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.

X-Linked TLR7 Deficiency Underlies Critical COVID-19 Pneumonia in a Male Patient with Ataxia-Telangiectasia.

BACKGROUND: Coronavirus disease 2019 (COVID-19) exhibits a wide spectrum of clinical manifestations, ranging from asymptomatic to critical conditions. Understanding the mechanism underlying life-threatening COVID-19 is instrumental for disease prevention and treatment in individuals with a high risk. OBJECTIVES: We aimed to identify the genetic cause for critical COVID-19 pneumonia in a patient with a preexisting inborn error of immunity (IEI). METHODS: Serum levels of specific antibodies against the virus and autoantibodies against type I interferons (IFNs) were measured. Whole exome sequencing was performed, and the impacts of candidate gene variants were investigated. We also evaluated 247 ataxia-telangiectasia (A-T) patients in the Iranian IEI registry. RESULTS: We report a 7-year-old Iranian boy with a preexisting hyper IgM syndrome who developed critical COVID-19 pneumonia. IgM only specific COVID-19 immune response was detected but no autoantibodies against type I IFN were observed. A homozygous deleterious mutation in the ATM gene was identified, which together with his antibody deficiency, radiosensitivity, and neurological signs, established a diagnosis of A-T. Among the 247 A-T patients evaluated, 36 had SARS-CoV-2 infection, but all had mild symptoms or were asymptomatic except the index patient. A hemizygous deleterious mutation in the TLR7 gene was subsequently identified in the patient. CONCLUSIONS: We report a unique IEI patient with combined ATM and TLR7 deficiencies. The two genetic defects underlie A-T and critical COVID-19 in this patient, respectively.

X-linked recessive TLR7 deficiency in ~1% of men under 60 years old with life-threatening COVID-19.

Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract.

Absence of Toll-like receptor 7 protects mice against Pseudomonas aeruginosa pneumonia.

Toll-like receptor 7 (TLR7) is a sensor of microbial ssRNA that participates in the immune response process in many diseases. We herein sought to establish the role of TLR7 in Pseudomonas aeruginosa pneumonia. Pneumonia model was created by intratracheally injecting Pseudomonas aeruginosa and the effects of TLR7 on survival, bacterial burden, lung pathology, cytokine and chemokine production, and pulmonary leukocyte recruitment were measured after Pseudomonas aeruginosa challenge. TLR7 expression was significantly elevated in WT mice after Pseudomonas aeruginosa infection. TLR7-/- mice demonstrated enhanced survival, bacterial clearance, leukocyte infiltration, and macrophages phagocytic activity, and decreased pathology and capillary leakage. Besides, improved survival and bacterial clearance were observed in WT mice treated with TLR7 antagonist IRS661. More importantly, lack of TLR7 suppressed pro-inflammatory cytokine production and induced anti-inflammatory cytokine production in mice lungs. Finally, neutralized IL-10 damaged the bacterial clearance ability of TLR7 deficient mice, leading to decreased survival. Collectively, absence of TLR7 provided protective effects during Pseudomonas aeruginosa pneumonia and suggested that TLR7 could act as a novel immune target to treat clinical cases with Pseudomonas aeruginosa pneumonia.

Effect of TLR7 gene expression mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention role of IFN-γ.

