Toll-like Receptor Agonists Research Articles

Albumin-binding toll-like receptor 7 agonist nanocomplexes elicit antitumor immunity for cancer immunotherapy

Toll-like receptor agonists have been extensively applied as immunostimulatory adjuvants because of their well-defined pharmacological mechanisms, while systemic administration of Toll-like receptor agonists have shown limited efficacy due to their unfavorable pharmacokinetic properties. Here we constructed an albumin-binding Toll-like receptor 7 agonist (Ab1V209) by conjugating the Toll-like receptor 7 agonist 1V209 with truncated Evans Blue for binding to endogenous albumin. The resulting Ab1V209 has the ability to hijack albumin and is retained in tumor tissuesfor a higher accumulation than 1V209. As monotherapy, the systemic administration of Ab1V209 alters the function of tumor-associated macrophages towards anti-tumor phenotype, and increases the infiltration of CD8+ T cells, leading to controlled tumor growth with minimal systemic toxicity. When used in combination with an mRNA cancer vaccine, the combination therapy activated dendritic cells to provide antigen presentation molecule and co-stimulatory molecule, resulting in a strong T cell activation and expansion. This study presents a strategy for the rational engineering of albumin-binding adjuvants to enhance cancer immunotherapy.

Macrophages as Potential Therapeutic Targets in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogenous malignant hemopathy, and although new drugs have emerged recently, current treatment options still show limited efficacy. Therapy resistance remains a major concern due to its contribution to treatment failure, disease relapse, and increased mortality among patients. The underlying mechanisms of resistance to therapy are not fully understood, and it is crucial to address this challenge to improve therapy. Macrophages are immune cells found within the bone marrow microenvironment (BMME), of critical importance for leukemia development and progression. One defining feature of macrophages is their plasticity, which allows them to adapt to the variations in the microenvironment. While this adaptability is advantageous during wound healing, it can also be exploited in cancer scenarios. Thus, clinical and preclinical investigations that target macrophages as a therapeutic strategy appear promising. Existing research indicates that targeting macrophages could enhance the effectiveness of current AML treatments. This review addresses the importance of macrophages as therapeutic targets including relevant drugs investigated in clinical trials such as pexidartinib, magrolimab or bexmarilimab, but also provides new insights into lesser-known therapies, like macrophage receptor with a collagenous structure (MACRO) inhibitors and Toll-like receptor (TLR) agonists.

Toll-like receptor 7 protects against intestinal inflammation and restricts the development of colonic tissue-resident memory CD8+ T cells.

The maintenance of intestinal homeostasis depends on a complex interaction between the immune system, intestinal epithelial barrier, and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover, persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7), which recognizes single-stranded RNA viruses, and ATG16L1, which facilitates the delivery of viral nucleic acids to the autolysosome endosome, are required for anti-viral immune responses. However, the role of the enteric virome in IBD is still poorly understood. Here, we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state, Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice, reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2 ) or dendritic cells (Atg16l1ΔCd11c ) leads to a similar increase of LP TRM. Furthermore, Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index, histoscore, and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. In conclusion, the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants.

A systematic review and risk of bias analysis of in vitro studies on trophoblast response to immunological triggers

An increasing amount of evidence suggests that immune responses may affect trophoblast functioning, which in turn may play a role in gestational disorders and fetal development. This systematic review offers the first summary of in vitro studies on the trophoblast response to immunological triggers, in conjunction with a risk of bias analysis.A search in Pubmed and Embase yielded 110 relevant studies. Primary trophoblasts were the most commonly used cell type, but trophoblast subtypes were not always defined. Similarly, the exact natures of trophoblast cell lines were sometimes unclear. Cytokines and Toll-like receptor agonists were often used as interventions, but most studies focused on a select few substances such as tumor necrosis factor-α and lipopolysaccharide. In regard to the outcome parameters, some important trophoblast functions, such as hormone production and barrier formation were underrepresented.Whether or not risk of bias was high varied strongly between types of bias. Risk of selection bias, for example, was usually low. However, none of the included studies mentioned blinding or plate randomization. Only a select few studies mentioned passage numbers, use of vehicle control or conflict of interest.In conclusion, better characterization of trophoblast subtypes and a broader range of studied interventions and outcome parameters would contribute to a more complete understanding of trophoblast responses to immune stimuli. Additionally, researchers are encouraged to replicate experiments and pay close attention when setting up and writing down methodologies, in order to improve the reproducibility and translatability of their work.

