TLR9 triggers MyD88-independent anti-inflammatory signaling in lupus.

Activation of Toll-like receptor 7 (TLR7) can induce lupus in mice, whereas activation of TLR9 can prevent it, even though both receptors interact with myeloid differentiation primary response gene 88 (MyD88) for downstream signaling. How TLR9 triggers anti-inflammatory responses in autoimmunity is unclear. Leibler et al. recently reported that TLR9 initiates anti-inflammatory signaling and inhibits lupus pathogenesis in a MyD88-independent but ligand-dependent manner.

Mechanisms and Effects of Activation of Innate Immunity by Mitochondrial Nucleic Acids.

In recent years, a growing number of roles have been identified for mitochondria in innate immunity. One principal mechanism is that translocation of mitochondrial nucleic acid species from the mitochondrial matrix to the cytosol and endolysosomal lumen in response to an array of microbial and non-microbial environmental stressors has been found to serve as a second messenger event in the cell signaling of the innate immune response. Thus, mitochondrial DNA and RNA have been shown to access the cytosol through several regulated mechanisms involving remodeling of the mitochondrial inner and outer membranes and to access lysosomes via vesicular transport, thereby activating cytosolic (e.g., cyclic GMP-AMP synthase [cGAS]; retinoic acid-inducible gene-I [RIG-I]-like receptors) and endolysosomal (Toll-like Receptor [TLR]7, -9) nucleic acid receptors that induce type I interferons and pro-inflammatory cytokines. In this mini-review, we discuss these molecular mechanisms of mitochondrial nucleic acid mislocalization and their roles in host defense, autoimmunity, and auto-inflammatory disorders. The emergent paradigm is one in which host-derived DNA interestingly serves as a signal amplifier in the innate immune response and also as an alarm signal for disturbances in organellar homeostasis. The apparent vast excess of mitochondria and mitochondrial DNA nucleoids per cell may thus serve to sensitize the cell response to stressors while ensuring an underlying reserve of intact mitochondria to sustain cellular metabolism. An improved understanding of these molecular mechanisms will hopefully afford future opportunities for therapeutic intervention in human disease.

An innate immune response to adeno-associated virus genomes decreases cortical dendritic complexity and disrupts synaptic transmission.

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.

Toll-like Receptor 9 Inhibition Mitigates Fibroproliferative Responses in Translational Models of Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-Like Receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA (mtDNA). OBJECTIVES: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially-available indirect inhibitors and a proprietary, selective direct small molecule inhibitor. METHODS: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. MEASUREMENTS AND MAIN RESULTS: In two independent IPF cohorts, plasma mtDNA activates TLR9 in a manner associated with the expression of MCP-1, IL-6, TNFα, and IP-10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via TGFß1 and stiff substrates, and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9 associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. CONCLUSIONS: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.

Near-infrared photoimmunotherapy as a complementary modality to in situ vaccine in a preclinical pancreatic cancer model.

Pancreatic cancer is one of the most refractory malignancies. In situ vaccines (ISV), in which intratumorally injected immunostimulatory adjuvants activate innate immunity at the tumor site, utilize tumor-derived patient-specific antigens, thereby allowing for the development of vaccines in patients themselves. Near-infrared photoimmunotherapy (NIR-PIT) is a novel therapy that selectively kills cancer cells exclusively in the NIR-irradiated region. Extending our previous research showing that ISV using the unique nanoparticulate Toll-like receptor 9 (TLR9) ligand K3-SPG induced effective antitumor immunity, here we incorporated NIR-PIT into K3-SPG-ISV so that local tumor destruction by NIR-PIT augments the antitumor effect of ISV. In the mouse model of pancreatic cancer, the combination of K3-SPG-ISV and CD44-targeting NIR-PIT showed synergistic systemic antitumor effects and enhanced anti-programmed cell death-1 (PD-1) blockade. Mechanistically, strong intratumoral upregulation of interferon-related genes and dependency on CD8+ T cells were observed, suggesting the possible role of interferon and cytotoxic T cell responses in the induction of antitumor immunity. Importantly, this combination induced immunological memory in therapeutic and neoadjuvant settings. This study represents the first attempt to integrate NIR-PIT with ISV, offering a promising new direction for cancer immunotherapy, particularly for pancreatic cancer.

