TLR3 Agonist Poly Research Articles

Acute effects of TLR3 agonist Poly(I:C) on bone marrow hematopoietic progenitor cells in mice

Hematopoietic progenitor cells (HPCs) in bone marrow with limited abilities for self-renewal and differentiation continuously supply hematopoietic cells through life. When suffering infection or inflammation, HPCs will actively proliferate to provide differentiated hematopoietic cells to maintain hematopoietic homeostasis. Poly(I:C), an agonist of TLR3, can specifically activate Type I interferon (IFN-I) signaling which exerts anti-inflammatory effects and influence hematopoiesis after infection. However, the effects of Poly(I:C)-induced IFN-I on the bone marrow hematopoietic system still deserve attention. In this study, our results revealed the efficacy of the IFN-I model, with a remarkably decrease in HPCs and a sharp elevation in LSKs numbers after single dose of Poly(I:C) injection. Apoptotic ratios of HPCs and LSKs significantly increased 48 h after Poly(I:C) treatment. Application of Poly(I:C) prompted the transition of HPCs and LSKs from G0 to G1 phases, potentially leading to the accelerated exhaustion of HPCs. From the cobblestone area-forming cell (CAFC) assay, we speculate that Poly(I:C) impairs the differentiation capacity of HPCs as well as their colony-forming ability. RT-qPCR and immunohistochemistry revealed significant upregulation of IFN-I associated genes and proteins following Poly(I:C) treatment. In conclusion, a single dose of Poly(I:C) induced an acute detrimental effect on HPCs within 48 h potentially due to TLR3 engagement. This activation cascaded into a robust IFN-I response emanating from the bone marrow, underscoring the intricate immunological dynamics at play following Poly(I:C) intervention.

Abstract PS07-06: A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention

Abstract Purpose: To evaluate the merits of a novel citrullinated-enolase 1 (ENO1) peptide-based vaccine for inducing an effective response against triple negative breast cancer (TNBC). Background: Targets for cancer vaccines have included mutated proteins and peptides, as well as proteins that are overexpressed in cancer but otherwise ubiquitous in their structure. However, protein modifications other than mutation may also induce an immune response. Several lines of evidence have demonstrated a role for the posttranslational modification (PTM) citrullination as inducing immunogenicity. Dysregulated protein citrullination by peptidyl arginine deiminases (PADI) has been associated with autoimmune diseases and is currently being explored for its relevance to cancer. Previously, our group established PADI2, which catalyzes the PTM from arginine to citrulline, to be highly expressed in TNBC compared to normal mammary and other tissues. We observed that PADI2-associated protein citrullination promotes antigenicity. Importantly, we demonstrated that protein citrullination, non-occurring in DNA and RNA-based vaccines, is highly cancer specific. On the basis of these findings, we developed a novel vaccine based on cancer-selective citrullinated-enolase 1 peptides (cit-ENO1) that were identified from mass spectrometry surfaceome and immunopeptidome data from breast cancer cells. We assessed the extent to which a citENO1 peptide based vaccine elicits a potent host response and attenuates tumor development in a syngeneic mouse model of TNBC. Design: To confirm that the cit-ENO1 peptide vaccine elicited an immune response, we immunized female B6129SF1/J mice with 10nmol cit-ENO1 peptides or 10nmol of corresponding unmodified peptides as the antigen, plus the TLR3 agonist poly I:C as an adjuvant (10mg), subcutaneously once a week, for three weeks. Saline or the adjuvant alone were used as controls. One week post the last round of immunization, draining lymph nodes (LNs) and immunized skin were harvested, and T cell phenotypes evaluated using flow-cytometry. To assess anti-cancer efficacy, female B6129SF1/J mice were immunized with cit-ENO1 peptides or unmodified peptides with poly I:C as an adjuvant followed by orthotopic implantation with murine BRCA1co/co; MMTV-Cre; p53+/- TNBC cells (1.5 x 106) and tumor growth as well as overall survival were assessed. Results: We observed that the cit-ENO1 peptide vaccination induced a statistically significantly greater percentage of activated CD8+ PD-1+ effector T cells as well as CD8+ CD44+ CD62L- effector memory T cells in the LNs compared with either the control groups or the unmodified ENO1 peptide. CD8+ PD-1+ T-cells were also found to be significantly elevated in immunized skin of cit-ENO1 vaccinated mice. Both cit-ENO1 and unmodified ENO1 vaccine increased CD4+ PD-1+ T cells from the LNs and immunized skin. Remarkably, the cit-ENO1 vaccine but not the unmodified-ENO1 vaccine significantly delayed tumor growth and markedly improved overall survival compared to respective controls (Logrank test for trend 2-sided p< 0.05). Conclusion: We have developed a novel vaccine based on cancer-selective cit-ENO1 peptides that stimulates a potent anti-cancer immune response and significantly delays tumor initiation and progression in a preclinical, immune competent mouse model of TNBC. Importantly, the anti-cancer effects were not observed with corresponding unmodified ENO1 peptides. Citation Format: Ricardo Leon Letelier, Hiroyuki Katayama, Yihui Chen, Yinning Cai, Fuchung Hsiao, Banu Arun, Samir Hanash. A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-06.

