Search Results (225)

Background: While mRNA vaccines effectively limit hospitalization and severe COVID-19 disease, the precise early innate immune mechanisms associated with their efficacy and reactogenicity remain underexplored. The identification of innate immune correlates prior to vaccination could provide mechanistic insights and potentially predict responses. Methods: We developed an in vitro model to study the innate immune activation of pre-vaccination peripheral blood mononuclear cells (PBMCs) collected from participants enrolled in a well-characterized COVID-19 BioNTech/Pfizer BNT162b2 vaccine (BNT162b2 vaccine) cohort. Pre-vaccination PBMCs were stimulated with empty lipid nanoparticle (LNP), mRNA-LNP, or Toll-like receptor (TLR) agonists. Using multiparameter spectral flow cytometry, we analyzed the baseline immune state, innate responsiveness to stimuli, and cytokine profiles of study participants. These pre-vaccination in vitro results were analyzed for correlations with post-vaccination symptoms and spike-specific IgG responses. Results: Baseline dendritic cell (DC) states inversely correlated with the magnitude of symptoms following BNT162b2 vaccination. Heightened conventional (cDC) and weaker plasmacytoid DC (pDC) responses to RNA stimuli correlated with the magnitude of an acute IgG response. IgG durability modestly correlated with a lower pDC state but higher cDC2 and monocyte baseline states and inversely correlated with TLR3 agonist responsiveness. Conclusions: The pre-vaccination assessment of innate immune function and resting states can be used to fit models potentially predictive of immunogenicity and reactogenicity to BNT162b2 vaccination. Pre-vaccination DC states may influence reactogenicity, while the response to RNA may impact antibody responses. Our data suggest that pre-vaccination assessment offers insights into the innate mechanisms driving mRNA vaccine responses and has predictive potential. Full article

Background/Objectives: Activation of the Toll-like receptor 7 (TLR-7) plays an important role in the pathogenesis of many autoimmune diseases and viral infections. Although we have previously observed inflammation-mediated dysregulation of placental transporters, the role of TLR-7 has not been examined. Using the TLR-7 agonist, imiquimod (IMQ), we evaluated transporter expression in IMQ-treated pregnant rats and ex vivo in cultured rat placental explants. Methods: We administered 5 mg/kg (IP) of IMQ to pregnant Sprague Dawley rats on gestational day (GD) 14. The expression levels of inflammatory biomarkers and transporters were measured in maternal and fetal tissues by qRT-PCR and immunodetection methods, and effects on the placental proteome were assessed using LC/MS/MS. The involvement of TLR-7 was confirmed in rat placental explants. Results: IMQ administration resulted in Irf7 induction and increased levels of IL-6, Tnf-α, and type-I/II interferon pathways in maternal liver and placenta, which is consistent with TLR-7 activation. Proteomic profiling revealed IMQ-mediated activation of pathways involved in immune response, vesicle trafficking, and oxidative stress. Significantly decreased placental, hepatic, and renal protein expression of P-glycoprotein (PGP) was seen in the IMQ group. Likewise, TLR-7 activation using single-stranded RNA resulted in an induction of inflammatory biomarkers and downregulation of PGP in rat placental explants. Conclusions: We demonstrated that the activation of TLR-7 signaling during pregnancy reduces the expression of PGP in placenta and maternal tissues. Further studies are warranted, as decreased protein expression could result in decreased activity and altered fetal exposure to its substrates. Full article

Respiratory viruses can cause life-threatening conditions such as sepsis and acute respiratory distress syndrome. However, vaccines and effective antivirals are available for only a limited number of infections. The majority of approved antivirals are direct-acting agents, which target viral proteins essential for infection. Unfortunately, mutations have already emerged that confer resistance to these antivirals. In addition, there is an urgent need for broad-spectrum antivirals to address the unpredictable emergence of new viruses with pandemic potential. One promising strategy involves modulating the innate immune response and cellular signaling. Imiquimod, a Toll-like receptor 7 (TLR7) agonist, has shown efficacy in murine models of influenza and respiratory syncytial virus (RSV). Additionally, it demonstrates antiviral activity against herpes simplex virus type 1 (HSV-1) and RSV independent of the TLR7/nuclear factor kappa B (NF-κB) pathway, with protein kinase A (PKA) as a crucial downstream effector. In this study, we demonstrate that imiquimod exhibits concentration-dependent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and canine coronavirus (CCoV) in epithelial cells, underscoring its broad-spectrum action against coronaviruses. Moreover, its anti-coronavirus effect appears to be independent of the TLR/NF-κB and PKA/exchange protein directly activated by cyclic adenosine monophosphate (EPAC) pathways and may instead be linked to the activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. The ability of imiquimod to inhibit coronavirus replication via the MEK/ERK pathway, coupled with its immunomodulatory properties, highlights its potential as a broad-spectrum antiviral. Full article

