Herpes simplex encephalitis due to a mutation in an E3 ubiquitin ligase.

Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Autophagy Deregulation in HIV-1-Infected Cells Increases Extracellular Vesicle Release and Contributes to TLR3 Activation.

Human immunodeficiency virus type 1 (HIV-1) infection can result in HIV-associated neurocognitive disorder (HAND), a spectrum of disorders characterized by neurological impairment and chronic inflammation. Combined antiretroviral therapy (cART) has elicited a marked reduction in the number of individuals diagnosed with HAND. However, there is continual, low-level viral transcription due to the lack of a transcription inhibitor in cART regimens, which results in the accumulation of viral products within infected cells. To alleviate stress, infected cells can release accumulated products, such as TAR RNA, in extracellular vesicles (EVs), which can contribute to pathogenesis in neighboring cells. Here, we demonstrate that cART can contribute to autophagy deregulation in infected cells and increased EV release. The impact of EVs released from HIV-1 infected myeloid cells was found to contribute to CNS pathogenesis, potentially through EV-mediated TLR3 (Toll-like receptor 3) activation, suggesting the need for therapeutics to target this mechanism. Three HIV-1 TAR-binding compounds, 103FA, 111FA, and Ral HCl, were identified that recognize TAR RNA and reduce TLR activation. These data indicate that packaging of viral products into EVs, potentially exacerbated by antiretroviral therapeutics, may induce chronic inflammation of the CNS observed in cART-treated patients, and novel therapeutic strategies may be exploited to mitigate morbidity.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons.

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

UPK3A+ umbrella cell damage mediated by TLR3-NR2F6 triggers programmed destruction of urothelium in Hunner-type interstitial cystitis/painful bladder syndrome.

Urothelial damage and barrier dysfunction emerge as the foremost mechanisms in Hunner-type interstitial cystitis/bladder pain syndrome (HIC). Although treatments aimed at urothelial regeneration and repair have been employed, their therapeutic effectiveness remains limited due to the inadequate understanding of specific cell types involved in damage and the lack of specific molecular targets within these mechanisms. Therefore, we harnessed single-cell RNA sequencing to elucidate the heterogeneity and developmental trajectory of urothelial cells within HIC bladders. Through reclustering, we identified eight distinct clusters of urothelial cells. There was a significant reduction in UPK3A+ umbrella cells and a simultaneous increase in progenitor-like pluripotent cells (PPCs) within the HIC bladder. Pseudotime analysis of the urothelial cells in the HIC bladder revealed that cells faced challenges in differentiating into UPK3A+ umbrella cells, while PPCs exhibited substantial proliferation to compensate for the loss of UPK3A+ umbrella cells. The urothelium in HIC remains unrepaired, despite the substantial proliferation of PPCs. Thus, we propose that inhibiting the pivotal signaling pathways responsible for the injury to UPK3A+ umbrella cells is paramount for restoring the urothelial barrier and alleviating lower urinary tract symptoms in HIC patients. Subsequently, we identified key molecular pathways (TLR3 and NR2F6) associated with the injury of UPK3A+ umbrella cells in HIC urothelium. Finally, we conducted in vitro and in vivo experiments to confirm the potential of the TLR3-NR2F6 axis as a promising therapeutic target for HIC. These findings hold the potential to inhibit urothelial injury, providing promising clues for early diagnosis and functional bladder self-repair strategies for HIC patients. © 2024 The Pathological Society of Great Britain and Ireland.

Motor, Cognitive, and Behavioral Impairment in TLR3 and TLR9 Deficient Male Mice: Insights into the Non-Immunological Roles of Toll-Like Receptors.