Enhanced Efficacy of a TLR3 Agonist Delivered by Cowpea Chlorotic Mottle Virus Nanoparticles

Intratumoral immunotherapies are those that are administered directly into a tumor to remodel the local tumor microenvironment and stimulate systemic anti‐tumor immunity. Small molecule toll‐like receptor (TLR) agonists are undergoing development as intratumoral immunotherapies, and here the TLR3 agonist poly(I:C) is considered. Because small molecule therapeutics often suffer rapid washout effects and ineffective immune cell uptake, poly(I:C) is encapsulated into nanoparticles derived from cowpea chlorotic mottle virus (CCMV). The particles (but not the separate components) stimulate the activity of macrophages in vitro and are able to reduce tumor growth and prolong survival in mouse models of colon cancer and melanoma. CCMV‐poly(I:C) is also combined with oxaliplatin and found the combination therapy to be even more potent, strongly inhibiting tumor growth and increasing survival. The analysis of immune markers reveals that CCMV‐poly(I:C) VLPs with oxaliplatin promoted the infiltration and activation of CD4+ and CD8+ cells and the production of IL‐4 and IFN‐γ, indicating a synergistic immunogenic effect. The combined treatment also enhances the rate of apoptosis and immunogenic cell death (ICD). The data support the development of combination therapies involving immunomodulatory plant virus nanoparticles and antineoplastic drugs to attack tumors directly and via the activation of innate and adaptive immune responses.

Abstract 4098: A citrullinated-ENO1 peptide vaccine delayed tumor growth and markedly improved overall survival in a mouse model of triple-negative breast cancer

