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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.

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.

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.)

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.

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.

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.)

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.)

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.)

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.)

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.)

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.)

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.

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.)

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.)

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.)

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.)

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.)

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.)

Cell-free DNA (cfDNA) released from dead cells could be a player in some autoimmune disorders by activating Toll-like receptor 9 (TLR9) and inducing proinflammatory cytokines. Cationic nanoparticles (cNPs) address cfDNA clearance, yet challenges persist, including toxicity, low specificity and ineffectiveness against endocytosed cfDNA. This study introduced pH-sensitive cNPs, reducing off-target effects and binding cfDNA at inflammatory sites. This unique approach inhibits the TLR9 pathway, offering a novel strategy for inflammation modulation. Synthesized cNPs, with distinct cationic moieties, exhibit varied pKa values, enhancing cfDNA binding. Comprehensive studies elucidate the mechanism, demonstrating minimal extracellular binding, enhanced endosomal DNA binding, and optimal tumor necrosis factor-α suppression. In a traumatic brain injury mice model, pH-sensitive cNPs effectively suppress inflammatory cytokines, highlighting their potential in acute inflammation regulation., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Smart nanorobots have emerged as novel drug delivery platforms in nanomedicine, potentially improving anti-cancer efficacy and reducing side effects. In this study, an intelligent tumor microenvironment-responsive nanorobot is developed that effectively delivers CpG payloads to Toll-like receptor 9 (TLR9)-positive tumors to induce autophagy-mediated cell death for immunotherapy. The nanorobots are fabricated by co-self-assembly of two amphiphilic triblock polymer peptides: one containing the matrix metallopeptidase 2 (MMP2)-cleaved GPLGVRGS motif to control the mechanical opening of the nanorobots and provide targeting capability for TLR-9-positive tumors and the other consisting of an arginine-rich GRRRDRGRS sequence that can condense nuclear acid payloads through electrostatic interactions. Using multiple tumor-bearing mouse models, it is investigated whether the intravenous injection of CpG-loaded nanorobots could effectively deliver CpG payloads to TLR-9-positive tumors and elicit anti-tumor immunity through TLR9 signaling and autophagy. Therefore, besides being a commonly used adjuvant for tumor vaccination, CpG-loaded nanorobots can effectively reprogram the tumor immunosuppressive microenvironment and suppress tumor growth and recurrence. This nanorobot-based CpG immunotherapy can be considered a feasible approach to induce anti-tumor immunity, showing great therapeutic potential for the future treatment of TLR9-positive cancers., (© 2023 Wiley‐VCH GmbH.)

We have previously shown that excessive activation of macrophage proinflammatory activity plays a key role in TCE-induced immune liver injury, but the mechanism of polarization is unclear. Recent studies have shown that TLR9 activation plays an important regulatory role in macrophage polarization. In the present study, we demonstrated that elevated levels of oxidative stress in hepatocytes mediate the release of mtDNA into the bloodstream, leading to the activation of TLR9 in macrophages to regulate macrophage polarization. In vivo experiments revealed that pretreatment with SS-31, a mitochondria-targeting antioxidant peptide, reduced the level of oxidative stress in hepatocytes, leading to a decrease in mtDNA release. Importantly, SS-31 pretreatment inhibited TLR9 activation in macrophages, suggesting that hepatocyte mtDNA may activate TLR9 in macrophages. Further studies revealed that pharmacological inhibition of TLR9 by ODN2088 partially blocked macrophage activation, suggesting that the level of macrophage activation is dependent on TLR9 activation. In vitro experiments involving the extraction of mtDNA from TCE-sensitized mice treated with RAW264.7 cells further confirmed that hepatocyte mtDNA can activate TLR9 in mouse peritoneal macrophages, leading to macrophage polarization. In summary, our study comprehensively confirmed that TLR9 activation in macrophages is dependent on mtDNA released by elevated levels of oxidative stress in hepatocytes and that TLR9 activation in macrophages plays a key role in regulating macrophage polarization. These findings reveal the mechanism of macrophage activation in TCE-induced immune liver injury and provide new perspectives and therapeutic targets for the treatment of OMDT-induced immune liver injury., 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 Authors. Published by Elsevier Inc. All rights reserved.)

