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Qingling Guo,1,2 Haili Qu,3 Hong Zhang,4 Xia Zhong4 1Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, People’s Republic of China; 2Department of Endocrinology, Shandong Provincial Hospital, Cheeloo College of Medicine,Shandong University, Jinan, 250012, People’s Republic of China; 3Department of Nursing, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, People’s Republic of China; 4Department of General Practice, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, People’s Republic of ChinaCorrespondence: Xia ZhongDepartment of General Practice, Shandong Provincial Hospital Affiliated to Shandong First Medical University, No. 324 Jingwu Road, Huaiyin Distinct, Jinan, Shandong Province, 250021, People’s Republic of ChinaEmail zxbs1231@126.comBackground: Prunella vulgaris L. (PV) has been used to treat autoimmune thyroiditis (AIT), but the underlying mechanism remains unknown. The present study was designed to evaluate the effect of PV on AIT and explore the role of high-mobility group box-1 (HMGB1) signaling in PV-mediated effects in vivo and in vitro.Methods: In the present study, bioactive components of PV were identified using UPLC-ESI-MS. The protective effects and potential mechanisms critical for the anti-inflammatory and immunomodulatory effects of PV in AIT were investigated in a rat model of thyroglobulin-induced experimental autoimmune thyroiditis (EAT) and in lipopolysaccharide (LPS)-induced thyroid follicular cells (TFCs).Results: The main bioactive compound identified in PV was rosmarinic acid. The thyroid volume, thyroiditis inflammation score and serum thyroglobulin antibody levels of EAT rats were attenuated by PV treatment (P< 0.01). In addition, PV significantly reduced the elevated levels of the proinflammatory cytokines TNF-α, IL-6, IL-1β and monocyte chemoattractant protein-1 (MCP-1) both in vivo (P< 0.01) and in vitro (P< 0.05). PV downregulated HMGB1 mRNA and protein expression, reduced HMGB1 secretion, and inhibited TLR9 signaling pathways (TLR9 and MyD88) in PV-treated EAT rats and TFCs. Moreover, PV reversed the increases in the numbers of splenic Th1, Th2, and Th17 cells. Finally, our results acquired following administration of ethyl pyruvate, an HMGB1 inhibitor, to splenocytes cultured in vitro supported the hypothesis that the HMGB1/TLR9 pathway is involved in the PV-mediated reductions in Th1, Th2 and Th17 cells.Conclusion: PV decreased the activity of the TLR9/MyD88 pathway and proinflammatory cytokines through HMGB1. In addition, we are the first to show that PV attenuated the HMGB1-induced increases in Th1, Th2 and Th17 cells in AIT models. These findings provide new evidence for the potential therapeutic value of PV as a treatment for AIT and other autoimmune diseases.Keywords: autoimmune thyroiditis, Prunella vulgaris L., HMGB1, TLR9

Abstract Background A low tissue oxygen level,

Qin Li,1,&ast; Jie Ren,2,&ast; Wei Liu,3 Guoqin Jiang,2 Rongkuan Hu1 1GenePharma Co., Ltd., Suzhou, 215125, Jiangsu, People’s Republic of China; 2Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215004, People’s Republic of China; 3Department of Pathology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, Jiangsu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Guoqin JiangDepartment of General Surgery, The Second Affiliated Hospital, Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu, People’s Republic of ChinaTel +86-512-67784797Email jiang_guoqin@163.comRongkuan HuGenePharma Co., Ltd., 199 Dongping Street, Suzhou, 215125, Jiangsu, People’s Republic of ChinaTel +86-512-86668828Email rkhu@mail.ustc.edu.cnPurpose: The purpose of our research was to identify and evaluate synthetic phosphorothioate-modified CPG oligodeoxynucleotides (CPG-ODNs) activating innate and adaptive immune responses. Furthermore, combined treatment with CpG and an mRNA cancer vaccine was evaluated in melanoma models as a therapeutic approach.Methods: A molecular assay was used to screen new CpG molecules; mouse modeling and pathological analysis were used to confirm the antitumor effect of CpG alone or in combination with an mRNA vaccine. Finally, safety was assessed by monitoring blood biochemistry.Results: We first screened and identified a new CpG-B class ODN (CpG2018B) that effectively stimulated type II interferons in both mouse plasmacytoid dendritic cells (pDCs) and human peripheral blood mononuclear cells (PBMCs). In addition, CpG2018B promoted cytokine production mainly via toll-like receptor 9 (TLR9) pathways. We further demonstrated that intratumoral (IT) injection of CpG2018B inhibited melanoma growth in syngeneic models and could turn “cold” tumors into “hot” tumors. Then, CpG2018B and an mRNA-based neoantigen cancer vaccine were encapsulated in lipid nanoparticles (LNPs) and intratumorally injected into melanoma mouse models. Interestingly, vaccination with CpG or the mRNA vaccine alone could inhibit tumor growth, while combination of CpG with the mRNA vaccine enhanced the antitumor effect. Finally, we described the long-term safety and tolerability of CpG2018B and mRNA therapy in mice model.Conclusion: We identified a novel CpG-B class ODN to promote the immune response, and CpG combined with mRNA cancer vaccines is an attractive candidate approach for immunostimulatory sequence (ISS)-based therapeutic strategies.Keywords: CpG, IFN-γ, TLR9, mRNA vaccine, neoantigen

Introduction: Systemic lupus erythematosus (SLE) is a chronic, inflammatory, multiorgan, systemic autoimmune disease. It is characterized by the high production of autoantibodies against nuclear compounds. TLRs (toll-like receptors 7/9) are pattern-recognition receptors that recognize nucleic acids and induce proinflammatory responses by activating NF-kB and producing type I interferon, which play a role in eliciting innate/adaptive immune responses and developing chronic inflammation. TLR7 and TLR9 single nucleotide polymorphisms (SNPs) have been linked to systemic lupus erythematosus in numerous studies (SLE). In this work, we wanted to evaluate and analyze single nucleotide polymorphisms (SNPs) in the TLR7 (rs3853839) and TLR9 (rs187084) genes among Egyptian SLE patients and healthy controls. Method: Whole blood samples were taken from 100 SLE patients and 100 controls; DNA was extracted and then processed for TLR7 rs3853839 and TLR9 rs187084 single nucleotide polymorphisms analysis by real-time polymerase chain reaction technology and restriction fragment-length polymorphism. We also assessed the association between TLR 7 and TLR 9 genes polymorphism with SLE clinical parameters. Results: Our results showed that TLR7 rs3853839 CG genotypes and G allele were significantly associated with SLE. Also, TLR7 rs3853839 genotypes and alleles were significantly associated with nephritis, arthritis, oral ulcers, and thrombocytopenia.Whereas genotypes and alleles of TLR9 were not significantly associated with the risk nor the clinical characteristics of SLE except for malar rash. Conclusion: In the investigated Egyptian cohort, our findings suggest that TLR7 rs3853839 gene polymorphisms increase the risk for SLE development and play a role in developing clinical characteristics, especially nephritis.

Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease that affects the salivary and lacrimal glands, as well as other organ systems like the lungs, kidneys and nervous system. SS can occur alone or in combination with another autoimmune disease, such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. The etiology of SS is unknown but recent studies have revealed the implication of the activation of innate immune receptors, including Toll-like receptors (TLRs), mainly through the detection of endogenous nucleic acids, in the pathogenesis of systemic autoimmune diseases. Studies on SS mouse models suggest that TLRs and especially TLR7 that detects single-stranded RNA of microbial or endogenous origin can drive the development of SS and findings in SS patients corroborate those in mouse models. In this review, we will give an overview of the function and signaling of nucleic acid-sensing TLRs, the interplay of TLR7 with TLR8 and TLR9 in the context of autoimmunity, summarize the evidence for the critical role of TLR7 in the pathogenesis of SS and present a possible connection between SARS-CoV-2 and SS.