Abstract Purpose: To evaluate the impact of a novel citrullinated-enolase 1 (cit-ENO1) peptide-based vaccine on the immune response, tumor growth and overall survival in a mouse model of triple-negative breast cancer (TNBC). Background: Cancer vaccines have consisted primarily of mutated or overexpressed proteins in cancer. We have uncovered dysregulated protein citrullination by peptidyl arginine deiminases (PADI) as a feature of triple negative breast cancer inducing immunogenicity to citrullinated proteins. On the basis of these findings, we investigated a novel vaccine based on cancer-selective cit-ENO1 peptides that we identified from mass spectrometry analysis of the surfaceome and immunopeptidome in TNBC. We assessed the extent to which the vaccine elicited an immune response and delayed tumor growth and improved overall survival in a syngeneic mouse model of TNBC. Experimental Design: We immunized female B6129SF1/J mice with cit-ENO1 peptides or corresponding unmodified ENO1 (unm-ENO1) peptides as antigens, together with the TLR3 agonist poly I:C as an adjuvant, once a week, for three weeks. PBS or poly I:C alone were used as controls. One week post the last round of immunization, draining lymph nodes (LN) and immunized skin were harvested, and T cell phenotypes evaluated using flow cytometry. To assess anti-cancer efficacy, mice were immunized with cit-ENO1 or unm-ENO1 peptides, followed by orthotopic implantation with murine BRCA1co/co; MMTV-Cre; p53+/- TNBC cells. Tumor growth and overall survival were assessed. Results: Cit-ENO1 peptide vaccination induced a statistically significantly greater percentage of activated CD8+ PD-1+ effector T cells as well as CD8+ CD44+ CD62L- effector memory T cells in the LNs compared with either the control groups or the unm-ENO1 peptide. CD8+ PD-1+ T-cells were also significantly elevated in immunized skin of cit-ENO1 vaccinated mice. Both cit-ENO1 and unm-ENO1 vaccine increased CD4+ PD-1+ T cells from the LNs and immunized skin. Remarkably, the cit-ENO1 vaccine but not the um-ENO1 vaccine significantly delayed tumor growth and markedly improved overall survival compared to respective controls. Conclusion: Cancer-selective cit-ENO1 peptides induced a potent anti-cancer immune response and significantly delayed tumor initiation and progression in a preclinical, immune competent mouse model of TNBC. Citation Format: Ricardo A. Leon Letelier, Hiroyuki Katayama, Yihui Chen, Yinning Cai, Fuchung Hsiao, Banu Arun, Samir Hanash. A citrullinated-ENO1 peptide vaccine delayed tumor growth and markedly improved overall survival in a mouse model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4098.

Poly(I:C)/c-GAMP/Alum ternary adjuvant system synergistically enhances the immunogenicity of glycopeptide antigen

The highly glycosylated mucin 1 (MUC1) serves as a promising target for cancer vaccines. However, the weak immunogenicity of MUC1 greatly impedes its application. Here, we covalently linked a MUC1 glycopeptide to carrier protein bovine serum albumin (BSA), and the resulting BSA-MUC1 conjugate was integrated with a ternary adjuvant system comprising TLR3 agonist poly(I:C), STING agonist c-GAMP, and Alum adjuvant. Compared to adjuvant-free and Alum-adjuvanted groups, the ternary adjuvant group induced 18.3- and 10.9-fold increases in MUC1-specific IgG titers, and effectively suppressed tumor growth. These findings may propose a straightforward yet highly effective strategy for designing vaccines, offering promising prospects in cancer treatment.

Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-β, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

Rhinovirus infection induces secretion of endothelin-1 from airway epithelial cells in both in vitro and in vivo models

BackgroundRhinovirus (RV) infection of airway epithelial cells triggers asthma exacerbations, during which airway smooth muscle (ASM) excessively contracts. Due to ASM contraction, airway epithelial cells become mechanically compressed. We previously reported that compressed human bronchial epithelial (HBE) cells are a source of endothelin-1 (ET-1) that causes ASM contraction. Here, we hypothesized that epithelial sensing of RV by TLR3 and epithelial compression induce ET-1 secretion through a TGF-β receptor (TGFβR)-dependent mechanism.MethodsTo test this, we used primary HBE cells well-differentiated in air–liquid interface culture and two mouse models (ovalbumin and house dust mite) of allergic airway disease (AAD). HBE cells were infected with RV-A16, treated with a TLR3 agonist (poly(I:C)), or exposed to compression. Thereafter, EDN1 (ET-1 protein-encoding gene) mRNA expression and secreted ET-1 protein were measured. We examined the role of TGFβR in ET-1 secretion using either a pharmacologic inhibitor of TGFβR or recombinant TGF-β1 protein. In the AAD mouse models, allergen-sensitized and allergen-challenged mice were subsequently infected with RV. We then measured ET-1 in bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR) following methacholine challenge.ResultsOur data reveal that RV infection induced EDN1 expression and ET-1 secretion in HBE cells, potentially mediated by TLR3. TGFβR activation was partially required for ET-1 secretion, which was induced by RV, poly(I:C), or compression. TGFβR activation alone was sufficient to increase ET-1 secretion. In AAD mouse models, RV induced ET-1 secretion in BALF, which positively correlated with AHR.ConclusionsOur data provide evidence that RV infection increased epithelial-cell ET-1 secretion through a TGFβR-dependent mechanism, which contributes to bronchoconstriction during RV-induced asthma exacerbations.

