Expression of Toll-like receptors in oral squamous cell carcinoma.

Almost 380,000 new cases of oral cancer were reported worldwide in 2020. Oral squamous cell carcinoma (OSCC) accounts for 90% of all types of oral cancers. Emerging studies have shown association of Toll-like receptors (TLRs) in carcinogenesis. The present study aimed to investigate the expression levels and tissue localization of TRL1 to TRL10 and NF-κB between OSCC and healthy oral mucosa, as well as effect of Candida colonization in TRL expression in OSCC. Full thickness biopsies and microbial samples from 30 newly diagnosed primary OSCC patients and 26 health controls were collected. The expression of TLR1 to TLR10 and NF-κB was analyzed by immunohistochemistry. Microbial samples were collected from oral mucosa to detect Candida. OSCC epithelium showed lower staining intensity of TRL1, TRL2 TRL5, and TRL8 as compared to healthy controls. Similarly, staining intensity of TRL3, TRL4, TRL7, and TRL8 were significantly decreased in basement membrane (BM) zone. Likewise, OSCC endothelium showed lower staining intensity of TLR4, TLR7 and TLR8. Expression of NF-κB was significantly stronger in normal healthy tissue compared to OSCC sample. Positive correlation was found between the expression of NF-κB, TRL9 and TRL10 in basal layer of the infiltrative zone OSCC samples (P = 0.04 and P = 0.002, respectively). Significant increase in TRL4 was seen in BM zone of sample colonized with Candida (P = 0.01). According to the limited number of samples, our data indicates downregulation of TLRs and NF-κB in OSCC, and upregulation of TLR4 expression with presence of Candida.

Decoding Toll-like receptors: Recent insights and perspectives in innate immunity.

Toll-like receptors (TLRs) are an evolutionarily conserved family in the innate immune system and are the first line of host defense against microbial pathogens by recognizing pathogen-associated molecular patterns (PAMPs). TLRs, categorized into cell surface and endosomal subfamilies, recognize diverse PAMPs, and structural elucidation of TLRs and PAMP complexes has revealed their intricate mechanisms. TLRs activate common and specific signaling pathways to shape immune responses. Recent studies have shown the importance of post-transcriptional regulation in TLR-mediated inflammatory responses. Despite their protective functions, aberrant responses of TLRs contribute to inflammatory and autoimmune disorders. Understanding the delicate balance between TLR activation and regulatory mechanisms is crucial for deciphering their dual role in immune defense and disease pathogenesis. This review provides an overview of recent insights into the history of TLR discovery, elucidation of TLR ligands and signaling pathways, and their relevance to various diseases.

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

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

Exploring the evolutionary mechanism of hepatitis B and gastric cancer based on Mendelian randomization combined with bioinformatics analysis.

Chronic hepatitis B virus infection (HBV) infection appears to be associated with extrahepatic cancers. This study aims to evaluate the causality and evolutionary mechanism of chronic HBV infection and gastric cancer through Mendelian randomization (MR) analysis and bioinformatics analysis. We conducted 2-sample MR to investigate the causal relationship between chronic HBV infection and gastric cancer. We identified 5 independent genetic variants closely associated with exposure (chronic HBV infection) as instrumental variables in a sample of 1371 cases and 2938 controls of East Asian descent in Korea. The genome wide association study (GWAS) data for the outcome variable came from the Japanese Biobank. Bioinformatics analysis was used to explore the evolutionary mechanism of chronic HBV infection and gastric cancer. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify key targets that are commonly associated with both diseases, and their biological functions were investigated. Multiple machine-learning models were employed to select hub genes. The MR analysis showed a positive causal relationship between chronic HBV infection and gastric cancer (IVW: OR = 1.165, 95% CI = 1.085-1.250, P < .001), and the result was robust in sensitivity analysis. According to the bioinformatics analysis, the 5 key targets were mainly enriched in Toll-like receptor signaling and PI3K-Akt signaling. Two hub genes, CXCL9 and COL6A2, were identified, and a high-performing predictive model was constructed. Chronic HBV infection is positively associated with gastric cancer, and the evolutionary mechanism may be related to Toll-like receptor signaling. Prospective studies are still needed to confirm these findings.

SESN1 functions as a new tumor suppressor gene via Toll-like receptor signaling pathway in neuroblastoma.

