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The pathway of Janus-activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) (termed as JAK2/STAT3) plays an active role in stroke-related inflammation induced by ischemic stress. Gastrodin, the primary compound in Gastrodia elata Bl, has been identified for its notable neuroprotective effects and demonstrated to ameliorate cerebral ischemia-reperfusion but its exact mechanisms governing this defense are still unclear. This study aims to investigate whether gastrodin can regulate mitochondrial function via the JAK2/STAT3 pathway to limit cerebral ischemia-reperfusion. In vivo, gastrodin significantly reduced infarct volume, improved neurobiological function, attenuated neuronal apoptosis, oxidative stress, mitochondrial impairment, mtDNA leakage, and inflammatory responses. At the cellular level, gastrodin administration rescued OGD/R-induced cell apoptosis, oxidative stress, and mitochondrial dysfunction. Mechanistically, gastrodin notably suppressed Toll-like receptor 9 (TLR9) expression, important for the recognition of disrupted endogenous DNA to produce inflammatory reactions. Furthermore, gastrodin mitigated inflammation by inhibiting JAK2/STAT3 signaling, influencing inflammatory factors to aggravate inflammation. Notably, the effects of gastrodin were abolished by Coumermycin A1 (C-A1), a JAK2 agonist, validating the role of JAK2/STAT3 signaling. In summary, gastrodin enhances the protective effect against mitochondrial damage in ischemic stroke by inhibiting JAK2/STAT3 signaling. Gastrodin is a possible therapy for cerebral ischemia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pneumonia is a worldwide threat to public health, demanding novel preventative and therapeutic strategies. The lung epithelium is a critical environmental interface that functions as a physical barrier to pathogen invasion while also actively sensing and responding to pathogens. We have reported that stimulating lung epithelial cells with a combination therapeutic consisting of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) induces synergistic pneumonia protection against a wide range of pathogens. We report here that mice deficient in TLR9, the previously described receptor for ODN, still displayed partial ODN-induced protection. This prompted us to seek an alternate ODN receptor, and we discovered by mass spectroscopy that the RNA sensor RIG-I could also bind DNA-like ODN. ODN binding by RIG-I resulted in MAVS-dependent pneumonia-protective signaling events. While RIG-I is essential to native defenses against viral infections, we report that therapeutic RIG-I activation with ODN promoted pathogen killing and host survival following both viral and bacterial challenges. These data indicate that maximal ODN-induced pneumonia protection requires activation of both the TLR9/MyD88 and RIG-I/MAVS signaling pathways. These findings not only identify what we believe to be a novel pattern recognition receptor for DNA-like molecules, but reveal a potential therapeutic strategy to protect susceptible individuals against lethal pneumonias during periods of peak vulnerability.

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4 + T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy., (© 2024. The Author(s).)

Complexes of extracellular nucleic acids (NAs) with endogenous proteins or peptides, such as LL37, break immune balance and cause autoimmune diseases, whereas NAs with arginine-enriched peptides do not. Inspired by this, we synthesize a polyarginine nanoparticle PEG-TK-NP Arg , which effectively inhibits Toll-like receptor-9 (TLR9) activation, in contrast to LL37. To explore the discrepancy effect of PEG-TK-NP Arg and LL37, we evaluate the periodic structure of PEG-TK-NP Arg -NA and LL37-NA complexes using small-angle X-ray scattering. LL37-NA complexes have a larger inter-NA spacing that accommodates TLR9, while the inter-NA spacing in PEG-TK-NP Arg -NA complexes mismatches with the cavity of TLR9, thus inhibiting an interaction with multiple TLR9s, limiting their clustering and damping immune induction. Subsequently, the inhibitory inflammation effect of PEG-TK-NP Arg is proved in an animal model of rheumatoid arthritis. This work on how the scavenger-NA complexes inhibit the immune response may facilitate proof-of-concept research translating to clinical application., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Lymphangioleiomyomatosis (LAM) is a devastating disease primarily found in women of reproductive age that leads to cystic destruction of the lungs. Recent work has shown that LAM causes immunosuppression and that checkpoint inhibitors can be used as LAM treatment. Toll-like receptor (TLR) agonists can also reactivate immunity, and the TLR9 agonist CpG oligodeoxynucleotide (CpG-ODN) has been effective in treating lung cancer in animal models. In this study, we investigated the use of TLR9 agonist CpG-ODN as LAM immunotherapy in combination with checkpoint inhibitor anti-PD1 and standard of care rapamycin, and determined the immune mechanisms underlying therapeutic efficacy. We used survival studies, flow cytometry, ELISA, and histology to assess immune response and survival after intranasal treatment with CpG-ODN in combination with rapamycin or anti-PD1 therapy in a mouse model of metastatic LAM. We found that local administration of CpG-ODN enhances survival in a mouse model of LAM. We found that a lower dose led to longer survival, likely because of fewer local side effects, but increased LAM nodule count and size compared with the higher dose. CpG-ODN treatment also reduced regulatory T cells and increased the number of T-helper type 17 cells as well as cytotoxic T cells. These effects appear to be mediated in part by plasmacytoid dendritic cells because depletion of plasmacytoid dendritic cells reduces survival and abrogates T-helper type 17 cell response. Finally, we found that CpG-ODN treatment is effective in early-stage and progressive disease and is additive with anti-PD1 therapy and rapamycin. In summary, we have found that TLR9 agonist CpG-ODN can be used as LAM immunotherapy and effectively synergizes with rapamycin and anti-PD1 therapy in LAM.