Revealing of TLR-9 gene polymorphisms by qPCR HRM technique and their influence on TLR-9 serum level in acute myeloid leukemia patients: Case-control study.

Acute myeloid leukemia (AML) is one of the most common and fatal malignancies that affect adults, which can quickly become aggressive if left untreated, and leukemia cells invade the bone marrow. TLR-9 is an innate immune cell receptor sensitive to various PAMPs and encoded by the TLR-9 gene. As is often known, genetic polymorphisms in any gene can help the development of the disease, and these three polymorphisms, rs187084, rs5743836, and rs352140 of TLR-9, have been studied in many different cancer disorders. Therefore, this study aimed to discover the multiple forms of a TLR-9 gene in a sample of Iraqi AML patients. A total of 120 participants in a case-control study were enrolled in the current study. Using CBC, some hematological parameters were evaluated, and the serum level of TLR-9 was assessed using the ELISA technique. DNA was extracted directly from blood, and a high-resolution melting (HRM) analysis was then carried out. The results revealed a significant difference in some blood parameters among patients and healthy control, while WBC and lymphocytes were without an evident difference between the two groups of the current investigation. The serum concentration of TLR-9 showed an elevated level in patients (P value < 0.01). Nonetheless, this increase was not affected by the genotype patterns of polymorphisms. According to the P-value, there was a significant difference in wild genotypes of the three polymorphisms (rs187084, rs5743836, and rs352140). At the same time, the odds ratio revealed the association with the disease as a protective factor. In contrast, there was a significant difference in the heterozygous and mutant genotypes of TLR-9 polymorphisms, though the odds ratio confirmed the association with the AML as a risk factor. The results of rs352140 were compatible with H.W.E since there were no significant differences between the observed and expected values for either patients or healthy controls. In contrast, the result of rs5743836 was not consistent with the HWE. Furthermore, although it corresponds with the healthy one, the finding of rs187084 conflicted with H.W.E. in the patient group. In conclusion, High serum levels of TLR-9 in patients could act as biomarkers for AML. The TLR-9 gene polymorphisms (rs187084, rs5743836, and rs352140) have been linked to an increased risk of AML and may impact the disease progression in the Iraqi population.

An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer.

OBJECTIVES: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

Epstein-Barr Virus DNA Exacerbates Arthritis in a Mouse Model via Toll-like Receptor 9.

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.

Massively recruited sTLR9+ neutrophils in rapidly formed nodules at the site of tumor cell inoculation and their contribution to a pro-tumor microenvironment.

Neutrophils exert either pro- or anti-tumor activities. However, few studies have focused on neutrophils at the tumor initiation stage. In this study, we unexpectedly found a subcutaneous nodule in the groin areas of mice inoculated with tumor cells. The nodule was developed 24 h after the inoculation, filled with tumor cells and massively recruited neutrophils, being designated as tumor nodules. 22% of the neutrophils in tumor nodules are surface TLR9 (sTLR9) expressing neutrophils (sTLR9+ neutrophils). With tumor progression, sTLR9+ neutrophils were sustainably increased in tumor nodules/tumor tissues, reaching to 90.8% on day 13 after inoculation, with increased expression of IL-10 and decreased or no expression of TNFα. In vivo administration of CpG 5805 significantly reduced sTLR9 expression of the sTLR9+ neutrophils. The reduction of sTLR9 on neutrophils in tumor nodules contributed to the induction of an anti-tumor microenvironment conductive to the inhibition of tumor growth. Overall, the study provides insights for understanding the role of sTLR9+ neutrophils in the tumor development, especially in the early stage.

Comprehensive lipidomics of lupus-prone mice using LC-MS/MS identifies the reduction of palmitoylethanolamide that suppresses TLR9-mediated inflammation.