AIMS: Neuroblastoma (NB) is the most common extracranial solid tumor in children, with a 5-year survival rate of <50% in high-risk patients. MYCN amplification is an important factor that influences the survival rate of high-risk patients. Our results indicated MYCN regulates the expression of SESN1. Therefore, this study aimed to investigate the role and mechanisms of SESN1 in NB. METHODS: siRNAs or overexpression plasmids were used to change MYCN, SESN1, or MyD88's expression. The role of SESN1 in NB cell proliferation, migration, and invasion was elucidated. Xenograft mice models were built to evaluate SESN1's effect in vivo. The correlation between SESN1 expression and clinicopathological data of patients with NB was analyzed. RNA-Seq was done to explore SESN1's downstream targets. RESULTS: SESN1 was regulated by MYCN in NB cells. Knockdown SESN1 promoted NB cell proliferation, cell migration, and cell invasion, and overexpressing SESN1 had opposite functions. Knockdown SESN1 promoted tumor growth and shortened tumor-bearing mice survival time. Low expression of SESN1 had a positive correlation with poor prognosis in patients with NB. RNA-Seq showed that Toll-like receptor (TLR) signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway in cancer were potential downstream targets of SESN1. Knockdown MyD88 or TLRs inhibitor HCQ reversed the effect of knockdown SESN1 in NB cells. High expression of SESN1 was significantly associated with a higher immune score and indicated an active immune microenvironment for patients with NB. CONCLUSIONS: SESN1 functions as a new tumor suppressor gene via TLR signaling pathway in NB.

The contribution of pattern recognition receptor signalling in the development of age related macular degeneration: the role of toll-like-receptors and the NLRP3-inflammasome.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss, characterised by the dysfunction and death of the photoreceptors and retinal pigment epithelium (RPE). Innate immune cell activation and accompanying para-inflammation have been suggested to contribute to the pathogenesis of AMD, although the exact mechanism(s) and signalling pathways remain elusive. Pattern recognition receptors (PRRs) are essential activators of the innate immune system and drivers of para-inflammation. Of these PRRs, the two most prominent are (1) Toll-like receptors (TLR) and (2) NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)-inflammasome have been found to modulate the progression of AMD. Mutations in TLR2 have been found to be associated with an increased risk of developing AMD. In animal models of AMD, inhibition of TLR and NLRP3 has been shown to reduce RPE cell death, inflammation and angiogenesis signalling, offering potential novel treatments for advanced AMD. Here, we examine the evidence for PRRs, TLRs2/3/4, and NLRP3-inflammasome pathways in macular degeneration pathogenesis.

Dietary Intervention with Whey Protein Concentrate Does Not Affect Toll-like Receptor Responses and Gene Expression Patterns in Peripheral Blood Mononuclear Cells of Healthy Volunteers.

Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants.

Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. In vitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Type I IFN in Glomerular Disease: Scarring beyond the STING.

The field of nephrology has recently directed a considerable amount of attention towards the stimulator of interferon genes (STING) molecule since it appears to be a potent driver of chronic kidney disease (CKD). STING and its activator, the cyclic GMP-AMP synthase (cGAS), along with intracellular RIG-like receptors (RLRs) and toll-like receptors (TLRs), are potent inducers of type I interferon (IFN-I) expression. These cytokines have been long recognized as part of the mechanism used by the innate immune system to battle viral infections; however, their involvement in sterile inflammation remains unclear. Mounting evidence pointing to the involvement of the IFN-I pathway in sterile kidney inflammation provides potential insights into the complex interplay between the innate immune system and damage to the most sensitive segment of the nephron, the glomerulus. The STING pathway is often cited as one cause of renal disease not attributed to viral infections. Instead, this pathway can recognize and signal in response to host-derived nucleic acids, which are also recognized by RLRs and TLRs. It is still unclear, however, whether the development of renal diseases depends on subsequent IFN-I induction or other processes involved. This review aims to explore the main endogenous inducers of IFN-I in glomerular cells, to discuss what effects autocrine and paracrine signaling have on IFN-I induction, and to identify the pathways that are implicated in the development of glomerular damage.

Mechanisms of mesothelial cell response to viral infections: HDAC1-3 inhibition blocks poly(I:C)-induced type I interferon response and modulates the mesenchymal/inflammatory phenotype.