Background: CpG oligonucleotides (ODNs) are synthetic single-stranded DNA sequences that act as immunostimulants. They have been increasingly used to treat several cancers; however, thrombocytopenia is a potential recognized side effect of some sequences., Objectives: We tested the ability of 2 CpG ODNs (ODN 2395 and ISIS 120704) to induce thrombocytopenia when administered to BALB/c mice and determined mechanisms associated with thrombocytopenia., Methods: BALB/c mice were prebled and then injected with titrated doses of CpG ODNs, and platelet counts were determined. The mice were treated with intravenous immunoglobulin (IVIg) or various inhibitors and antagonists of toll-like receptor 9 (TLR9) and spleen tyrosine kinase (Syk) to determine their effects on thrombocytopenia., Results: Compared with saline-treated mice or mice treated with 2'-O-methoxyethyl-modified antisense ODN, both ODN 2395 and ISIS 120704 induced acute dose-dependent thrombocytopenia within 3 and 24 hours, respectively. The thrombocytopenia was associated with significant increases in plasma monocyte chemoattractant protein 1. IVIg administration significantly rescued the CpG ODN-induced thrombocytopenia, as did treatment with either a Syk inhibitor or TLR9 antagonists. In vitro, CpG ODN could activate human platelets and this correlated significantly with enhanced IVIg- and Syk-dependent phagocytosis by THP-1 monocytes., Conclusion: These results suggest that CpG ODNs induce acute inflammatory-associated (IVIg-sensitive) thrombocytopenia that can be alleviated by Syk- or TLR9-blockade, and an IVIg- and Syk-dependent platelet clearance pathway appears primarily responsible for the thrombocytopenia., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neutrophil cationic proteins (NCPs) are a group of granule antimicrobial and inflammatory proteins released by activated neutrophils. These proteins primarily function via their positively charged structure, which facilitates interactions with bacterial membranes and the formation of immunogenic DNA complexes, thereby contributing to the initiation of wound repair in injured skin. After analyzing the structural properties of secreted neutrophil granule proteins, we identified OLFM4 as the only negatively charged molecule that interferes with NCP oligomerization. Through this interference, OLFM4 can inhibit neutrophil-mediated bacterial killing and DNA complex-dependent activation of Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDCs) and neutrophils. While addition of exogenous OLFM4 blocks these processes, OLFM4 inhibition enhances neutrophil-dependent bacterial killing and DNA complex formation, ultimately leading to accelerated closure of skin wounds., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

(1) Introduction: Toll-like receptors (TLRs) are key in immune response by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In gastric cancer (GC), TLR2, TLR3, TLR4, and TLR9 are crucial for modulating immune response and tumor progression. (2) Objective: This study aimed to assess the percentage of dendritic cells and monocytes expressing TLR2, TLR3, TLR4, and TLR9, along with the concentration of their soluble forms in the serum of GC patients compared to healthy volunteers. Factors such as disease stage, tumor type, age, and gender were also analyzed. (3) Materials and Methods: Blood samples from newly diagnosed GC patients and healthy controls were immunophenotyped using flow cytometry to assess TLR expression on dendritic cell subpopulations and monocytes. Serum-soluble TLRs were measured by ELISA. Statistical analysis considered clinical variables such as tumor type, stage, age, and gender. (4) Results: TLR expression was significantly higher in GC patients, except for TLR3 on classical monocytes. Soluble forms of all TLRs were elevated in GC patients, with significant differences based on disease stage but not tumor type, except for serum TLR2, TLR4, and TLR9. (5) Conclusions: Elevated TLR expression and soluble TLR levels in GC patients suggest a role in tumor pathogenesis and progression, offering potential biomarkers and therapeutic targets.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose expression regulates immune cell differentiation. Single-cell transcriptomic profiling was used to ascertain the heterogeneity of AHR expression in human B cell subpopulations. We identified a unique population of B cells marked by expression of AHR, CD9, and myeloid genes such as CD14 and CXCL8. Results were confirmed directly in human PBMCs and purified B cells at the protein level. TLR9 signaling induced CD14, CD9, and IL-8 protein expression in CD19+ B cells. CD14-expressing CD9+ B cells also highly expressed AHR and atypical B cell markers such as CD11c and TBET. In patients with active lupus disease, CD14+ and CD9+ B cells are dysregulated, with loss of CD9+ B cells strongly predicting disease severity and demonstrating the relevance of CD9+ B cells in systemic lupus erythematosus and autoimmune disease., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Glioblastoma multiforme (GBM) remains incurable despite multimodal treatments after surgical debulking. Almost all patients with GBM relapse within a narrow margin (2-3 cm) of the initial resected lesion due to the unreachable residual cancerous cells. Here, a completely biodegradable microneedle for surgical cavity delivery glioblastoma-associated macrophages (GAMs)-activating immune nano-stimulator that mitigates glioblastoma relapse is reported. The residual tumor lesion-directed biocompatible microneedle releases the nano-stimulator and toll-like receptor 9 agonist in a controlled manner until the microneedles completely degrade over 1 week, efferently induce in situ phonotypic shifting of GAMs from anti- to pro-inflammatory and the tumor recurrence is obviously inhibited. The implantable microneedles offer a significant improvement over conventional transdermal ones, as they are 100% degradable, ensuring safe application within surgical cavities. It is also revealed that the T cells are recruited to the tumor niche as the GAMs initiate anti-tumor response and eradicate residual GBM cells. Taken together, this work provides a potential strategy for immunomodulating the postoperative tumor niche to mitigate tumor relapse in GBM patients, which may have broad applications in other malignancies with surgical intervention., (© 2024 Wiley‐VCH GmbH.)