ABSTRACT Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-β, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention. IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-β, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.

In lifestyle-related diseases, such as cardiovascular, metabolic, respiratory, and kidney diseases, chronic inflammation plays a causal role in their pathogenesis; however, underlying mechanisms of sterile chronic inflammation are not well-understood. Previous studies have confirmed the damage of cells in these organs in the presence of various risk factors such as diabetes, dyslipidemia, and cigarette smoking, releasing various endogenous ligands for pattern recognition receptors. These studies suggested that nucleic acids released from damaged tissues accumulate in these tissues, acting as an endogenous ligand. Undamaged DNA is an integral factor for the sustenance of life, whereas, DNA fragments, especially those from pathogens, are potent activators of the inflammatory response. Recent studies have indicated that inflammatory responses such as the production of type I interferon (IFN) induced by DNA-sensing mechanisms which contributes to self-defense system in innate immunity participates in the progression of inflammatory diseases by the recognition of nucleic acids derived from the host, including mitochondrial DNA (mtDNA). The body possesses several types of DNA sensors. Toll-like receptor 9 (TLR9) recognizes DNA fragments in the endosomes. In addition, the binding of DNA fragments in the cytosol activates cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS), resulting in the synthesis of the second messenger cyclic GMP-AMP (cGAMP). The binding of cGAMP to stimulator of interferon genes (STING) activates NF-κB and TBK-1 signaling and consequently the production of many inflammatory cytokines including IFNs. Numerous previous studies have demonstrated the role of DNA sensors in self-defense through the recognition of DNA fragments derived from pathogens. Beyond the canonical role of TLR9 and cGAS-STING, this review describes the role of these DNA-sensing mechanism in the inflammatory responses caused by endogenous DNA fragments, and in the pathogenesis of lifestyle-related diseases.

Acute lung injury(ALI)/acute respiratory distress syndrome(ARDS) is a form of acute-onset hypoxemic respiratory failure characterised by an acute, diffuse, inflammatory lung injury, and increased alveolar-capillary permeability, which is caused by a variety of pulmonary or nonpulmonary insults. Recently, aberrant mitochondria and mitochondrial DNA(mtDNA) level are associated with the development of ALI/ARDS, and plasma mtDNA level shows the potential to be a promising biomarker for clinical diagnosis and evaluation of lung injury severity. In mechanism, the mtDNA and its oxidised form, which are released from impaired mitochondria, play a crucial role in the inflammatory response and histopathological changes in the lung. In this review, we discuss mitochondrial outer membrane permeabilisation (MOMP), mitochondrial permeability transition pore(mPTP), extracellular vesicles (EVs), extracellular traps (ETs), and passive release as the principal mechanisms for the release of mitochondrial DNA into the cytoplasm and extracellular compartments respectively. Further, we explain how the released mtDNA and its oxidised form can induce inflammatory cytokine production and aggravate lung injury through the Toll-like receptor 9(TLR9) signalling, cytosolic cGAS-stimulator of interferon genes (STING) signalling (cGAS-STING) pathway, and inflammasomes activation. Additionally, we propose targeting mtDNAmediated inflammatory pathways as a novel therapeutic approach for treating ALI/ARDS. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of oral cancer in developing countries and its burden correlates with exposure to tobacco and excessive alcohol consumption. Toll like receptors (TLRs) are major sensors of inflammatory stimuli, from both microbial and sterile causes and as such, they have been related to tumor progression and metastasis. Here, we evaluated the expression of TLR2, 4 and 9 as well as CD3+, CD8+ and Granzyme B+ cell infiltration by immunohistochemistry in oral samples of 30 patients with OSCC, classified according to their consumption of alcohol. Our findings indicate that there is a significant association between heavy alcohol consumption and tumors with higher expression levels of TLR9. Moreover, patients with TLR9high tumors, as well as those who indicated high consumption of alcohol exhibited a diminished overall survival. TCGA data analysis indicated that TLR9high tumors express a significant increase in some genes related with the oral cavity itself, inflammation and tumor promotion. Our analysis of tumor infiltrating leukocytes demonstrated that the major differences perceived in heavy alcohol consumers was the location of CD8+ T cells infiltrating the tumor, which showed lower numbers intratumorally. Our data suggest the existence of a pathogenic loop that involves alcohol consumption, high TLR9 expression and the immunophenotype, which might have a profound impact on the progression of the disease. [ABSTRACT FROM AUTHOR]

CD71+ erythroid cells (CECs) were only known as erythrocyte progenitors not so long ago. In present times, however, they have been shown to be active players in immune regulation, especially in immunosuppression by the means of ROS, arginase-1 and arginase-2 production. Here, we uncover organ-of-origin differences in cytokine gene expression using NanoString and protein production using Bio-Plex between CECs from healthy human adult bone marrow and from human fetal liver parenchyma. Namely, healthy human adult bone marrow CECs both expressed and produced IFN-a, IL-1b, IL-8, IL-18 and MIF mRNA and protein, while human fetal liver parenchyma CECs expressed and produced IFN-a, IL15, IL18 and TNF-b mRNA and protein. We also detected TLR2 and TLR9 gene expression in both varieties of CECs and TLR1 and NOD2 gene expression in human fetal liver parenchyma CECs only. These observations suggest that there might be undiscovered roles in immune response for CECs. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) play critical roles in the first line of host defense against pathogens through recognition of pathogen-associated molecular patterns and initiation of the innate immune responses. The proper localization of TLRs in specific subcellular compartments is crucial for their ligand recognition and downstream signaling to ensure appropriate responses against pathogens while avoiding erroneous or excessive activation. Several TLRs, including TLR7 and TLR9 but not TLR4, depend on UNC93B1 for their proper intracellular localization and signaling. Accumulating evidence suggest that UNC93B1 differentially regulates its various client TLRs, but the specific mechanisms by which UNC93B1 controls individual TLRs are not well understood. Protein N-glycosylation is one of the most frequent and important post-translational modification that occurs in membrane-localized or secreted proteins. UNC93B1 was previously shown to be glycosylated at Asn251 and Asn272 residues. In this study, we investigated whether N-glycosylation of UNC93B1 affects its function by comparing wild type and glycosylation-defective mutant UNC93B1 proteins. It was found that glycosylation of Asn251 and Asn272 residues can occur independently of each other and mutation of neither N251Q or N272Q in UNC93B1 altered expression and localization of UNC93B1 and TLR9. In contrast, CpG DNA-stimulated TLR9 signaling was severely inhibited in cells expressing UNC93B1(N272Q), but not in cells with UNC93B1(N251Q). Further, it was found that glycosylation at Asn272 of UNC93B1 is essential for the recruitment of MyD88 to TLR9 and the subsequent downstream signaling. On the other hand, the defective glycosylation at Asn272 did not affect TLR7 signaling. Collectively, these data demonstrate that the glycosylation at a specific asparagine residue of UNC93B1 is required for TLR9 signaling and the glycosylation status of UNC93B1 differently affects activation of TLR7 and TLR9. [ABSTRACT FROM AUTHOR]