Despite broad anti-cancer efficacy as Toll-Like Receptor (TLR) 7/8 agonists, imidazoquinolines remain limited in use via systemic administration or in situ vaccination therapies due to inflammatory toxicity. One approach to address this challenge involves better targeting the action of imidazoquinolines by caging them as glycoconjugate prodrugs. Within cancer cells, imidazoquinoline glycoconjugates are activated by hydrolases prior to efflux by ABC transport proteins, where they then elicit tumoricidal effects from the assistance of bystander immune cells, such as tumor-infiltrating lymphocytes and associated macrophages, in local proximity. While this concept of Bystander-Assisted ImmunoTherapy (BAIT) has been established at a molecular level in vitro, tolerability or efficacy of BAIT has not been reported in vivo. Here, we evaluate the MTD and tumor growth delay efficacy of a lead BAIT prodrug (BAIT628) in a male C57BL/6 mouse TRAMP-C2 prostate cancer model to further establish this methodology. Overall, we find that systemic BAIT628 is well tolerated at over 5-fold the dose-limiting inflammatory toxicity of the parent imidazoquinoline (up to 5 mg/mouse/day I.P. for 10 days). Analyzing serum cytokines reveals that IL-10 production, elicited by the mannoside caging group, likely contributes to the enhanced MTD. Using BAIT628 as an in situ vaccination immunotherapy (seven times over 3 weeks) resulted in significant tumor growth delay and increased survival, both alone and in combination with a murinized α-PD-L1 checkpoint blockade. The tumor histology of tumor-infiltrating immune cell subsets (CD4⁺, CD8⁺, CD11c⁺) reveals significant increases in CD11c⁺ populations, consistent with TLR7/8 agonism. Overall, BAIT628 is well tolerated and exhibits significant efficacy in the TRAMP-C2 model. These results demonstrate how the BAIT approach can optimize imidazoquinolines for in vivo tolerability and subsequent efficacy as cancer immunotherapeutics. Full article

The activation of immunosuppressed antigen-presenting cells (APCs) in the tumor microenvironment is a key goal in modern cancer immunotherapy. Our laboratory utilizes a liposome-based immunotherapy platform that binds endogenous complement to deliver antigen, adjuvant, and therapeutic agents to APCs in vivo. The liposomes contain external linker groups, which readily bind complement protein C3, and mediate liposomal uptake via complement receptor 3 into APCs. To test the ability of a model antigen to bind to these external linker groups on C3-liposomes and elicit a robust adaptive immune response, we conjugated a modified ovalbumin peptide (OVA-C) to the liposomes and incorporated a toll-like receptor (TLR) 4 agonist, monophosphoryl lipid A (MPLA), in the liposomal membrane. Adaptive immune responses from C57BL/6 mice were analyzed by ELISA and ELISpot. Mice vaccinated with OVA-C liposomes elicited significantly greater humoral and cellular adaptive responses relative to controls. Furthermore, female mice vaccinated with MPLA + OVA-C liposomes produced significantly more IgG antibodies than males vaccinated with the same liposomes. In conclusion, antigen binding on the exterior of C3-liposomes markedly improves antigen loading efficiency and still allows for complement C3-targeted delivery to APCs. These data demonstrate the initiation of a robust cellular and humoral immune response using a new liposomal delivery platform. Full article

Exopolysaccharides (EPSs) produced by probiotic bacteria have garnered attention due to their effects on the gut health of humans and animals. The nutrients that probiotics access during their growth are essential for producing beneficial effects on host health. Direct immunomodulatory effects of graminan-type fructans (GTFs) from Agave tequilana through toll-like receptors (TLRs) have been demonstrated. However, the immunomodulatory effects of these fructans, mediated through the EPSs produced by the probiotics cultivated with them, remain unexplored. We explored the immunomodulatory effects of lactic acid bacteria (LAB) strains isolated from swine and their EPSs, based on the GTFs used as carbon sources during their growth. While the LAB strains activated the NF-κB pathway independently of the GTF source, their EPSs activated it in a GTF source-dependent manner. LAB activation through TLR2 showed a GTF source dependency, whereas their EPSs activated TLR2 independently of the GTF source. The LAB and their EPSs activated TLR4 in a GTF source-dependent manner. Both the LAB and their EPSs inhibited the activation of TLR2 and TLR4 agonists, which exhibited a strong dependence on the GTF source. The strength of GTF C’s immunomodulatory effects on LAB illustrates its specificity, its impact on the EPS structure, and its biological effects. Our results support the promising health benefits of this synbiotic model for swine health and lowering inflammation. Full article

