontal region. The diagnosis of multiphasic disseminated encephalomyelitis (MDEM) triggered by herpes simplex virus was made. Herpes virus is a neurotropic virus that can cause a wide variety of neurological infection-triggered autoimmune disorders and that is particularly damaging to the central nervous system in situations of impaired immune system. TLR3 is expressed in astrocytes and dendritic cells of the central nervous system and is essential for natural immunity to herpes simplex. TLR3-deficient patients have already been described with herpes simplex encephalitis. TLR3 deficiency may predispose and explain autoimmune and demyelinating manifestations induced by herpes virus. The association of multiphasic disseminated encephalomyelitis triggered by herpes virus in a patient with TLR3 deficiency has not been previously reported in the literature.

Toll-Like Receptor 3 Deficiency Leads to Altered Immune Responses to Chlamydia trachomatis Infection in Human Oviduct Epithelial Cells.

Reproductive tract pathology caused by Chlamydia trachomatis infection is an important global cause of human infertility. To better understand the mechanisms associated with Chlamydia-induced genital tract pathogenesis in humans, we used CRISPR genome editing to disrupt Toll-like receptor 3 (TLR3) function in the human oviduct epithelial (hOE) cell line OE-E6/E7 in order to investigate the possible role(s) of TLR3 signaling in the immune response to Chlamydia Disruption of TLR3 function in these cells significantly diminished the Chlamydia-induced synthesis of several inflammation biomarkers, including interferon beta (IFN-ß), interleukin-6 (IL-6), interleukin-6 receptor alpha (IL-6Rα), soluble interleukin-6 receptor beta (sIL-6Rß, or gp130), IL-8, IL-20, IL-26, IL-34, soluble tumor necrosis factor receptor 1 (sTNF-R1), tumor necrosis factor ligand superfamily member 13B (TNFSF13B), matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-3. In contrast, the Chlamydia-induced synthesis of CCL5, IL-29 (IFN-λ1), and IL-28A (IFN-λ2) was significantly increased in TLR3-deficient hOE cells compared to their wild-type counterparts. Our results indicate a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw that Chlamydia infection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity, such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3, in hOE cells; however, their expression levels were significantly dysregulated in TLR3-deficient hOE cells. Finally, we demonstrate using hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial lipopolysaccharide (LPS) within Chlamydia inclusions, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection.

Toll-like receptors 2 and 7 mediate coagulation activation and coagulopathy in murine sepsis.

BACKGROUND: Sepsis is a life-threatening condition often manifested as marked inflammation and severe coagulopathy. Toll-like receptors (TLRs) play a pivotal role in inflammation, organ dysfunction and mortality in animal sepsis. OBJECTIVES: To investigate the role of TLR signaling in mediating sepsis-induced coagulopathy (SIC) in a mouse model. METHODS: Polymicrobial sepsis was created by cecal ligation and puncture (CLP) or fecal slurry peritoneal injection. To quantify global clotting function, two viscoelastic assays were performed with rotational thromboelastometry, and the results were presented as maximum clot firmness (MCF): (a) EXTEM to test tissue factor (TF)-initiated clot formation; and (b) FIBTEM to test EXTEM in the presence of a platelet inhibitor, cytochalasin D. Plasma coagulation factors were quantified with ELISA. TF gene expression and protein expression were determined with real-time quantitative reverse transcription PCR and flow cytometry, respectively. RESULTS: Between 4 and 24 hours after CLP surgery, wild-type mice showed significant MCF reduction in both EXTEM and FIBTEM tests. This was accompanied by marked thrombocytopenia and a significant increase in the levels of plasminogen activator inhibitor-1, plasma TF, and D-dimer. In comparison, TLR2-/- and TLR7-/- CLP mice showed preserved MCF and platelet counts, and near-normal plasma TF levels. Bone marrow-derived macrophages treated with a TLR2 agonist Pam3cys-Ser-(Lys)4 (Pam3cys) or a TLR7 agonist (R837) showed marked increases in TF gene expression and protein expression. MicroRNA-146a, a newly identified proinflammatory mediator that is upregulated during sepsis, induced TF production via a TLR7-dependent mechanism. CONCLUSIONS: Murine sepsis leads to an increased procoagulant response, thrombocytopenia, and global coagulopathy. TLR2 and TLR7 play an important role in procoagulant production and in SIC.

Severe influenza pneumonitis in children with inherited TLR3 deficiency.

Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.

Angiotensin II-induced hypertension and cardiac hypertrophy are differentially mediated by TLR3- and TLR4-dependent pathways.