Infectious peritonitis is a leading cause of peritoneal functional impairment and a primary factor for therapy discontinuation in peritoneal dialysis (PD) patients. Although bacterial infections are a common cause of peritonitis episodes, emerging evidence suggests a role for viral pathogens. Toll-like receptors (TLRs) specifically recognize conserved pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and fungi, thereby orchestrating the ensuing inflammatory/immune responses. Among TLRs, TLR3 recognizes viral dsRNA and triggers antiviral response cascades upon activation. Epigenetic regulation, mediated by histone deacetylase (HDAC), has been demonstrated to control several cellular functions in response to various extracellular stimuli. Employing epigenetic target modulators, such as epidrugs, is a current therapeutic option in several cancers and holds promise in treating viral diseases. This study aims to elucidate the impact of TLR3 stimulation on the plasticity of human mesothelial cells (MCs) in PD patients and to investigate the effects of HDAC1-3 inhibition. Treatment of MCs from PD patients with the TLR3 agonist polyinosinic:polycytidylic acid (Poly(I:C)), led to the acquisition of a bona fide mesothelial-to-mesenchymal transition (MMT) characterized by the upregulation of mesenchymal genes and loss of epithelial-like features. Moreover, Poly(I:C) modulated the expression of several inflammatory cytokines and chemokines. A quantitative proteomic analysis of MCs treated with MS-275, an HDAC1-3 inhibitor, unveiled altered expression of several proteins, including inflammatory cytokines/chemokines and interferon-stimulated genes (ISGs). Treatment with MS-275 facilitated MMT reversal and inhibited the interferon signature, which was associated with reduced STAT1 phosphorylation. However, the modulation of inflammatory cytokine/chemokine production was not univocal, as IL-6 and CXCL8 were augmented while TNF-α and CXCL10 were decreased. Collectively, our findings underline the significance of viral infections in acquiring a mesenchymal-like phenotype by MCs and the potential consequences of virus-associated peritonitis episodes for PD patients. The observed promotion of MMT reversal and interferon response inhibition by an HDAC1-3 inhibitor, albeit without a general impact on inflammatory cytokine production, has translational implications deserving further analysis.

Endosomal Toll-Like Receptors as Therapeutic Targets for Autoimmune Diseases.

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) reside in the endosomal compartment of innate immune cells, such as macrophages and dendritic cells. NAs transported to the endosomal compartment are degraded by DNases and RNases. Degradation products, including single-stranded DNA, oligoRNA, and nucleosides, are recognized by TLR7, TLR8, and TLR9 to drive the defense responses against pathogens. NA degradation influences endosomal TLR responses by generating and degrading TLR ligands. TLR ligand accumulation because of impaired NA degradation causes constitutive TLR activation, leading to autoinflammatory and autoimmune diseases. Furthermore, some genes associated with these diseases promote endosomal TLR responses. Therefore, endosomal TLRs are promising therapeutic targets for TLR-mediated inflammatory diseases, and novel drugs targeting TLRs are being developed.

Protocol for localized macrophage stimulation with small-molecule TLR agonist via fluidic force microscopy.

Here, we present a protocol to deliver nanoliter volumes of Toll-like receptor (TLR) agonist onto a culture of nuclear factor κB (NF-κB) reporter macrophages using fluidic force microscopy and a micron-scale probe. We describe steps for quantifying the dose of agonist by modeling their diffusion with experimental inputs. We then detail procedures for quantifying and categorizing macrophage responses to individual and varied doses and combining agonist concentration and macrophage response to analyze the NF-κB response to localized TLR stimulation. For complete details on the use and execution of this protocol, please refer to Mulder et al. (2024).1.

Brucella infection and Toll-like receptors.

Brucella consists of gram-negative bacteria that have the ability to invade and replicate in professional and non-professional phagocytes, and its prolonged persistence in the host leads to brucellosis, a serious zoonosis. Toll-like receptors (TLRs) are the best-known sensors of microorganisms implicated in the regulation of innate and adaptive immunity. In particular, TLRs are transmembrane proteins with a typical structure of an extracellular leucine-rich repeat (LRR) region and an intracellular Toll/interleukin-1 receptor (TIR) domain. In this review, we discuss Brucella infection and the aspects of host immune responses induced by pathogens. Furthermore, we summarize the roles of TLRs in Brucella infection, with substantial emphasis on the molecular insights into its mechanisms of action.

Impact of Toll-Like Receptors (TLRs) and TLR Signaling Proteins in Trigeminal Ganglia Impairing Herpes Simplex Virus 1 (HSV-1) Progression to Encephalitis: Insights from Mouse Models.