OBJECTIVE: To explore the mechanism of TLR7 mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention effect of IFN-γ in the process. MATERIALS AND METHODS: The experimental animals were 70 C57BL/6J female mice of clean grade, which were divided into 7 groups according to different treatment protocols, including Normal group, Asthma group, Model+1-MT group, Model+IFN-γ group, Model+TLR7 agonist group, TLR7 deficient group, and Model+TLR7 deficient group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissues. The positive expression rates of TLR7, p-IKKα and NF-κBp65 were detected by immunohistochemistry. bronchoalveolar lavage fluid (BALF) cells were classified and counted. The contents of interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ in BALF supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Following isolation, culture and plasmid construction of airway smooth muscle cells (ASMCS) from normal mice and asthmatic mice, cells were transfected and divided into the Control group, pcDNA-TLR7 NC group, siRNA-TLR7 NC group, pcDNA-TLR7 group, siRNA-TLR7 group, Asatone group, Triptolide group, and pcDNA-TLR7 +Asatone group. The expression of TLR7, IDO, p-IKKα and NF-κBp65 was detected by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to detect the proliferation of ASMCS. The cell cycle and apoptosis of ASMCS were detected by flow cytometry. RESULTS: HE staining showed successful modeling of asthma. Immunohistochemical test showed that the positive expression rate of TLR7 in the Asthma group was significantly decreased, and that of IKKα and NF-κBp65 was significantly increased, with significantly increased IL-4, IL-10, IL-12 and IFN-γ levels (all p<0.05). Model+1-MT group and Model+TLR7 deficient group had a large number of inflammatory cell infiltration, increased IL-4, IL-10, IL-12 and IFN-γ levels, decreased expression levels of TLR7 and IDO, and increased expression of p-IKKα and NF-κBp65 (all p<0.05); while the opposites results were detected in Model+IFN-γ group and Model+TLR7 agonist group (all p<0.05). Cell transfection experiments revealed that pcDNA-TLR7 group and Triptolide group had increased TLR7 expression while decreased p-IKKα and NF-κBp65, decreased proliferation level, and increased cell apoptosis (all p<0.05); while the contrary results were found in siRNA-TLR7 group and Asatone group (all p<0.05); yet without significant difference in pcDNA-TLR7+Asatone group (all p>0.05). CONCLUSIONS: Upregulation of TLR7 can inhibit the activation of NF-κB signaling pathway, reduces airway inflammation, inhibits ASMCS proliferation and thus promotes cell apoptosis in asthmatic mice. Besides, IFN-γ can exert a protective role in suppressing the progression of inflammation in asthma.

TLR7 Expression Aggravates Invasive Pulmonary Aspergillosis by Suppressing Anti-Aspergillus Immunity of Macrophages.

Toll-like receptors (TLRs) play a critical role in early immune recognition of Aspergillus, which can regulate host defense during invasive pulmonary Aspergillosis (IPA). However, the role of TLR7 in the pathogenesis of IPA remains unknown. In this study, an in vivo model of IPA was established to investigate the contribution of TLR7 to host anti-Aspergillus immunity upon invasive pulmonary Aspergillus fumigatus infection. The effects of TLR7 on phagocytosis and killing capacities of A. fumigatus by macrophages and neutrophils were investigated in vitro We found that TLR7 knockout mice exhibited lower lung inflammatory response and tissue injury, higher fungal clearance, and greater survival in an in vivo model of IPA compared with wild-type mice. TLR7 activation by R837 ligand led to wild-type mice being more susceptible to invasive pulmonary Aspergillus fumigatus infection. Macrophages, but not neutrophils, were required for the protection against IPA observed in TLR7 knockout mice. Mechanistically, TLR7 impaired phagocytosis and killing of A. fumigatus by macrophages but not neutrophils. Together, these data identify TLR7 as an important negative regulator of anti-Aspergillus innate immunity in IPA, and we propose that targeting TLR7 will be beneficial in the treatment of IPA.

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.

Toll-like receptor 7 deficiency mitigates hyperoxia-induced acute lung injury in mice.