Higher toll-like receptor 3 expression in umbilical cord blood B cells than in adult blood B cells

Background Umbilical cord blood (UCB), which is considered a rich source of stem cells, has been used for applications in different clinical settings. Therefore, it is crucial to examine the toll-like receptor (TLR) expression levels in UCB B cells as compared to adult blood cells. Aim To determine the phenotypes of B cells in UCB and to investigate their expression of TLR3 as compared to adult blood. Patients and methods Samples of UCB were collected (n=20) after delivery, and peripheral blood samples were collected from female healthy volunteers (n=10) in K2EDTA tubes. Cells were washed twice, then stained using anti-CD19 and anti-TLR3. The samples were acquired by flow cytometry to assess the phenotype of B cells and their expression of TLR3. Besides, the liver and kidney functions were assessed. Results The relative number of CD19+ cells showed lower numbers (5.35%) in UCB than adult blood (15.64%). Additionally, the absolute number of CD19+ cells showed lower numbers by two-fold in cord blood than in adult blood. The relative expression of TLR3 on CD19+ cells showed lower expression in UCB as compared to adult blood by 5.8-fold. However, the absolute number of TLR3+CD19+ was higher in UCB than in adult blood by two-fold. The liver and kidney function showed normal values as investigated the enzyme activity of aspartate aminotransferase, alanine aminotransferase, bilirubin, and creatinine in both cord and adult. Conclusion B cells express lower TLR3 in cord blood than in adult blood. The data from this study open new avenues for the manipulation of cord blood by TLR agonists for clinical application.

Lipopolysaccharide Induces Trained Innate Immune Tolerance in the Heart Through Interferon Signaling in a Model of Stress-Induced Cardiomyopathy.

Although the ability of the heart to adapt to environmental stress has been studied extensively, the molecular and cellular mechanisms responsible for cardioprotection are not yet fully understood. We administered Toll-like receptor (TLR) agonists or a diluent to wild-type mice and assessed their potential to induce cardiac protection against injury from a high intraperitoneal dose of isoproterenol (ISO) administered 7 days later. Cardioprotective effects were analyzed through serum cardiac troponin I levels, immune cell profiling via flow cytometry, echocardiography, and multiomic single-nuclei RNA and ATAC sequencing. Pretreatment with the TLR4 agonist lipopolysaccharide (LPS), but not TLR1/2 or TLR3 agonists, conferred cardioprotection against ISO, as demonstrated by reduced cardiac troponin I leakage, decreased inflammation, preservation of cardiac structure and function, and improved survival. Remarkably, LPS-induced tolerance was reversed by β-glucan treatment. Multiomic analysis showed that LPS-tolerized hearts had greater chromatin accessibility and upregulated gene expression compared to hearts treated with LPS and β-glucan (reverse-tolerized). The LPS tolerance was associated with upregulation of interferon response pathways across various cell types, including cardiac myocytes and stromal cells. Blocking both type 1 and type 2 interferon signaling eliminated LPS-induced tolerance against ISO, while pretreatment with recombinant type 1 and 2 interferons conferred cardiac protection. Multiomic sequencing further revealed enhanced cytoprotective signaling in interferon-treated hearts. Analysis of cell-cell communication networks indicated increased autocrine signaling by cardiac myocytes, as well as greater paracrine signaling between stromal cells and myeloid cells, in LPS-tolerized versus reverse-tolerized hearts. LPS pretreatment confers cardiac protection against ISO-induced injury through TLR4 mediated type 1 and 2 interferon signaling, consistent with trained innate immune tolerance. The observation that LPS-induced protection in cardiac myocytes involves both cell-autonomous and non-cell-autonomous mechanisms underscores the complexity of innate immune tolerance in the heart, warranting further investigation into this cardioprotective phenotype. What is new?: The Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) confers cardiac protection against isoproterenol-mediated injury in a manner consistent with trained innate immune tolerance, which is reversed by β-glucan treatment.Activation of type 1 and 2 interferon signaling, which is downstream of Toll-like receptor 4, is necessary and sufficient for LPS-induced cardiac protection.LPS-tolerized hearts show heightened autocrine signaling by cardiac myocytes and, to a greater degree, increased cell-cell communication between cardiac myocytes and stromal and myeloid cells compared to reverse-tolerized hearts.What are the clinical implications?: TLR4 and interferon signaling play key roles in the establishment of cardiac protection and LPS-induced trained innate immune tolerance.The protective effects of LPS are mediated by cell-autonomous and non-cell-autonomous mechanisms, suggesting that a deeper understanding of the molecular and cellular signatures of innate immune tolerance is required for the development of targeted approaches to modulate trained innate immunity, and consequently cytoprotection, in the heart.