Despite the safety profile of subunit vaccines, the inferior immunogenicity hinders their application in the nasal cavity. This study introduces a novel antigen delivery and adjuvant system utilizing mucoadhesive chitosan-catechol (Chic) on silica spiky nanoparticles (Ssp) to enhance immunity through multiple mechanisms. The Chic functionalizes the Ssp surface and incorporates with SARS-CoV-2 spike protein receptor-binding domain (RBD) and toll-like receptor (TLR)9 agonist unmethylated cytosine-guanine (CpG) motif, forming uniform virus-like nanoparticles (Ssp-Chic-RBD-CpG) via electrostatic and covalent interactions. Ssp-Chic-RBD-CpG, mimicking the morphology and function of inactive virions, effectively prolongs the retention time of RBD in the nasal mucosa by 3.92-fold compared to RBD alone, enhances the maturation of dendritic cells (DCs), and facilitates the antigen trafficking to the draining lymph nodes, which subsequently induces a stronger mucosal immunity. Mechanistically, the enhanced chemokine chemokine (C-C motif) ligand 20 (CCL20)-driven DCs recruitment and maturation by Ssp-Chic-RBD-CpG are evidenced by a cell co-culture model. In addition, the overexpression of TLR4/9 and activation of MYD88/NF-κB signaling pathway in activation of DCs are observed. Proof of principle is obtained for RBD, but similar delivery mechanisms can be applied in other protein-based subunit vaccines as well when intranasal administration is needed., (© 2024 Wiley‐VCH GmbH.)

Human cells contain two types of adenosine deaminases (ADA) each with unique properties: ADA1, which is present in all cells where it modulates intracellular functions and extracellular signaling, and ADA2, which is secreted by immune cells. The exact intracellular functions of ADA2 remain undetermined and less defined than those of ADA1. ADA2 has distinct characteristics, such as low adenosine affinity, heparin-binding ability, and putative lysosomal entry. Here, we confirm that ADA2 is a lysosomal protein that binds toll-like receptor 9 (TLR9) agonists, specifically CpG oligodeoxynucleotides (CpG ODNs). We show that interferon-alpha (IFN-α) is secreted in response to TLR9 activation by CpG ODNs and natural DNA and markedly increases when ADA2 expression is downregulated in plasmacytoid dendritic cells (pDCs). Additionally, the pretreatment of pDCs with RNA further stimulates IFN-α secretion by pDCs after activation with CpG ODNs. Our findings indicate that ADA2 regulates TLR9 responses to DNA in activated pDCs. In conclusion, decreasing ADA2 expression or blocking it with specific oligonucleotides can enhance IFN-α secretion from pDCs, improving immune responses against intracellular infections and cancer., (© 2024. Higher Education Press.)

Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.

The apoptosis-prone property of alveolar epithelial cells plays a crucial role in pulmonary fibrosis(PF), but the role of pyroptosis in it is still unclear. Toll-like receptor 9(TLR9) has been reported to play a vital role in the pathogenesis of many diseases. However, the effect of TLR9 on alveolar epithelial cells in PF has not been fully elucidated. Gene expression microarray related to Idiopathic pulmonary fibrosis(IPF) was obtained from the Gene Expression Omnibus(GEO) database. In the mouse model of bleomycin-induced PF, adeno-associated virus(AAV6) was used to interfere with TLR9 to construct TLR9 knockdown mice to study the role of TLR9 in PF, and the specific mechanism was studied by intratracheal instillation of NLR family pyrin domain containing 3(NLRP3) activator. In vitro experiments were performed using A549 cells. Bleomycin-induced pyroptosis in the lung tissue of PF mice increased, and TLR9 protein levels also increased, especially in alveolar epithelial cells. The levels of fibrosis and pyroptosis in lung tissue of TLR9 knockdown mice were improved. We found that TLR9 can bind to the NLRP3, thereby increasing the activation of the NLRP3/caspase-1 inflammasome pathway. When we use the NLRP3 activator, the levels of fibrosis and pyroptosis in lung tissue of TLR9 knockout mice can be counteracted. Pyroptosis of alveolar epithelial cells plays a vital role in PF, and TLR9 can promote NLRP3-mediated pyroptosis of alveolar epithelial cells to aggravate the progression of PF and may become a feasible target for the prevention and treatment of PF., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation., (© 2024 The Author(s). Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Background & Aims: Toll-like receptor 9 (Tlr9) is a pathogen recognition receptor detecting unmethylated DNA derivatives of pathogens and damaged host cells. It is therefore an important modulator of innate immunity. Here we investigated the role of Tlr9 in fibrogenesis and progression of hepatocellular carcinoma in chronic liver disease., Materials and Methods: We treated mice with a constitutive deletion of Tlr9 (Tlr9 -/- ) with DEN/CCl 4 for 24 weeks. As a second model, we used hepatocyte-specific Nemo knockout (Nemo Δhepa ) mice and generated double knockout (Nemo Δhepa Tlr9 -/- ) animals., Results: We show that Tlr9 is in the liver primarily expressed in Kupffer cells, suggesting a key role of Tlr9 in intercellular communication during hepatic injury. Tlr9 deletion resulted in reduced liver fibrosis as well as tumor burden. We observed down-regulation of hepatic stellate cell activation and consequently decreased collagen production in both models. Tlr9 deletion was associated with decreased apoptosis and compensatory proliferation of hepatocytes, modulating the initiation and progression of hepatocarcinogenesis. These findings were accompanied by a decrease in interferon-β and an increase in chemokines having an anti-tumoral effect., Conclusions: Our data define Tlr9 as an important receptor involved in fibrogenesis, but also in the initiation and progression of hepatocellular carcinoma during chronic liver diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Both we and others have found that RBC counts are significantly lower in older compared to younger. However, when gender is factored in, a significant age-related decrease of RBC counts is observed only in men but not in women., Methods: qPCR and confocal microscopy were used to detect the presence of mtDNA in RBCs. Flow cytometry and specific inhibitors were used to determine how RBCs uptake cf-mtDNA. The peripheral blood was collected from 202 young adults and 207 older adults and RBC and plasma were isolated. The levels of TLR9 + RBCs and apoptotic RBCs after uptake of cf-mtDNA by RBCs were measured by flow cytometry. The kit detects changes in SOD and MDA levels after cf-mtDNA uptake by RBCs. Young RBCs (YR) and old RBCs (OR) from single individuals were separated by Percoll centrifugation., Results: We found a significant decrease in RBC counts and a significant increase in the RDW with aging only in men. We also found that significantly elevated mtDNA content in RBCs was observed only in men during aging and was not found in women. Further studies demonstrated that RBCs could take up cf-mtDNA via TLR9, and the uptake of mtDNA might lead to a decrease in the RBC number and an increase in RDW due to an increase of oxidative stress., Conclusions: The RBC mtDNA content might be a potential marker of RBC aging and the elevated RBC mtDNA content might be the cause of faster senescence in males than females., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier B.V.)