HIV protease inhibitor saquinavir inhibits toll-like receptor 4 activation by targeting receptor dimerization

ObjectiveToll-like receptor 4 (TLR4) is crucial in induction of innate immune response through recognition of invading pathogens or endogenous alarming molecules. Ligand-triggered dimerization of TLR4 is essential for the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Saquinavir (SQV), an FDA-approved HIV protease inhibitor, has been shown to attenuate the activation of NF-κB induced by HMGB1 by blocking TLR4-MyD88 association in proteasome independent pathway. This study aims to define whether SQV is an HMGB1-specific and MyD88-dependent TLR4 signaling inhibitor and which precise signaling element of TLR4 is targeted by SQV.Materials and methodsPMA differentiated human THP-1 macrophages or reconstituted HEK293 cells were pretreated with SQV before stimulated by different TLR agonists. TNF-α level was evaluated through ELISA assay. NF-κB activation was analyzed using NF-κB SEAP reporting system. The levels of MyD88/TRIF pathways-related factors were examined by immunoblot. TLR4 endocytosis was assessed by immunocytochemistry. TLR4 dimerization was determined using immunoprecipitation between different tagged TLR4 and an in silico molecular docking experiment was performed to explore the possible binding site of SQV on its target.ResultsOur data showed that SQV suppresses both MyD88- and TRIF-dependent pathways in response to lipopolysaccharide (LPS), a critical sepsis inducer and TLR4 agonist, leading to downregulation of NF-κB and IRF3. SQV did not suppress MyD88-dependent pathway triggered by TLR1/2 agonist Pam3csk4. In the only TRIF-dependent pathway, SQV did not alleviate IRF3 phosphorylation induced by TLR3 agonist Poly(I:C). Furthermore, dimerization of TLR4 following LPS or HMGB1 stimulation was decreased by SQV.ConclusionWe concluded that TLR4 receptor complex is one of the mammalian targets of SQV, and TLR4-mediated immune responses and consequent risk for uncontrolled inflammation could be modulated by FDA-approved drug SQV.

Abstract 673: CHROMR Governs Interferon Signaling And Cholesterol Homeostasis In Human Atherosclerotic Plaque

Long noncoding RNAs (lncRNA) have emerged as critical regulators of gene expression, yet their potential contribution to human disease remains poorly understood. We previously identified the primate-specific lncRNA CHROMR as a key regulator of cholesterol homeostasis. Here, we report a novel function for CHROMR in controlling interferon signaling, which is known to regulate innate and adaptive immune responses that drive atherosclerosis. Using single cell RNA-sequencing of human atheroma, we observed robust expression of CHROMR in plaque immune cell populations, which correlated strongly with the expression of two subsets of interferon-stimulated genes (ISG). Interrogation of these genesets using DisGeNET, a comprehensive catalogue of genes and variants associated to human diseases, revealed associations with atherosclerosis and coronary artery disease. Differential expression analysis of CHROMR -expressing versus non-expressing immune cells showed higher levels of ISGs particularly in the monocyte-macrophage cluster. To assess the impact of CHROMR depletion on inflammatory gene expression, we knocked down CHROMR in THP1 macrophages using GapmeR antisense oligonucleotides (or controls) and treated with the TLR3 agonist poly(I:C) to stimulate the interferon response. Transcriptome profiling and unsupervised hierarchical clustering of genes differentially expressed in CHROMR -sufficient and -depleted macrophages revealed that CHROMR silencing markedly repressed expression of key ISGs, including MX1 , IFI44L , STAT1 , and CXCL and CCL family chemokines ( CXCL10 , CXCL11, CCL2 ). Conversely, overexpression of CHROMR in THP1 macrophages increased expression of these ISGs. Furthermore, CHROMR depletion in macrophages reduced histone acetylation at regulatory regions of ISG loci. Mechanistically, we showed that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional co-repressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Taken together, our work unveils a critical role for CHROMR in coordinating interferon signaling responses in macrophages and suggests that targeting this lncRNA may reduce inflammation in atherosclerotic plaques.