BACKGROUND: Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to infection or injury. Recent studies have uncovered their intriguing functions as moonlighting proteins involved in various biological processes, including development, learning, and memory. However, the specific functions of individual TLRs are still largely unknown. AIMS: We investigated the effects of TLR3 and TLR9 receptor deficiency on motor, cognitive, and behavioral functions during development using genetically modified male mice of different ages. METHODS: We evaluated the motor coordination, anxiety-like behavior, spatial learning, and working memory of male mice lacking the TLR3 and TLR9 genes at different ages (two, four, six, and eight months) using the rotarod, open field, water maze, and T-maze tests. RESULTS: We observed that the deletion of either TLR3 or TLR9 resulted in impaired motor performance. Furthermore, young TLR3-deficient mice exhibited reduced anxiety-like behavior and spatial learning deficits; however, their working memory was unaffected. In contrast, young TLR9-knockout mice showed hyperactivity and a tendency toward decreased working memory. CONCLUSIONS: These findings provide valuable insights into the broader roles of the TLR system beyond the innate immune response, revealing its involvement in pathways associated with the central nervous system. Importantly, our results establish a strong association between the endosomal receptors TLR3 and TLR9 and the performance of motor, cognitive, and behavioral tasks that change over time. This study contributes to the growing body of research on the multifaceted functions of TLRs and enhances our understanding of their participation in non-immune-related processes.

TMEM2 suppresses TLR3-mediated IFN-ß/ISG56/CXCL10 expression in BEAS-2B bronchial epithelial cells.

BACKGROUND: Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-ß and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. METHODS AND RESULTS: TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-ß. The expression of TMEM2, IFN-ß, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-ß, CXCL10, and ISG56, while IFN-ß-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. CONCLUSIONS: TMEM2 knockdown enhanced TLR3-mediated IFN-ß, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.

RNA is a pro-apoptotic target of cisplatin in cancer cell lines and C. elegans.

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

Endoplasmic reticulum stress enhances the expression of TLR3-induced TSLP by airway epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Discovery of an ellipticine derivative as TLR3 inhibitor against influenza A virus and SARS-CoV-2.

Influenza and COVID-19 continue to pose global threats to public health. Classic antiviral drugs have certain limitations, coupled with frequent viral mutations leading to many drugs being ineffective, the development of new antiviral drugs is urgent. Meanwhile, the invasion of influenza virus can cause an immune response, and an excessive immune response can generate a large number of inflammatory storms, leading to tissue damage. Toll-like receptor 3 (TLR3) recognizes virus dsRNA to ignite the innate immune response, and inhibit TLR3 can block the excess immune response and protect the host tissues. Taking TLR3 as the target, SMU-CX1 was obtained as the specific TLR3 inhibitor by high-throughput screening of 15,700 compounds with IC50 value of 0.11 µM. Its anti-influenza A virus activity with IC50 ranged from 0.14 to 0.33 µM against multiple subtypes of influenza A virus and also showed promising anti-SARS-CoV-2 activity with IC50 at 0.43 µM. Primary antiviral mechanism study indicated that SMU-CX1 significantly inhibited PB2 and NP protein of viruses, it can also inhibit inflammatory factors in host cells including IFN-ß, IP-10 and CCL-5. In conclusion, this study demonstrates the potential of SMU-CX1 in inhibiting IAV and SARS-CoV-2 activity, thereby offering a novel approach for designing antiviral drugs against highly pathogenic viruses.

Possible involvement of DExD/H box helicase 60 in synovial inflammation of rheumatoid arthritis: role of toll-like receptor 3 signaling.

BACKGROUND: Innate immunity is known to be implicated in the etiology of synovitis in rheumatoid arthritis (RA). However, details of the molecular mechanisms have not been fully clarified. DExD/H-box helicase 60 (DDX60), a putative RNA helicase, is of consequence in anti-viral innate immune reactions followed by inflammation. Although DDX60 is involved in the pathogenesis of autoimmune diseases such as systemic lupus nephritis, the role of DDX60 in RA has not been elucidated. The objective of this study was to examine the expression and the role of DDX60 in RA synovial inflammation. METHODS AND RESULTS: DDX60 protein expression was investigated by immunohistochemistry in synovial tissues resected from 4 RA and 4 osteoarthritis (OA) patients. We found that synovial DDX60 expression was more intense in RA than in OA. Treatment of human rheumatoid fibroblast-like synoviocytes in culture with polyinosinic-polycytidylic acid, a Toll-like receptor 3 (TLR3) ligand, increased DDX60 protein and mRNA expression. A knockdown experiment of DDX60 using RNA interference revealed a decrease in the expression of poly IC-induced C-X-C motif chemokine ligand 10 (CXCL10) which induces lymphocyte chemotaxis. CONCLUSIONS: The synovial DDX60 was more expressed in RA patients than in OA. In human RFLS, DDX60 stimulated by TLR3 signaling affected CXCL10 expression. DDX60 may contribute to synovial inflammation in RA.

