TLR4 Research Articles

Systemic chemokine-modulatory regimen combined with neoadjuvant chemotherapy in patients with triple-negative breast cancer

BackgroundHigher cytotoxic T lymphocyte (CTL) numbers in the tumor microenvironment (TME) predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) and positive long-term outcomes in triple-negative breast cancer (TNBC). pCR...

TLR3 activation enhances antitumor effects of sorafenib in hepatocellular carcinoma by activating NK cell functions through ERK and NF-κB pathways

Background Sorafenib is a standard therapeutic agent for advanced hepatocellular carcinoma (HCC). However, its efficacy is moderate, as the survival of patients is prolonged for only a few months, and the response rate is low. The mechanism of low efficacy remains unclear. In this study, we investigated the effect of Toll-like receptor 3 (TLR3) on the effects of sorafenib on HCC. Methods Polyinosinic-polycytidylic acid [poly(I: C)] was used as a double-stranded RNA analog and TLR3 agonist in subsequent experiments. After orthotopic implantation of HCC tumors in BALBc nu/nu or C57BL/6 mice, survival time, tumor growth, and metastasis in the abdomen and lungs were analyzed. Flow cytometry and cytotoxicity assays were used to analyze NK cells isolated from the spleen or peripheral blood. ELISA was used to detect the expression of plasma interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1. In addition, the expression of phosphorylated-extracellular regulated kinase 1/2 (pERK1/2), phosphorylated-protein kinase B (pAKT), ERK1/2 and AKT was analyzed by Western blotting. Results Sorafenib reduced the number and activity of NK cells in tumor-bearing mice and simultaneously decreased the levels of MCP-1 and IFN-γ in the plasma. The combination of sorafenib and poly(I: C) synergistically inhibited tumor growth and metastasis in tumor xenograft mice and prolonged survival. Poly(I: C) not only exerts a direct inhibitory effect on tumor growth and metastasis by targeting the TLR3 receptor on tumor cells but also facilitates the proliferation and activation of NK cells, indirectly impeding tumor progression. Mechanistically, poly(I: C) decreased the sorafenib-induced inhibition of ERK phosphorylation and increased the phosphorylation of IκB in NK cells, thereby enhancing NK cell function. Conclusion Activation of TLR3 can enhance the antitumor effect of sorafenib on HCC. The combination of a TLR3 activator and sorafenib may be a new strategy for the treatment of HCC.

Targeting mast cell activation alleviates anti-MHC I antibody and LPS-induced TRALI in mice by pharmacologically blocking the TLR3 and MAPK pathway

Transfusion-related lung injury (TRALI) poses a significant risk following blood transfusion and remains the primary cause of transfusion-related morbidity and mortality, primarily driven by the activation of immune cells through anti-major histocompatibility complex class I (anti-MHC I) antibody. However, it remains to be defined how immune microenvironmental cue contributes to TRALI. Here, we uncover that activated mast cells within the immune microenvironment promote lung inflammation and injury in antibody-mediated TRALI, both in vitro and in vivo. This was demonstrated by co-culturing lipopolysaccharide (LPS)-pretreated mast cell line with anti-MHC I antibody and establishing a “two-hit” TRALI mouse model through intratracheal injection of LPS followed by tail-vein injection of anti-MHC I antibody. Importantly, mast cell-deficient KitW-sh/W-sh mice exhibited markedly reduced lung inflammation and injury responses in antibody-mediated TRALI compared with wild-type mice. Mechanistically, activation of toll-like receptor 3 (TLR3)/mitogen-activated protein kinase (MAPK) signaling pathway in mast cells contributes to the enhanced production of proinflammatory factors. These excessive proinflammatory factors produced by activated mast cells contribute to lung inflammation and injury in antibody-mediated TRALI. Pharmacologically targeting the TLR3/MAPK pathway to inhibit mast cell activation normalizes the proinflammatory microenvironment and alleviates lung inflammation and injury in the preclinical TRALI mouse model. Overall, we find that activation of mast cells via the TLR3/MAPK pathway contributes to lung inflammation and injury in antibody-mediated TRALI, providing novel insights into its underlying mechanisms. Furthermore, targeting activated mast cells and the associated pathway offers potential therapeutic strategies for antibody-mediated TRALI.

