A carrier-free nanovaccine combined with cancer immunotherapy overcomes gemcitabine resistance.

Drug resistance is a significant challenge in cancer chemotherapy and is a primary factor contributing to poor recovery for cancer patients. Although drug-loaded nanoparticles have shown promise in overcoming chemotherapy resistance, they often carry a combination of drugs and require advanced design and manufacturing processes. Furthermore, they seldom approach chemotherapy-resistant tumors from an immunotherapy perspective. In this study, we developed a therapeutic nanovaccine composed solely of chemotherapy-induced resistant tumor antigens (CIRTAs) and the immune adjuvant Toll-like receptor (TLR) 7/8 agonist R848 (CIRTAs@R848). This nanovaccine does not require additional carriers and has a simple production process. It efficiently delivers antigens and immune stimulants to dendritic cells (DCs) simultaneously, promoting DCs maturation. CIRTAs@R848 demonstrated significant tumor suppression, particularly when used in combination with the immune checkpoint blockade (ICB) anti-PD-1 (αPD-1). The combined therapy increased the infiltration of T cells into the tumor while decreasing the proportion of regulatory T cells (Tregs) and modulating the tumor microenvironment, resulting in long-term immune memory. Overall, this study introduces an innovative strategy for treating chemotherapy-resistant tumors from a novel perspective, with potential applications in personalized immunotherapy and precision medicine.

Role of toll-like receptor 4/mutant myeloid differentiation primary response 88/nuclear factor kappa-B mediated inflammation in diabetes mellitus with Northwest dryness syndrome.

OBJECTIVE: To investigate the role of toll-like receptor 4 (TLR4)/mutant myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa-B (NF-κB) signaling pathway-mediated inflammation in diabetes mellitus with Northwest dryness syndrome. METHODS: Rats were randomly divided into the normal control, type 2 diabetes (T2DM) model, Northwest dryness syndrome + T2DM (Northwest dryness), and simple internal dampness + T2DM (internal dampness) groups. Enzyme-linked immunosorbent assay was used to detect biochemical indexes and inflammatory factors. The histopathological observation was performed. Quantitative real-time polymerase chain reaction and Western blot analysis were used to detect the mRNA and protein expression levels, respectively. RESULTS: Compared with the T2DM group, the glycosylated hemoglobin A1c, insulin, glucose tolerance, the homeostasis model assessment of insulin resistance, tumor necrosis factor-α, interleukin 1ß, interleukin 16, malondialdehyde, blood lipid, alanine aminotransferase, and aspartate aminotransferase were significantly elevated in the internal dampness group. Their levels were significantly elevated in the Northwest dryness group than in the T2DM and internal dampness groups. The superoxide dismutase, glutathione peroxidase, liver glycogen, and organ-to-weight ratio were significantly declined in the internal dampness group and the Northwest dryness group than in the T2DM group. However, these levels were elevated in the Northwest dryness group than in the internal dampness group. Moreover, the mRNA expression levels of interferon regulatory factor 5 and NF-κB p65, and the protein expression levels of TLR4, MyD88, and NF-κB were significantly higher in the internal dampness and the Northwest dryness groups than the T2DM group. Additionally, the mRNA and protein levels were significantly higher in the Northwest dryness group than in the internal dampness group. CONCLUSION: Northwest dryness syndrome-mediated TLR4/MyD88/NF-κB pathway and chronic inflammation might be associated with the occurrence and development of T2DM.

Activated sympathetic nerve post stroke downregulates Toll-like receptor 5 and disrupts the gut mucosal barrier.

The gut permeability significantly increases after ischemic stroke, partly due to disrupted mucosal barrier, but the mechanism remains elusive. Here, we found that the mucus disruption starts at 2 h post stroke, whereas goblet cell functions remain intact. Meanwhile, the flagellated bacteria Helicobacter thrives and penetrates in the mucus layer. Elimination of the mucosal microbiota or transplantation of Helicobacter in germ-free mice reveals an important role of the mucosal microbiota in mucus disruption. The bacterial invasion is due to downregulated Toll-like receptor 5 (TLR5) and its downstream products flagellin-specific IgA and antimicrobial peptides. Knockdown of intestinal TLR5 increases the abundance of flagellated bacteria and exacerbates mucus injury. Intestinal TLR5 is downregulated by the activation of sympathetic nerve. Serum noradrenaline level is positively associated with flagellin level in patients with stroke and patients' prognosis. These findings reveal a neural pathway in which the sympathetic nerve disrupts the mucosal barrier, providing potential therapeutic targets for stroke injury.