Nucleic acid sensing Toll-like receptors 3 and 9 play complementary roles in the development of bacteremia after nasal colonization associated with influenza co-infection.

Streptococcus pneumoniae can cause mortality in infant, elderly, and immunocompromised individuals owing to invasion of bacteria to the lungs, the brain, and the blood. In building strategies against invasive infections, it is important to achieve greater understanding of how the pneumococci are able to survive in the host. Toll-like receptors (TLRs), critically important components in the innate immune system, have roles in various stages of the development of infectious diseases. Endosomal TLRs recognize nucleic acids of the pathogen, but the impact on the pneumococcal diseases of immune responses from signaling them remains unclear. To investigate their role in nasal colonization and invasive disease with/without influenza co-infection, we established a mouse model of invasive pneumococcal diseases directly developing from nasal colonization. TLR9 KO mice had bacteremia more frequently than wildtype in the pneumococcal mono-infection model, while the occurrence of bacteremia was higher among TLR3 KO mice after infection with influenza in advance of pneumococcal inoculation. All TLR KO strains showed poorer survival than wildtype after the mice had bacteremia. The specific and protective role of TLR3 and TLR9 was shown in developing bacteremia with/without influenza co-infection respectively, and all nucleic sensing TLRs would contribute equally to protecting sepsis after bacteremia.

Lipid Nanoparticle Encapsulation Empowers Poly(I:C) to Activate Cytoplasmic RLRs and Thereby Increases Its Adjuvanticity.

Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.

The Functional Mechanisms of Toll-Like Receptor 3 and Its Implications in Digestive System Tumors.

Toll-like receptor 3 (TLR3) is a prominent member of the Toll-like receptor (TLR) family and has the ability to recognize and bind intracellular double-stranded RNA (dsRNA). Once triggered by a viral infection or other pathological condition, TLR3 activates immune cells and induces the production of interferons and other immune response molecules. Additionally, TLR3 is considered an important immune modulator, as it can regulate cell apoptosis and promote anticancer immunity. The investigation and application of TLR3 agonists in digestive system tumors have attracted widespread attention and are regarded as a promising cancer treatment strategy with potential clinical applications. TLR3 expression levels are generally elevated in most digestive system tumors, and higher TLR3 expression is associated with a better prognosis. Therefore, TLR3 has emerged as a novel therapeutic target for digestive system tumors. It has been used in combination with chemotherapy, radiotherapy, and targeted therapy and demonstrated excellent efficacy and tolerability. This has provided new ideas and hopes for the treatment of digestive system tumors. This review discusses the mechanisms of TLR3 and its frontier research in digestive system tumors.

The effect of TLR3 priming conditions on MSC immunosuppressive properties.

BACKGROUND: Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory properties, making them suitable for cell therapy. Toll-like receptors (TLRs) in MSCs respond to viral load by secreting immunosuppressive or proinflammatory molecules. The expression of anti-inflammatory molecules in MSCs can be altered by the concentration and duration of exposure to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)). This study aimed to optimize the preconditioning of MSCs with poly(I:C) to increase immunosuppressive effects and to identify MSCs with activated TLR3 (prMSCs). METHODS: Flow cytometry and histochemical staining were used to analyze MSCs for immunophenotype and differentiation potential. MSCs were exposed to poly(I:C) at 1 and 10 µg/mL for 1, 3, and 24 h, followed by determination of the expression of IDO1, WARS1, PD-L1, TSG-6, and PTGES2 and PGE2 secretion. MSCs and prMSCs were cocultured with intact (J-) and activated (J+) Jurkat T cells. The proportion of proliferating and apoptotic J+ and J- cells, IL-10 secretion, and IL-2 production after cocultivation with MSCs and prMSCs were measured. Liquid chromatography-mass spectrometry and bioinformatics analysis identified proteins linked to TLR3 activation in MSCs. RESULTS: Poly(I:C) at 10 µg/mL during a 3-h incubation caused the highest expression of immunosuppression markers in MSCs. Activation of prMSCs caused a 18% decrease in proliferation and a one-third increase in apoptotic J+ cells compared to intact MSCs. Cocultures of prMSCs and Jurkat cells had increased IL-10 and decreased IL-2 in the conditioned medium. A proteomic study of MSCs and prMSCs identified 53 proteins with altered expression. Filtering the dataset with Gene Ontology and Reactome Pathway revealed that poly(I:C)-induced proteins activate the antiviral response. Protein‒protein interactions by String in prMSCs revealed that the antiviral response and IFN I signaling circuits were more active than in native MSCs. prMSCs expressed more cell adhesion proteins (ICAM-I and Galectin-3), PARP14, PSMB8, USP18, and GBP4, which may explain their anti-inflammatory effects on Jurkat cells. CONCLUSIONS: TLR3 activation in MSCs is dependent on exposure time and poly(I:C) concentration. The maximum expression of immunosuppressive molecules was observed with 10 µg/mL poly(I:C) for 3-h preconditioning. This priming protocol for MSCs enhances the immunosuppressive effects of prMSCs on T cells.