Using a dual immunoinformatics and bioinformatics approach to design a novel and effective multi-epitope vaccine against human torovirus disease

Human Torovirus (HToV), a member of the Coronaviridae family, causes severe enteric diseases with no specific medication available. To develop novel preventative measures, we employed immunoinformatics techniques to design a multi-epitope-based subunit vaccine (HToV-MEV) triggering diverse immune responses. We selected non-allergenic, non-toxic, and antigenic epitopes from structural polyproteins, joined them with suitable linkers, and added an adjuvant 50S ribosomal L7/L12 peptide. The vaccine's solubility score of 0.903678 and physiochemical properties were found effective. Molecular dynamics simulations and free energy calculations revealed strong binding affinity for Toll-like receptor 3 (TLR-3), with a lowest energy score of −303.88, indicating high affinity. In-silico cloning and codon optimization showed significant production potential in E. coli. Immune simulations predicted a human immunological response. Our results are promising, but subsequent in vivo research is recommended. The HToV-MEV vaccine design demonstrates potential for preventing HToV-related diseases, and further investigation is warranted to explore its therapeutic applications.

Cylindromatosis lysine 63 deubiquitinase (CYLD) suppress TLR3-mediated CCL5 expression in human renal proximal tubular epithelial cells.

One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-β (IFN-β). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs). Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-β, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-β or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/β (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression. CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.

Sodium aescinate alleviates neuropathic pain through suppressing OGT-mediated O-GlcNAc modification of TLR3 to inactivate MAPK signaling pathway

Neuropathic pain results from damage to nerves or the brain, and is characterized by symptoms such as allodynia, spontaneous pain, and hyperalgesia. The causes of this type of pain are intricate, which can make it difficult to treat. Sodium aescinate (SA), a natural extract from horse chestnut tree seeds, has been shown to act as a neuroprotector by inhibiting microglia activation. This study aims to explore the therapeutic potential of SA for neuropathic pain and the molecular mechanisms regulated by SA treatment. Through in vivo animal models and experiments, we found that SA treatment significantly reduced mechanical allodynia and heat hyperalgesia in neuropathic pain models. Additionally, SA inhibited O-GlcNAc-transferase (OGT)-induced O-GlcNAcylation (O-GlcNAc) modification in neuropathic pain mice. OGT overexpression could impede the therapeutic effects of SA on neuropathic pain. Further investigation revealed that Toll-like receptor 3 (TLR3), stabilized by OGT-induced O-GlcNAc modification, could activate the Mitogen activated protein kinase (MAPK) signaling pathway. Further in vivo experiments demonstrated that TLR3-mediated p38 mitogen-activated protein kinase (p38MAPK) activation is involved in SA-mediated relief of neuropathic pain. In conclusion, this study uncovers a novel molecular pathway deactivated by SA treatment in neuropathic pain.

Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release invivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation invitro (P<0.01). We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Poly (I:C) increases the expression of galectin 1, 3, 9 and HGF genes in exosomes isolated from human Wharton's jelly mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are commonly employed as a powerful tool for the treatment of immune-mediated problems owing to their capacity to regulate the immune system and differentiate into different tissues. Researchers use mesenchymal stem cell products given the limitations associated with the application of MSCs. Exosomes are nanometer vesicles derived from MSCs that are used in cell-free therapy. Inflammatory environmental conditions, such as stimulation of Toll-like receptor 3 (TLR-3), has the ability to adjust the immune-regulating properties and anti-inflammatory function of mesenchymal stem cells and their exosomes. Galectins and hepatocyte growth factor (HGF) are known as immunomodulatory factors in mesenchymal stem cells. This study was designed to examine the expression of galectin-1, galectin-3, galectin-9, and HGF genes in exosomes isolated from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) after stimulation with Poly (I:C) (Polyinosinic:polycytidylic acid sodium salt). MethodsTo begin, the explant technique was used to extract mesenchymal stem cells from human umbilical cord Wharton's jelly. Then, the stem cells were stimulated using Poly (I:C) at three time intervals of 12, 24 and 48 h. Exosomes secreted from the supernatant of cells were extracted and exosome confirmation tests, including Scanning electron microscopy (SEM), Dynamic light scattering (DLS) and Flow cytometry were performed. Finally, the expression of galectin-1, galectin-3, galectin-9, and HGF genes in exosomes was evaluated by Real-Time PCR at three time intervals of 12, 24 and 48 h after stimulation. ResultsThe findings of our study indicated that following stimulation with Poly (I:C), the expression of galectin-9 and HGF (P < 0.05) genes was markedly higher than in the control group after 12 h. After 24 h, the expression of galectin-9 (P < 0.01), galectin-3 and HGF (P < 0.05) increased; the expression of galectin-1, galectin-3, (P < 0.05), galectin-9 and HGF genes (p < 0.01) significantly increased compared to the control group after 48 h. ConclusionTLR3 stimulation can increase the expression of galectins and HGF genes in exosomes derived from hWJ-MSCs and may be improve the immunosuppressive abilities of exosomes.

In silico design of multi-epitope vaccines against the hantaviruses by integrated structural vaccinology and molecular modeling approaches.

Hantaviruses are single-stranded RNA viruses belonging to the family Bunyaviridae that causes hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) worldwide. Currently, there is no effective vaccination or therapy available for the treatment of hantavirus, hence there is a dire need for research to formulate therapeutics for the disease. Computational vaccine designing is currently a highly accurate, time and cost-effective approach for designing effective vaccines against different diseases. In the current study, we shortlisted highly antigenic proteins i.e., envelope, and nucleoprotein from the proteome of hantavirus and subjected to the selection of highly antigenic epitopes to design of next-generation multi-epitope vaccine constructs. A highly antigenic and stable adjuvant was attached to the immune epitopes (T-cell, B-cell, and HTL) to design Env-Vac, NP-Vac, and Com-Vac constructs, which exhibit stronger antigenic, non-allergenic, and favorable physiochemical properties. Moreover, the 3D structures were predicted and docking analysis revealed robust interactions with the human Toll-like receptor 3 (TLR3) to initiate the immune cascade. The total free energy calculated for Env-Vac, NP-Vac, and Com-Vac was -50.02 kcal/mol, -24.13 kcal/mol, and -62.30 kcal/mol, respectively. In silico cloning, results demonstrated a CAI value for the Env-Vac, NP-Vac, and Com-Vac of 0.957, 0.954, and 0.956, respectively, while their corresponding GC contents were 65.1%, 64.0%, and 63.6%. In addition, the immune simulation results from three doses of shots released significant levels of IgG, IgM, interleukins, and cytokines, as well as antigen clearance over time, after receiving the vaccine and two booster doses. Our vaccines against Hantavirus were found to be highly immunogenic, inducing a robust immune response that demands experimental validation for clinical usage.

Upregulation of key factors of viral entry of corona- and influenza viruses upon TLR3-signaling in cells from the respiratory tract and clinical treatment options by 1,8-Cineol.