In specific pathological conditions, addressing liver injury may yield favorable effects on renal function through the phenomenon of liver-kidney crosstalk. Mitochondrial DNA (mtDNA) possesses the capability to trigger downstream pathways of inflammatory cytokines, ultimately leading to immune-mediated organ damage. Consequently, understanding the intricate molecular mechanisms governing mtDNA involvement in diseases characterized by liver-kidney crosstalk is of paramount significance. This study seeks to elucidate the role of mtDNA in conditions marked by liver-kidney crosstalk. In previous clinical cases, it has been observed that patients with Trichloroethylene Hypersensitivity Syndrome (TCE-HS) who experience severe liver injury often also exhibit renal injury. In this study, patients diagnosed with trichloroethylene hypersensitivity syndrome were recruited from Shenzhen Occupational Disease Control Center. And Balb/c mice were treated with trichloroethylene. The correlation between liver and kidney injuries in patients with TCE-HS was assessed using Enzyme-Linked Immunosorbent Assay (ELISA). Alterations in mtDNA levels were examined in mouse hepatocytes, red blood cells (RBCs), and renal tubular epithelial cells utilizing immunofluorescence and PCR techniques. TCE-sensitized mice exhibited a significant increase in reactive oxygen species (ROS) and the opening of the mitochondrial permeability transition pore in hepatocytes, resulting in the release of mtDNA. Furthermore, heightened levels of mtDNA and Toll-like Receptor 9 (TLR9) expression were observed in RBCs. Additional experiments demonstrated elevated expression of TLR9 and its downstream mediator MyD88 in renal tubule epithelial cells of TCE-sensitized mice. In vitro investigations confirmed that mtDNA activates the TLR9 pathway in TCMK-1 cells. Collectively, these results suggest that mtDNA released from mitochondrial damage in hepatocytes is carried by RBCs to renal tubular epithelial cells and mediates inflammatory injury in renal tubular epithelial cells through activation of the TLR9 receptor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Immune checkpoint protein V-domain immunoglobulin suppressor of T cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. Previous mechanistic studies have indicated that VISTA impairs the toll-like receptor (TLR)-mediated activation of myeloid antigen-presenting cells, promoting the expansion of myeloid-derived suppressor cells, and suppressing tumor-reactive cytotoxic T cell function., Methods: The aim of this study was to develop a dual-action lipid nanoparticle (dual-LNP) coloaded with VISTA-specific siRNA and TLR9 agonist CpG oligonucleotide. We used three murine preclinical tumor models, melanoma YUMM1.7, melanoma B16F10, and colon carcinoma MC38 to assess the functional synergy of the two cargoes of the dual LNP and therapeutic efficacy., Results: The dual-LNP synergistically augmented antitumor immune responses and rejected large established tumors whereas LNPs containing VISTA siRNA or CpG alone were ineffective. In comparison with therapies using the soluble CpG and a VISTA-specific monoclonal antibody, the dual-LNP demonstrated superior therapeutic efficacy yet with reduced systemic inflammatory cytokine production. In three murine models, the dual-LNP treatment achieved a high cure rate. Tumor rejection was associated with influx of immune cells to tumor tissues, augmented dendritic cell activation, production of proinflammatory cytokines, and improved function of cytotoxic T cells., Conclusions: Our studies show the dual-LNP ensured codelivery of its synergistic cargoes to tumor-infiltrating myeloid cells, leading to simultaneous silencing of VISTA and stimulation of TLR9. As a result, the dual-LNP drove a highly potent antitumor immune response that rejected large aggressive tumors, thus may be a promising therapeutic platform for treating immune-cold tumors., Competing Interests: Competing interests: LLW is an inventor involved with the commercial development of VISTA with ImmuNext (Lebanon, NH). The other authors declare no potential conflicts of interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. Here we leverage the wound-induced hair neogenesis (WIHN) models in adult mice to explore the correlation between degree of damage and the healing process and outcome. The multimodal analysis, in combination with single-cell RNA sequencing help to explore the difference in wounds of gentle and heavy damage degrees, identifying the potential role of toll-like receptor 9 (TLR9) in sensing the injury and regulating the immune reaction by promoting the migration of γδT cells. The TLR9 deficient mice or wounds injected with TLR9 antagonist have greatly impaired healing and lower WIHN levels. Inhibiting the migration of γδT cells or knockout of γδT cells also suppress the wound healing and regeneration, which can't be rescued by TLR9agonist. Finally, the amphiregulin (AREG) is shown as one of most important effectors secreted by γδT cells and keratinocytes both in silicon or in the laboratory, whose expression influences WIHN levels and the expression of stem cell markers. In total, our findings reveal a previously unrecognized role for TLR9 in sensing skin injury and influencing the tissue repair and regeneration by modulation of the migration of γδT cells, and identify the TLR9-γδT cells-areg axis as new potential targets for enhancing tissue regeneration., (© 2024. The Author(s).)