Herpes simplex virus 1 (HSV-1) or simplexvirus humanalpha 1 is a neurotropic virus that is responsible for orofacial infections in humans. More than 70% of the world's population may have seropositivity for HSV-1, and this virus is a leading cause of sporadic lethal encephalitis in humans. The role of toll-like receptors (TLRs) in defending against HSV-1 infection has been explored, including the consequences of lacking these receptors or other proteins in the TLR pathway. Cell and mouse models have been used to study the importance of these receptors in combating HSV-1, how they relate to the innate immune response, and how they participate in the orchestration of the adaptive immune response. Myeloid differentiation factor 88 (MyD88) is a protein involved in the downstream activation of TLRs and plays a crucial role in this signaling. Mice with functional MyD88 or TLR2 and TLR9 can survive HSV-1 infection. However, they can develop encephalitis and face a 100% mortality rate in a dose-dependent manner when MyD88 or TLR2 plus TLR9 proteins are non-functional. In TLR2/9 knockout mice, an increase in chemokines and decreases in nitric oxide (NO), interferon (IFN) gamma, and interleukin 1 (IL-1) levels in the trigeminal ganglia (TG) have been correlated with mortality.

Toll-like receptor signalling as a cannabinoid target.

Toll-like receptors (TLRs) have become a focus in biomedicine and biomedical research given the roles of this unique family of innate immune proteins in immune activation, infection, and autoimmunity. It is evident that TLR dysregulation, and subsequent alterations in TLR-mediated inflammatory signalling, can contribute to disease pathogenesis, and TLR targeted therapies are in development. This review highlights evidence that cannabinoids are key regulators of TLR signalling. Cannabinoids include component of the plant Cannabis sativa L. (C. sativa), synthetic and endogenous ligands, and overall represent a class of compounds whose therapeutic potential and mechanism of action continues to be elucidated. Cannabinoid-based medicines are in the clinic, and are furthermore under intense investigation for broad clinical development to manage symptoms of a range of disorders. In this review, we present an overview of research evidence that signalling linked to a range of TLRs is targeted by cannabinoids, and such cannabinoid mediated effects represent therapeutic avenues for further investigation. First, we provide an overview of TLRs, adaptors and key signalling events, alongside a summary of evidence that TLRs are linked to disease pathologies. Next, we discuss the cannabinoids system and the development of cannabinoid-based therapeutics. Finally, for the bulk of this review, we systematically outline the evidence that cannabinoids (plant-derived cannabinoids, synthetic cannabinoids, and endogenous cannabinoid ligands) can cross-talk with innate immune signalling governed by TLRs, focusing specifically on each member of the TLR family. Cannabinoids should be considered as key regulators of signalling controlled by TLRs, and such regulation should be a major focus in terms of the anti-inflammatory propensity of the cannabinoid system.

Zeolitic imidazolate frameworks activate endosomal Toll-like receptors and potentiate immunogenicity of SARS-CoV-2 spike protein trimer.

Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework-8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.

Recent advances in different interactions between toll-like receptors and hepatitis B infection: a review.

Hepatitis B virus (HBV) B infections remain a primary global health concern. The immunopathology of the infection, specifically the interactions between HBV and the host immune system, remains somewhat unknown. It has been discovered that innate immune reactions are vital in eliminating HBV. Toll-like receptors (TLRs) are an essential category of proteins that detect pathogen-associated molecular patterns (PAMPs). They begin pathways of intracellular signals to stimulate pro-inflammatory and anti-inflammatory cytokines, thus forming adaptive immune reactions. HBV TLRs include TLR2, TLR3, TLR4, TLR7 and TLR9. Each TLR has its particular molecule to recognize; various TLRs impact HBV and play distinct roles in the pathogenesis of the disease. TLR gene polymorphisms may have an advantageous or disadvantageous efficacy on HBV infection, and some single nucleotide polymorphisms (SNPs) can influence the progression or prognosis of infection. Additionally, it has been discovered that similar SNPs in TLR genes might have varied effects on distinct populations due to stress, diet, and external physical variables. In addition, activation of TLR-interceded signaling pathways could suppress HBV replication and increase HBV-particular T-cell and B-cell reactions. By identifying these associated polymorphisms, we can efficiently advance the immune efficacy of vaccines. Additionally, this will enhance our capability to forecast the danger of HBV infection or the threat of dependent liver disease development via several TLR SNPs, thus playing a role in the inhibition, monitoring, and even treatment guidance for HBV infection. This review will show TLR polymorphisms, their influence on TLR signaling, and their associations with HBV diseases.

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

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

LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.