Aging probably is the most complexed process in biology. It is manifested by a variety of hallmarks. These hallmarks weave a network of aging; however, each hallmark is not uniformly strong for the network. It is the weakest link determining the strengthening of the network of aging, or the maximum lifespan of an organism. Therefore, only improvement of the weakest link has the chance to increase the maximum lifespan but not others. We hypothesize that mitochondrial dysfunction is the weakest link of the network of aging. It may origin from the innate intramitochondrial immunity related to the activities of pathogen DNA recognition receptors. These receptors recognize mtDNA as the PAMP or DAMP to initiate the immune or inflammatory reactions. Evidence has shown that several of these receptors including TLR9, cGAS and IFI16 can be translocated into mitochondria. The potentially intramitochondrial presented pathogen DNA recognition receptors have the capacity to attack the exposed second structures of the mtDNA during its transcriptional or especially the replicational processes, leading to the mtDNA mutation, deletion, heteroplasmy colonization, mitochondrial dysfunction, and alterations of other hallmarks, as well as aging. Pre-consumption of the intramitochondrial presented pathogen DNA recognition receptors by medical interventions including development of mitochondrial targeted small molecule which can neutralize these receptors may retard or even reverse the aging to significantly improve the maximum lifespan of the organisms., 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. Published by Elsevier B.V.)

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS-STING (for cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N 6 -methyladenosine (m 6 A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m 6 A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m 6 A modification sites. Knockdown of METTL3, an m 6 A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m 6 A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m 6 A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m 6 A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m 6 A modification system., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in widespread applications from basic research to clinics as therapeutic agents for cancer immunotherapy, viral infection, allergic diseases and asthma since their discovery in 1995. The major factors to consider for clinical translation using CpG motifs are the protection of CpG ODNs from DNase degradation and the delivery of CpG ODNs to the Toll-like receptor-9 expressed human B-cells and plasmacytoid dendritic cells. Therefore, great efforts have been devoted to the advances of efficient delivery systems for CpG ODNs. In this review, we outline new horizons and recent developments in this field, providing a comprehensive summary of the nanoparticle-based CpG delivery systems developed to improve the efficacy of CpG-mediated immune responses, including DNA nanostructures, inorganic nanoparticles, polymer nanoparticles, metal-organic-frameworks, lipid-based nanosystems, proteins and peptides, as well as exosomes and cell membrane nanoparticles. Moreover, future challenges in the establishment of CpG delivery systems for immunotherapeutic applications are discussed. We expect that the continuously growing interest in the development of CpG-based immunotherapy will certainly fuel the excitement and stimulation in medicine research.

Toll-like receptor 9 (TLR-9) is a protein that helps our immune system identify specific DNA types. Upon detection, CpG oligodeoxynucleotides signal the immune system to generate cytokines, essential proteins that contribute to the body's defence against infectious diseases. Native phosphodiester type B CpG ODNs induce only Interleukin-6 with no effect on interferon-α. We prepared silicon quantum dots containing different surface charges, such as positive, negative, and neutral, using amine, acrylate-modified Plouronic F-127, and Plouronic F-127. Then, class B CpG ODNs are loaded on the surface of the prepared SiQDs. The uptake of ODNs varies based on the surface charge; positively charged SiQDs demonstrate higher adsorption compared to SiQDs with negative and neutral surface charges. The level of cytokine production in peripheral blood mononuclear cells was found to be associated with the surface charge of SiQDs prior to the binding of the CpG ODNs. Significantly higher levels of IL-6 and IFN-α induction were observed compared to neutral and negatively charged SiQDs loaded with CpG ODNs. This observation strongly supports the notion that the surface charge of SiQDs effectively regulates cytokine induction., (© 2024. The Author(s).)

Epstein-Barr virus (EBV) DNA is known to be shed upon reactivation of latent EBV. Based on our previous findings linking Toll-like receptor-9 (TLR9) to an EBV DNA-driven surge in IL-17A production, we aimed to examine the therapeutic potential of TLR9 inhibition in EBV DNA-exacerbated arthritis in a collagen-induced arthritis (CIA) mouse model. C57BL/6J mice were administered either collagen, EBV DNA + collagen, EBV DNA + collagen + TLR9 inhibitor, or only the TLR9 inhibitor. After 70 days, paw thicknesses, clinical scores, and gripping strength were recorded. Moreover, affected joints, footpads, and colons were histologically scored. Furthermore, the number of cells co-expressing IL-17A, IFN-γ, and FOXP3 in joint sections was determined by immunofluorescence assays. Significantly decreased paw thicknesses, clinical scores, and histological scores with a significantly increased gripping strength were observed in the group receiving EBV DNA + collagen + TLR9 inhibitor, compared to those receiving EBV DNA + collagen. Similarly, this group showed decreased IL-17A+ IFN-γ+, IL-17A+ FOXP3+, and IL-17A+ IFN-γ+ FOXP3+ foci counts in joints. We show that inhibiting TLR9 limits the exacerbation of arthritis induced by EBV DNA in a CIA mouse model, suggesting that TLR9 could be a potential therapeutic target for rheumatoid arthritis management in EBV-infected individuals.