At the end of the 2019 coronavirus pandemic (COVID-19), highly contagious variants of coronaviruses had emerged. Together with influenza viruses, different variants of the coronavirus currently cause most colds and require appropriate drug treatment. We have investigated the expression of important factors for the replication of these viruses, namely transmembrane protease serine subtype 2 (TMPRSS2), neuropilin1 (NRP1), and angiotensin converting enzyme 2 (ACE2) or tumor necrosis factor-α (TNF-α) after toll like receptor-3 (TLR-3) stimulation using RT-qPCR and flow cytometry (FC) analysis. As model served primary fibroblasts derived from the lung and nasal cavity, as well as epidermal stem cells and fully matured respiratory epithelium. The stimulated cell cultures were treated with pharmaceuticals (Dexamethasone and Enzalutamide) and the outcome was compared with the phytomedicine 1,8-Cineol. The stimulation of TLR3 is sufficient to induce the expression of exactly those targets that were highly expressed in the corresponding culture type, specifically ACE2 and TMPRSS2 in respiratory epithelial stem cells and NRP1 in fibroblast cells. It seems this self-perpetuating cycle of infection-driven upregulation of key viral replication factors by the innate immune system represents an evolutionary advantage for viruses using these transcripts as viral replication factors. Likewise, to the standard pharmaceuticals with proven clinical efficiency, 1,8-Cineol was able to disrupt this self-perpetuating cycle. Considering the minor side effects and negligible pharmacological interactions with other drugs, it is conceivable that an adjuvant or combinatorial therapy with 1,8-Cineol for respiratory diseases caused by corona- or influenza viruses would be beneficial.

Aluminum oxyhydroxide-Poly(I:C) combination adjuvant with balanced immunostimulatory potentials for prophylactic vaccines

The development of high-purity antigens promotes the urgent need of novel adjuvant with the capability to trigger high levels of immune response. Polyinosinic-polycytidylic (Poly(I:C)) is a synthetic double-stranded RNA (dsRNA) that can engage Toll-like receptor 3 (TLR3) to initiate immune responses. However, the Poly(I:C)-induced toxicity and inefficient delivery prevent its applications. In our study, combination adjuvants are formulated by aluminum oxyhydroxide nanorods (AlOOH NRs) and Poly(I:C), named Al-Poly(I:C), and the covalent interaction between the two components is further demonstrated. Al-Poly(I:C) mediates enhanced humoral and cellular immune responses in three antigen models, i.e., HBsAg virus-like particles (VLPs), human papilloma virus (HPV) VLPs and varicella-zoster virus (VZV) glycoprotein E (gE). Further mechanistic studies demonstrate that the dose and molecular weight (MW) of Poly(I:C) determine the physicochemical properties and adjuvanticity of the Al-Poly(I:C) combination adjuvants. Al-Poly(I:C) with higher Poly(I:C) dose promotes antigen-bearing dendritic cells (DCs) recruitment and B cells proliferation in lymph nodes. Al-Poly(I:C) formulated with higher MW Poly(I:C) induces higher activation of helper T cells, B cells, and CTLs. This study demonstrates that Al-Poly(I:C) potentiates the humoral and cellular responses in vaccine formulations. It offers insights for adjuvant design to meet the formulation requirements in both prophylactic and therapeutic vaccines.

Blockade of endolysosomal acidification suppresses TLR3-mediated proinflammatory signaling in airway epithelial cells

BackgroundEndolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm. ObjectivesHere we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants. MethodsHSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release. ResultsWe show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein. ConclusionThese findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs. Clinical implicationThese findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.

The Mucus-Binding Factor Mediates Lacticaseibacillus rhamnosus CRL1505 Adhesion but Not Immunomodulation in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 possesses immunomodulatory activities in the gastrointestinal and respiratory tracts when administered orally. Its adhesion to the intestinal mucosa does not condition its beneficial effects. The intranasal administration of L. rhamnosus CRL1505 is more effective than the oral route at modulating immunity in the respiratory tract. Nonetheless, it has not yet been established whether the adherence of the CRL1505 strain to the respiratory mucosa is needed to provide the immune benefits to the host. In this study, we evaluated the role of adhesion to the respiratory mucosa of the mucus-binding factor (mbf) knock-out L. rhamnosus CRL1505 mutant (Δmbf CRL1505) in the context of a Toll-like receptor 3 (TLR3)-triggered innate immunity response. In vitro adhesion studies in porcine bronchial epitheliocytes (PBE cells) indicated that L. rhamnosus Δmbf CRL1505 adhered weakly compared to the wild-type strain. However, in vivo studies in mice demonstrated that the Δmbf CRL1505 also reduced lung damage and modulated cytokine production in the respiratory tract after the activation of TLR3 to a similar extent as the wild-type strain. In addition, the mutant and the wild-type strains modulated the production of cytokines and antiviral factors by alveolar macrophages in the same way. These results suggest that the Mbf protein is partially involved in the ability of L. rhamnosus CRL1505 to adhere to the respiratory epithelium, but the protein is not necessary for the CRL1505 strain to exert its immunomodulatory beneficial effects. These findings are a step forward in the understanding of molecular interactions that mediate the beneficial effects of nasally administered probiotics.