Ethnopharmacological Relevance: Zhilong Huoxue Tongyu Capsule (ZL) is clinically prescribed for acute ischemic stroke (AIS). However, only a few studies have addressed the mechanisms of ZL in treating AIS., Aim of the Study: To explore the underlying mechanism of macrophage polarization and inflammation mediated by ZL, and to provide a reference for AIS treatment., Materials and Methods: Sixteen SD rats were fed with different dose of ZL (0, 0.4, 0.8, and 1.6 g/kg/d) for 4 days to prepare ZL serum. After 500 ng/mL lipopolysaccharide (LPS) stimulation, RAW264.7 cells were administrated with ZL serum. Then, experiments including ELISA, flow cytometry, real-time quantitative PCR and Western blot were performed to verify the effects of ZL on macrophage polarization and inflammation. Next, let-7i inhibitor was transfected in RAW264.7 cells when treated with LPS and ZL serum to verify the regulation of ZL on the let-7i/TLR9/MyD88 signaling pathway. Moreover, the interaction between let-7i and TLR9 was confirmed by the dual-luciferase assay., Results: ZL serum significantly decreased the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and increased the expression of IL-10 and transforming growth factor β1 (TGF-β1) of LPS stimulated-macrophages. Furthermore, ZL serum polarized macrophages toward M2, decreased the expressions of TLR9, MyD88, and iNOS, as well as increased the expressions of let-7i, CHIL3, and Arginase-1. It is worth mentioning that the effect of ZL serum is dose-dependent. However, let-7i inhibitor restored all the above effects in LPS stimulated-macrophages. In addition, TLR9 was the target of let-7i., Conclusions: ZL targeted let-7i to inhibit TLR9 expression, thereby inhibiting the activation of the TLR9/MyD88 pathway, promoting the M2 polarization, and inhibiting the development of inflammation in AIS., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, the upper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B (K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing and immunomodulatory activity were assessed by hematoxylin-eosin and AZAN staining, as well as immunohistochemistry. One week following the operation, inflammatory reactions occurred in all of the experimental groups; however, re-revascularization and newly formed hard tissue deposition were observed in the pulp chamber of all groups at week 2. A positive trend in the expression of immune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest that synthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage-TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulate the repair process and hard tissue formation. Further studies are needed to determine the effects of current immunomodulatory agents in vitro and in vivo and develop treatment strategies for dental tissue regeneration.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

Background: Dendritic cells (DCs) regulate the immune response associated with T lymphocytes, but their role in stroke remains unclear. In this study, we investigated the causal relationship between DCs and T-cell response in intracerebral hemorrhage (ICH) by focusing on TLRs (toll-like receptors) that may modulate the function of DCs., Methods: We studied the effects of TLR4, TLR2, and TLR9 on DC-mediated T-cell response and the outcomes of ICH using male C57BL/6 and CD11c-DTx (diphtheria toxin) receptor mice. We administered specific agents intraperitoneally or orally and evaluated the results using flow cytometry, real-time polymerase chain reaction, Western blotting, immunofluorescence staining, histopathology, and behavioral tests., Results: TLR4 and TLR2 activation induces DC maturation and reduces the ratio of regulatory T to T-helper 17 cells in the brain and periphery after ICH. When either of these receptors is activated, it can worsen neuroinflammation and exacerbate ICH outcomes. TLR9 also promotes DC maturation, stabilizing the number of DCs, particularly conventional DCs. TLR9 has the opposite effects on regulatory T/T-helper 17 balance, neuroinflammation, and ICH outcomes compared with TLR4 and TLR2. Upon stimulation, TLR4 and TLR9 may achieve these effects through the p38-MAPK (p38-mitogen-activated protein kinase)/MyD88 (myeloid differentiation primary response gene 88) and indoleamine 2,3-dioxygenase 1 (IDO1)/GCN2 (general control nonderepressible 2) signaling pathways, respectively. DCs act as intermediaries for TLR-mediated T-cell response., Conclusions: TLR-mediated opposing effects of DCs on T-cell response may provide novel strategies to treat ICH., Competing Interests: None.

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.

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation., (Copyright © 2024 by The American Association of Immunologists, Inc.)

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.

Chagas disease is caused by the intracellular protozoan parasite Trypanosoma cruzi . This disease affects mainly rural areas in Central and South America, where the insect vector is endemic. However, this disease has become a world health problem since migration has spread it to other continents. It is a complex disease with many reservoirs and vectors and high genetic variability. One of the host proteins involved in the pathogenesis is SLAMF1. This immune receptor acts during the infection of macrophages controlling parasite replication and thus affecting survival in mice but in a parasite strain-dependent manner. Therefore, we studied the role of SLAMF1 by quantitative proteomics in a macrophage in vitro infection and the different responses between Y and VFRA strains of Trypanosoma cruzi . We detected different significant up- or downregulated proteins involved in immune regulation processes, which are SLAMF1 and/or strain-dependent. Furthermore, independently of SLAMF1, this parasite induces different responses in macrophages to counteract the infection and kill the parasite, such as type I and II IFN responses, NLRP3 inflammasome activation, IL-18 production, TLR7 and TLR9 activation specifically with the Y strain, and IL-11 signaling specifically with the VFRA strain. These results have opened new research fields to elucidate the concrete role of SLAMF1 and discover new potential therapeutic approaches for Chagas disease.