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

Introduction: 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. Methods: 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. Results: 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. Discussion: 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. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=323122.

TLR2 promotes traumatic deep venous thrombosis of the lower extremity following femoral fracture by activating the NF­κB/COX­2 signaling pathway in rats.

Endothelial cells (ECs) are crucial for maintaining the integrity of blood vessel walls and reducing thrombosis. Deep venous thrombosis (DVT) is a common thrombotic disease and its diagnosis and treatment remain at the stage of coagulation function examination and post-onset treatment. Thus, identifying the pathogenesis of DVT is important. The present study investigated the significance of the Toll-like receptor 2 (TLR2)/nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2) signaling pathway in a human umbilical vein EC (HUVECs) oxygen glucose deprivation (OGD) model and femoral fractures were induced in anesthetized rats using a quantifiable impact device delivering 5 J of energy to each side of the proximal outer thigh, followed by external fixation with a hip spica cast to create a traumatic deep venous thrombosis (TDVT) animal model. Rats were subjected to quantitative impact fixation to establish a TDVT model. The rats were treated with a TLR2 agonist (Pam3CSK4) and a TLR2 inhibitor (C29) via intraperitoneal injection and thrombus formation was examined. HUVECs were subjected to OGD and treated with Pam3CSK4 or C29 and cell viability and apoptosis were assessed. Western blotting, immunofluorescence and reverse transcription-quantitative PCR were used to examine the inflammatory responses and signaling pathways. In vivo experiments showed that Pam3CSK4 promoted thrombus formation and increased the mRNA and protein expression of NF-κB, COX-2, Tissue factor (TF), IL-6 and P-selectin compared with the model and C29 groups. In vitro experiments showed that Pam3CSK4 treatment resulted in a higher number of apoptotic cells than C29 treatment and that it increased the levels of NF-κB, COX-2, IL-6 and P-selectin, whereas C29 decreased them. Thus, TLR2 promotes the inflammatory response in EC through the NF-κB/COX-2 signaling pathway, which may lead to EC apoptosis and the occurrence of TDVT.

Modulation of chemotherapy-induced peripheral neuropathy by JZL195 through glia and the endocannabinoid system.

Chemotherapy-induced peripheral neuropathy (CIPN) used to treat cancer, is a significant side effect with a complex pathophysiology, and its mechanisms remain unclear. Recent research highlights neuroinflammation, which is modulated by the endocannabinoid system (ECS) and associated with glial activation, and the role of toll-like receptor 4 (TLR4) in CIPN. This study aimed to investigate the effects of JZL195, an inhibitor of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and explore the connection between cannabinoid receptors and TLR4 in glial cells. A CIPN animal model was developed using cisplatin-injected male C57BL/6 mice. Mechanical and cold allodynia were assessed through von Frey and acetone tests. Western blot analysis was used to examine the expression of catabolic enzymes, cannabinoid receptors, glial cells, and neuroinflammatory factors in the dorsal root ganglia (DRGs) and spinal cord. Immunohistochemistry was used to investigate the colocalization of cannabinoid receptors and TLR4 in glial cells. JZL195 alleviated pain by inhibiting FAAH/MAGL, modulating the ECS and neuroinflammatory factors, and suppressing glial cell activity. Additionally, cannabinoid receptors and TLR4 colocalized with astrocytes and microglia in the spinal cord. This study highlights the therapeutic potential of JZL195 in modulating the ECS and suggests a correlation between cannabinoid receptors and TLR4 in spinal glial cells, providing insight into alleviating pain and neuroinflammation in CIPN.

Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2'O-methylation.

TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2'O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8.

25-hydroxycholesterol promotes brain cytokine production and leukocyte infiltration in a mouse model of lipopolysaccharide-induced neuroinflammation.