The Protective Effects of Goitrin on LPS-Induced Septic Shock in C57BL/6J Mice via Caspase-11 Non-Canonical Inflammasome Inhibition.

Septic shock is defined as a subset of sepsis, which is associated with a considerably high mortality risk. The caspase-11 non-canonical inflammasome is sensed and activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis, it plays a critical role in septic shock. However, there are few known drugs that can control caspase-11 non-canonical inflammasome activation. We report here that goitrin, an alkaloid from Radix Isatidis, shows protective effects in LPS-induced septic shock and significant inhibitory effect in caspase-11 non-canonical inflammasome pathway. Male C57BL/6J were injected intraperitoneally with LPS (20 mg/kg) to induce experimental septic shock. The results demonstrated that the survival rates of mice pretreated with goitrin or Toll-like receptor 4 (TLR4) inhibitor TKA-242 increased, and LPS-induced hypothermia and lung damage improved by inhibiting inflammatory response. Elucidating the detailed mechanism, we surprisingly found goitrin is really different from TAK-242, it independent of the TLR4 signal activation, but significantly inhibited the activation of caspase-11 non-canonical inflammasome, including cleaved caspase-11 and N-terminal fragment of gasdermin D (GSDMD-NT). Furthermore, with a nonlethal dose of the TLR3 agonist poly(I:C)-primed and subsequently challenged with LPS to induce caspase-11-mediated lethal septic shock, the efficacy of goitrin had been verified. Those results revealed the effect of goitrin in protective against LPS-induced septic shock via inhibiting caspase-11 non-canonical inflammasome, which provided a new therapeutic strategy for clinical treatment of septic shock.

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils.

Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality.

Graphene oxide exposure suppresses immune responses and increases the sensitivities of zebrafishes to lipopolysaccharides via the down-regulation of Toll-like receptors

Besides the roles in regulating immune responses, recent studies have also suggested a pivotal role of Toll-like receptors (TLRs) in lipid metabolism. Previously we reported that graphene oxide (GO) suppresses TLR signaling pathways. In this study, we further investigated the in vivo toxicity of GO in zebrafish larvae via TLRs. Zebrafish embryos (5 h post fertilization; hpf) were exposed to 0.01, 0.1, 1 or 10 mg/L GO for up to 5 days, and the results revealed that GO induced developmental toxicity, in the form of delayed hatching time and increased deformity, mortality and heart rate; blood flow and linear velocity, however, remained unchanged. Meanwhile, the locomotor activities of larvae were also increased with GO exposure. As the possible mechanism for these effects, GO exposure significantly decreased TLR1, TLR4 and TLR5 expression, leading to suppressed expression of immune genes, including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor α (TNFα). Furthermore, the expression of perilipin 2 (PLIN2) was also decreased, which consequently led to decreased lipid droplet (LD) staining and triglyceride (TG) content. Interestingly, GO-induced deformity was significantly increased by TLR2/TLR4 agonist lipopolysaccharides (LPS), but less effectively promoted by TLR3 agonist poly inosinic: cytidylic acid (IC). The results from this study suggested that GO exposure induces developmental toxicity to zebrafish larvae via the suppression of TLR signaling pathways, likely due to immune inhibition and dysfunction of LD biogenesis.