INTRODUCTION: Toll-like receptor (TLR) 7 is an important mediator in inflammation. However, its role in hyperoxia-induced acute lung injury (HALI) remains to be elucidated. METHODS: C57BL/6 wild-type and C57BL/6 background TLR 7 deficiency mice were exposed to hyperoxia to stimulate HALI in airtight cages. Animals were sacrificed at 72 h post hyperoxia or room air exposure. Lung injury indicators were measured. Moreover, soluble epoxide hydrolase (sEH) activity was detected by a 14, 15-EET/DHET ELISA kit. Activation of activator protein (AP)-1 and nuclear factor kappa-B (NF-κB) was detected with enzyme linked immunosorbent assay kits. RESULTS: Our data revealed that pulmonary histological assay and wet to dry weight ratio, myeloperoxidase and malondialdehyde activity were reduced in TLR 7 deficiency mice compared with wild-type mice. Moreover, hyperoxia-caused elevation of sEH activity was reduced in TLR 7 deficiency mice. Transcription factors AP-1 activation was significantly inhibited in TLR 7 deficiency mice compared with wild-type mice. Similarly, the activation of NF-κB was reduced in TLR 7 deficiency mice. Tumor necrosis factor-α and interleukin-1ß, potent proinflammatory cytokines, were reduced in TLR 7 deficiency mice. CONCLUSION: TLR 7 deficiency is associated with inhibition of inflammation in HALI in mice.

Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.

PA-X antagonises MAVS-dependent accumulation of early type I interferon messenger RNAs during influenza A virus infection.

The sensing of viral nucleic acids by the innate immune system activates a potent antiviral response in the infected cell, a key component of which is the expression of genes encoding type I interferons (IFNs). Many viruses counteract this response by blocking the activation of host nucleic acid sensors. The evolutionarily conserved influenza A virus (IAV) protein PA-X has been implicated in suppressing the host response to infection, including the expression of type I IFNs. Here, we characterise this further using a PA-X-deficient virus of the mouse-adapted PR8 strain to study activation of the innate immune response in a mouse model of the early response to viral infection. We show that levels of Ifna4 and Ifnb1 mRNAs in the lungs of infected mice were elevated in the absence of PA-X and that this was completely dependent on MAVS. This therefore suggests a role for PA-X in preventing the accumulation of early type I IFN mRNAs in the lung during IAV infection.

Toll-like receptor 7 deficiency promotes survival and reduces adverse left ventricular remodelling after myocardial infarction.

AIMS: The Toll-like receptor 7 (TLR7) is an intracellular innate immune receptor activated by nucleic acids shed from dying cells leading to activation of the innate immune system. Since innate immune system activation is involved in the response to myocardial infarction (MI), this study aims to identify if TLR7 is involved in post-MI ischaemic injury and adverse remodelling after MI. METHODS AND RESULTS: TLR7 involvement in MI was investigated in human tissue from patients with ischaemic heart failure, as well as in a mouse model of permanent left anterior descending artery occlusion in C57BL/6J wild type and TLR7 deficient (TLR7-/-) mice. TLR7 expression was up-regulated in human and mouse ischaemic myocardium after MI. Compared to wild type mice, TLR7-/- mice had less acute cardiac rupture associated with blunted activation of matrix metalloproteinase 2, increased expression of tissue inhibitor of metalloproteinase 1, recruitment of more myofibroblasts, and the formation of a myocardial scar with higher collagen fibre density. Furthermore, inflammatory cell influx and inflammatory cytokine expression post-MI were reduced in the TLR7-/- heart. During a 28-day follow-up after MI, TLR7 deficiency resulted in less chronic adverse left ventricular remodelling and better cardiac function. Bone marrow (BM) transplantation experiments showed that TLR7 deficiency in BM-derived cells preserved cardiac function after MI. CONCLUSIONS: In acute MI, TLR7 mediates the response to acute cardiac injury and chronic remodelling probably via modulation of post-MI scar formation and BM-derived inflammatory infiltration of the myocardium.

Mer Receptor Tyrosine Kinase Signaling Prevents Self-Ligand Sensing and Aberrant Selection in Germinal Centers.