Background: The oral squamous cell carcinoma (OSCC) composes about 90% of all head and neck cancers. The toll-like receptor (TLR)+ immune cells have potential of invasion and malignancy transformation. The aim of this study was assessment of possible associations between clinicopathological indices and TLR2 and TLR9 gene expression in OSCC. Methods: Forty-two OSCC samples with related healthy margins including 25 early and 17 advanced stages were gathered. The samples were classified histologically from grade I to II. The expression of TLR2 and TLR2 was evaluated by Real-time PCR. The patient's disease-free survival (DFS) and overall survival (OS) were analyzed using SPSS V.23 software. Results: The expression of TLR2 and TLR9 genes in tumor tissues (especially in grade I and II) were higher than healthy surgical margin tissue (p< 0.001). TLR9 expression in grade II was statistically significant than grade I in tumor tissue (p< 0.001). TLR9 expression in advanced stage was statistically significant in compare to early stage (p= 0.012). In advanced stage both overall survival (p= 0.029) and disease-free survival (p= 0.012) were statistically lower than early stage. The follow-up time to recurrence in advanced stage was statistically lower than early stage (p= 0.007). Conclusions: Overexpression of TLRs 2, 9 play role in the pathogenesis and tumor development of OSCC and can be applied as biomarker in prognostic approaches. [ABSTRACT FROM AUTHOR]

(1) Background: Porcine Parvovirus (PPV) is a single-stranded DNA virus without envelope which causes great harm in relation to porcine reproductive disorders in clinic. Cluster of Differentiation 38 (CD38) is a transmembrane protein widely existing in mammals. Its various functions make it a very popular research object, including in the viral infection field. (2) Methods: Western blotting and an EdU Cell Proliferation Kit were used to evaluate the effect of CD38-deficient cells. Relative quantitative real-time RT-PCR was used to detect the transcription levels of cytokines after PPV infection. The renilla luciferase reporter gene assay was used to verify the activation function of CD38 on downstream factors. The fluorescence probe method was used to detect the level of intracellular reactive oxygen species (ROS). (3) Results: This study found that the loss of CD38 function inhibited the up-regulated state of Toll-like Receptor 9 (TLR9), Interferon-α (IFN-α), and Myxovirus Resistance 1 (Mx1) after PPV infection. The luminescence of the group transfected with both CD38 expression plasmid and TLR9 promoter renilla luciferase reporter plasmid was significantly up-regulated compared with the control, suggesting that CD38 may activate the promoter of TLR9. In addition, CD38 deficiency not only activated the transcription of Sirtuin-1 (SIRT1), but also inhibited ROS level and the transcription of NLR Family Pyrin Domain Containing 3 (NLRP3). (4) Conclusion: (i) CD38 may participate in the TLR9/IFN-α/Mx1 pathway by activating the expression of TLR9 after PPV infected PK-15 cells; (ii) CD38 may activate the NLRP3/CASP1 pathway by increasing ROS level; (iii) CD38 deficiency activates the expression of SIRT1 and can prevent the normal proliferation of PPV. [ABSTRACT FROM AUTHOR]

Abstract Background Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. Results We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. Conclusions The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of oral cancer in developing countries and its burden correlates with exposure to tobacco and excessive alcohol consumption. Toll like receptors (TLRs) are major sensors of inflammatory stimuli, from both microbial and sterile causes and as such, they have been related to tumor progression and metastasis. Here, we evaluated the expression of TLR2, 4 and 9 as well as CD3+, CD8+ and Granzyme B+ cell infiltration by immunohistochemistry in oral samples of 30 patients with OSCC, classified according to their consumption of alcohol. Our findings indicate that there is a significant association between heavy alcohol consumption and tumors with higher expression levels of TLR9. Moreover, patients with TLR9high tumors, as well as those who indicated high consumption of alcohol exhibited a diminished overall survival. TCGA data analysis indicated that TLR9high tumors express a significant increase in some genes related with the oral cavity itself, inflammation and tumor promotion. Our analysis of tumor infiltrating leukocytes demonstrated that the major differences perceived in heavy alcohol consumers was the location of CD8+ T cells infiltrating the tumor, which showed lower numbers intratumorally. Our data suggest the existence of a pathogenic loop that involves alcohol consumption, high TLR9 expression and the immunophenotype, which might have a profound impact on the progression of the disease.

Summary: Background: Type I interferon (IFN-I) production by plasmacytoid dendritic cells (pDCs) occurs during viral infection, in response to Toll-like receptor 7 (TLR7) stimulation and is more vigorous in females than in males. Whether this sex bias persists in ageing people is currently unknown. In this study, we investigated the effect of sex and aging on IFN-α production induced by PRR agonist ligands. Methods: In a large cohort of individuals from 19 to 97 years old, we measured the production of IFN-α and inflammatory cytokines in whole-blood upon stimulation with either R-848, ODN M362 CpG-C, or cGAMP, which activate the TLR7/8, TLR9 or STING pathways, respectively. We further characterized the cellular sources of IFN-α. Findings: We observed a female predominance in IFN-α production by pDCs in response to TLR7 or TLR9 ligands. The higher TLR7-driven IFN-α production in females was robustly maintained across ages, including the elderly. The sex-bias in TLR9-driven interferon production was lost after age 60, which correlated with the decline in circulating pDCs. By contrast, STING-driven IFN-α production was similar in both sexes, preserved with aging, and correlated with circulating monocyte numbers. Indeed, monocytes were the primary cellular source of IFN-α in response to cGAMP. Interpretation: We show that the sex bias in the TLR7-induced IFN-I production is strongly maintained through ages, and identify monocytes as the main source of IFN-I production via STING pathway. Funding: This work was supported by grants from Région Occitanie/Pyrénées-Méditerranée (#12052910, Inspire Program #1901175), University Paul Sabatier, and the European Regional Development Fund (MP0022856).

Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization. Extracellular signal-regulated kinase (ERK) signaling and β2-integrin upregulation, early responses to TLR7 and TLR9 ligands, are defective in Rab27a deficiency. CpG-stimulated Rab27a-deficient neutrophils present increased tumor necrosis factor alpha (TNF-α) secretion and decreased secretion of a selected group of mediators, including interleukin (IL)-10. In vivo , CpG-challenged Rab27a-null mice show decreased production of type I interferons (IFNs) and IFN-γ, and the IFN-α secretion defect is confirmed in Rab27a-null plasmacytoid dendritic cells. Our findings have significant implications for immunodeficiency, inflammation, and CpG adjuvant vaccination. [Display omitted] • Rab27a deficiency causes retention of eTLRs in LC3B/LAMP1+ amphisomes • Delayed CpG internalization impaired early eTLR signaling in Rab27a-null neutrophils • Rab27a is required for IL-10 and IFN-α secretion independently of eTLR signaling • CpG-challenged Rab27a-null mice show decreased type I IFN secretion Yu et al. demonstrate that, in the absence of Rab27a, endosomal Toll-like receptors are retained at amphisomes and CpG internalization is delayed. CpG-stimulated Rab27a-deficient neutrophils present selective defects in cytokine secretion. Defective nucleic acid sensing and altered cytokine secretion in Rab27a deficiency contribute to infection susceptibility and inflammatory disease. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9−/− mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9−/− mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection. [ABSTRACT FROM AUTHOR]