Epstein-Barr virus (EBV), a Herpesviridae family member, is associated with an increased risk of autoimmune disease development in the host. We previously demonstrated that EBV DNA elevates levels of the pro-inflammatory cytokine IL-17A and that inhibiting Toll-like receptor (TLR) 3, 7, or 9 reduces its levels. Moreover, this DNA exacerbated colitis in a mouse model of inflammatory bowel disease (IBD). In the study at hand, we examined whether inhibition of TLR3, 7, or 9 alleviates this exacerbation. Mice were fed 1.5% dextran sulfate sodium (DSS) water and administered EBV DNA. Then, they were treated with a TLR3, 7, or 9 inhibitor or left untreated. We also assessed the additive impact of combined inhibition of all three receptors. Mice that received DSS, EBV DNA, and each inhibitor alone, or a combination of inhibitors, showed significant improvement. They also had a decrease in the numbers of the pathogenic colonic IL-17A+IFN-γ+ foci. Inhibition of all three endosomal TLR receptors offered no additive benefit over administering a single inhibitor. Therefore, inhibition of endosomal TLRs reduces EBV DNA exacerbation of mouse colitis, offering a potential approach for managing IBD patients infected with EBV.

Toll-like receptors (TLRs) have a critical role in recognizing pathogenic patterns and initiating immune responses against TB and HIV. Previously, studies described the gene expression of TLRs in patients with TB and HIV. Here, we demonstrated TLRs protein expressions and their association with clinical status and plasma markers in TB, HIV, and TB/HIV coinfection. The phenotyping of TLR2, TLR4, and TLR9 on CD14+ monocytes and their subsets were determined by multicolor flow cytometry. Host plasma biomarkers and microbial indices were measured using Luminex Multiplex assay and standard of care tools, respectively. TLR2 expression significantly enhanced in TB, slightly increased in HIV but slightly reduced in TB/HIV coinfection compared to apparently health controls (HC). On the other hand, TLR4 expression was significantly increased in TB, HIV, and TB/HIV compared to HC. Expression of TLR4 was equally enhanced on classical and intermediate monocytes while higher TLR2 expression on intermediate than classical monocytes. TLR4 had a positive correlation pattern with plasma biomarkers while TLR2 had an inverse correlation pattern. TLR4 is associated with disease severity while TLR2 is with the immune-competent status of patients. Our findings demonstrated that the pattern of TLR expression is disease as well as monocyte subset specific and distinct factors drive these differences., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2024 Wegene Tamene et al.)

Introduction: Toll-like- receptors (TLR) control important aspects of innate and adaptive immune responses. Renal cells are among the non-immune cells that express (TLR). Therefore, their activation might be implicated in renal tubulo-interstitial injury., Aim: The study aimed to compare TLR9 expression in patients with primary membranous nephropathy (MN) to patients with lupus membranous nephropathy., Methods: Kidney sections from 10 Lupus nephritis (LN) patients and ten patients with primary MN were analyzed by immunohistochemistry using anti-human TLR9 antibody., Results: Results showed that TLR9 expression was weak and exclusively tubular in primary MN patients' biopsies. There was a significant difference between LN patients' biopsies and primary MN patients' biopsies. TLR9 expression was more diffused in LN patients' specimen than in those with primary MN., Conclusion: This study focuses on molecular level pathogenesis of MN. The data suggest that the receptors TLR9 may play role in tubulointerstitial injury in the pathogenesis of LN but not primary membranous nephropathy.

Objective: This study aimed to investigate the possible role of granulin (GRN) in activating the TLR9-IFN-α pathway in renal tubular epithelial cells (RTECs) and explore clues that RTECs regulate the micro-environment of inflammatory response in lupus nephritis (LN)., Methods: Renal sections from 57 LN patients and 30 non-LN patients were sampled for histological study, and GRN overexpression RTECs were applied for cytological study., Results: In the histological study, GRN is highly expressed in LN RTECs with tubulointerstitial inflammation (TII) and well co-localized with TLR9. ROC analysis suggested a potential relationship between GRN expression in RTECs and therapeutic response. Moreover, IFN-α also highly expressed in LN RTECs with TII, and the intensity of IFN-α is positively correlated with the co-localization intensity of GRN and TLR9. In the cytological study, LN serum, especially serum from LN with TII, activates the expression of TLR9 in RTECs, and GRN engages the interaction of TLR9 to activate the expression of IFN-α in RTECs. While TLR9 inhibitors can suppress the expression of IFN-α in RTECs, the degree of inhibition is dose-dependent., Conclusion: The expression of GRN in RTECs is associated with interstitial inflammation and therapeutic response. GRN may mediate the activation of the TLR9-IFN-α pathway in RTECs and involve in the micro-environment of inflammatory response in LN., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.