Mer tyrosine kinase (Mer) signaling maintains immune tolerance by clearing apoptotic cells (ACs) and inducing immunoregulatory signals. We previously showed that Mer-deficient mice (Mer-/-) have increased germinal center (GC) responses, T cell activation, and AC accumulation within GCs. Accumulated ACs in GCs can undergo necrosis and release self-ligands, which may influence the outcome of a GC response and selection. In this study, we generated Mer-/- mice with a global MyD88, TLR7, or TLR9 deficiency and cell type-specific MyD88 deficiency to study the functional correlation between Mer and TLRs in the development of GC responses and autoimmunity. We found that GC B cell-intrinsic sensing of self-RNA, but not self-DNA, released from dead cells accumulated in GCs drives enhanced GC responses in Mer-/- mice. Although self-ligands directly affect GC B cell responses, the loss of Mer in dendritic cells promotes enhanced T cell activation and proinflammatory cytokine production. To study the impact of Mer deficiency on the development of autoimmunity, we generated autoimmune-prone B6.Sle1b mice deficient in Mer (Sle1bMer-/-). We observed accelerated autoimmunity development even under conditions where Sle1bMer-/- mice did not exhibit increased AC accumulation in GCs compared with B6.Sle1b mice, indicating that Mer immunoregulatory signaling in APCs regulates B cell selection and autoimmunity. We further found significant expansion, retention, and class-switching of autoreactive B cells in GCs under conditions where ACs accumulated in GCs of Sle1bMer-/- mice. Altogether, both the phagocytic and immunomodulatory functions of Mer regulate GC responses to prevent the development of autoimmunity.

Induction of Type I Interferon through a Noncanonical Toll-Like Receptor 7 Pathway during Yersinia pestis Infection.

Yersinia pestis causes bubonic, pneumonic, and septicemic plague, diseases that are rapidly lethal to most mammals, including humans. Plague develops as a consequence of bacterial neutralization of the host's innate immune response, which permits uncontrolled growth and causes the systemic hyperactivation of the inflammatory response. We previously found that host type I interferon (IFN) signaling is induced during Y. pestis infection and contributes to neutrophil depletion and disease. In this work, we show that type I IFN expression is derived from the recognition of intracellular Y. pestis by host Toll-like receptor 7 (TLR7). Type I IFN expression proceeded independent of myeloid differentiation factor 88 (MyD88), which is the only known signaling adaptor for TLR7, suggesting that a noncanonical mechanism occurs in Y. pestis-infected macrophages. In the murine plague model, TLR7 was a significant contributor to the expression of serum IFN-ß, whereas MyD88 was not. Furthermore, like the type I IFN response, TLR7 contributed to the lethality of septicemic plague and was associated with the suppression of neutrophilic inflammation. In contrast, TLR7 was important for defense against disease in the lungs. Together, these data demonstrate that an atypical TLR7 signaling pathway contributes to type I IFN expression during Y. pestis infection and suggest that the TLR7-driven type I IFN response plays an important role in determining the outcome of plague.

Toll-like receptor 7 deficiency protects apolipoprotein E-deficient mice from diet-induced atherosclerosis.

Toll-like receptor 7 (TLR7) mediates autoantigen and viral RNA-induced cytokine production. Increased TLR7 expression in human atherosclerotic lesions suggests its involvement in atherogenesis. Here we demonstrated TLR7 expression in macrophages, smooth muscle cells (SMCs), and endothelial cells from mouse atherosclerotic lesions. To test a direct participation of TLR7 in atherosclerosis, we crossbred TLR7-deficient (Tlr7 -/-) mice with apolipoprotein E-deficient (Apoe -/-) mice and produced Apoe -/- Tlr7 -/- and Apoe -/- Tlr7 +/+ littermates, followed by feeding them an atherogenic diet to produce atherosclerosis. Compared to Apoe -/- Tlr7 +/+ mice, Apoe -/- Tlr7 -/- mice showed reduced aortic arch and sinus lesion areas. Reduced atherosclerosis in Apoe -/- Tlr7 -/- mice did not affect lesion macrophage-positive area and CD4+ T-cell number per lesion area, but reduced lesion expression of inflammatory markers major histocompatibility complex-class II and IL6, lesion matrix-degrading proteases cathepsin S and matrix metalloproteinase-9, and systemic serum amyloid A levels. TLR7 deficiency also reduced aortic arch SMC loss and lesion intima and media cell apoptosis. However, TLR7 deficiency did not affect aortic wall elastin fragmentation and collagen contents, or plasma lipoproteins. Therefore, TLR7 contributes to atherogenesis in Apoe -/- mice by regulating lesion and systemic inflammation. A TLR7 antagonist may mitigate atherosclerosis.