Background: Mucin-1 (MUC1) is a glycoprotein that is hypoglycosylated and overexpressed in most adenocarcinomas, making it a promising target for cancer vaccines. Our group previously demonstrated that C3 (OPSS)-liposomes enhance antigen uptake by antigen-presenting cells (APCs) via the complement C3 pathway and, when combined with toll-like receptor (TLR) agonists, reduce tumor growth in murine cancer models. Methods: In the present study, we evaluate the immunogenicity of MUC1 peptide vaccines encapsulated in C3-liposomes, with and without TLR agonists, using MUC1-tolerant transgenic mice challenged with Lewis lung carcinoma (LLC.MUC1) cells. To assess vaccine effectiveness, tumor volumes were measured, and flow cytometry and ELISA and ELISPOT assays were used to assess the immune response. Results: Both male and female C57BL/6 transgenic mice vaccinated with MUC1 C3-liposomes developed significantly smaller tumors than those vaccinated with free MUC1 peptide or PBS. Notably, a sex-dependent response emerged in mice vaccinated with MUC1 C3-liposomes with TLR agonists (TLR4, TLR7/8, and TLR9); male mice exhibited greater tumor suppression than females. Flow cytometry analysis revealed that female mice had significantly higher levels of CD11b⁺, LY6C⁺, and LY6G⁺ MDSC cells, suggesting a potential mechanism for the sex difference. Additionally, MUC1 C3-liposome vaccination elicited robust adaptive immune responses, including significantly higher levels of IFN-γ-producing T cells and MUC1-specific IgG antibodies compared to non-encapsulated MUC1 or TLR adjuvant-only formulations. Conclusions: These findings underscore the potential of C3-liposome-based antigen vaccines to enhance anti-tumor immunity and highlight the impact of sex differences in vaccine efficacy. Full article

Nickel oxide nanoparticles (NiONPs) can induce liver fibrosis, and their mechanism may be related to non-coding RNA, nuclear receptor signal transduction and ferroptosis, but the regulatory relationship between them is not clear. In this study, we aimed to investigate the role of hsa_circ_0001944 in regulating the Farnesol X receptor (FXR)/Toll-like receptor 4 (TLR4) pathway and ferroptosis in NiONPs-induced collagen deposition. We observed decreased FXR expression, increased TLR4 expression and alterations in ferroptosis features in both the rat liver fibrosis and the LX-2 cell collagen deposition model. To investigate the regulatory relationship among FXR, TLR4 and ferroptosis, we treated LX-2 cells with FXR agonist (GW4064), TLR4 inhibitor (TAK-242) and ferroptosis agonist (Erastin) combined with NiONPs. The results showed that TAK-242 alleviated collagen deposition by increasing ferroptosis features. Furthermore, GW4064 reduced the expression of TLR4, increased the ferroptosis features and alleviated collagen deposition. The results indicated that FXR inhibited the expression of TLR4 and enhanced the ferroptosis features, which were involved in the process of collagen deposition in LX-2 cells induced by NiONPs. Subsequently, we predicted that hsa_circ_0001944 might regulate FXR through bioinformatics analysis, and found NiONPs reduced the expression of hsa_circ_0001944 in LX-2 cells. Overexpression of hsa_circ_0001944 increased FXR level, reduced TLR4 level, increased the ferroptosis features and alleviated collagen deposition in LX-2 cells. In summary, we demonstrated that hsa_circ_0001944 regulates the FXR/TLR4 pathway and ferroptosis alleviate collagen formation induced by NiONPs. Full article