Toll-like receptors (TLR) are key components of the innate immune system that elicit inflammatory responses through the adaptor proteins myeloid differentiation protein 88 (MyD88) and Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-ß (TRIF). Previously, we demonstrated that TRIF mediates the signaling of angiotensin II (ANG II)- induced hypertension and cardiac hypertrophy. Since TRIF is activated selectively by TLR3 and TLR4, our goals in this study were to determine the roles of TLR3 and TLR4 in mediating ANG II-induced hypertension and cardiac hypertrophy, and associated changes in proinflammatory gene expression in heart and kidney. In wild-type (WT) mice, ANG II infusion (1,000 ng·kg-1·min-1 for 3 wk) increased systolic blood pressure and caused cardiac hypertrophy. In ANG II-infused TLR4-deficient mice (Tlr4del), hypertrophy was significantly attenuated despite a preserved or enhanced hypertensive response. In contrast, in TLR3-deficient mice (Tlr3-/-), both ANG II-induced hypertension and hypertrophy were abrogated. In WT mice, ANG II increased the expression of several proinflammatory genes in hearts and kidneys that were attenuated in both TLR4- and TLR3-deficient mice compared with WT. We conclude that ANG II activates both TLR4-TRIF and TLR3-TRIF pathways in a nonredundant manner whereby hypertension is dependent on activation of the TLR3-TRIF pathway and cardiac hypertrophy is dependent on both TLR3-TRIF and TLR4-TRIF pathways. NEW & NOTEWORTHY Angiotensin II (ANG II)-induced hypertension is dependent on the endosomal Toll-like receptor 3 (TLR3)-Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-ß (TRIF) pathway of the innate immune system but not on cell membrane localized TLR4. However, ANG II-induced cardiac hypertrophy is regulated by both TLR4-TRIF and TLR3-TRIF pathways. Thus, ANG II-induced rise in systolic blood pressure is independent of TLR4-TRIF effect on cardiac hypertrophy. The TLR3-TRIF pathway may be a potential target of therapeutic intervention.

TLR3 deficiency exacerbates the loss of epithelial barrier function during genital tract Chlamydia muridarum infection.

PROBLEM: Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact. METHOD OF STUDY: We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot. RESULTS: qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect. CONCLUSION: Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.

Both maternal and placental toll-like receptor activation are necessary for the full development of proteinuric hypertension in mice.

OBJECTIVE: Innate immune system activation and excessive inflammation contributes to hypertension during pregnancy (HTN-preg). Activation of Toll-like receptors (TLRs), the primary innate immune system sensor, is evident in women with HTN-preg and is sufficient to induce pregnancy-dependent, proteinuric hypertension in animals. However, whether HTN-preg is a maternal disease, a placental disease, or both is unclear. We hypothesized that activation of TLR3, the double-stranded RNA sensor, in both maternal systemic and placental cells would be necessary for the full development of HTN-preg in mice. STUDY DESIGN: Various mating schemes generated pregnant mice that lacked TLR3 in maternal cells, paternally-derived placental cells, and both. Mice were then injected with a TLR3 agonist on days 13, 15, and 17 of pregnancy. MAIN OUTCOME MEASURES: Blood pressure, urinary protein excretion, fetal development, maternal vascular endothelial function, and immune system activation were all assessed and compared between groups. RESULTS: Pregnant mice lacking TLR3 in maternal cells as well as pregnant mice lacking TLR3 in placental cells had significantly attenuated increases in systolic blood pressure, urinary protein excretion, fetal demise, and endothelial dysfunction compared to wild-type pregnant mice following TLR3 activation. Pregnant mice lacking TLR3 in both maternal systemic and placental cells were completely resistant to the hypertension, proteinuria, fetal demise, endothelial dysfunction, splenomegaly, and increases in pro-inflammatory immune cells induced by TLR3 activation. CONCLUSIONS: These data suggest that both maternal and placental TLR3 activation are crucial for the full development of HTN-preg and that TLR3 antagonists may be beneficial in some women with HTN-preg.

Loss of toll-like receptor 3 aggravates hepatic inflammation but ameliorates steatosis in mice.

The importance of toll-like receptor (TLR) 4 in the pathogenesis of steatohepatitis has been well documented; however, little is known about the role of TLR3. In this study, we determined whether the depletion of TLR3 modulated hepatic injury in mice and further aimed to provide mechanistic insights into the TLR3-mediated modulation of diet-induced hepatic inflammation and fat accumulation. Hepatic steatosis and inflammatory response were induced by feeding wild-type (WT) or TLR3 knockout mice a high-fat diet for 8 weeks. Primary liver resident cells, including hepatocytes, Kupffer cells, and hepatic stellate cells (HSCs), were treated with palmitic acid. TLR3 knockout mice fed a high-fat diet showed severe hepatic inflammation accompanied by nuclear factor-κB and IRF3 activation, which is mainly induced by the activation of Kupffer cells. Decreased TLR4 expression was restored in hepatic mononuclear cells and Kupffer cells in TLR3 knockout mice compared to that in the WT. Moreover, hepatic steatosis was decreased in TLR3 knockout mice. Hepatocytes from TLR3 knockout mice exhibited reduced expression of cannabinoid receptors. HSCs from TLR3 knockout mice showed decreased expression of the enzymes involved in endocannabinoid synthesis. In conclusion, this study suggests that the selective modulation of TLR3 could be a novel therapeutic target for the treatment of hepatic inflammation and steatosis.