Rab GTPases not only play a crucial role in the exocytosis and membrane transport of all eukaryotic cells, but are also gradually being found to be involved in innate immunity in mammals. However, the immunological function of Rab3a in fish remains unclear. In this study, two members of Rab3a, CiRab3aa and CiRab3ab, were cloned from grass carp (Ctenopharyngodon idella) for the first time. It was observed that CiRab3aa exhibited a prompt response to grass carp reovirus (GCRV) infection, and suppressing the expression of the CiRab3aa effectively inhibited GCRV replication, whereas the expression level of the CiRab3ab gene did not affect GCRV replication. Furthermore, overexpression of CiRab3aa significantly suppressed the mRNA levels of TLR9 , TNF-α , IL-1β , IL-6 , IL-10 , ERK , and IFN-I following stimulation with CpG ODN 1670 A, suggesting that CiRab3aa is a negative regulator of TLR9 signaling pathway. In conclusion, this study not only revealed functional differences between CiRab3aa and CiRab3ab in grass carp immunity, providing new sights into understanding their function in teleost fish, but also provided a new target for the treatment strategy of grass carp hemorrhagic disease. These results will help address the harm and economic losses caused by GCRV infection in grass carp breeding industry. • CiRab3aa and CiRab3ab genes were cloned and characterized in grass carp. • There are 7 amino acid differences in the Rab domain of Rab3aa and Rab3aa protein. • Knockdown of CiRab3aa significantly inhibited the replication of GCRV. • CiRab3aa was a negative regulator of the TLR9 signaling pathway. [ABSTRACT FROM AUTHOR]

• A mutation at the 359th amino acid of TLR9 inhibits the expansion of splenic T cells during Trypanosoma brucei infection. • The MAPK signaling pathway was suppressed and cytokine production reduced in Tlr9M7Btlr mice during trypanosome infection. • Trypanosome DNA activates the MAPK signaling pathways through TLR9. Trypanosoma brucei , a causative agent of human and animal trypanosomiasis, regularly switches its major surface antigen to avoid elimination by the immune system. Toll-like receptor 9 (TLR9) is a key modulator for resistance to host-infective trypanosomes; however, the underlying molecular mechanism remains indistinct. Thus, we first approached the issue using Tlr9-mutant mice that render them non-responsive to TLR9 agonists. After infection, T cells in the spleens of Tlr9-mutant mice were analyzed by flow cytometry and a reduction in CD8+, CD4+ T, and NKT cells was observed in Tlr9-mutant mice compared to WT mice. We further found that the responses of inflammatory cytokines in the sera were reduced in Tlr9-mutant mice after T. brucei infection. The underlying molecular mechanism was that T. b. brucei DNA activated TLR9, which consequently upregulated the expression of p38 and ERK/MAPK, resulting in host resistance to trypanosome infection. In conclusion, these findings provide novel insights into the TLR9-mediated host responses to trypanosome infection. [ABSTRACT FROM AUTHOR]

IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV infection have remained elusive. Therefore, in this study, we explore the molecular mechanisms underlying IgA secretion in response to infection, with a particular focus on PEDV, a devastating enteric virus affecting global swine production. Our investigation begins by examining changes in IgA concentrations in both serum and small intestinal contents following PEDV infection in 2- and 4-week-old pigs. Remarkably, a significant increase in IgA levels in these older pigs post-infection were observed. To delve deeper into the regulatory mechanisms governing IgA secretion in response to PEDV infection, isolated porcine intestinal B cells were co-cultured with monocytes derived DCs (Mo-DCs) in vitro. In the intestinal DC-B cell co-cultures, IgA secretion was found to increase significantly after PEDV infection, as well as upregulating the expression of AID, GLTα and PSTα reflecting isotype switching to IgA. In addition, the expression of TLR9 was upregulated in these cultures, as determined by RT-qPCR and western blotting. Moreover, our findings extend to in vivo observations, where we detected higher levels of TLR9 expression in the ileum of pig post PEDV infection. Collectively, our results highlight the ability of PEDV to stimulate the generation of IgA, particularly in elder pigs, and identify TLR9 as a critical mediator of IgA production within the porcine intestinal microenvironment during PEDV infection. • IgA expression in both serum and feces of pigs shows a notable increase following PEDV infection. • PEDV infection induces the CSR of IgA in pigs. • TLR9 is identified as a key mediator of IgA production in an in vitro coculture of DCs and B cells. • TLR9 abundance is higher in pigs after PEDV infection. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy in humans and dogs. Several studies disclosed some similarities between the two species, including the constitutive activation of the NF-κB pathway as a fundamental underlying pathogenetic mechanism. In humans, the downregulation of IL-1R8 is implicated in DLBCL development, but its role in dogs has not been explored so far. To gain insight into the pathogenesis of this tumor in dogs, we evaluated the mRNA and protein expression of IL-1R8 in 12 control lymph nodes obtained from dogs not bearing tumors and from 50 dogs with DLBCL. Moreover, we analyzed through qRT-PCR the expression of TLR7, TLR9, MYC, and p52 genes that are known to be involved in the IL-1R8 regulatory network. IL-1R8 and p52 were downregulated in DLBCLs compared to control lymph nodes (p < 0.001), while a higher expression of TLR7, TLR9, and MYC was observed in DLBCLs (p < 0.01). Immunohistochemistry confirmed the gene expression results, revealing a significantly lower IL-1R8 staining score in DLBCLs compared to control lymph nodes (p < 0.0001). Taken together, these results suggest that IL-1R8 downregulation may represent one of the mechanisms driving DLBCL pathogenesis in dogs, mainly through the dysregulation of the Toll-like/interleukin receptors signaling cascade and the aberrant activation of the classical NF-κB pathway. [ABSTRACT FROM AUTHOR]

DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors. [ABSTRACT FROM AUTHOR]

Simple Summary: Despite the successful introduction of targeted therapies, Chronic Lymphocytic Leukemia (CLL) remains incurable. This is thought to be partially due to the pro-survival and anti-apoptotic signaling that CLL cells receive from the lymph node microenvironment. Therefore, inhibition of CLL migration into the lymph nodes is an attractive therapeutic option. Here, our aim was to gain a further understanding of what transcriptomic and miRNomic changes drive CLL migration and, from this, select promising therapeutic targets. We identified focal adhesion kinase (FAK) as one such potential target and demonstrated that inhibition of FAK in primary CLL samples effectively reduces both CXCL12 induced migration and invasion in vitro. Successful inhibition of CLL migration could increase the sensitivity of CLL cells to the currently used targeted therapeutics and therefore improve patient outcomes. The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL. [ABSTRACT FROM AUTHOR]

Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated TLR9 and STING gene expression while blocking RIG-I and AIM2 expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients' blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia. [ABSTRACT FROM AUTHOR]

Mucositis is a common gastrointestinal complication in cancer patients undergoing chemoradiotherapy, including oral mucositis and gastrointestinal mucositis, with clinical manifestations of oral ulcers, vomiting, diarrhea and pain that seriously reduce the quality of life of patients and even affect anticancer therapy. Toll-like receptor (TLR) are important receptors involved in innate immunity and in the development of chemoradiation-induced mucositis by mediating the effect between microorganisms and the host. A comprehensive understanding of the role of TLR in mucositis is helpful to guide the prevention and treatment of mucositis. This paper reviews the available studies on TLR and mucositis. The results of the literature review indicate that different TLR have different roles in chemoradiation-induced mucositis: TLR2 is an important receptor in the inflammatory cascade of chemoradiation-induced mucositis; TLR4 activation can increase gastrointestinal mucosal inflammation and lead to oral epithelial ulceration; TLR5 agonists can reduce the degree of radiation-induced mucositis damage; and antagonizing or knocking out TLR9 can reduce chemoradiation-induced gastrointestinal mucositis. However, no TLR agonists or inhibitors have yet been applied in clinical practice, and additional studies are needed to explore the role of different TLR in mucositis in the future to provide a reference for the precise prevention and treatment of chemoradiation-induced mucositis.