Phase 2 randomized study of rintatolimod following FOLFIRINOX in patients with locally advanced pancreatic adenocarcinoma.

TPS4214 Background: The immunosuppressive microenvironment of pancreatic ductal adenocarcinoma (PDAC) is a driver of its lethality and resistance to immunotherapy. Strategies which modulate the immunosuppressive milieu have the potential to improve the anti-tumor response and improve outcomes in patients with PDAC. Rintatolimod is a synthetic double-stranded RNA molecule that activates the toll-like receptor 3 (TLR3) present on dendritic cells, B-cells, natural killer cells and pancreatic epithelial cells. TLR3 stimulation may increase cytotoxic T-cell activity in PDAC through dendritic cell maturation, antigen priming and downstream proliferation of cytotoxic T-cells. Twenty-seven patients were treated with rintatolimod in a single center Named Patient Program at Erasmus University Medical Center in Rotterdam between January 2017 and February 2019. All had locally advanced or metastatic PDAC which had not progressed on FOLFIRINOX (median 8 cycles). Maintenance rintatolimod, administered intravenously twice weekly for up to 18 weeks, was generally well tolerated. The most common toxicities were grade 1-2 myalgia (30%), chills (52%) and fatigue (30%); no toxicities ≥ grade 3 were observed. Progression-free and overall survival (from cycle 1 FOLFIRINOX) were 13 and 19 months. Methods: This is a phase 2 randomized, open-label, multicenter clinical trial. The primary objective is to assess the efficacy of rintatolimod compared to observation in subjects with locally advanced PDAC which has not progressed after FOLFIRINOX. Eligible patients have completed at least 4 months of FOLFIRINOX, have no evidence of progressive disease, have measurable disease by RECIST v1.1, Karnofsky Performance Status ≥80 and have adequate organ function. Subjects with resected PDAC or metastatic disease are not eligible. Subjects are randomized 2:1 to rintatolimod vs. observation. Rintatolimod is administered intravenously twice weekly until disease progression in the experimental arm. Cross-sectional imaging is performed every 8 weeks in both arms. The primary endpoint is progression-free survival, as assessed by the investigator. Secondary endpoints include overall survival, objective response rate, duration of response and safety, as assessed by CTCAE v5.0. Exploratory endpoints include quality of life and changes in immune cell composition and T-cell response in the peripheral blood to characterize the immunomodulatory effects of rintatolimod. The planned sample size is 90 subjects. The final analysis will be performed after 64 events (disease progression or death) have been observed. The study is powered to detect a hazard ratio of 0.47 for median PFS with 80% power and significance level of 5%. Clinical trial information: NCT05494697 .

Effect of Toll-like Receptor-3 Antagonist on Viral Asthma Exacerbations Via a TLR3/dsRNA Complex Pathway