Cosmetically Applicable Soluble Agonists for Toll-like Receptor 2 Produced by Fermentation of Asparagus Extract Supplemented with Skimmed Milk Using Lactobacillus delbrueckii subsp. lactis TL24 Consist of Molecules Larger than 100 kDa and Can Be Stabilized by Lyophilization with Dextrin.

Cosmetically applicable soluble agonists for Toll-like receptor 2 (TLR2), which can strengthen skin barrier function, were produced by fermentation of asparagus (Asparagus officinalis L.) extract supplemented with skimmed milk using Lactobacillus delbrueckii subsp. lactis TL24. Their molecular size was estimated to be >100 kDa. Their TLR2-stimulating activity was stable over 1 year at 4 °C, but it decreased by more than 95% within 10 and 4 months at 25 °C and 40 °C, respectively. The possibility of stabilization of TLR2-stimulating activity by powdering was tested, and we found that lyophilization with 10% or a higher amount of dextrin could stabilize the activity even at 40 °C. The powdered fermented product dose-dependently stimulated TLR2. It augmented the formation of tight junctions in normal human keratinocytes, as detected by fluorescence staining of occludin and ZO-1, whereas their protein and gene expression levels did not increase, suggesting that a change in subcellular localization of these proteins without significant changes in their amounts might be responsible. The powder nature has some benefits over the aqueous, besides stability, e.g., it can be dissolved just before application, allowing fresh material to be used each time, and it may widen a range of cosmetic applications in non-aqueous types of cosmetics.

Rice Husk Silica Liquid Enhances Autophagy and Reduces Overactive Immune Responses via TLR-7 Signaling in Lupus-Prone Models.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by widespread inflammation and multi-organ damage. Toll-like receptor 7 (TLR-7) and autophagy have been implicated in SLE pathogenesis. Rice husk silica liquid (RHSL) has shown potential for modulating inflammatory responses, but its effects on SLE have not been thoroughly investigated. This study aims to evaluate the impact of RHSL on immune responses and autophagy in cell culture experiments, focusing on its effects on TLR-7 signaling, cytokine production, and autophagy modulation. RAW264.7 cells and human peripheral blood mononuclear cells (PBMCs) from healthy donors and SLE patients were used. Cells were stimulated with LPS or TLR-7 agonists and treated with RHSL. Cell viability was assessed, and cytokine levels (TNF-α and IL-6) were measured by ELISA. Autophagy-related proteins (LC3II, ATG5-ATG12) were analyzed by Western blotting. The effect of autophagy inhibition was studied using 3-methyladenine (3-MA). A concentration of 100 μg/mL RHSL did not affect cell viability but significantly reduced the TNF-α production in TLR-7 agonist-stimulated RAW264.7 cells (compared to TLR-7 alone, 3.41 ± 0.54 vs. 6.72 ± 0.07 folds) and PBMCs (compared to TLR-7 alone, 0.97 ± 0.19 vs. 1.40 ± 0.33 folds). RHSL enhanced autophagy, as evidenced by increased LC3II (4.35 ± 1.08 folds) and ATG5-ATG12 (7.07 ± 1.30 folds) conjugation in both RAW264.7 cells and SLE patient-derived PBMCs. The reduction in TNF-α production by RHSL was attenuated by 3-MA, indicating that autophagy plays a role in this process. RHSL also inhibited the translocation of phosphorylated NF-κB into the nucleus, suggesting a mechanism for its anti-inflammatory effects. RHSL exhibits potential as an immunomodulatory agent in SLE by enhancing autophagy and modulating TLR-7 signaling pathways. These findings suggest that RHSL could offer therapeutic benefits for managing inflammatory responses in SLE and warrant further investigation into its clinical applications.