Neuroinflammation has been implicated in the pathogenesis of several neurologic and psychiatric disorders. Microglia are key drivers of neuroinflammation and, in response to different inflammatory stimuli, overexpress a proinflammatory signature of genes. Among these, Ch25h is a gene overexpressed in brain tissue from Alzheimer's disease as well as various mouse models of neuroinflammation. Ch25h encodes cholesterol 25-hydroxylase, an enzyme upregulated in activated microglia under conditions of neuroinflammation, that hydroxylates cholesterol to form 25-hydroxycholesterol (25HC). 25HC can be further metabolized to 7α,25-dihydroxycholesterol, which is a potent chemoattractant of leukocytes. We have previously shown that 25HC increases the production and secretion of the proinflammatory cytokine, IL-1ß, by primary mouse microglia treated with lipopolysaccharide (LPS). In the present study, wildtype (WT) and Ch25h-knockout (KO) mice were peripherally administered LPS to induce an inflammatory state in the brain. In LPS-treated WT mice, Ch25h expression and 25HC levels increased in the brain relative to vehicle-treated WT mice. Among LPS-treated WT mice, females produced significantly higher levels of 25HC and showed transcriptomic changes reflecting higher levels of cytokine production and leukocyte migration than WT male mice. However, females were similar to males among LPS-treated KO mice. Ch25h-deficiency coincided with decreased microglial activation in response to systemic LPS. Proinflammatory cytokine production and intra-parenchymal infiltration of leukocytes were significantly lower in KO compared to WT mice. Amounts of IL-1ß and IL-6 in the brain strongly correlated with 25HC levels. Our results suggest a proinflammatory role for 25HC in the brain following peripheral administration of LPS.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling.

BACKGROUND: Neuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation. METHODS: To track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation. RESULTS: We observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88. CONCLUSIONS: Circulating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

Fosaprepitant improves cyclophosphamide-induced bladder damage by alleviating inflammatory response in mice.

Inhibition of inflammatory process is a key therapeutic target for the treatment of interstitial cystitis (IC). Recent reports indicate that neurokinin 1 receptor (NK1R) antagonists have beneficial roles in inflammatory-based diseases. Herein, we investigate the protective effects of fosaprepitant (FOS), a NK1R antagonist, in cyclophosphamide (CP)-induced cystitis. The cystitis model was established multiple CP (80 mg/kg; i.p.) injection one day apart, and mice were treated with FOS (20 and 60 mg/kg/day; i.p.) for seven consecutive days. Detrusor contractility, vesical vascular permeability, myeloperoxidase (MPO) activity and protein expression levels of the TLR4 pathway were evaluated in mice bladder. Carbachol and electric field stimulation-evoked contractions of detrusor strips were significantly increased in CP-treated mice, which was significantly attenuated by FOS (60 mg/kg/day) treatment (p˂0.001, p˂0.05). Notably, vesical vascular permeability was markedly impaired in CP-induced cystitis, that was restored by FOS (60 mg/kg/day) treatment (p˂0.01). MPO activity was significantly increased in cystitis group whereas FOS (20 and 60 mg/kg/day) treatment remarkably suppressed MPO activity in bladder tissue (p˂0.001). Although TLR4 expression increased with cystitis, MyD88 and p-NFκBSer536/total NFκB did not change, FOS (20 and 60 mg/kg/day) treatment caused a dramatic decrease in TLR4 expression (p˂0.001), indicating the anti-inflammatory effect of FOS. In conclusion, FOS improved detrusor overactivity and inflammatory response by inhibiting MPO activity and TLR4 expression, resulting in functional and histological recovery in CP-induced cystitis.

Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages.