Background: The Toll-like receptor-3 (TLR3) ligand Poly(I:C) has been shown to induce a viral aggravation of severe asthma by identifying double-stranded RNA (dsRNA). This study aimed to evaluate the therapeutic role of the TLR3/dsRNA complex inhibitor-calbiochem compound in the treatment of Poly(I:C)-induced viral asthma exacerbations through the ovalbu-min-induced asthma model in Swiss albino mice. Methods: Poly(I:C) and Ovalbumin drugs were injected in mice to sensitize (i.p. on 0, 7, and 14th day) and challenge (i.n. on the 21st and 22nd days). In contrast, the treatment drug TLR3/dsRNA complex inhibitor-calbiochem was given on the 21st and 22nd days intraperitoneally within the study period. In-vivo measurements were carried out in BALF and serum for pro-inflammatory cytokines, inflammatory leukocyte counts, lactate dehydrogenase (LDH) and nitrite levels, lungs/body weight index, and lung tissue histopathology using H and E staining in mice airways. Results: High levels of cytokines (NF-κB, IL-1β, IL-5, RANTES, MIP-2, and MCP-1) are seen in groups exposed to OVA and Poly (I:C). Further, inflammatory leukocyte cell counts, lung-body weight (LW/BW) index, airway hyperresponsiveness (AHR), and lung tissue damage sug-gest exacerbations in mice airways. On the other hand, TLR3/dsRNA complex inhibitor-calbio-chem and dexamethasone significantly reversed these changes toward normal levels. Conclusions: These results suggest that the novel compound TLR3/dsRNA complex inhibitor-calbiochem has a better therapeutic role than dexamethasone for managing inflammatory char-acteristics in asthmatic mice lungs and is a potent target for viral asthma exacerbations

Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated mesenchymal stromal cells reveals induction of T-cell response pathways.

To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology. 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4). Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints. Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20). Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Retinoic Acid Neutralizes the Effects of Herpes Simplex Virus Type 1-Infected Cell Protein 0 (ICP0) in Retinal Pigment Epithelial Cells.

Herpes simplex virus (HSV) infection of the cornea, uvea, and retina is the leading infectious cause of blindness worldwide. This study examined the effects of retinoic acid (RA) on the protein levels of interleukin (IL)-17A and IL-23 cytokines with known proinflammatory effects and toll-like receptor 3 (TLR3) messenger RNA (mRNA) expression in retinal pigment epithelial (ARPE-19) cells treated with HSV-1-infected cell protein 0 (ICP0). We used 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide assay to calculate the half maximal inhibitory concentration (IC50) doses of RA and ICP0 in ARPE-19 cells. At the end of 24 hours, protein levels of IL-17A and IL-23 were analyzed using enzyme-linked immunosorbent assay. TLR3 mRNA expression levels were also calculated using reverse transcription-polymerase chain reaction (RT-PCR). RA administration decreased IL-17A levels, which were elevated by ICP0. The IL-23 levels were similar between the ICP0-treated and control groups, but the difference was significant between the ICP0-treated group and RA+ICP0 combination. These results showed that RA can significantly increase IL-23 levels in the presence of ICP0. Although ICP0 dramatically increased TLR3 mRNA expression compared with that in the control group, the RA+ICP0 combination returned TLR3 mRNA expression to a level similar to that in the control group (P = 0.419). RA may potentially neutralize HSV-1 ICP0 negative effects in ARPE-19 cells.

Soluble form of the MDA5 protein in human sera

Viral double-stranded RNA (dsRNA) is sensed by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including melanoma differentiation-associated gene 5 (MDA5). MDA5 recognizes the genome of dsRNA viruses and replication intermediates of single-stranded RNA viruses. MDA5 also plays an important role in the development of autoimmune diseases, such as Aicardi-Goutieres syndrome and type I diabetes. Patients with dermatomyositis with serum MDA5 autoantibodies (anti-CADM-140) are known to have a high risk of developing rapidly progressive interstitial lung disease and poor prognosis. However, there have been no reports on the soluble form of MDA5 in human serum. In the present study, we generated in-house monoclonal antibodies (mAbs) against human MDA5. We then performed immunohistochemical analysis and sensitive sandwich immunoassays to detect the MDA5 protein using two different mAbs (clones H27 and H46). As per the immunohistochemical analysis, the MDA5 protein was moderately expressed in the alveolar epithelia of normal lungs and was strongly expressed in the cytoplasm of lymphoid cells in the tonsils and acinar cells of the pancreas. Interestingly, soluble MDA5 protein was detectable in the serum, but not in the urine, of healthy donors. Soluble MDA5 protein was also detectable in the serum of patients with dermatomyositis. Immunoblot analysis showed that human cells expressed a 120 kDa MDA5 protein, while the 60 kDa MDA5 protein increased in the supernatant of peripheral mononuclear cells within 15 min after MDA5 agonist/double-strand RNA stimulation. Hydrogen deuterium exchange mass spectrometry revealed that an anti-MDA5 mAb (clone H46) bound to the epitope (415QILENSLLNL424) derived from the helicase domain of MDA5. These results indicate that a soluble MDA5 protein containing the helicase domain of MDA5 could be rapidly released from the cytoplasm of tissues after RNA stimulation.