Relevance. Human immunodeficiency virus (HIV) infection is an independent factor in reduced fertility and a risk factor for miscarriage. There are some date an endometrial receptivity of HIV-infected patients has changed that plays an important role in embryo invasion, but the true reasons for the decrease in fertility rate in HIV infection remain unknown. Aim. Study of the expression of CD20, CD56 and TLR9 antigens on uterine epithelial cells of HIV-infected patients and the effectiveness of treatment for chronic endometritis by sodium nucleospermate. Materials and methods. This parallel-group study was done at two centres in the Russia. Participants were adults women aged 26 to 49 years (mean age 33.352.9 years), who were HIV-infected (n=12) and HIV-negative (22). An immunocytochemical study of endometrial biopsies taken on the 710th day of the menstrual cycle before and after treatment was done. The course of treatment with sodium nucleospermate was 42 days. Results. The expression level of CD56 and TLR9 in HIV-infected patients was 7.640.92% and 0.330.18%, respectively, and significantly differed from the expression levels in HIV-seronegative patients. There was a decrease in the expression levels of CD20 and CD56 and an increase in the expression levels of TLR9 in all groups of patients after treatment with sodium nucleospermate. Conclusion. A decrease TLR9 expression on uterine epithelial cells in HIV-infected patients showing lack of ability of innate immunity to eliminate pathogens associated with subclinical inflammation and it correlates with an increase in the expression of markers of chronic endometritis.

Purpose: In HT29 colon cancer cells, a close interplay between self-DNA-induced TLR9 signaling and autophagy response was found, with remarkable effects on cell survival and differentiation. IGF1R activation drives the development and malignant progression of colorectal cancer. IGF1R inhibition displays a controversial effect on autophagy. The interrelated roles of IGF1R inhibition and TLR9/autophagy signaling in HT29 cancer cells have not yet been clarified. In our study, we aimed to investigate the complex interplay of IGF1R inhibition and TLR9/autophagy signaling in HT29 cells.Methods: HT29 cells were incubated with tumor-originated self-DNA with or without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine), and TLR9 (ODN2088), respectively. Cell proliferation and metabolic activity measurements, direct cell counting, NanoString and Taqman gene expression analyses, immunocytochemistry, WES Simple Western blot, and transmission electron microscopy investigations were performed.Results: The concomitant use of tumor-derived self-DNA and IGF1R inhibitors displays anti-proliferative potential, which can be reversed by parallel TLR9 signaling inhibition. The distinct effects of picropodophyllin, ODN2088, and chloroquine per se or in combination on HT29 cell proliferation and autophagy suggest that either the IGF1R-associated or non-associated autophagy machinery is “Janus-faced” regarding its actions on cell proliferation. Autophagy, induced by different combinations of self-DNA and inhibitors is not sufficient to rescue HT29 cells from death but results in the survival of some CD133-positive stem-like HT29 cells.Conclusion: The creation of new types of combined IGF1R, autophagy, and/or TLR9 signaling inhibitors would play a significant role in the development of more personalized anti-tumor therapies for colorectal cancer.

ABSTRACT Upon infection, DNA viruses can be sensed by pattern recognition receptors (PRRs), leading to the activation of type I and III interferons to block infection. Therefore, viruses must inhibit these signaling pathways, avoid being detected, or both. Papillomavirus virions are trafficked from early endosomes to the Golgi apparatus and wait for the onset of mitosis to complete nuclear entry. This unique subcellular trafficking strategy avoids detection by cytoplasmic PRRs, a property that may contribute to the establishment of infection. However, as the capsid uncoats within acidic endosomal compartments, the viral DNA may be exposed to detection by Toll-like receptor 9 (TLR9). In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we showed that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts. Finally, we demonstrated that the cancer-associated human papillomaviruses show a reduction in CpG dinucleotides within a TLR9 recognition complex. IMPORTANCE Viruses must avoid detection by the innate immune system. In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we demonstrated that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts.

The innate immune system detects diverse microbial species with a limited repertoire of immune receptors that recognize nucleic acids. The cost of this immune surveillance strategy is the potential for inappropriate recognition of self-derived nucleic acids and subsequent autoimmune disease. The relative expression of two closely related receptors, Toll-like receptor (TLR) 7 and TLR9, is balanced to allow recognition of microbial nucleic acids while limiting recognition of self-derived nucleic acids. Situations that tilt this balance toward TLR7 promote inappropriate responses, including autoimmunity; therefore, tight control of expression is critical for proper homeostasis. Here we report that differences in codon bias limit TLR7 expression relative to TLR9. Codon optimization of Tlr7 increases protein levels as well as responses to ligands, but, unexpectedly, these changes only modestly affect translation. Instead, we find that much of the benefit attributed to codon optimization is actually the result of enhanced transcription. Our findings, together with other recent examples, challenge the dogma that codon optimization primarily increases translation. We propose that suboptimal codon bias, which correlates with low guanine-cytosine (GC) content, limits transcription of certain genes. This mechanism may establish low levels of proteins whose overexpression leads to particularly deleterious effects, such as TLR7.

Background: Toll-like receptors (TLRs) are critical sensors for the conservation of bacterial molecules and play a key role in host defense against pathogens. The effect of TLRs on the maintenance of diabetic nephropathy (DN) and resistance to infection has been investigated; however, the detailed effects of TLR9 on DN development remain elusive. Methods: We performed quantitative reverse transcription-polymerase chain reaction and western blotting to detect TLR9 expression levels in the kidneys of experimental mice (db/db) and high-glucose-treated mouse mesangial cell strains (MCs). Results: TLR9 expression was found to be remarkably upregulated in the kidneys of experimental mice (db/db) and MCs cultivated under hyperglycemic conditions. Moreover, knockdown of TLR9 could restrain NF-kB viability and downregulate the NLRP3 inflammasome in high glucose-treated MCs. TLR9 inhibition also alleviated inflammation and apoptosis, which was reversed by the addition of the NF-κB activator, betulinic acid. Furthermore, depleted TLR9 levels restrained NF-κB viability and NLRP3 expression and reduced kidney inflammation, microalbuminuria discharge, blood sugar level, and glomerular damage in experimental mice (db/db) kidneys. Conclusions These findings offer novel insights into the regulation of TLR9 via the nuclear factor-kB/NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome inflammation pathways in DN progression. [ABSTRACT FROM AUTHOR]

Objective: Numerous studies have shown that mitochondrial DNA (mtDNA) can trigger innate immune signaling, and exercise can induce mitochondrial stress. Therefore, this study is aimed at investigating the influence of different types of acute exercise on the innate immune signaling triggered by mtDNA. Methods: Male C57BL/6 mice (n = 18) were randomly and equally divided into three groups. They were control group, acute moderate-intensity endurance exercise group (AMIE), and 3-day exhaustive exercise group (EE) respectively. Mice were sacrificed immediately after exercise. The spleen, liver, and blood were taken for analysis. Results: The amount of mtDNA in the liver cytoplasm and plasma was significantly decreased after AMIE (p < .05). However, the amount of mtDNA in plasma was increased after EE (p < .05). The mRNA expression of TFAM, and most TLR9 and cGAS/STING signaling pathway-related genes in the liver and spleen was markedly elevated, whereas the expression of those genes in leukocytes was reduced after AMIE. Furthermore, AMIE significantly decreased the protein expression of NLRP3 inflammasome in the liver (p < .05) and STING in spleen (p < .01). Also, AMIE and EE caused a drop in circulating IFN-α levels (p < .05). Conclusion: A single bout of moderate-intensity exercise reduces mtDNA-induced innate immune signaling and suppresses inflammatory responses by decreasing hepatic cytoplasmic and circulating mtDNA. However, repeated bouts of exhaustive exercise stimulate innate immune signaling by increasing levels of circulating mtDNA. [ABSTRACT FROM AUTHOR]