TLR3 activation with poly I:C exacerbates escalated alcohol consumption in dependent male C57BL/6J mice

ABSTRACT Background: Activation of TLR3 receptors, which are sensitive to viral infection, has emerged as a possible mechanism that increases alcohol intake in rodents. Objectives: These studies examined whether a history of ethanol dependence exacerbated the increase in drinking driven by the TLR3 agonist poly I:C. Methods: Male C57BL/6J mice (>10 per group) were given access to ethanol (20% v/v) 2 hours a day following a history of home cage drinking or after having been rendered ethanol-dependent using a chronic intermittent ethanol (CIE) vapor model. After testing multiple doses, a 5 mg/kg repeated poly I:C challenge was used to probe the effects of repeated immune challenge, alone or in conjunction with repeated cycles of CIE, on voluntary drinking. An ethanol (12% v/v) operant self-administration model was used to test the effects of poly I:C on stress-induced reinstatement of ethanol seeking and consumption. Results: Poly I:C in naive animals resulted in transient, modest increases in ethanol intake in the home cage and in self-administration (p < 0.05). However, poly I:C challenge resulted in sensitized stress-induced ethanol consumption and evoked a strong and persistent escalation of drinking in mice with a history of dependence (p < 0.05 for both). Conclusion: Activation of viral immune defense may affect ethanol consumption in dependence and sensitivity to future stressors. As patients who suffer from alcohol use disorder are at a heightened risk for viral infection, this interaction could generate risk factors for exacerbating behaviors associated with Alcohol Use Disorders via an immune mechanism.

A microarray patch SARS-CoV-2 vaccine induces sustained antibody responses and polyfunctional cellular immunity

Sustainable global immunization campaigns against COVID-19 and other emerging infectious diseases require effective, broadly deployable vaccines. Here, we report a dissolvable microarray patch (MAP) SARS-CoV-2 vaccine that targets the immunoresponsive skin microenvironment, enabling efficacious needle-free immunization. Multicomponent MAPs delivering both SARS-CoV-2 S1 subunit antigen and the TLR3 agonist Poly(I:C) induce robust antibody and cellular immune responses systemically and in the respiratory mucosa. MAP vaccine-induced antibodies bind S1 and the SARS-CoV-2 receptor-binding domain, efficiently neutralize the virus, and persist at high levels for more than a year. The MAP platform reduces systemic toxicity of the delivered adjuvant and maintains vaccine stability without refrigeration. When applied to human skin, MAP vaccines activate skin-derived migratory antigen-presenting cells, supporting the feasibility of human translation. Ultimately, this shelf-stable MAP vaccine improves immunogenicity and safety compared to traditional intramuscular vaccines and offers an attractive alternative for global immunization efforts against a range of infectious pathogens.

Enhancing the immune effect of oHSV-1 therapy through TLR3 signaling in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults, with patients having a low overall survival rate. Oncolytic viruses (OVs) have been shown effective as monotherapy or combined with immunotherapy in the treatment of UM. Oncolytic herpes simplex type I virus (oHSV-1) was found to alter gene expression and immune function in UMs. We investigated whether a combination treatment would be more effective in treating UM and reactive immune cells. RNA sequencing analysis were used to identify the effect of oHSV-1 infection in UM cells and protein changes were validated by western blot. Cell viability assays were performed through UM cell lines (MUM2B, 92.1, and MP41) and retinal pigment epithelial cell line (ARPE-19) to identify the efficacy and safety of the combination treatment. Western blot, qRT-PCR, cell viability assay and immunocytochemistry were performed to discover the reactivation of immune cells (U937 and HMC3). Through RNA sequencing analysis and in vitro molecular biology assays, this study tested the ability of oHSV-1 combined with the TLR3 agonist poly(I:C) to re-activate the TLR3 meditated NF-ƙB signaling pathway and further increase the anti-tumor activity of UM cells and macrophages, including the stimulation of macrophage polarization and proliferation. These findings indicate that the treatment of UM with a combination of oHSV-1 and poly(I:C) generates immune responses and enhances anti-tumoral activity, suggesting the need for further investigations and clinical trials of this combination.