Safety and efficacy of toll-like receptor agonists as therapeutic agents and vaccine adjuvants for infectious diseases in animals: a systematic review.

Strengthening global health security relies on adequate protection against infectious diseases through vaccination and treatment. Toll-like receptor (TLR) agonists exhibit properties that can enhance immune responses, making them potential therapeutic agents or vaccine adjuvants. We conducted an extensive systematic review to assess the efficacy of TLR agonists as therapeutic agents or vaccine adjuvants for infectious diseases and their safety profile in animals, excluding rodents and cold-blooded animals. We collected qualitative and available quantitative data on the efficacy and safety outcomes of TLR agonists and employed descriptive analysis to summarize the outcomes. Among 653 screened studies, 51 met the inclusion criteria. In this review, 82% (42/51) of the studies used TLR agonists as adjuvants, while 18% (9/51) applied TLR agonist as therapeutic agents. The predominant TLR agonists utilized in animals against infectious diseases was CpG ODN, acting as a TLR9 agonist in mammals, and TLR21 agonists in chickens. In 90% (46/51) of the studies, TLR agonists were found effective in stimulating specific and robust humoral and cellular immune responses, thereby enhancing the efficacy of vaccines or therapeutics against infectious diseases in animals. Safety outcomes were assessed in 8% (4/51) of the studies, with one reporting adverse effects. Although TLR agonists are efficacious in enhancing immune responses and the protective efficacy of vaccines or therapeutic agents against infectious diseases in animals, a thorough evaluation of their safety is imperative to in-form future clinical applications in animal studies. https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=323122.

A Pooled Analysis of Eight Clinical Studies Suggests a Link Between Influenza-Like Symptoms and Pharmacodynamics of the Toll-Like Receptor-7 Agonist Vesatolimod.

Vesatolimod is a Toll-like receptor-7 (TLR7) agonist in clinical development as part of a combination regimen for human immunodeficiency virus (HIV) cure. Influenza-like symptoms associated with TLR7-mediated immune activation have been reported in clinical trials of vesatolimod. Therefore, a broader understanding of the safety profile of vesatolimod and association with dose and mechanism of action will help inform future clinical studies. In this analysis, data on flu-like adverse events of interest (AEIs) were pooled from eight clinical studies in which 606 participants either received single or multiple doses of vesatolimod (0.3-12mg; n = 505) or placebo (n = 101). Vesatolimod pharmacokinetics, inflammatory responses, and pharmacodynamics were assessed. The incidence of flu-like AEIs was higher with vesatolimod versus placebo (19% [96/505] vs. 8% [8/101]) and increased with vesatolimod dose and exposure. Most flu-like AEIs with vesatolimod were grade 1 or 2 severity (55% [53 of 96] grade 1; 35% [34 of 96] grade 2) with onset primarily after the first and second dose. Occurrence of flu-like AEIs after doses 1-3 was predictive of reoccurrence after later doses. Dose-dependent elevations of pharmacodynamic biomarkers (interferon-stimulated gene 15, 2'-5'-oligoadenylate synthetase 1, myxovirus resistance-1, interferon-α, interleukin-1 receptor antagonist, interferon-γ-induced protein 10, interferon-inducible T-cell-α chemoattractant) observed in participants with flu-like AEIs suggest a link with vesatolimod mechanism of action. Flu-like AEIs associated with vesatolimod administration were typically mild but increased with exposure, which may be predicted by the response to initial doses. The data suggest that adaptive clinical monitoring could help maximize pharmacodynamic responses and balance adverse events in future clinical trials of vesatolimod.