The aberrant secretion of proinflammatory cytokines by immune cells is the principal cause of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Toll-like receptor 7 (TLR7) and TLR9, sequestered to the endosomal compartment of dendritic cells and macrophages, are closely associated with the initiation and progression of these diseases. Therefore, the development of drugs targeting dysregulated endosomal TLRs is imperative to mitigate systemic inflammation. Here, we applied the principles of computer-aided drug discovery to identify a novel low-molecular-weight compound, TLR inhibitory compound 10 (TIC10), and its potent derivative (TIC10g), which demonstrated dual inhibition of TLR7 and TLR9 signaling pathways. Compared to TIC10, TIC10g exhibited a more pronounced inhibition of the TLR7- and TLR9-mediated secretion of the proinflammatory cytokine tumor necrosis factor-α in a mouse macrophage cell line and mouse bone marrow dendritic cells in a concentration-dependent manner. While TIC10g slightly prevented TLR3 and TLR8 activation, it had no impact on cell surface TLRs (TLR1/2, TLR2/6, TLR4, or TLR5), indicating its selectivity for TLR7 and TLR9. Additionally, mechanistic studies suggested that TIC10g interfered with TLR9 activation by CpG DNA and suppressed downstream pathways by directly binding to TLR9. Western blot analysis revealed that TIC10g downregulated the phosphorylation of the p65 subunit of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated kinase, p38-MAPK, and c-Jun N-terminal kinase. These findings indicate that the novel ligand, TIC10g, is a specific dual inhibitor of endosomal TLRs (TLR7 and TLR9), disrupting MAPK- and NF-κB-mediated proinflammatory gene expression.

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8 + T cell activation. Alternatively, pDCs may also promote CD8 + T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response.

Introduction: Targeted protein degradation represents a promising therapeutic approach, while diabetic cardiomyopathy (DCM) arises as a consequence of aberrant insulin secretion and impaired glucose and lipid metabolism in the heart.., Objectives: Considering that the Toll-like receptor 9 (TLR9) signaling pathway plays a pivotal role in regulating energy metabolism, safeguarding cardiomyocytes, and influencing glucose uptake, the primary objective of this study was to investigate the impact of TLR9 on diabetic cardiomyopathy (DCM) and elucidate its underlying mechanism., Methods: Mouse model of DCM was established using intraperitoneal injection of STZ, and mice were transfected with adeno-associated virus serotype 9-TLR9 (AAV9-TLR9) to assess the role of TLR9 in DCM. To explore the mechanism of TLR9 in regulating DCM disease progression, we conducted interactome analysis and employed multiple molecular approaches., Results: Our study revealed a significant correlation between TLR9 expression and mouse DCM. TLR9 overexpression markedly mitigated cardiac dysfunction, myocardial fibrosis, oxidative stress, and apoptosis in DCM, while inflammation levels remained relatively unaffected. Mechanistically, TLR9 overexpression positively modulated mitochondrial bioenergetics and activated the AMPK-PGC1a signaling pathway. Furthermore, we identified Triad3A as an interacting protein that facilitated TLR9's proteasomal degradation through K48-linked ubiquitination. Inhibiting Triad3A expression improved cardiac function and pathological changes in DCM by enhancing TLR9 activity., Conclusions: The findings of this study highlight the critical role of TLR9 in maintaining cardiac function and mitigating pathological alterations in diabetic cardiomyopathy. Triad3A-mediated regulation of TLR9 expression and function has significant implications for understanding the pathogenesis of DCM. Targeting TLR9 and its interactions with Triad3A may hold promise for the development of novel therapeutic strategies for diabetic cardiomyopathy. Further research is warranted to fully explore the therapeutic potential of TLR9 modulation in the context of cardiovascular diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

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.

There are two known mechanisms by which natural killer (NK) cells recognize and kill diseased targets: (i) direct killing and (ii) antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated an indirect NK cell activation strategy for the enhancement of human NK cell killing function. We did this by leveraging the fact that toll-like receptor 9 (TLR9) agonism within pools of human peripheral blood mononuclear cells (PBMCs) results in a robust interferon signaling cascade that leads to NK cell activation. After TLR9 agonist stimulation, NK cells were enriched and incorporated into assays to assess their ability to kill tumor cell line targets. Notably, differential impacts of TLR9 agonism were observed-direct killing was enhanced while ADCC was not increased. To ensure that the observed differential effects were not attributable to differences between human donors, we recapitulated the observation using our Natural Killer-Simultaneous ADCC and Direct Killing Assay (NK-SADKA) that controls for human-to-human differences. Next, we observed a treatment-induced decrease in NK cell surface CD16-known to be shed by NK cells post-activation. Given the essential role of CD16 in ADCC, such shedding could account for the observed differential impact of TLR9 agonism on NK cell-mediated killing capacity., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells. Toll-like receptor 9 (TLR9) plays a role in autoimmune diseases, and B cell-specific TLR9 deficiency delays T1D development. Gut microbiota are implicated in T1D, although the relationship is complex. However, the impact of B cell-specific deficiency of TLR9 on intestinal microbiota and the impact of altered intestinal microbiota on the development of T1D are unclear., Objectives: This study investigated how gut microbiota and the intestinal barrier contribute to T1D development in B cell-specific TLR9-deficient NOD mice. Additionally, this study explored the role of microbiota in immune regulation and T1D onset., Methods: The study assessed gut permeability, gene expression related to gut barrier integrity, and gut microbiota composition. Antibiotics depleted gut microbiota, and fecal samples were transferred to germ-free mice. The study also examined IL-10 production, Breg cell differentiation, and their impact on T1D development., Results: B cell-specific TLR9-deficient NOD mice exhibited increased gut permeability and downregulated gut barrier-related gene expression. Antibiotics restored gut permeability, suggesting microbiota influence. Altered microbiota were enriched in Lachnospiraceae, known for mucin degradation. Transferring this microbiota to germ-free mice increased gut permeability and promoted IL-10-expressing Breg cells. Rag -/- mice transplanted with fecal samples from Tlr9 fl/fl Cd19 -Cre + mice showed delayed diabetes onset, indicating microbiota's impact., Conclusion: B cell-specific TLR9 deficiency alters gut microbiota, increasing gut permeability and promoting IL-10-expressing Breg cells, which delay T1D. This study uncovers a link between TLR9, gut microbiota, and immune regulation in T1D, with implications for microbiota-targeted T1D therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Huang, Peng, Wang, Wong, Wang, Wang and Wen.)