Transcriptome profiling of Madin-Darby bovine kidney cells uncover differences in the susceptibility of cattle to Toxoplasma gondii and Neospora caninum.

Toxoplasma gondii and Neospora caninum are two major apicomplexan protozoan parasites with heteroxenous life cycles and worldwide distributions. The transplacental transmission of N. caninum causes bovine abortion, which resulting in serious economic losses to the dairy industry. Although T. gondii was also reported to cause abortions in pregnant woman and small ruminants, scarce cases about the symptom to the host cattle and the causality remains unknown. In this study, transcriptome analysis of Madin Darby bovine kidney (MDBK) cells infected with T. gondii and N. caninum was performed to uncover the differences in susceptibility of cattle to the two parasites. The results showed that 256 and 2225 differentially expressed genes (DEGs) were detected in cells infected with N. caninum and T. gondii, respectively. Moreover, significant biological differences were revealed by the functional analysis including GO and KEGG enrichment. One serpin peptidase inhibitor (SEPRINA14), which is associated with immunosuppression during pregnancy, was found to significantly decrease in cells infected with N. caninum and increase in cells infected with T. gondii-infected cells. Pattern recognition receptors TLR3 and NOD2 were also significantly upregulated in N. caninum-infected MDBK cells, but not in T. gondii. They could induce an increased inflammatory response leading to severe tissue damage. In addition, the interleukin 12 receptor subunit beta 2 (IL12ß2), which plays an essential role in Th1 and Th2 cell differentiation and inflammatory bowel disease, was also markedly upregulated in the N. caninum infected cells, which led to an imbalance in the Th1 and Th2 cells by promoting the Th1 cellular response. Altogether, our findings recognized a new understanding on the differences between T. gondii and N. caninum infection of MDBK cells, where SEPRINA14, TLR3, NOD2, and IL12ß2 may be the key genes that affect the difference in susceptibility of cattle to T. gondii and N. caninum, especially in pregnant animals. This study provides more clues as to why N. caninum is more likely to cause abortions in cattle.

TLR3 Gene Polymorphism in HCV Infection in the Kazakh Population of Western Kazakhstan.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) infection is a common cause of cirrhosis worldwide, leading to significant economic and social burdens. Approximately 170 million people (3% of the population) are infected with HCV, with the risk of developing complications such as cirrhosis and hepatocellular carcinoma. In the United States, HCV is the main cause of liver cirrhosis, accounting for 26% of cases. Recent studies have shown an increase in the proportion of HCV-related liver cirrhosis. MATERIALS AND METHODS: A total of 102 patients with chronic hepatitis C in the reactivation phase from the Atyrau and Aktobe regional hepatology centers, who had not previously received antiviral therapy, were examined. A control group, matched by gender and age, included 127 practically healthy individuals of Kazakh nationality. All patients underwent a comprehensive examination, which included a complete blood count, a biochemical blood analysis and PCR for HCV. Venous blood samples were taken from all subjects for molecular genetic analysis. Genotyping of TLR3 polymorphism (rs5743312, rs5743305, rs3775291, rs5743311, rs1879026) was performed using real-time PCR. Thes study is a case control study. RESULTS: In patients with cirrhosis of the liver resulting from chronic hepatitis C (HCV), the results of biochemical analysis were statistically significantly higher than in patients with HCV without liver cirrhosis: the levels of total bilirubin (p 0.017*), alkaline phosphatase (p 0.022*), and gamma-glutamyl transferase (0.041*) were elevated. The results indicated that the CC genotype of TLR3 rs1879026 was associated with the development and chronicity of HCV infection compared to practically healthy individuals (p=0.001). In the distribution of genotypes and alleles for rs5743312, rs5743305, rs3775291, and rs5743311, no significant differences were found between patients with HCV and the healthy control group. CONCLUSION: The TLR3 rs1879026 gene polymorphism plays a significant role in the predisposition to HCV infection in the Kazakh population of the Aktobe and Atyrau regions.