Mu-Opioid Receptor (MOR) Dependence of Pain in Chemotherapy-Induced Peripheral Neuropathy.

We recently demonstrated that transient attenuation of Toll-like receptor 4 (TLR4) in dorsal root ganglion (DRG) neurons, can both prevent and reverse pain associated with chemotherapy-induced peripheral neuropathy (CIPN), a severe side effect of cancer chemotherapy, for which treatment options are limited. Given the reduced efficacy of opioid analgesics to treat neuropathic, compared with inflammatory pain, the cross talk between nociceptor TLR4 and mu-opioid receptors (MORs), and that MOR and TLR4 agonists induce hyperalgesic priming (priming), which also occurs in CIPN, we determined, using male rats, whether (1) antisense knockdown of nociceptor MOR attenuates CIPN, (2) and attenuates the priming associated with CIPN, and (3) CIPN also produces opioid-induced hyperalgesia (OIH). We found that intrathecal MOR antisense prevents and reverses hyperalgesia induced by oxaliplatin and paclitaxel, two common clinical chemotherapy agents. Oxaliplatin-induced priming was also markedly attenuated by MOR antisense. Additionally, intradermal morphine, at a dose that does not affect nociceptive threshold in controls, exacerbates mechanical hyperalgesia (OIH) in rats with CIPN, suggesting the presence of OIH. This OIH associated with CIPN is inhibited by interventions that reverse Type II priming [the combination of an inhibitor of Src and mitogen-activated protein kinase (MAPK)], an MOR antagonist, as well as a TLR4 antagonist. Our findings support a role of nociceptor MOR in oxaliplatin-induced pain and priming. We propose that priming and OIH are central to the symptom burden in CIPN, contributing to its chronicity and the limited efficacy of opioid analgesics to treat neuropathic pain.

A Photoactivatable Self-Assembled Nanoagonist for Synergistic Therapy against Pancreatic Ductal Adenocarcinoma.

Immunotherapy has revolutionized the cancer treatment paradigm, yet efficient immunotherapeutic responses against immune-cold/desert cancers remain challenging. Herein, we report that photoactivatable nanoagonists yield a potent antitumor synergy of photoimmunotherapy against pancreatic ductal adenocarcinoma (PDAC). The nanoagonist was fabricated by assembling an amphiphilic boron dipyrromethene-derived polymer conjugated with a Toll-like receptor agonist via a photocleavable linker and stimulator of interferon genes agonist. The nanoagonist enables light-induced generation of reactive oxygen species and on-demand release of the agonists to yield synergistic photoimmunotherapy. The produced tumor antigens promote dendritic cell maturation, which is further amplified by these agonists for eliciting adaptive immunity, accompanied by apparently abscopal and long-term memory effects. The nanoagonist further alleviates the fibrosis of tumor stroma and the immunosuppressive microenvironment, leading to the deep infiltrations of clinically used therapeutics and immune cells to yield preferable combinational treatments against PDAC models. These results provide valuable insights into activatable nanoparticles for cancer therapy against immune-desert cancers.

The Protective Effects of Mcl-1 on Mitochondrial Damage and Oxidative Stress in Imiquimod-Induced Cancer Cell Death.