Toll-like receptors (TLRs) on macrophages sense microbial components and trigger the production of numerous cytokines and chemokines that mediate the inflammatory response to infection. Although many of the components required for the activation of the TLR pathway have been identified, the mechanisms that appropriately regulate the magnitude and duration of the response and ultimately restore homeostasis are less well understood. Furthermore, a growing body of work indicates that TLR signaling reciprocally interacts with other fundamental cellular processes, including lipid metabolism but only a few specific molecular links between immune signaling and the macrophage lipidome have been studied in detail. Oxysterol-binding protein (Osbp) is the founding member of a family of lipid-binding proteins with diverse functions in lipid sensing, lipid transport, and cell signaling but its role in TLR responses is not well defined. Here, we demonstrate that altering the state of Osbp with its natural ligand, 25-hydroxycholesterol (25HC), or pharmacologically, sustains and thereby amplifies Tlr4-induced cytokine production in vitro and in vivo. CRISPR-induced knockdown of Osbp abrogates the ability of these ligands to sustain TLR responses. Lipidomic analysis suggested that the effect of Osbp on TLR signaling may be mediated by alterations in triglyceride production and treating cells with a Dgat1 inhibitor, which blocks triglyceride production and completely abrogates the effect of Osbp on TLR signaling. Thus, Osbp is a sterol sensor that transduces perturbations of the lipidome to modulate the resolution of macrophage inflammatory responses.

GSDMD is associated with survival in human breast cancer but does not impact anti-tumor immunity in a mouse breast cancer model.

Inflammation plays a pivotal role in cancer development, with chronic inflammation promoting tumor progression and treatment resistance, whereas acute inflammatory responses contribute to protective anti-tumor immunity. Gasdermin D (GSDMD) mediates the release of pro-inflammatory cytokines such as IL-1ß. While the release of IL-1ß is directly linked to the progression of several types of cancers, the role of GSDMD in cancer is less clear. In this study, we show that GSDMD expression is upregulated in human breast, kidney, liver, and prostate cancer. Higher GSDMD expression correlated with increased survival in primary breast invasive carcinoma (BRCA), but not in liver hepatocellular carcinoma (LIHC). In BRCA, but not in LIHC, high GSDMD expression correlated with a myeloid cell signature associated with improved prognosis. To further investigate the role of GSDMD in anticancer immunity, we induced breast cancer and hepatoma tumors in GSDMD-deficient mice. Contrary to our expectations, GSDMD deficiency had no effect on tumor growth, immune cell infiltration, or cytokine expression in the tumor microenvironment, except for Cxcl10 upregulation in hepatoma tumors. In vitro and in vivo innate immune activation with TLR ligands, that prime inflammatory responses, revealed no significant difference between GSDMD-deficient and wild-type mice. These results suggest that the impact of GSDMD on anticancer immunity is dependent on the tumor type. They underscore the complex role of inflammatory pathways in cancer, emphasizing the need for further exploration into the multifaceted effects of GSDMD in various tumor microenvironments. As several pharmacological modulators of GSDMD are available, this may lead to novel strategies for combination therapy in cancer.

Association of TLR4 polymorphisms (Asp299Gly and Thr399Ile) with sepsis: a meta-analysis and trial sequence analysis.

Several investigations have been carried out to explore the genetic association of TLR4 codon variants, specifically Asp299Gly and Thr399Ile, and susceptibility to sepsis, but the results have been contradictory. The present study aimed to conduct a meta-analysis to draw a definitive conclusion regarding the role of TLR4 genetic variants (Asp299Gly and Thr399Ile) in sepsis. A thorough literature search was conducted using the PubMed, Scopus, and Science Direct databases. The inclusion and exclusion criteria were established to ensure the accuracy of the data. The Comprehensive Meta-Analysis Software v4 was utilized to perform the meta-analysis and related analyses. A total of 13 studies were analyzed, including 2328 sepsis cases and 2495 healthy controls for the TLR4 Asp299Gly variant. Eight studies provided genotype data for the rs4986791 polymorphism. The Asp299Gly variant showed a marginal protective effect in the allele (p = 0.08, odds ratio = 0.71) and dominant (p = 0.09, odds ratio = 0.71) genetic models, although it was not statistically significant. The trial sequential analysis indicated that further case-control studies are necessary to draw definitive conclusions about the TLR4 polymorphisms in sepsis. The TLR4 Asp299Gly variant may have a protective effect against sepsis. However, additional research with larger sample sizes across diverse populations is required to validate this finding.

Effects of toll like receptor 4 (TLR4) and toll like receptor 2 (TLR 2) gene polymorphisms on clinical outcomes in acute non-biliary pancreatitis patients.