• TAL induced TLR3 and TLR9 activation. • The stimulation of TLR3 or TLR9 signaling induced more apoptosis via the overexpression of caspase-3 and caspase-8. • The stimulation of TLR3 or TLR9 with TAL treatment could overcome TAL resistance. Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) induce cell death by inhibiting the repair of DNA strand breaks binding to PARP and regulate immune cells functions. Toll-like receptors (TLRs) mediate the tumor microenvironment through the modulation of proinflammatory cytokines and chemokines. In this context, this study addressed the relationship between the efficacy of talazoparib (TAL) as a PARPi and the activation of TLR3 or TLR9 by Polyinosinic:polycytidylic acid (Poly I:C) or CpG oligodeoxynucleotides (CpG-ODN) stimulation, respectively in triple negative breast cancer (TNBC). TAL alone and the combination of TAL with Poly I:C or CpG-ODN induced cell death were analyzed by water-soluble tetrazolium salt 1 (WST-1), Annexin V analysis, acridine orange staining and mRNA levels of caspase-3 and caspase-8 in HCC1937 and HCC1937-R (TAL resistant) TNBC cells. Additionally, the expression of TLR3, TLR9 and interferon regulatory factor 7 (IRF7) was observed with immunofluorescence staining and western blot analysis. Our findings showed that TAL induced TLR3 and TLR9 activation and acted in synergy with TLR3 and TLR9 agonists in TNBC cells. The stimulation of TLR3 or TLR9 and TAL treatment caused significantly more apoptosis in TNBC cells through the over-expression of caspase-3 and caspase-8. Additionally, TAL combined with Poly I:C or CpG-ODN more increased TLR3, TLR9 and IRF7 protein levels in HCC1937 cells and treatment with TAL and Poly I:C had greater potential for overcoming TAL resistance. In conclusion, the combination of PARPi with TLR agonists may be a new therapeutic combined strategy for the effective immunotherapy of TNBC. [ABSTRACT FROM AUTHOR]

Objective: Due to its well-known anti-inflammatory property, oxymatrine (OMT) has received more attention on the aspect of treating ulcerative colitis. Although efforts have been undertaken to understand the therapeutic mechanism of OMT on ulcerative colitis (UC), the remedial principle is still ambiguous. Numerous studies have shown that TLR9/Myd88/NF-κB signal pathway played a key role in the pathogenesis of UC. Moreover, TLR9/Myd88/NF-κB signal pathway is a part of the most important pathways for regulating the immune response. Methods: We explored the influence of OMT with different dosages on UC by establishing a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. Moreover, the participation of TLR9/Myd88/NF-κB signal pathway and whether OMT protects against UC though targeting this pathway are further studied. Results: Our data revealed that OMT could significantly relieve the symptom of TNBS-induced colitis in rats by reactivating the tight junction protein and, more important, by inhibiting the activation of TLR9/Myd88/NF-κB pathway and protein expression levels of its downstream inflammatory factors. Conclusion: OMT could relieve colitis in rat models by impacting tight junction proteins' TLR9/Myd88/NF-κB signal pathways and activity. [ABSTRACT FROM AUTHOR]

Introduction: Imiquimod plays an important role in the management of condyloma and premalignant lesions. Successively, an increase of hypopigmented lesions following imiquimod application has been reported. However, the mechanisms of imiquimod on melanocytes remain unclear. This study was designed to assess the effect of Imiquimod on the functions of melanocytes in vitro. Methods: Primary cultured melanocytes were isolated from normal control skin tissue. After incubation with imiquimod for 48 h in vitro, cell viability was analyzed by cell counting kit‐8 assay. Apoptosis was detected using the Annexin V‐fluorescein‐5‐isothiocyanate flow cytometry assay. Melanin content and tyrosinase activity in melanocytes were measured by colorimetric method and the modified dopachrome method. The production of inflammatory cytokine interleukin 8 (IL‐8), IL‐6, and soluble ICAM‐1 (soluble Intercellular Adhesion Molecule‐1[sICAM‐1]) in melanocytes were measured by enzyme‐linked immunosorbent assay (ELISA). Toll‐like receptor 7 (TLR7), toll‐like receptor 9 (TLR9) protein, and autophagy‐related proteins microtubule‐associated protein 1A/1B‐light chain 3 (LC3‐II), p62, mechanistic target of rapamycin (mTOR), and Atg5 were assessed using western blot analysis. Results: Imiquimod significantly inhibited the activity of tyrosinase activity and decreased melanin content in melanocytes and significantly increased apoptosis and IL‐6, IL‐8, and sICAM‐1 production in melanocytes. Moreover, the expression of TLR7 and TLR9 proteins were significantly increased, and the expression of mTOR, p62 protein were markedly decreased, but the expression of LC3II/I and Atg5 protein were significantly increased in melanocytes after incubating with imiquimod. Conclusions: This study shows that imiquimod directly inhibits melanogenesis and increases melanocyte apoptosis rates. These effects combined with the upregulation of TLR7 and TLR9 together with increased autophagy activity and inflammatory cytokines production, might be the main reasons leading to hypopigmented lesions after imiquimod application. [ABSTRACT FROM AUTHOR]

Abstract Background Glioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells. However, identification of the signaling molecules and underlying mechanisms has not been completely elucidated. Methods Human samples and glioma cell lines were cultured in vitro to determine the effects of adenovirus (ADV) infection by sphere formation, RT-qPCR, western blotting, FACS and immunofluorescence. For in vivo analysis, mouse intracranial tumor model was applied. Bioinformatics analysis, gene knockdown by siRNA, RT-qPCR and western blotting were applied for further mechanistic studies. Results Infection of patient-derived glioma cells with ADV increases the formation of tumor spheres. ADV infection upregulated stem cell markers and in turn promoted the capacities of self-renewal and multi-lineage differentiation of the infected tumor spheres. These ADV infected tumor spheres had stronger potential to form xenograft tumors in immune-compromised mice. GSCs formation could be promoted by ADV infection via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1. Knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, HMGB1, a damage associated molecular pattern (DAMP) that triggers TLR signaling, also upregulated stemness markers in glioma cells. Conclusion ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling. Video abstract

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is the most common malignancy caused by EBV infection. Toll-like receptors (TLRs) as major components of innate immune system are crucial in the development of inflammatory processes and carcinogenesis. The aim of our study was to evaluate tissue and serum level of TLR9 in EBV-positive and EBV-negative gastric cancer patients. The study involved 30 EBV(+) and 30 EBV(-) patients. EBV DNA was detected in fresh frozen tumor tissue. In serum samples TLR9 level, transforming growth factor β (TGFβ), interleukin 10 (IL-10) and antibodies against EBV were detected using ELISA tests. TLR9 level was also measured in homogenate of tumour tissue. TLR9 level was statistically lower in EBV(+) patients both in serum and tissue, with statistically higher level in tissue than in serum. Lower level of TLR9 was accompanied by higher level of Epstein-Barr virus capsid antigen (EBVCA), Epstein-Barr virus nuclear antigen (EBNA) and early antigen (EA). A lower level of TLR9 was detected in patients with poorly differentiated cancer (G3) and greater lymph nodes involvement (N3-N4). Lower level of TLR9 in patients with EA may point to TLR9 role in reactivation of EBV infection.