Lipid mediators are known to play crucial roles not only in the onset of the inflammatory response but also in the induction of resolution of inflammation. Here, we report that palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, can suppress the inflammation induced by Toll-like receptor (TLR) signaling both in vitro and in vivo. PEA was found to be significantly reduced in the serum and spleen of lupus-prone MRL/lpr mice analyzed by lipidomics. PEA suppressed pro-inflammatory cytokine production in a mouse macrophage cell line stimulated with TLR ligands such as lipopolysaccharide, peptidoglycan, poly (I:C), imiquimod, and CpG-ODN. PEA also inhibited both mRNA and protein levels of IL-6 in bone marrow-derived dendritic cells (BMDCs) and B cells stimulated with CpG-ODN. Augmentation of cell surface CD86 and CD40 on BMDCs and B cells, IgM production, and cell proliferation of B cells in response to CpG-ODN were attenuated by PEA. Moreover, PEA treatment significantly reduced mortality and serum IL-6 levels in mice injected with CpG-ODN plus D-galactosamine. Taken together, PEA ameliorates inflammation induced by TLR signaling, which could be a novel therapeutic target for inflammatory disorders.

BAX as the mediator of C-MYC sensitizes acute lymphoblastic leukemia to TLR9 agonists.

BACKGROUND: The prognosis of B-cell acute lymphoblastic leukemia (B-ALL) has improved significantly with current first-line therapy, although the recurrence of B-ALL is still a problem. Toll-like receptor 9 (TLR9) agonists have shown good safety and efficiency as immune adjuvants. Apart from their immune regulatory effect, the direct effect of TLR9 agonists on cancer cells with TLR9 expression cannot be ignored. However, the direct effect of TLR9 agonists on B-ALL remains unknown. METHODS: We discussed the relationship between TLR9 expression and the clinical characteristics of B-ALL and explored whether CpG 685 exerts direct apoptotic effect on B-ALL without inhibiting normal B-cell function. By using western blot, co-immunoprecipitation, immunofluorescence co-localization, and chromatin immunoprecipitation, we explored the mechanism of the apoptosis-inducing effect of CpG 685 in treating B-ALL cells. By exploring the mechanism of CpG 685 on B-ALL, the predictive biomarkers of the efficacy of CpG 685 in treating B-ALL were explored. These efficiencies were also confirmed in mouse model as well as clinical samples. RESULTS: High expression of TLR9 in B-ALL patients showed good prognosis. C-MYC-induced BAX activation was the key to the effect of CpG oligodeoxynucleotides against B-ALL. C-MYC overexpression promoted P53 stabilization, enhanced Bcl-2 associated X-protein (BAX) activation, and mediated transcription of the BAX gene. Moreover, combination therapy using CpG 685 and imatinib, a BCR-ABL kinase inhibitor, could reverse resistance to CpG 685 or imatinib alone by promoting BAX activation and overcoming BCR-ABL1-independent PI3K/AKT activation. CONCLUSION: TLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. CpG 685 monotherapy might be applicable to Ph- B-ALL patients with C-MYC overexpression and without BAX deletion. CpG 685 may also serve as an effective combinational therapy against Ph+ B-ALL.

Systemic lupus erythematosus and toll-like receptor 9 polymorphisms: A meta-analysis of genetic association studies.

OBJECTIVE: This study aimed to examine whether polymorphisms in toll-like receptor 9 (TLR9) contribute to vulnerability to systemic lupus erythematosus (SLE). METHODS: We searched the MEDLINE, Embase, and Web of Science databases for relevant articles. We conducted a meta-analysis to examine the association between the TLR9 rs187084, rs352139, rs352140, and rs5743836 polymorphisms and SLE risk. RESULTS: A total of 5447 patients with SLE and 6588 control participants across 26 trials from 24 articles were included. The TLR9 rs187084 T allele was significantly associated with SLE (odds ratio, 1.146; 95% confidence interval, 1.033-1.273; p = 0.010). In a meta-analysis, the TLR9 rs187084 T allele was associated with SLE in the Asian population but not in the Arab population, demonstrating the existence of ethnicity-specific effects. Using homozygote contrast and recessive models, the researchers also found that the TLR9 rs187084 T/C polymorphism was associated with SLE. The TLR9 rs352139 G allele was not associated with SLE in this meta-analysis. After accounting for ethnic differences, we found that the TLR9 rs352139 G allele was not associated with SLE in Asians and Arabs. Furthermore, homozygote contrast and dominant models found no association between the TLR9 rs352139 G/A polymorphism and SLE. TLR9 polymorphisms at rs352140 and rs5743836 were not associated with an increased risk of SLE in any genetic prediction model, including people of Asian, European, or Latin American ancestry. CONCLUSIONS: SLE susceptibility is associated with the TLR9 rs187084 polymorphism in the Asian population and the rs187084, rs352139, rs352140, and rs5743836 polymorphisms in the Asian, European, and Latin American populations, respectively.