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of FDA-approved neuron-targeting gene therapies. Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex. Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers, is not restricted to a particular capsid serotype, transgene, promoter, or production facility, and cannot be explained by responses to surgery or transgene expression. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents and an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors. The AAV genome is rich in unmethylated CpG DNA, which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9), and acutely blocking TLR9 preserves dendritic complexity and AMPA receptor subunit composition in AAV-injected mice. These results reveal unexpected impacts of an immune response to the AAV genome on neuronal structure and function and identify approaches to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Promising outcomes have been observed in multiple myeloma (MM) with the use of immunotherapies, specifically chimeric antigen receptor T (CAR-T) cell therapy. However, a portion of MM patients do not respond to CAR-T therapy, and the reasons for this lack of response remain unclear. The objective of this study was to investigate the impact of miR-34a on the immunosuppressive polarization of macrophages obtained from MM patients., Methods: The levels of miR-34a and TLR9 (Toll-like receptor 9) were examined in macrophages obtained from both healthy individuals and patients with MM. ELISA was employed to investigate the cytokine profiles of the macrophage samples. Co-culture experiments were conducted to evaluate the immunomodulatory impact of MM-associated macrophages on CAR-T cells., Results: There was an observed suppressed activation of macrophages and CD4 + T lymphocytes in the blood samples of MM patients. Overexpression of miR-34a in MM-associated macrophages dampened the TLR9 expression and impaired the inflammatory polarization. In both the co-culture system and an animal model, MM-associated macrophages suppressed the activity and tumoricidal effect of CAR-T cells in a miR-34a-dependent manner., Conclusion: The findings imply that targeting the macrophage miR-34a/TLR9 axis could potentially alleviate the immunosuppression associated with CAR-T therapy in MM patients., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

The bleomycin-induced scleroderma model is a well-established and dependable method for creating a mouse model of SSc (systemic sclerosis). In the field of skin connective tissue diseases, increasing evidence from clinical and animal experiments suggests that TLRs (Toll-like receptors) play an important role in several diseases. This study aimed to determine the role of TLR7 (Toll-like receptor 7) and TLR9 (Toll-like receptor 9) in the mechanisms of immune abnormalities and fibrosis in SSc. This study used TLR7-KO mice (TLR7-knockout mice with a balb/c background) and TLR9-KO mice (TLR9-knockout mice with a balb/c background) as well as WT mice (wild-type balb/c mice). All three kinds of mice were induced by BLM (bleomycin) in a scleroderma model as the experimental group; meanwhile, WT mice treated with PBS (phosphate-buffered saline) were used as the control group. We analyzed the fibrotic phenotype and the immunological abnormality phenotype of TLR7-deficient and TLR9-deficient mice in the SSc disease model using flow cytometry, RT-PCR (reverse transcription-polymerase chain reaction), a histological examination, and IHC (immunohistochemical staining). In a mouse model of SSc disease, the deletion of TLR7 attenuated skin and lung fibrosis, while the deletion of TLR9 exacerbated skin and lung fibrosis. The deletion of TLR7 resulted in a relative decrease in the infiltration and expression of various pro-inflammatory and fibrotic cells and cytokines in the skin. On the other hand, the deletion of TLR9 resulted in a relative increase in the infiltration and expression of various pro-inflammatory and cytokine-inhibiting cells and cytokines in the skin. Under the influence of pDCs (plasmacytoid dendritic cells), the balances of Beff/Breg (IL-6 + CD19 + B cell/IL-10 + CD19 + B cell), Th17/Treg (IL-17A + CD4 + T cell/Foxp3 + CD25 + CD4 + T cell), M1/M2 (CD86 + macrophage/CD206 + macrophage), and Th1/Th2 (TNFα + CD3 + CD4 + T cell/IL-4 + CD3 + CD4 + T cell) were biased towards the suppression of inflammation and fibrosis as a result of the TLR7 deletion. Comparatively, the balance was biased towards promoting inflammation and fibrosis due to the TLR9 deletion. In the SSc model, TLR7 promoted inflammation and fibrosis progression, while TLR9 played a protective role. These results suggest that TLR7 and TLR9 play opposite roles in triggering SSc to produce immune system abnormalities and skin fibrosis.

Plasmacytoid dendritic cells (pDCs) are strongly implicated as a major source of IFN-I in systemic lupus erythematosus (SLE), triggered through TLR-mediated recognition of nucleic acids released from dying cells. However, relatively little is known about how TLR signaling and IFN-I production are regulated in pDCs. In this article, we describe a role for integrin αvβ3 in regulating TLR responses and IFN-I production by pDCs in mouse models. We show that αv and β3-knockout pDCs produce more IFN-I and inflammatory cytokines than controls when stimulated through TLR7 and TLR9 in vitro and in vivo. Increased cytokine production was associated with delayed acidification of endosomes containing TLR ligands, reduced LC3 conjugation, and increased TLR signaling. This dysregulated TLR signaling results in activation of B cells and promotes germinal center (GC) B cell and plasma cell expansion. Furthermore, in a mouse model of TLR7-driven lupus-like disease, deletion of αvβ3 from pDCs causes accelerated autoantibody production and pathology. We therefore identify a pDC-intrinsic role for αvβ3 in regulating TLR signaling and preventing activation of autoreactive B cells. Because αvβ3 serves as a receptor for apoptotic cells and cell debris, we hypothesize that this regulatory mechanism provides important contextual cues to pDCs and functions to limit responses to self-derived nucleic acids., (Copyright © 2024 by The American Association of Immunologists, Inc.)