Mitochondria, vital organelles that generate ATP, determine cell fate. Dysfunctional and damaged mitochondria are fragmented and removed through mitophagy, a mitochondrial quality control mechanism. The FDA-approved drug IMQ, a synthetic agonist of Toll-like receptor 7, exhibits antitumor activity against various skin malignancies. We previously reported that IMQ promptly reduced the level of the antiapoptotic Mcl-1 protein and that Mcl-1 overexpression attenuated IMQ-triggered apoptosis in skin cancer cells. Furthermore, IMQ profoundly disrupted mitochondrial function, promoted mitochondrial fragmentation, induced mitophagy, and caused cell death by generating high levels of ROS. However, whether Mcl-1 protects mitochondria from IMQ treatment is still unknown. In this study, we demonstrated that Mcl-1 overexpression induced resistance to IMQ-induced apoptosis and reduced both IMQ-induced ROS generation and oxidative stress in cancer cells. Mcl-1 overexpression maintained mitochondrial function and integrity and prevented mitophagy in IMQ-treated cancer cells. Furthermore, IL-6 protected against IMQ-induced apoptosis by increasing Mcl-1 expression and attenuating IMQ-induced mitochondrial fragmentation. Mcl-1 overexpression ameliorates IMQ-induced ROS generation and mitochondrial fragmentation, thereby increasing mitochondrial stability and ultimately attenuating IMQ-induced cell death. Investigating the roles of Mcl-1 in mitochondria is a potential strategy for cancer therapy development.

Abstract 15: Cell-Free Mitochondrial DNA Drives Endothelial Cell Activation and Vascular Dysfunction via Toll-Like Receptor 9 and Inflammasome Pathway Activation

Immune system activation has been shown to play an important role in the pathogenesis of hypertension and substantial evidence has been highlighting the importance of inflammasomes in vascular disease progression. Mitochondria damage leads to mitochondrial DNA (mtDNA) releasing into the cytoplasm, which, in turn, triggers the activation of leucine-rich-containing (NLR) family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes, thereby exacerbating inflammation. However, whether mtDNA release-dependent exacerbation of inflammasomes drives endothelial cell activation and vascular damage remains to be elucidated. We hypothesize that cytosolic mtDNA release triggers endothelial cell activation and inflammation via Toll-Like Receptor 9 (TLR9) and inflammasome pathway activation, leading to vascular dysfunction. To assess this hypothesis, a concentration-response curve to endothelin-1 (ET-1) was performed in mesenteric resistance arteries (MRAs) isolated from wild-type and TLR9 knockout C57 mice in the presence or absence of mtDNA (4ug/mL) for one hour. To test inflammasome pathway activation, vascular smooth muscle cells (VSMC) and endothelial cells (EC) were incubated with TLR9 agonist (ODN2395, 3uM) or mtDNA (4ug/mL) and co-incubated with TLR9/AIM2 inhibitor (ODN2088, 2uM) or NLRP3 inhibitor (MCC950, 10uM) for 24 hours. Protein expression was assessed via Western blot and immunophenotyping was performed by flow cytometry. Data are presented as mean ± S.E.M and significance was set at p<0.05. After incubation with mtDNA, MRAs from wild-type mice presented an increased contractile response to ET-1 which was prevented in TLR9 KO mice. In VSMC, TLR9 expression increased after incubation with ODN2395 (vehicle: 1.2 ± 0.2 vs ODN2395: 4 ± 1.5) or mtDNA (vehicle: 1.2 ± 0.2 vs mtDNA: 4.2 ± 1.2) while co-incubation with ODN2088 reversed this effect. Additionally, mtDNA promoted increased TNF-alpha (vehicle: 1 ± 0.1 vs mtDNA: 2.6 ± 0.5) and IL-6 (vehicle: 1 ± 0.1 vs mtDNA: 4.6 ± 0.3) expression in VSMC which was abolished in the presence of ODN2088. In EC, flow cytometry indicated that incubation with mtDNA promoted a 4-fold increase in the expression of CD31+ and a 2-fold increase in CD54+ cells while co-incubation with ODN2088 or MCC950 reversed this effect. Altogether these data suggest that cell-free mtDNA triggers the innate immune response via TLR9 and inflammasomes driving EC activation, vascular dysfunction, and inflammation.

TLR agonists promote formation of Tertiary Lymphoid Structure and improve anti-glioma immunity.