Neoantigen Immunotherapeutic-Gel Combined with TIM-3 Blockade Effectively Restrains Orthotopic Hepatocellular Carcinoma Progression.

Herein, we integrate the Hepa1-6 liver cancer-specific neoantigen, toll-like receptor 9 agonist and stimulator of interferon genes agonist by silk-hydrogel package, and combine with TIM-3 blockade to elicit robust antitumor immunity for effectively suppressing orthotopic hepatocellular carcinoma (HCC) progression. Unlike intradermal injection of simple mixed components with short-term immune protection, the neoantigen immunotherapeutic-gels evoke long-term immune protection to achieve significant prophylactic and therapeutic activity against HCC through only one-shot administration without any side effects. Notably, the synergized immunotherapy by further combining NGC-gels with TIM-3 antibody significantly reduces regulatory T-cells and increases the IFN-γ and IL-12p70 levels in tumor tissues for promoting the infiltration of IFN-γ+CD8+T-cells and 41BB+CD8+T-cells to achieve complete remission (4/7) and prevent pulmonary metastasis in orthotopic HCC, and establish long-term memory against tumor rechallenge with remarkably longer survival time (180 days). Overall, this study provides an attractive and promising synergistic strategy for HCC immunotherapy with possible clinical translation prospects.

Genetic diversity of Toll-like receptor 9 in swamp eels (Monopterus albus).

The swamp eel, Monopterus albus, is an important aquaculture species in Asia (mainly China) whose production has seriously suffered from infectious diseases. In spite of the critical requirement for aquaculture practices, to date there is scant information on its immune defence. Here, the genetic characteristics of Toll-like receptor 9 (TLR9), which plays crucial roles in the initiation of host defence against microbial invasion, were analysed. It exhibits a striking lack of genetic variation resulting from a recent demographic bottleneck. A comparison with the homologue of M. javanensis revealed that replacement but not silent differences have nonrandomly accumulated in the coding sequences at the early stage following their split from a common ancestor. Furthermore, the replacements relevant to the type II functional divergence have mainly occurred in structural motifs mediating ligand recognition and receptor homodimerization. These results provide hints to understand the diversity-based strategy of TLR9 in the arms race against pathogens. Furthermore, the findings reported here give credence to the importance of basic immunology knowledge, especially for the key elements, in genetic engineering and breeding for disease resistance in the eel and other fishes.

Regulation of Cathelicidin Antimicrobial Peptide (CAMP) Gene Expression by TNFα and cfDNA in Adipocytes.