Introduction: In this retrospective study, it was aimed to evaluate effects of Toll Like Receptor 4 (TLR4) and Toll Like Receptor 2 (TLR 2) gene polymorphisms on clinical outcomes in acute non-biliary pancreatitis patients. Methods: A total of 108 acute non-biliary pancreatitis patients (ANBP) were retrospectively subjected to the study. Gender, age, number of attacks, hospitalization duration, amylase, lipase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), leukocyte, C-reactive protein (CRP), total bilirubin, direct bilirubin, Atlanta score, ultrasonography (USG), Computer Tomography (CT) and patient outcome differences between TLR 4 Rs4986790, TLR 4 Rs4986791 and TLR 2 groups were evaluated. Results: According to TLR 4 Rs4986790 groups, females were significantly common in AA sequence (AA) group with statistically significant difference (p<0.05). Leukocyte mean of AG sequence (AG) group was significantly higher than of AA group (p<0.05). All parameter differences between TLR 4 Rs4986791 and TLR 2 groups were statistically insignificant (p>0.05). there was a statistically significant correlation between TLR 4 Rs4986790 and gender (r=0.265; p<0.01), Leukocyte (r=0.200; p<0.05) and Pseudocyst (r=0.203; p<0.05). TLR 4 Rs4986790 gene polymorphism had significant effect on leukocyte level in acute non-biliary pancreatitis patients (OR: -0.1.900; p<0.05). Predictive value of leukocyte for TLR 4 Rs4986790 was statistically significant (Area Under Curve: 0.624; p<0.05). For 7.65 leukocyte cut off value, sensitivity for AA gene polymorphism was 84.2% and specificity was 40.5. Conclusion: Although the clinical and outcome parameters of ANBP patients in terms of TLR 4 Rs4986791 and TLR 2 do not show significant differences, research findings point to the diagnostic value of patients' leukocyte parameters in determining TLR-4 Rs4986790 ploimorphism groups.

Immunoactive signatures of circulating tRNA- and rRNA-derived RNAs in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA sequencing (RNA-seq) method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5'-phosphate and 3'-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5'-tRNA halves containing a 2',3'-cyclic phosphate. We discovered a remarkable accumulation of the 5'-half derived from tRNAValCAC in plasma from COPD patients, whereas the 5'-tRNAGlyGCC half is predominant in healthy donors. Further, the 5'-tRNAValCAC half activates human macrophages via Toll-like receptor 7 and induces cytokine production. Additionally, we identified circulating rRNA-derived fragments that were upregulated in COPD patients and demonstrated their ability to induce cytokine production in macrophages. Our findings provide evidence of circulating, immune-active sncRNAs in patients with COPD, suggesting that they serve as inflammatory mediators in the pathogenesis of COPD.

Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1.

Background: Mesenchymal stem/stromal cells (MSCs) maintain tissue homeostasis in response to microenvironmental perturbations. Toll-like receptors (TLRs) are key sensors for exogenous and endogenous signals produced during injury. In this study, we aimed to investigate whether TLRs affect the homeostatic functions of MSCs after injury. Methods: We examined the expression of TLR2, TLR3 and TLR4 in MSCs, and analyzed the functional significance of TLR2 activation using single-cell RNA sequencing. Additionally, we investigated the effects and mechanisms of TLR2 and its downstream activation in MSCs on the MSCs themselves, on monocytes/macrophages, and in a mouse model of sterile injury-induced inflammatory corneal angiogenesis. Results: MSCs expressed TLR2, which was upregulated by monocytes/macrophages. Activation of TLR2 in MSCs promoted their immunoregulatory and angiostatic functions in monocytes/macrophages and in mice with inflammatory corneal angiogenesis, whereas TLR2 inhibition attenuated these functions. Single-cell RNA sequencing revealed AKR1C1, a gene encoding aldo-keto reductase family 1 member C1, as the most significantly inducible gene in MSCs upon TLR2 stimulation, though its stimulation did not affect cell compositions. AKR1C1 protected MSCs against ferroptosis, increased secretion of anti-inflammatory cytokines, and enhanced their ability to drive monocytes/macrophages towards immunoregulatory phenotypes, leading to the amelioration of inflammatory corneal neovascularization in mice. Conclusion: Our findings suggest that activation of TLR2-AKR1C1 signaling in MSCs serves as an important pathway for the survival and homeostatic activities of MSCs during injury.

Protective effects of insulin on dry eye syndrome via TLR4/NF-κB pathway: based on network pharmacology and in vitro experiments validation.