Glioma, characterized by limited lymphocytic infiltration, constitutes an "immune-desert" tumor displaying insensitivity to various immunotherapies. This study aims to explore therapeutic strategies for inducing tertiary lymphoid structure (TLS) formation within the glioma microenvironment (GME) to transition it from an immune-resistant to an activated state. TLS formation in GME was successfully induced by intracranial administration of Toll-like receptor (TLR) agonists (OK-432, TLR2/4/9 agonist) and glioma antigens (i.c. αTLR-mix). We employed staining analysis, antibody neutralization, single-cell RNA sequencing (scRNA-Seq), and BCR/TCR sequencing to investigate the underlying mechanisms of TLS formation and its role in anti-glioma immunity. Additionally, a preliminary translational clinical study was conducted. TLS formation correlated with increased lymphocyte infiltration in GME and led to improved prognosis in glioma-bearing mice. In the study of TLS induction mechanisms, certain macrophages/microglia and Th17 displayed markers of "LTo" and "LTi" cells, respectively, interaction through LTα/β-LTβR promoted TLS induction. Post-TLS formation, CD4+ and CD8+ T cells but not CD19+ B cells contributed to anti-glioma immunity. Comparative analysis of B/T cells between brain and lymph node showed that brain B/T cells unveiled switch from naïve to mature, some B cells highlighted an enrichment of CSR-associated genes, V gene usage and clonotype bias were observed. In related clinical studies, i.c. αTLR-mix treatment exhibited tolerability, and chemokines/cytokines assay provided preliminary evidence supporting TLS formation in GME. TLS induction in GME enhanced anti-glioma immunity, improved the immune microenvironment, and controlled glioma growth, suggesting potential therapeutic avenues for treating glioma in the future.

Synergistic immune augmentation enabled by covalently conjugating TLR4 and NOD2 agonists

Enhancing the efficacy of subunit vaccines relies significantly on the utilization of potent adjuvants, particularly those capable of triggering multiple immune pathways. To achieve synergistic immune augmentation by Toll-like receptor 4 agonist (TLR4a) and nucleotide-binding oligomerization-domain-containing protein 2 agonist (NOD2a), in this work, we conjugated RC529 (TLR4a) and MDP (NOD2a) to give RC529-MDP, and evaluated its adjuvanticity for OVA antigen. Compared to the unconjugated RC529+MDP, RC529-MDP remarkably enhanced innate immune responses with 6.8-fold increase in IL-6 cytokine, and promoted the maturation of antigen-presenting cells (APCs), possibly because of the conjugation of multiple agonists ensuring their delivery to the same cell and activation of various signaling pathways within that cell. Furthermore, RC529-MDP improved OVA-specific antibody response, T cells response and the memory T cells ratio relative to the unconjugated mixture. Therefore, covalently conjugating TLR4 agonist and NOD2 agonist was an effective strategy to enhance immune responses, providing the potential to design and develop more effective vaccines.

Maternal immune activation by toll-like receptor 7 agonist during mid-gestation increases susceptibility to blood-brain barrier leakage after puberty