CpG oligodeoxynucleotides (ODNs), as a novel type of adjuvant with immunomodulatory effects, are recognized by Toll-like receptors (TLRs) in Litopenaeus vannamei. In the present study, eleven LvTLRs-pCMV recombinants (rLvTLRs) were constructed to investigate the relationships between various CpG ODNs and different LvTLRs in human embryonic kidney 293T (HEK293T) cells, which was further confirmed by bio-layer interferometry (BLI) technique. The results of dual luciferase reporter assay showed that every LvTLR could activate multiple downstream genes, mainly including NF-κB, CREB, ISRE, IL-6-promoter, TNF-α-promoter and Myc, thereby inducing main signaling pathways in shrimps. Most CpG ODNs possessed affinities to more than one LvTLR, while each LvTLR could recognize multiple CpG ODNs, and the widely recognized ligands within CpG ODNs are A-class and B-class. Moreover, BLI analysis showed that CpG 2216, Cpg 2006, CpG 2143 and CpG 21425 exhibited dose-dependent affinity to the expressed TLR protein, which were consistent with the results in HEK293T cells. It suggested that the interactions of CpG ODNs with LvTLRs were indispensable for the immune regulation triggered by CpG ODNs, and these findings would lay foundations for studying the activations of LvTLRs to immune signaling pathways and shedding lights on the immune functions and mechanisms of CpG ODNs., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hepatic ischemia/reperfusion injury (IRI) remains a severe threat during liver surgery and transplantation, accounting for unfavorable clinical outcomes. Modafinil (MOD), a wakefulness-inducing compound, is increasingly disclosed to protect against IRI. However, the specific literatures covering the association between MOD and hepatic IRI are few. Here, this paper is committed to unraveling the role and response mechanism of MOD in hepatic IRI. After the establishment of hepatic IRI mice model and cell model, relevant assay kits measured the concentrations of biochemical indicators of hepatotoxicity and hematoxylin and eosin staining estimated liver morphology. Enzyme-linked immunosorbent assay, reverse-transcription quantitative polymerase chain reaction, and western blot evaluated inflammatory levels. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling assay and western blot appraised apoptosis. Western blot also analyzed the expression of Toll-like receptor 9 (TLR9)/myeloid differentiation primary response gene 88 (MyD88)/p38 signaling-associated proteins. Cell counting kit-8 method judged cell viability. MOD was discovered to mitigate liver dysfunction and morphological damage, inflammatory response, apoptosis in vivo and improve cell viability, suppress inflammatory response and apoptosis in vitro. In addition, MOD inactivated TLR9/Myd88/p38 signaling both in vitro and in vivo. Further, TLR9 elevation reversed the inhibitory role of MOD in inflammatory response and cell apoptosis in vitro. Anyway, MOD blocked TLR9/Myd88/p38 signaling to exhibit anti-inflammatory and anti-apoptotic properties in hepatic IRI., (© 2024 Wiley Periodicals LLC.)

Background: Periodontitis is one of the most common chronic oral inflammatory diseases. Over the past decade, herpes viruses, particularly Epstein-Barr virus (EBV), have been considered promising pathogenic candidates for periodontitis. However, the specific mechanism by which EBV contributes to the development of periodontitis is still unknown. This study aimed to explore the mechanism of EBV underlying the inflammatory response in human gingival fibroblasts (HGFs)., Materials and Methods: HGFs were stimulated with different concentrations of EBV (10 4 , 10 5 , 10 6 , 10 7 , and 10 8 DNA copies/mL) for 0, 8, 24, or 48 hours. The mRNA levels of interleukin (IL)-1β, tumour necrosis factor-α (TNF-α), IL-8, monocyte chemoattractant protein-1 (MCP-1), and Toll-like receptor 9 (TLR9) were measured using quantitative real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assays (ELISAs) were performed for determining the mRNA and protein levels of IL-1β, TNF-α, IL-8, and MCP-1. Real-time PCR and ELISA were performed to determine the protein levels of IL-1β, TNF-α, IL-8, and MCP-1. Activation of the TLR9/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) pathway was evaluated using western blotting., Results: The expressions of IL-1β, TNF-α, IL-8, and MCP-1 were significantly upregulated in HGFs under EBV stimulation in a concentration- and time-dependent manner. EBV promoted TLR9 and MyD88 expression and induced NF-κB transcription. On the contrary, the upregulation of these factors and the activation of NF-κB pathway were drastically inhibited by TLR9 antagonists., Conclusions: Our findings demonstrate that EBV promotes the production of inflammatory cytokines IL-1β and TNF-α and chemokines IL-8 and MCP-1 in HGFs through the TLR9/MyD88/NF-κB pathway., Competing Interests: Conflict of interest None disclosed., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs., (© 2024. The Author(s).)

Toll-like receptor 9 (TLR9) recognizes bacterial, viral and self DNA and play an important role in immunity and inflammation. However, the role of TLR9 in obesity is less well-studied. Here, we generate B-cell-specific Tlr9-deficient (Tlr9 fl/fl /Cd19Cre +/- , KO) B6 mice and model obesity using a high-fat diet. Compared with control mice, B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation, weight gain, and impaired glucose and insulin tolerance. Furthermore, the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced, and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition, gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing, we identify altered gut microbial communities including reduced Lachnospiraceae, which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9, Irf4 and Il-10 interconnecting metabolic homeostasis, with the function of B and T cells, and gut microbiota in obesity., (© 2024. The Author(s).)

Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However, FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here, we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore, using single-cell RNA sequencing, we found two subsets of FRCs (CD55 hi and CD55 lo ) in the mesenteric FALC. The CD55 hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55 lo FRCs were enriched in gene expression related to immune response. Interestingly, we found that TLR9 is dominantly expressed in the CD55 lo subset. Activation of TLR9 signaling suppressed proliferation, cytokine production, and retinoid metabolism in the CD55 lo FRC, but not CD55 hi FRC. Notably, we found that adoptive transfer of Tlr9 -/- CD55 lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9 -/- CD55 hi FRC. Furthermore, we identified CD55 hi and CD55 lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55 lo subset. Therefore, inhibition of TLR9 in the CD55 lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Jiang, Li, Huang, Jayakumar, Billiar and Deng.)