Inhibitory effects of Belamcanda extract on inflammatory response and antiviral mechanism in H9N2 Avian influenza virus: insights from in vitro and in vivo studies

Avian influenza, particularly the H9N2 subtype, presents significant challenges to poultry health, underscoring the need for effective antiviral interventions. This study explores the antiviral capabilities of Belamcanda extract, a traditional Chinese medicinal herb, against H9N2 Avian Influenza Virus (AIV) in Specific Pathogen-Free (SPF) chicks. Through a comprehensive approach, we evaluated the impact of the extract on cytokine modulation and crucial immunological signaling pathways, essential for understanding the host-virus interaction. Our findings demonstrate that Belamcanda extract significantly modulates the expression of key inflammatory cytokines, including Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 (IL-1), Interleukin-2 (IL-2), and Interleukin-6 (IL-6), which are pivotal to the host's response to H9N2 AIV infection. Western blot analysis further revealed that the extract markedly reduces the expression of critical immune signaling molecules such as Toll-Like Receptor 3 (TLR3), TIR-domain-containing adapter-inducing interferon-β (TRIF), and Nuclear Factor Kappa B (NF-κB). These insights into the mechanisms by which Belamcanda extract influences host immune responses and hinders viral replication highlight its potential as an innovative antiviral agent for poultry health management. The study advances our comprehension of natural compounds' antiviral mechanisms and lays the groundwork for developing strategies to manage viral infections in poultry. The demonstrated ability of Belamcanda extract to modulate immune responses and inhibit viral replication establishes it as a promising candidate for future antiviral therapy development, especially in light of the need for effective treatments against evolving influenza virus strains and the critical demand for enhanced poultry health management strategies.

An LGR6 frameshift variant abrogates receptor expression on select leukocyte subsets and is associated with viral infections

AbstractThe leucine-rich repeat-containing G-protein–coupled receptor 6 (LGR6) was recently identified as the cognate receptor for the proresolving mediator maresin 1 (MaR1). To address the biological role of LGR6 in humans, we investigated the functional impact of a genetic variant in the gene encoding for LGR6, which is predicted to lead to a frameshift mutation in one of the receptor isoforms, on both receptor expression and immune cell responses. In neutrophils, monocytes, and natural killer (NK) cells from volunteers homozygous for this variant, we found a significant downregulation in the expression of LGR6 when compared with controls without the variant; whereas the LGR6 expression was essentially similar in monocyte-derived macrophages and CD8+ T cells. Functionally, loss of LGR6 expression was linked with a decreased ability of neutrophils and monocytes to phagocytose bacteria. We observed an increase in neutrophil chemotaxis and leukotriene B4 production and increased expression of activation markers, including markers for platelet-leukocyte phagocyte heterotypic aggregates, such as CD41, in neutrophils and monocytes from the variant group. Using data from the UK Biobank, we found that at a population level the rs4266947 variant, which is in high linkage disequilibrium with rs74355478, was associated with a higher incidence of viral infections. Intriguingly, neutrophils, NK cells, and CD8+ T cells from volunteers with the LGR6 variant displayed altered viral responses when stimulated with Toll-like receptor 3 (TLR3), TLR7/TLR8, and TLR9 agonists. Together, these findings shed new light on the cell type–specific regulation of LGR6 expression and the role of this receptor in directing host immune responses.