Understanding the complex interactions between metabolism and the immune system ("metaflammation") is crucial for the identification of key immunomodulatory factors as potential therapeutic targets in obesity and in cardiovascular diseases. Cathelicidin antimicrobial peptide (CAMP) is an important factor of innate immunity and is expressed in adipocytes. CAMP, therefore, might play a role as an adipokine in metaflammation and adipose inflammation. TNFα, cell-free nucleic acids (cfDNA), and toll-like receptor (TLR) 9 are components of the innate immune system and are functionally active in adipose tissue. The aim of the present study was to investigate the impact of TNFα and cfDNA on CAMP expression in adipocytes. Since cfDNA acts as a physiological TLR9 agonist, we additionally investigated TLR9-mediated CAMP regulation in adipocytes and adipose tissue. CAMP gene expression in murine 3T3-L1 and human SGBS adipocytes and in murine and human adipose tissues was quantified by real-time PCR. Adipocyte inflammation was induced in vitro by TNFα and cfDNA stimulation. Serum CAMP concentrations in TLR9 knockout (KO) and in wildtype mice were quantified by ELISA. In primary adipocytes of wildtype and TLR9 KO mice, CAMP gene expression was quantified by real-time PCR. CAMP gene expression was considerably increased in 3T3-L1 and SGBS adipocytes during differentiation. TNFα significantly induced CAMP gene expression in mature adipocytes, which was effectively antagonized by inhibition of PI3K signaling. Cell-free nucleic acids (cfDNA) significantly impaired CAMP gene expression, whereas synthetic agonistic and antagonistic TLR9 ligands had no effect. CAMP and TLR9 gene expression were correlated positively in murine and human subcutaneous but not in intra-abdominal/visceral adipose tissues. Male TLR9 knockout mice exhibited lower systemic CAMP concentrations than wildtype mice. CAMP gene expression levels in primary adipocytes did not significantly differ between wildtype and TLR9 KO mice. These findings suggest a regulatory role of inflammatory mediators, such as TNFα and cfDNA, in adipocytic CAMP expression as a novel putative molecular mechanism in adipose tissue innate immunity.

Fibronectin-EDA accumulates via reduced ubiquitination downstream of Toll-like receptor 9 activation in SSc-ILD fibroblasts.

Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of systemic sclerosis-associated interstitial lung disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-like receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several damage-associated molecular pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform fibronectin-extra domain A (FN-EDA) are prominent in pathological extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway.

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d-type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self-nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Variability of endogenous deoxyribonuclease activity and its pathophysiological consequences.

Deoxyribonucleases (DNases) are enzymes that cleave DNA. Some DNases are secreted outside of cells where they can cleave extracellular DNA (ecDNA). High concentration of ecDNA is associated with diseases such as sepsis, preeclampsia, and systemic lupus. DNA can be released from dying cells and is immunogenic. DNases cleave ecDNA and might prevent activation of the immune system. Low DNase activity could be disadvantageous in diseases where high amounts of ecDNA are released from dying cells. The relationship between DNase activity and ecDNA remains unknown. The lack of standard values in DNase activity makes the studies difficult to compare. This review focuses on summarizing methods for DNase activity measurements, the possible implication of decreased DNase activity in diseases, and the impact on diseases associated with a high concentration of ecDNA.

Mitochondrial DNA as a Possible Ligand for TLR9 in Irinotecan-induced Small Intestinal Mucositis.

Cancer chemotherapy and radiotherapy may result in mucositis characterized by stem cell damage and inflammation in the gastrointestinal tract. The molecular mechanisms underlying this pathology remain unknown. Based on the assumption that mitochondrial CPG-DNA (mtDNA) released and sensed by TLR9 could underlie mucositis pathology, we analyzed the mtDNA levels in sera as well as inflammatory and disease parameters in the small intestine from wild-type (WT) and TLR9-deficient mice (TLR9-/-) in an experimental model of intestinal mucositis induced by irinotecan. Additionally, we verified the ability of WT and TLR9-/- macrophages to respond to CpG-DNA in vitro. WT mice injected with irinotecan presented a progressive increase in mtDNA in the serum along with increased hematocrit, shortening of small intestine length, reduction of intestinal villus:crypt ratio and increased influx of neutrophils, which were followed by higher expression of Nlrp3 and Casp1 mRNA and increased IL-1ß levels in the ileum when compared to vehicle-injected mice. TLR9-deficient mice were protected in all these parameters when compared to WT mice. Furthermore, TLR9 was required for the production of IL-1ß and NO after macrophage stimulation with CpG-DNA. Overall, our findings show that the amount of circulating free CpG-DNA is increased upon chemotherapy and that TLR9 activation is important for NLRP3 inflammasome transcription and further IL-1ß release, playing a central role in the development of irinotecan-induced intestinal mucositis. We suggest that TLR9 antagonism may be a new therapeutic strategy for limiting irinotecan-induced intestinal inflammation.