HIV-1 infection is successfully treated by antiretroviral therapy; however, it is not curative as HIV-1 remains present in the viral reservoir. A strategy to eliminate the viral reservoir relies on the reactivation of the latent provirus to subsequently trigger immune-mediated clearance. Here, we investigated whether the activation of Toll-like receptor 8 (TLR8) or RIG-I-like receptor (RLR) together with the latency reversal agent (LRA) second mitochondrial-derived activator of caspases mimetics (SMACm) leads to HIV-1 reservoir reduction and antiviral immune activation. The TLR8 and RLR agonist elicited a robust pro-inflammatory cytokine response in PBMCs from both PWH and uninfected people. Notably, co-stimulation with SMACm specifically enhanced TLR8 induced pro-inflammatory cytokine as well as CD8 T cell responses. Ex vivo treatment of PBMCs from PWH with SMACm significantly decreased the size of the inducible HIV-1 reservoir, whereas targeting TLR8 or RLR reduced the HIV-1 reservoir in 50% of PWH ex vivo. Although co-stimulation with TLR8/RLR agonists further reduced the HIV-1 reservoir in 25% of PWH ex vivo, effectively inducing antiviral immunity may help eliminate reactivated HIV-1 cells in vivo. Our findings strongly suggest that LRAs can be used in combination with agonists for pattern recognition receptors to reactivate HIV-1 and induce antiviral immunity. Full article

Immunosenescence, a process with a dysfunctional immune response that may favor infection is associated with an increase in inflammatory responses mediated by proinflammatory cytokines, characteristic of inflammaging. Aging and immunosenescence have a relationship relating to oxidative stress and inflammaging. Therefore, natural antioxidant compounds could be candidates for the control of the oxidative process. Our purpose was to evaluate the effect of resveratrol (Resv) on the antioxidant, antiviral, and anti-inflammatory responses induced by toll-like receptors (TLRs) 3, 4, and 7/8 agonists stimulation on peripheral blood mononuclear cells (PBMCs) of elderly and healthy female individuals (63–82 years old) and young and healthy female individuals (21–31 years old). Our data show that Resv may upregulate antioxidant factor expression, such as catalase (CAT) and SIRT1, in response to TLR4 and TLR7/8 agonists, similarly in both young and aged groups. Moreover, the Resv anti-inflammatory effect was detected by inhibiting IL-1β, TNF-α, and IL-10 secretion levels, as well as by the chemokines CCL2 and CCL5, induced by TLR4 and TLR7/8 stimulation. Curiously, Resv decreased antiviral genes, such as MxA, STING, and IRF7 expression, possibly by reducing the inflammatory effects of interferon-induced genes. Taken together, our results demonstrate the ability of Resv to stimulate antioxidant factors, leading to a downmodulation of the inflammatory response induced by innate immune stimulation. These findings point out Resv as a strategy to control the upregulation of inflammatory response, even in elderly individuals. Full article

Endosomal toll-like receptors (TLRs) TLR7, TLR8, and TLR9 play an important role in systemic lupus erythematosus (SLE) pathogenesis. The proteolytic processing of these receptors in the endolysosome is required for signaling in response to DNA and single-stranded RNA, respectively. Targeting this proteolytic processing may represent a novel strategy to inhibit TLR-mediated pathogenesis. Human alpha 1 antitrypsin (hAAT) is a protease inhibitor with anti-inflammatory and immunoregulatory properties. However, the effect of hAAT on endosomal TLRs remains elusive. In this study, we first tested the effect of hAAT on TLR9 signaling in dendritic cells (DCs). We showed that hAAT inhibited TLR9-mediated DC activation and cytokine production. Human AAT also lowered the expressions of interferon signature genes. Western blot analysis showed that hAAT reduced the expression of the active form (cleaved) of TLR9 in DCs, indicating a novel mechanism of hAAT function in the immune system. We next tested the effect of hAAT on TLR7/8 signaling. Similar to the effect on TLR9 signaling, hAAT also inhibited R848 (TLR7 and 8 agonist)-induced DC activation and functions and lowered the expressions of interferon signature genes. Our in vivo studies using hAAT transgenic mice also showed that hAAT attenuated R848-induced pathogenesis. Specifically, hAAT completely blocked the R848 induction of germinal center T cells (GC T), B cells (GC B), and plasma cells (GC PCs), as well as T follicular T helper cells (T_FH), which are all critical in lupus development. These data demonstrated that hAAT inhibited TLR7/8 and TLR9 signaling pathways, which are critical for lupus development. These findings not only advanced the current knowledge of hAAT biology, but also implied an insight into the clinical application of hAAT. Full article