TLR9 Regulates the NF-κB-NLRP3-IL-1ß Pathway Negatively in Salmonella-Induced NKG2D-Mediated Intestinal Inflammation.

TLRs are key sensors for conserved bacterial molecules and play a critical role in host defense against invading pathogens. Although the roles of TLRs in defense against pathogen infection and in maintaining gut immune homeostasis have been studied, the precise functions of different TLRs in response to pathogen infection in the gut remain elusive. The present study investigated the role of TLR signaling in defense against the Gram-negative bacterial pathogen Salmonella typhimurium The results indicated that TLR9-deficient mice were more susceptible to S. typhimurium infection compared with wild-type and TLR2- or TLR4-deficient mice, as indicated by more severe intestinal damage and the highest bacterial load. TLR9 deficiency in intestinal epithelial cells (IECs) augmented the activation of NF-κB and NLRP3 inflammasomes significantly, resulting in increased secretion of IL-1ß. IL-1ß increased the expression of NKG2D on intestinal intraepithelial lymphocytes and NKG2D ligands on IECs, resulting in higher susceptibility of IECs to cytotoxicity of intestinal intraepithelial lymphocytes and damage to the epithelial barrier. We proposed that TLR9 regulates the NF-κB-NLRP3-IL-1ß pathway negatively in Salmonella-induced NKG2D-mediated intestinal inflammation and plays a critical role in defense against S. typhimurium infection and in the protection of intestinal integrity.

Toll-like receptor 3 deficiency decreases epileptogenesis in a pilocarpine model of SE-induced epilepsy in mice.

OBJECTIVE: Epilepsy affects 60 million people worldwide. Despite the development of antiepileptic drugs, up to 35% of patients are drug refractory with uncontrollable seizures. Toll-like receptors (TLRs) are central components of the nonspecific innate inflammatory response. Because TLR3 was recently implicated in neuronal plasticity, we hypothesized that it may contribute to the development of epilepsy after status epilepticus (SE). METHODS: To test the involvement of TLR3 in epileptogenesis, we used the pilocarpine model for SE in TLR3-deficient mice and their respective wild-type controls. In this model, a single SE event leads to spontaneous recurrent seizures (SRS). Two weeks after SE, mice were implanted with wireless electroencephalography (EEG) transmitters for up to 1 month. The impact of TLR3 deficiency on SE was assessed using separate cohorts of mice regarding EEG activity, seizure progression, hippocampal microglial distribution, and expression of the proinflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)ß. RESULTS: Our data indicate that TLR3 deficiency reduced SRS, microglial activation, and the levels of the proinflammatory cytokines TNFα and IFNß, and increased survival following SE. SIGNIFICANCE: This study reveals novel insights into the pathophysiology of epilepsy and the contribution of TLR3 to disease progression. Our results identify the TLR3 pathway as a potential future therapeutic target in SE.

--LUBAC deficiency perturbs TLR3 signaling to cause immunodeficiency and autoinflammation.

The linear ubiquitin chain assembly complex (LUBAC), consisting of SHANK-associated RH-domain-interacting protein (SHARPIN), heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), and HOIL-1-interacting protein (HOIP), is a critical regulator of inflammation and immunity. This is highlighted by the fact that patients with perturbed linear ubiquitination caused by mutations in the Hoip or Hoil-1 genes, resulting in knockouts of these proteins, may simultaneously suffer from immunodeficiency and autoinflammation. TLR3 plays a crucial, albeit controversial, role in viral infection and tissue damage. We identify a pivotal role of LUBAC in TLR3 signaling and discover a functional interaction between LUBAC components and TLR3 as crucial for immunity to influenza A virus infection. On the biochemical level, we identify LUBAC components as interacting with the TLR3-signaling complex (SC), thereby enabling TLR3-mediated gene activation. Absence of LUBAC components increases formation of a previously unrecognized TLR3-induced death-inducing SC, leading to enhanced cell death. Intriguingly, excessive TLR3-mediated cell death, induced by double-stranded RNA present in the skin of SHARPIN-deficient chronic proliferative dermatitis mice (cpdm), is a major contributor to their autoinflammatory skin phenotype, as genetic coablation of Tlr3 substantially ameliorated cpdm dermatitis. Thus, LUBAC components control TLR3-mediated innate immunity, thereby preventing development of immunodeficiency and autoinflammation.