Abstract Objectives Cardiac surgery can lead to post-operative end-organ complications secondary to activation of systemic inflammatory response. We hypothesize that surgical trauma or cardiopulmonary bypass (CPB) may initiate systemic inflammatory response via release of mitochondrial DNA (mtDNA) signaling Toll-like receptor 9 (TLR9) and interleukin-6 production (IL-6). Materials and methods The role of TLR9 in systemic inflammatory response in cardiac surgery was studied using a murine model of sternotomy and a porcine model of sternotomy and CPB. mtDNA and IL-6 were measured with and without TLR9-antagonist treatment. To study ischemia-reperfusion injury, we utilized an ex-vivo porcine kidney model. Results In the rodent model (n = 15), circulating mtDNA increased 19-fold (19.29 ± 3.31, p

Abstract Background Acute myeloid leukemia (AML) can modulate toll-like receptor-9 (TLR9) expression and activation. This study was conducted to elucidate the expression of TLR9 in AML patients and its relation to the prognosis of the disease. Results The study included 40 newly diagnosed AML patients managed in the hospital in addition to 20 sex and age matched normal volunteers as control. TLR9 expression assay was conducted on peripheral blood samples of AML cases before the start of treatment as well as the controls by immunophenotyping. TLR9 expression was ranging from 0.10 to 2.40% in AML patients with higher expression among the control, ranging from 0.94 to 8.25%. The median TLR9 expression in AML patients was significantly lower with advanced cytogenetic risk score. It is not significantly differing in relation to patients’ sex, age group, and FAB type of AML. However, significant lower median expression was found in relation to clinical outcome. TLR9 expression ≤ 1% showed lower median overall survival time when compared to those with > 1% expression. Conclusion This study concluded that AML patients express TLR9 in leukemic cells with very low percentage. This expression was negatively related to the clinical outcome.

Summary: Background: Type I IFN (IFN-I) is a family of cytokines involved in the pathogenesis of autoimmune and autoinflammatory diseases such as psoriasis. SIDT1 is an ER-resident protein expressed in the lymphoid lineage, and involved in anti-viral IFN-I responses in vivo, through an unclear mechanism. Herein we have dissected the role of SIDT1 in the natural IFN-producing cells, the plasmacytoid dendritic cells (pDC). Methods: The function of SIDT1 in pDC was determined by silencing its expression in human primary pDC and GEN2.2 cell line. SIDT1 role in vivo was assessed using the imiquimod-induced psoriasis model in the SIDT1-deficient mice (sidt1−/−). Findings: Silencing of SIDT1 in GEN2.2 led to a blockade of the IFN-I response after stimulation of TLR7 and TLR9, without affecting the pro-inflammatory responses or upregulation of maturation markers. We found that SIDT1 migrates from the ER to the endosomal and lysosomal compartments together with TLR9 after CpG stimulation, participating in the access of the TLR9-CpG complex to lysosome-related vesicles, and therefore mediating the activation of TBK1 and the nuclear migration of IRF7, but not of NF-κB. sidt1−/− mice showed a significant decrease in severity parameters of the imiquimod-induced acute psoriasis-like model, associated with a decrease in the production of IFN-I and IFN-dependent chemokines. Interpretation: Our findings indicate that SIDT1 is at the cross-road between the IFN-I and the proinflammatory pathways and constitutes a promising drug target for psoriasis and other diseases mediated by IFN-I responses. Funding: This work was supported by the Consejería de Salud y Familias de la Junta de Andalucía (PIER_S1149 and C2_S0050) and Instituto de Salud Carlos III (PI18/00082 and PI21/01151), partly supported by European FEDER funds, and prior funding to MEAR from the Alliance for Lupus Research and the Swedish Research Council.

Temporomandibular joint osteoarthritis (TMJ OA) is characterized by the degeneration of cartilage and subchondral bone. In this study, we observed a significant increase in cell-free DNA (cfDNA) levels during the progression of TMJ OA. Bioinformatics analysis identified TLR9 as a pivotal molecule in TMJ OA pathogenesis. The polyamidoamine (PAMAM) dendrimer characterized by a well-structured, highly branched, and reactive nature, exhibits robust binding and clearance capabilities for cfDNA. However, the abundant amino groups on the surface of PAMAM lead to its inherent toxicity. To mitigate this, PEG-5000 was conjugated to the surface of PAMAM dendrimers, enhancing safety. Our results indicate that PEG-PAMAM effectively inhibits the upregulation of the TLR9 protein in TMJ OA, significantly suppressing the activation of the p-IκBα/p-NF-κB signaling pathway and subsequently decreasing chondrocyte inflammation and apoptosis, as evidenced by both in vivo and in vitro experiments. We conclude that PEG-PAMAM is a safe and effective material for in vivo applications, offering a promising therapeutic strategy for TMJ OA by targeting cfDNA clearance.

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo . At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation ( Irf8, Cebpa, Itgam ) and antigen-presentation ( Ciita, Il12a, B2m )-related genes with concomitant reduction of leukemia-promoting Runx1 . Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo , IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition., Competing Interests: M.K. is an inventor on the patents that cover the design of CpG-STAT3d ODNs. M.K. serves on the Scientific Advisory Board of Scopus Biopharma and its subsidiary Duet Biotherapeutics with stock options and sponsored research., (© 2024 The Author(s).)

Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA (gDNA). Previous bioinformatic studies have demonstrated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here, we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. Utilizing reporter cell lines, we demonstrate that purified gDNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion, exacerbated by the inhibition of lipooligosaccharide biosynthesis, and is significantly altered during the induction of aberrance/persistence. Our observations support the hypothesis that chlamydial gDNA is released during the conversion between the pathogen's replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly. Given that C. trachomatis inclusions do not co-localize with TLR9-containing vacuoles in the pro-monocytic cell line U937, our findings also hint that chlamydial gDNA is capable of egress from the inclusion, and traffics to TLR9-containing vacuoles via an as yet unknown pathway., Competing Interests: The authors declare no conflict of interest.

Monkeypox virus (MPV) is closely related to the smallpox virus, and previous data from Africa suggest that the smallpox vaccine (VARV) is at least 85% effective in preventing MPV. No multi-epitope vaccine has yet been developed to prevent MPV infection. In this work, we used in silico structural biology and advanced immunoinformatic strategies to design a multi-epitope subunit vaccine against MPV infection. The designed vaccine sequence is adjuvanted with CpG-ODN and includes HTL/CTL epitopes for similar proteins between vaccinia virus (VACV) that induced T-cell production in vaccinated volunteers and the first draft sequence of the MPV genome associated with the suspected outbreak in several countries, May 2022. In addition, the specific binding of the modified vaccine and the immune Toll-like receptor 9 (TLR9) was estimated by molecular interaction studies. Strong interaction in the binding groove as well as good docking scores confirmed the stringency of the modified vaccine. The stability of the interaction was confirmed by a classical molecular dynamics simulation and normal mode analysis. Then, the immune simulation also indicated the ability of this vaccine to induce an effective immune response against MPV. Codon optimization and in silico cloning of the vaccine into the pET-28a (+) vector also showed its expression potential in the E. coli K12 system. The promising data obtained from the various in silico studies indicate that this vaccine is effective against MPV. However, additional in vitro and in vivo studies are still needed to confirm its efficacy.Communicated by Ramaswamy H. Sarma.