Toll-like receptor (TLR)7 and TLR9 agonists enhance interferon (IFN) beta-1a's immunoregulatory effects on B cells in patients with relapsing-remitting multiple sclerosis (RRMS).

We report that B cells from patients with RRMS have decreased endogenous IFN-ß secretion and deficient IFN receptor (IFNAR)1/2 and TLR7 gene expression in comparison to healthy controls (HCs), which may contribute to disregulation of cytokine secretion by B cells. We propose that TLR7 and TLR9 stimulation with loxorubin (LOX) and CpG, in combination with exogenous IFN-ß may effectively reconstitute endogenous IFN-ß production deficit and induce the secretion of immunoregulatory cytokines by B cells. Both LOX/IFN-ß and CpG/IFN-ß in-vitro treatments of B cells from RRMS patients induced higher endogenous IFN-ß gene expression in comparison to the exogenous IFN-ß alone. CpG/IFN-ß combination induced higher secretion of IL-10, TGF-ß, and IL-27 in comparison to stimulation with IFN-ß. Our study provides a basis for future clinical studies employing IFN-ß and TLR7/9 agonists, which may enhance the resolution of the inflammatory response in RRMS.

Cardiac RNA induces inflammatory responses in cardiomyocytes and immune cells via Toll-like receptor 7 signaling.

We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes and immune cells and contributes to myocardial ischemia/reperfusion injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect is unknown. Here we hypothesize that exRNA directly induces inflammation through specific Toll-like receptors (TLRs). To test the hypothesis, we treated rat neonatal cardiomyocytes, mouse bone marrow-derived macrophages (BMDM), or mouse neutrophils with RNA (2.5-10 µg/ml) isolated from rat cardiomyocytes or the hearts from mouse, rat, and human. We found that cellular RNA induced production of several cytokines such as macrophage inflammatory protein-2 (MIP-2), ILs, TNFα, and the effect was completely diminished by RNase, but not DNase. The RNA-induced cytokine production was partially inhibited in cells treated with TLR7 antagonist or genetically deficient in TLR7. Deletion of myeloid differentiation primary response protein 88 (MyD88), a downstream adapter of TLRs including TLR7, abolished the RNA-induced MIP-2 production. Surprisingly, genetic deletion of TLR3 had no impact on the RNA-induced MIP-2 response. Importantly, extracellular RNA released from damaged cardiomyocytes also induced cytokine production. Finally, mice treated with 50 µg of RNA intraperitoneal injection exhibited acute peritonitis as evidenced by marked neutrophil and monocyte migration into the peritoneal space. Together, these data demonstrate that exRNA of cardiac origin exhibits a potent pro-inflammatory property in vitro and in vivo and that exRNA induces cytokine production through TLR7-MyD88 signaling.

TLR9 signalling in microglia attenuates seizure-induced aberrant neurogenesis in the adult hippocampus.

Pathological conditions such as epilepsy cause misregulation of adult neural stem/progenitor populations in the adult hippocampus in mice, and the resulting abnormal neurogenesis leads to impairment in learning and memory. However, how animals cope with abnormal neurogenesis remains unknown. Here we show that microglia in the mouse hippocampus attenuate convulsive seizure-mediated aberrant neurogenesis through the activation of Toll-like receptor 9 (TLR9), an innate immune sensor known to recognize microbial DNA and trigger inflammatory responses. We found that microglia sense self-DNA from degenerating neurons following seizure, and secrete tumour necrosis factor-α, resulting in attenuation of aberrant neurogenesis. Furthermore, TLR9 deficiency exacerbated seizure-induced cognitive decline and recurrent seizure severity. Our findings thus suggest the existence of bidirectional communication between the innate immune and nervous systems for the maintenance of adult brain integrity.

Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses.

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

[Effect of toll-like receptor (TLR) 7 deficiencies on the in vivo immune response against Schistosoma japonicum].

OBJECTIVE: To explore the toll-like receptor 7 knocked out (TLR7-/-) mice immune response against Schistosoma japonicum. METHODS: C57BL/6 mice (WT) and TLR7-/- mice (TLR7-/-) were infected with 20 S. japonicum cercariae via shaved abdomen. There were nine mice in each group. At 6 weeks post-infection, mice were sacrificed. Adult worms were harvested by perfusion of the portal venous system, and the number of adult worms was determined. At the time of perfusion, livers were collected, weighed, and digested overnight with 5% potassium hydroxide, and eggs were counted. In addition, spleens were aseptically harvested when WT and TLR7-/- mice were sacrificed at day zero and 6 weeks after S. japonicum infection. After 72 hours of the co-culture with or without S. japonicum eggs, the culture supernatants were collected for cytokine assays by ELISA assay. RESULTS: At 6 weeks after infection, there was no significant difference in number of worms [(10.5 +/- 3.3) vs (9.8 +/- 5.2)] and eggs per gram of liver tissue [(38 251.9 +/- 4 891.5) vs (38 160.9 +/- 3 341.0)] between WT and TLR7-/- mice. As for Th1/Th2 cytokine secretion from spleen cells, the levels of TNF-alpha [(43.7 +/- 9.8) pg/ml] and INF-gamma [(215.2 +/- 35.4) pg/ml] from TLR7-/- infected mice were lower than those of WT infected mice[(63.4 +/- 22.9) pg/ml, (383.5 +/- 253.3) pg/ml]. For Th2 cytokines detection, the production of IL-10 [(1702.6 +/- 572.3) pg/ml] and IL-4 [(59.5 +/- 10.1) pg/ml] from TLR7-/- mice were higher than those of WT mice [(595.2 +/- 386.3) pg/ml, (8.3 +/- 0.9) pg/ml] (P < 0.05, P < 0.01), while IL-4 level [(63.9 +/- 33.9) pg/ml] from TLR7-/- infected mice was higher than those of WT infected mice [(23.3 +/- 11.5) pg/ml]. CONCLUSION: TLR7-/- mice has a dominant Th2 response under the normal state. The absence of TLR7 does not influence the immune response against S. japonicum infection at 6 weeks post-infection.

Extracellular microRNAs activate nociceptor neurons to elicit pain via TLR7 and TRPA1.

Intracellular microRNAs (miRNAs) are key regulators of gene expression. The role of extracellular miRNAs in neuronal activation and sensory behaviors are unknown. Here we report an unconventional role of extracellular miRNAs for rapid excitation of nociceptor neurons via toll-like receptor-7 (TLR7) and its coupling to TRPA1 ion channel. miRNA-let-7b induces rapid inward currents and action potentials in dorsal root ganglion (DRG) neurons. These responses require the GUUGUGU motif, only occur in neurons coexpressing TLR7 and TRPA1, and are abolished in mice lacking Tlr7 or Trpa1. Furthermore, let-7b induces TLR7/TRPA1-dependent single-channel activities in DRG neurons and HEK293 cells overexpressing TLR7/TRPA1. Intraplantar injection of let-7b elicits rapid spontaneous pain via TLR7 and TRPA1. Finally, let-7b can be released from DRG neurons by neuronal activation, and let-7b inhibitor reduces formalin-induced TRPA1 currents and spontaneous pain. Thus, secreted extracellular miRNAs may serve as novel pain mediators via activating TLR7/TRPA1 in nociceptor neurons.