Somatic ablation of IKKβ in liver and leukocytes is not tolerated in obese mice but hepatic IKKβ deletion improves fatty liver and insulin sensitivity.

The kinase IKKβ controls pro-inflammatory gene expression, and its activity in the liver and leukocytes was shown to drive metabolic inflammation and insulin resistance in obesity. However, it was also proposed that liver IKKβ signaling protects obese mice from insulin resistance and endoplasmic reticulum (ER) stress by increasing XBP1s protein stability. Furthermore, mice lacking IKKβ in leukocytes display increased lethality to lipopolysaccharides. This study aims at improving our understanding of the role of IKKβ signaling in obesity. We induced IKKβ deletion in hematopoietic cells and liver of obese mice by Cre-LoxP recombination, using an INF-inducible system, or a liver-specific IKKβ deletion in obese mice by adenovirus delivery of the Cre recombinase. The histopathological, immune, and metabolic phenotype of the mice was characterized. IKKβ deletion in the liver and hematopoietic cells was not tolerated in mice with established obesity exposed to the TLR3 agonist poly(I:C) and exacerbated liver damage and ER-stress despite elevated XBP1s. By contrast, liver-specific ablation of IKKβ in obese mice reduced steatosis and improved insulin sensitivity in association with increased XBP1s protein abundance and reduced expression of de-novo lipogenesis genes. We conclude that IKKβ blockage in liver and leukocytes is not tolerated in obese mice exposed to TLR3 agonists. However, selective hepatic IKKβ ablation improves fatty liver and insulin sensitivity in association with increased XBP1s protein abundance and reduced expression of lipogenic genes.

Profiling Differential Effects of 5 Selective Serotonin Reuptake Inhibitors on TLRs-Dependent and -Independent IL-6 Production in Immune Cells Identifies Fluoxetine as Preferred Anti-Inflammatory Drug Candidate

Excessive proinflammatory cytokine production induced by abnormal activation of Toll-like receptor (TLR) signaling, for example, by SARS-CoV-2 infection, can cause a fatal cytokine storm. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and fluvoxamine, used to treat depression, were recently reported to reduce the risk of severe disease in patients with coronavirus disease 2019 (COVID-19), but the mechanisms of the anti-inflammatory effects of SSRIs, and which SSRI would be most suitable as an anti-inflammatory drug, remain unclear. Here, we examined the inhibitory effects of 5 FDA-approved SSRIs, paroxetine, fluoxetine, fluvoxamine, sertraline and escitalopram, on the production of interleukin-6 (IL-6) induced by stimulation with multiple TLR agonists in murine macrophages and dendritic cells, and on the production of cytokines induced by concanavalin A in murine lymphocytes. In J774.1 murine macrophage cells, pretreatment with SSRIs significantly suppressed IL-6 release induced by TLR3 agonist poly(I:C), TLR4 agonist LPS or TLR9 agonist CpG ODN, but did not affect IL-6 release induced by TLR7 agonists imiquimod or resiquimod. In accordance with the results obtained in J774.1 cells, pretreatment with SSRIs also suppressed IL-6 release induced by a TLR3, TLR4 or TLR9 agonist in bone marrow-derived dendritic cells and peritoneal cells of C57BL/6 mice. On the other hand, interestingly, sertraline alone among the SSRIs amplified IL-6 production induced by TLR7 agonists in murine dendritic cells, though not in macrophages. Concanavalin A-induced production of IL-6 or IL-2 in murine lymphocytes was suppressed by SSRIs, suggesting that SSRIs also inhibit TLRs-independent IL-6 production. Since SSRIs suppressed both IL-6 production induced by multiple TLR agonists in macrophages or dendritic cells and TLR-independent IL-6 production in lymphocytes, they are promising candidates for treatment of patients with cytokine storm, which is mediated by overactivation of multiple TLRs in a complex manner, leading to the so-called IL-6 amplifier, an IL-6 overproduction loop. However, the 5 SSRIs examined here all showed different effects. Overall, our results suggest that fluoxetine may be the most promising candidate as an anti-inflammatory drug. An examination of the structural requirements indicated that the N-methyl group of fluoxetine has a critical role in the inhibition of IL-6 production.