Toll-Like Receptor 3 mRNA Expression of Peripheral Blood Mononuclear Cells Identifies Kidney Recipients with Potential for Improved Graft Performance.

BACKGROUND Toll-like receptor 3 expression is detected both on the cell membrane and in endosomes of peripheral blood mononuclear cells (PBMC). Our goal in this study was to determine to what extent a single, baseline measurement of non-stimulated PBMC TLR3-mRNA can be related to baseline GFR (b-GFR) and post-follow-up-GFR (F-up-GFR) of a kidney transplant (KT) and baseline immunosuppression. MATERIAL AND METHODS In non-stimulated PBMC we investigated averaged mRNA expression of Toll-like receptor 3. A total of 133 patients were enrolled; the median of months after KT surgery was 11.4, with median F-up at 21.3 months. A favorable course (FCF) was determined if F-up-eGFR improved. An unfavorable course (UCF) was determined if F-up-eGFR was lower at the end of the observation. RESULTS The highest TLR3-mRNA expression was at b-GFR grade 3b; it was moderately higher at b-GFR grade 3a, and marginally higher at b-GFR grades 1+2. Most of the FCF group had b-GFR grade 3b, less frequent obesity, more effective immunosuppression, and much higher TLR3-mRNA (59% of cases were in the high-TLR3 area). Both delayed graft function (DGF) and TLR3-mRNA range below the median for the entire KT cohort (low-TLR3 area) had a negative association with b-GFR. The UCF group had more frequent DGFs and obesity, less effective immunosuppression, and lower TLR3-mRNA. CONCLUSIONS In patients with GFR grade 3, high levels of TLR3-mRNA are associated with improved graft efficacy. In patients with impaired graft function, low TLR3- mRNA expression reduces the likelihood of improved renal graft function.

Activation of Toll-like receptor 3 inhibits HIV infection of human iPSC-derived microglia.

As a key immune cell in the brain, microglia are essential for protecting the central nervous system (CNS) from viral infections, including HIV. Microglia possess functional Toll-like receptor 3 (TLR3), a key viral sensor for activating interferon (IFN) signaling pathway-mediated antiviral immunity. We, therefore, studied the effect of poly (I:C), a synthetic ligand of TLR3, on the activation of the intracellular innate immunity against HIV in human iPSC-derived microglia (iMg). We found that poly (I:C) treatment of iMg effectively inhibits HIV infection/replication at both mRNA and protein levels. Investigations of the mechanisms revealed that TLR3 activation of iMg by poly (I:C) induced the expression of both type I and type III IFNs. Compared with untreated cells, the poly (I:C)-treated iMg expressed significantly higher levels of IFN-stimulated genes (ISGs) with known anti-HIV activities (ISG15, MxB, Viperin, MxA, and OAS-1). In addition, TLR3 activation elicited the expression of the HIV entry coreceptor CCR5 ligands (CC chemokines) in iMg. Furthermore, the transcriptional profile analysis showed that poly (I:C)-treated cells had the upregulated IFN signaling genes (ISG15, ISG20, IFITM1, IFITM2, IFITM3, IFITM10, APOBEC3A, OAS-2, MxA, and MxB) and the increased CC chemokine signaling genes (CCL1, CCL2, CCL3, CCL4, and CCL15). These observations indicate that TLR3 is a potential therapy target for activating the intracellular innate immunity against HIV infection/replication in human microglial cells. Therefore, further studies with animal models and clinical specimens are necessary to determine the role of TLR3 activation-driven antiviral response in the control and elimination of HIV in infected host cells.