Maternal immune activation (MIA), a maternal stressor, increases risk for neuropsychiatric diseases, such as Major Depressive Disorder in offspring. MIA of toll-like receptor 7 (TLR7) initiates an immune response in mother and fetuses in a sex-selective manner. The paraventricular nucleus of the hypothalamus (PVN), a brain region that is sexually dimorphic and regulates hypothalamic-pituitary-adrenal (HPA) stress responses, have been tied to stress-related behaviors (i.e., depression, anxiety, social impairments). The current study characterized the sex-selective impact of mid-gestational TLR7 activation on PVN vasculature of adult offspring based on a prior study of excess prenatal glucocorticoid stress. The PVN of offspring were evaluated to determine if fetal MIA impacted vascular leakage in the brains of adult mice with or without restraint stress. Timed-pregnant female mice were administered the TLR7 agonist Resiquimod (RQ) or saline vehicle on embryonic day (E) 12.5. Basal and restraint stress-induced corticosterone was measured to examine changes in stress response. Mice were perfused transcardially with fluorescein isothiocyanate (FITC) to assess blood vessel integrity. Sections with FITC-labeled blood vessels through the PVN of offspring were immunolabeled for Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet) and IBA-1 (microglia). MIA with RQ led to elevated levels of plasma corticosterone 60-minutes after restraint in offspring, suggesting prenatal RQ impairs glucocorticoid negative feedback. Blood-brain barrier integrity was assessed. Adult offspring of RQ injected dams showed greater leakage in the PVN (greater in males than females). GFAP+ colocalization with FITC-labeled vessels was lower in the PVN of offspring from RQ treated dams, potentially contributing to the observed increased FITC leakage. Microglia were examined in relation to the vasculature as an indicator of a neuroimmune response. Data show IBA-1+ cells greater in size and number in the PVN with closer proximity to blood vessels after maternal injection of RQ in a male-selective manner. Microglia were unchanged in females from RQ-treated dams but were smaller in size after restraint. This study provides support for sex-selective influences of fetal immune antecedents for altered brain vascular and blood brain barrier development and adult neuroendocrine function that could indicate a PVN locus for increased susceptibility for adult disorders.

The Expression of Toll-like Receptors in Cartilage Endplate Cells: A Role of Toll-like Receptor 2 in Pro-Inflammatory and Pro-Catabolic Gene Expression.

The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. Gene expression of TLR1-10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. The expression of TLR1-10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC.

Endosomal Toll-Like Receptors intermediate negative impacts of viral diseases, autoimmune diseases, and inflammatory immune responses on the cardiovascular system.

Cardiovascular disease (CVD) is the leading cause of morbidity globally, with chronic inflammation as a key modifiable risk factor. Toll-like receptors (TLRs), pivotal components of the innate immune system, including TLR-3, -7, -8, and -9 within endosomes, trigger intracellular cascades, leading to inflammatory cytokine production by various cell types, contributing to systemic inflammation and atherosclerosis. Recent research highlights the role of endosomal TLRs in recognizing self-derived nucleic acids during sterile inflammation, implicated in autoimmune conditions like myocarditis. This review explores the impact of endosomal TLRs on viral infections, autoimmunity, and inflammatory responses, shedding light on their intricate involvement in cardiovascular health and disease by examining literature on TLR-mediated mechanisms and their roles in CVD pathophysiology. Removal of endosomal TLRs mitigates myocardial damage and immune reactions, applicable in myocardial injury. Targeting TLRs with agonists enhances innate immunity against fatal viruses, lowering viral loads and mortality. Prophylactic TLR agonist administration upregulates TLRs, protecting against fatal viruses and improving survival. TLRs play a complex role in CVDs like atherosclerosis and myocarditis, with therapeutic potential in modulating TLR reactions for cardiovascular health.

Vascular injury derived apoptotic exosome-like vesicles trigger autoimmunity

According to a central tenet of classical immune theory, a healthy immune system must avoid self-reactive lymphocyte clones but we now know that B cells repertoire exhibit some level of autoreactivity. These autoreactive B cells are thought to rely on self-ligands for their clonal selection and survival. Here, we confirm that healthy mice exhibit self-reactive B cell clones that can be stimulated in vitro by agonists of toll-like receptor (TLR) 1/2, TLR4, TLR7 and TLR9 to secrete anti-LG3/perlecan. LG3/perlecan is an antigen packaged in exosome-like structures released by apoptotic endothelial cells (ApoExos) upon vascular injury. We demonstrate that the injection of ApoExos in healthy animals activates the IL-23/IL-17 pro-inflammatory and autoimmune axis, and produces several autoantibodies, including anti-LG3 autoantibodies and hallmark autoantibodies found in systemic lupus erythematosus. We also identify γδT cells as key mediators of the maturation of ApoExos-induced autoantibodies in healthy mice. Altogether we show that ApoExos released by apoptotic endothelial cells display immune-mediating functions that can stimulate the B cells in the normal repertoire to produce autoantibodies. Our work also identifies TLR activation and γδT cells as important modulators of the humoral autoimmune response induced by ApoExos.