Human metapneumovirus (HMPV) is a significant respiratory pathogen, particularly in vulnerable populations. Background: No vaccine for the prevention of HMPV is currently licensed, although several subunit vaccines are in development. Saponin-based adjuvant systems (AS), including QS-21, have transformed the field of subunit vaccines by dramatically increasing their potency and efficacy, leading to the development of several licensed vaccines. However, naturally sourced tree bark-extracted QS-21 faces supply and manufacturing challenges, hindering vaccine development. Objective: This study reports on an alternative plant cell culture system for the consistent production of highly pure QS-21. Method: We evaluated the efficacy of cultured plant cell (cpc)-produced QS-21 in a novel HMPV vaccine, formulating a recombinant pre-fusion stabilized HMPV F protein (preF) with cpcQS-21 and a synthetic toll-like receptor 4 (TLR4) agonist adjuvant formulation. Results: In mice, TLR4 agonist containing adjuvant formulations with plant cell-produced QS-21 performed equally to licensed adjuvant AS01 containing tree-bark-extracted QS-21 and demonstrated a significant increase in immunogenicity against HMPV preF compared to the unadjuvanted control. Conclusion: Our findings pave the way for a reliable, scalable, and sustainable source of pure QS-21, enabling the development of highly effective HMPV and other vaccines with significant public health impact. Full article

Plasmacytoid dendritic cells (pDCs) express Toll-like receptor 7 (TLR7) in the endosomes, recognize viral single-stranded RNA (ssRNA), and produce significant amounts of interferon (IFN)-α. Bovine lactoferrin (LF) enhances the response of IFN regulatory factors followed by the activation of IFN-sensitive response elements located in the promoter regions of the IFN-α gene and IFN-stimulated genes in the TLR7 reporter THP-1 cells in the presence of R-848, a TLR7 agonist. In ex vivo experiments using human peripheral blood mononuclear cells, LF enhances IFN-α levels in the supernatant in the presence of R-848. Additionally, it increases the expression of IFN-α, human leukocyte antigen (HLA)-DR, and CD86 in pDCs; HLA-DR and CD86 in myeloid dendritic cells; CD69 in CD56 dim natural killer and T killer cells; and IFN-γ in T helper type 1 and B cells in the presence of R-848. The inhibition of phagocytosis or neutralization of nucleolin, a receptor of LF, suppresses LF incorporation into pDCs. These results suggest that pDCs incorporate LF through phagocytosis or nucleolin-mediated endocytosis, and LF enhances TLR7 response in the endosome and subsequent IFN signaling pathway and activates innate and adaptive immune cells. We anticipate that LF modulates antiviral immunity against environmental ssRNA viruses and contributes to homeostasis. Full article

RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as “dysregulated immunity” is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity. Full article

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder traditionally characterised by the presence of amyloid beta (Aβ) plaques and neurofibrillary tau tangles in the brain. However, emerging research has highlighted additional metabolic hallmarks of AD pathology. These include the metabolic reprogramming of microglia in favour of glycolysis over oxidative phosphorylation. This shift is attributed to an ‘M1′-like pro-inflammatory phenotype, which exacerbates neuroinflammation and contributes to neuronal damage. The urea cycle also presents as an altered metabolic pathway in AD, due to elevated urea levels and altered expression of urea cycle enzymes, metabolites, and transporters in the brain. However, to date, these changes remain largely unexplored. Methods: This study focuses on understanding the effects of extracellular urea and urea transporter-B (UT-B) inhibition on inflammatory changes in lipoteichoic acid (LTA)-stimulated BV2 microglia and on the viability of SH-SY5Y neuronal cells under oxidative stress and neurotoxic conditions. Results: In BV2 microglia, UT-B inhibition demonstrated a notable anti-inflammatory effect by reducing the formation of nitric oxide (NO) and the expression of tumour necrosis factor α (TNFα) and CCL2 in response to stimulation with the toll-like receptor (TLR)2 agonist, lipoteichoic acid (LTA). This was accompanied by a reduction in extracellular urea and upregulation of UT-B expression. The application of exogenous urea was also shown to mediate the inflammatory profile of BV2 cells in a similar manner but had only a modest impact on UT-B expression. While exposure to LTA alone did not alter the microglial metabolic profile, inhibition of UT-B upregulated the expression of genes associated with both glycolysis and fatty acid oxidation. Conversely, neither increased extracellular urea nor UT-B inhibition had a significant impact on cell viability or cytotoxicity in SH-SY5Y neurones exposed to oxidative stressors tert-butyl hydroperoxide (t-BHP) and 6-hydroxydopamine (6-OHDA). Conclusions: This study further highlights the involvement of urea transport in regulating the neuroinflammation associated with AD. Moreover, we reveal a novel role for UT-B in maintaining microglial metabolic homeostasis. Taken together, these findings contribute supporting evidence to the regulation of UT-B as a therapeutic target for intervention into neuroinflammatory and neurodegenerative disease. Full article