Immunization with CSP and a RIG-I Agonist is Effective in Inducing a Functional and Protective Humoral Response Against Plasmodium.

Malaria is a major public health concern, as a highly effective human vaccine remains elusive. The efficacy of a subunit vaccine targeting the most abundant protein of the sporozoite surface, the circumsporozoite protein (CSP) has been hindered by difficulties in generating an effective humoral response in both quantity and quality. Using the rodent Plasmodium yoelii model we report here that immunization with CSP adjuvanted with 5’ppp-dsRNA, a RIG-I agonist, confers early and long-lasting sterile protection in mice against stringent sporozoite and mosquito bite challenges. The immunization induced high levels of antibodies, which were functional in targeting and killing the sporozoites and were sustained over time through the accumulation of long-lived plasma cells in the bone marrow. Moreover, 5’ppp-dsRNA-adjuvanted immunization with the CSP of P. falciparum was also significantly protective against challenges using a transgenic PfCSP-expressing P. yoelii parasite. Conversely, using the TLR3 agonist poly(A:U) as adjuvant resulted in a formulation that despite inducing high antibody levels was unable to generate equally functional antibodies and was, consequently, less protective. In conclusion, we demonstrate that using 5’ppp-dsRNA as an adjuvant to vaccines targeting CSP induces effective anti-Plasmodium humoral immunity.

STING activation under pre-existing inflammatory conditions causes severe skin disease

Abstract The activation of the stimulator of interferon genes 1 (Sting1) pathway causes rapid induction of type I IFNs. Recently, different STING agonists are being proposed as therapeutics for controlling viral infections like those caused by the SARS-CoV-2. Considering that excessive STING activation leads to autoimmunity, this study was undertaken to investigate the effects of STING activation under pre-existing systemic inflammatory conditions induced by a viral infection. Female BALB/c mice were treated (Tx) with TLR3 agonist poly(IC), which was followed 24h later by subcutaneous injection of STING agonist diamidobenzimidazole (diAbZi). Only diAbZi or vehicle Tx mice were used as controls. Mice Tx with poly(IC) and diAbZi showed the highest systemic levels of type I IFNs, IL-6, and TNFα. Within 10 days, the salivary glands showed increased ILC1, monocytes, and CD8+ T cells. However, glandular function was not affected. Five days after Tx, except for the vehicle controls, all mice showed differing extents of redness, hair loss, and ulceration at the injection site. By day 10, diABZi alone Tx mice showed either normal skin or a small scar formation. In contrast, mice Tx with poly IC and diABZi, showed extensive hair loss, erythema, and ulceration. In addition, hematoxylin and eosin-stained skin sections showed extensive neutrophilic infiltration at the site of ulceration, epidermal thickening, and patchy lymphocytic infiltrate that extended into the subdermal layers. Our data demonstrate that STING activation under systemic inflammatory conditions, such as a viral infection, can lead to severe skin disease. Therefore, caution needs to be exercised in employing STING agonists as therapeutics for treating SARS-CoV-2.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer

ObjectiveStroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy.DesignTo address the lack of efficacious therapeutic...