Toll-like receptor 9 (TLR9) is an intracellular innate immunity receptor that plays a vital role in chronic inflammation and in recognizing pathogenic and self-DNA in immune complexes. This activation of intracellular signaling leads to the transcription of either immune-related or malignancy genes through specific transcription factors. Thus, it has been hypothesized that TLR9 may cause glioma. This article reviews the roles of TLR9 in the pathogenesis of glioma and its related signaling molecules in either defending or promoting glioma. TLR9 mediates the invasion-induced hypoxia of brain cancer cells by the activation of matrix metalloproteinases (2, 9, and 13) in brain tissues. In contrast, the combination of the TLR9 agonist CpG ODN to radiotherapy boosts the role of T cells in antitumor effects. The TLR9 agonist CpG ODN 107 also enhances the radiosensitivity of human glioma U87 cells by blocking tumor angiogenesis. CpG enhances apoptosis in vitro and in vivo. Furthermore, it can enhance the antigen-presenting capacity of microglia, switch immune response toward CD8 T cells, and reduce the number of CD4CD25 Treg cells. CpG ODN shows promise as a potent immunotherapeutic drug against cancer, but specific cautions should be taken when activating TLR9, especially in the case of glioblastoma.

Renal involvement is an important cause of morbidity and mortality in systemic lupus erythematosus (SLE). The present study included patients with recently diagnosed Class III and Class IV lupus nephritis (LN) treated by Rheumatology who, upon the detection of alterations in their kidney function, were referred to Nephrology for the joint management of both medical specialties. The purpose of this study was to compare the plasma expression of Toll-Like Receptor 7 (TLR7) and TLR9 in healthy control (HC) subjects and newly diagnosed Class III and Class IV LN patients with 12-month follow-ups. The plasma expression of TLR7 and TLR9 proteins was determined by the ELISA method. A significant increase in the expression of TLR7 protein was found in Class III LN in the basal determination compared to the expression in the HC ( p = 0.002) and at 12 months of follow-up ( p = 0.03) vs. HC. The expression of TLR9 showed a behavior opposite to that of TLR7. TLR9 showed decreased protein expression in LN Class III patients' baseline and final measurements. The result was similar in the basal and final determinations of LN Class IV compared to the expression in HC. A significant decrease in SLEDAI -2K was observed at 12 months of follow-up in patients in Class III ( p = 0.01) and Class IV ( p = 0.0001) of LN. Complement C3 levels improved significantly at 12-month follow-up in Class IV patients ( p = 0.0001). Complement C4 levels decreased significantly at 12-month follow-up in LN Class III compared to baseline ( p = 0.01). Anti-DNA antibodies decreased significantly at 12 months of follow-up in Class IV LN ( p = 0.01). A significant increase in proteinuria was found at 12 months of follow-up in Class III LN, compared to the baseline determination ( p = 0.02). In LN Class IV, proteinuria decreased at 12 months of follow-up compared to baseline ( p = 0.0001). Albuminuria decreased at 12 months of follow-up in LN Class IV ( p = 0.006). Class IV LN, albuminuria also decreased at 12 months of follow-up ( p = 0.009). Hematuria persisted in all patients and the glomerular filtration rate did not change. Three Class IV patients died before 12 months of follow-up from various causes. In conclusion, although the rheumatologic data appeared to improve, the renal function data remained inconsistent. Decreased expression of TLR9 and increased expression of TLR7 could be useful in the early diagnosis of Class III and Class IV LN is correct.

Purpose: Interleukin-37 (IL-37) is an anti-inflammatory cytokine that inhibits a broad spectrum of inflammatory responses in various human cells, including neutrophils, macrophages, and endothelial cells. The aim of this study was to identify the role of IL-37 in toll-like receptor 9 (TLR9) signaling in human macrophages. Materials and Methods: Human macrophage U937 cells treated with CpG-oligonucleotides (CpG-ODN), recombinant IL-37, or dexamethasone were used in an in vitro study. IL-37 small interfering RNA (siRNA) and TLR9 siRNA were used to silence endogenous IL-37 and TLR9, respectively. Expression levels of phosphorylated nuclear factor-κB (NF-κB), IκBα, IL-37, IL-1β, tumor necrosis factor-α (TNF-α), and IL-6 protein were assessed by real-time quantitative polymerase chain reaction and Western blotting. CpG-ODN-mediated IL-37 expression stimulated by dexamethasone was detected using immunofluorescent analysis. Results: U937 cells treated with CpG-ODN induced activation of the NF-κB pathway and increased the expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6, but reduced that of IL-37. Recombinant IL-37 attenuated phosphorylation of NF-κB and IκBα and the expression of IL-1β, TNF-α, and IL-6 stimulated by CpG-ODN. Human macrophages transfected with IL-37 siRNA augmented the expression of IL-1β, TNF-α, and IL-6 mRNA and protein in cells treated with CpG-ODN. Dexamethasone markedly inhibited expression of pro-inflammatory cytokines in U937 cells, whereas IL-37 expression was increased with the addition of dexamethasone. Inflammatory responses elicited by CpG-ODN were dependent on an MyD88-TRAF6 pathway. IL-37 inhibited CpG-ODN-induced ubiquitination of TRAF6 in U937 macrophages. Conclusion: IL-37 inhibits CpG-ODN-mediated inflammatory responses through regulation of a TRAF6- NF-κB pathway in human macrophages. [ABSTRACT FROM AUTHOR]

IRF8 is a key regulator of innate immunity receptor signaling and plays diverse functions in the development of hematopoietic cells. The effects of IRF8 on hematopoietic stem cells (HSCs) are still unknown. Here, it is demonstrated that IRF8 deficiency results in a decreased number of long‐term HSCs (LT‐HSCs) in mice. However, the repopulation capacity of individual HSCs is significantly increased. Transcriptomic analysis shows that IFN‐γ and IFN‐α signaling is downregulated in IRF8‐deficient HSCs, while their response to proinflammatory cytokines is unchanged ex vivo. Further tests show that Irf8−/− HSCs can not respond to CpG, an agonist of Toll‐like receptor 9 (TLR9) in mice, while long‐term CpG stimulation increases wild‐type HSC abundance and decreases their bone marrow colony‐forming capacity. Mechanistically, as the primary producer of proinflammatory cytokines in response to CpG stimulation, dendritic cells has a blocked TLR9 signaling due to developmental defect in Irf8−/− mice. Macrophages remain functionally intact but severely reduce in Irf8−/− mice. In NK cells, IRF8 directly regulates the expression of Tlr9 and its deficiency leads to no increased IFNγ production upon CpG stimulation. These results indicate that IRF8 regulates HSCs, at least in part, through controlling TLR9 signaling in diverse innate immune cells. [ABSTRACT FROM AUTHOR]

Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Up to 40% of IgAN patients develop end-stage kidney disease after 15–20 years. Despite the poor prognosis associated with this multifactorial disease, no clear treatment strategy has been identified, primarily due to the lack of understanding of its pathogenesis. Clinical observations indicate that aberrant IgAN immune systems, rather than intrinsic renal abnormalities, may be involved in its pathogenesis. Moreover, nephritogenic IgA and its related immune complexes are considered to be produced not only in the mucosa, but also in systemic immune sites, such as the bone marrow; however, there are numerous challenges to understanding this dynamic and complex immune axis in humans. Thus, several investigators have used experimental animal models. Although there are inter-strain differences in IgA molecules and immune responses between humans and rodents, animal models remain a powerful tool for investigating IgAN's pathogenesis, and the subsequent development of effective treatments. Here, we introduced some classical models of IgAN with or without genetic manipulation and recent translational approaches with some promising models. This includes humanized mouse models expressing human IgA1 and human IgA Fc receptor (CD89) that develops spontaneously the disease. Pre-clinical studies targeting IgA1 are discussed. Together, animal models are very useful tools to study pathophysiology and to validate new therapeutic approaches for IgAN. [ABSTRACT FROM AUTHOR]