Dry eye syndrome (DES) is a multifactorial ocular surface disease and represents one of the most prevalent ophthalmic disorders. Insulin is an important metabolism-regulating hormone and a potential antioxidant with critical biological roles as anti-inflammatory and anti-apoptotic. However, its mechanism of action remains unknown. In this study, we used network pharmacology techniques and conducted cell experiments to investigate the protective effect of insulin on human corneal epithelial cells (HCECs). Eighty-seven common targets of insulin and DES were identified from the database. KEGG pathway enrichment analysis suggested that insulin may be crucial in regulating the toll-like receptor (TLR) signaling pathway by targeting key targets such as IL-6 and TNF. In cell experiments, insulin promoted HCECs proliferation, improved their ability to migrate, and inhibited apoptosis. Western blot and enzyme-linked immunosorbent assay (ELISA) also confirmed the upregulation of the expression of inflammatory factors such as IL-1ß, IL-6, and proteins related to the TLR4/NF-κB signaling pathway. However, the expression of these proteins was inhibited by insulin administration. Our results preliminarily verified insulin may exert a protective role on HCECs under hyperosmotic condition, which offered a novel perspective for the clinical management of this condition.

A comprehensive 26-color immunophenotyping panel to study the role of the gut-liver axis in chronic liver diseases.

The gut-liver axis includes the bidirectional communication between the gut and the liver, and thus covers signals from liver-to-gut and from gut-to-liver. Disruptions of the gut-liver axis have been associated with the progression of chronic liver diseases, including alcohol-related and metabolic dysfunction-associated steatotic liver disease and cholangiopathies. Immune cells and their expression of pattern recognition receptors, activation markers or immune checkpoints might play an active role in the communication between gut and liver. Here, we present a 26-color full spectrum flow cytometry panel for human cells to decipher the role of circulating immune cells in gut-liver communication during the progression of chronic liver diseases in a non-invasive manner, which has been optimized to be used on patient-derived whole blood samples, the most abundantly available clinical material. Our panel focuses on changes in pattern recognition receptors, including toll-like receptors (TLRs) or Dectin-1, and also includes other immunomodulatory molecules such as bile acid receptors and checkpoint molecules. Moreover, this panel can be utilized to follow the progression of chronic liver diseases and could be used as a tool to evaluate the efficiency of therapeutic targets directed against microbial mediators or modulating immune cell activation.

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

INTRODUCTION: 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. METHODS: 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 (0.3-12 mg; n = 505) or placebo (n = 101). Vesatolimod pharmacokinetics, inflammatory responses, and pharmacodynamics were assessed. RESULTS: 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. CONCLUSIONS: 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.

Pulmonary Administration of TLR2/6 Agonist after Allergic Sensitization Inhibits Airway Hyper-Responsiveness and Recruits Natural Killer Cells in Lung Parenchyma.

Asthma is a chronic lung disease with persistent airway inflammation, bronchial hyper-reactivity, mucus overproduction, and airway remodeling. Antagonizing T2 responses by triggering the immune system with microbial components such as Toll-like receptors (TLRs) has been suggested as a therapeutic concept for allergic asthma. The aim of this study was to evaluate the effect of a TLR2/6 agonist, FSL-1 (Pam2CGDPKHPKSF), administered by intranasal instillation after an allergic airway reaction was established in the ovalbumin (OVA) mouse model and to analyze the role of natural killer (NK) cells in this effect. We showed that FSL-1 decreased established OVA-induced airway hyper-responsiveness and eosinophilic inflammation but did not reduce the T2 or T17 response. FSL-1 increased the recruitment and activation of NK cells in the lung parenchyma and modified the repartition of NK cell subsets in lung compartments. Finally, the transfer or depletion of NK cells did not modify airway hyper-responsiveness and eosinophilia after OVA and/or FSL-1 treatment. Thus, the administration of FSL-1 reduces airway hyper-responsiveness and bronchoalveolar lavage eosinophilia. However, despite modifications of their functions following OVA sensitization, NK cells play no role in OVA-induced asthma and its inhibition by FSL-1. Therefore, the significance of NK cell functions and localization in the airways remains to be unraveled in asthma.