Homotypic targeting of immunomodulatory nanoparticles for enhanced peripheral and central immunity.

OBJECTIVES: Synthetic oligodeoxynucleotides (ODNs) that contain unmethylated cytosine-phosphate-guanine (CpG) motifs serve as immune adjuvants in disease treatment. However, the poor cell permeability and safety concerns limit their medical applications, and biocompatible strategies for efficient delivery of functional CpG ODNs are highly desirable. MATERIALS AND METHODS: Self-assembled, cell membrane-coated CpG nanoparticles (NP) are prepared, and their physicochemical properties are characterized. The uncoated and membrane-coated CpG NP are compared for their biocompatibility, cellular uptake kinetics, endocytic pathways, subcellular localization, and immunostimulatory activities in macrophages and microglia. RESULTS: Macrophage- or microglia-derived cell membrane camouflaging alters the endocytic pathways of CpG NP, promotes their targeted delivery to the cells with homologous membrane, ensures their endosomal localization, and enhances their immunomodulatory effects. CONCLUSIONS: We design a type of biomimetic NP consisting of self-assembled CpG NP core and cell membrane shell, and demonstrate its advantages in the modulation of peripheral and central immune cells. Our study provides a new strategy for the application of CpG ODNs.

TLR9 signalling inhibits Plasmodium liver infection by macrophage activation.

Recognition of pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs) plays a pivotal role in first-line pathogen defense. TLRs are also likely triggered during a Plasmodium infection in vivo by parasite-derived components. However, the contribution of innate responses to liver infection and to the subsequent clinical outcome of a blood infection is not well understood. To assess the potential effects of enhanced TLR-signalling on Plasmodium infection, we systematically examined the effect of agonist-primed immune responses to sporozoite inoculation in the P. berghei/C57Bl/6 murine malaria model. We could identify distinct stage-specific effects on the course of infection after stimulation with two out of four TLR-ligands tested. Priming with a TLR9 agonist induced killing of pre-erythrocytic stages in the liver that depended on macrophages and the expression of inducible nitric oxide synthase (iNOS). These factors have previously not been recognized as antigen-independent effector mechanisms against Plasmodium liver stages. Priming with TLR4 and -9 agonists also translated into blood stage-specific protection against experimental cerebral malaria (ECM). These insights are relevant to the activation of TLR signalling pathways by adjuvant systems of antimalaria vaccine strategies. The protective role of TLR4-activation against ECM might also explain some unexpected clinical effects observed with pre-erythrocytic vaccine approaches.

TLR activation, immune response and viral protection elicited in cattle by a commercial vaccine against Bovine Herpesvirus-1.

The innate and acquired immune response induced by a commercial inactivated vaccine against Bovine Herpesvirus-1 (BoHV-1) and protection conferred against the virus were analyzed in cattle. Vaccination induced high levels of BoHV-1 antibodies at 30, 60, and 90 days post-vaccination (dpv). IgG1 and IgG2 isotypes were detected at 90 dpv, as well as virus-neutralizing antibodies. An increase of anti-BoHV-1 IgG1 in nasal swabs was detected 6 days post-challenge in vaccinated animals. After viral challenge, lower virus excretion and lower clinical score were observed in vaccinated as compared to unvaccinated animals, as well as BoHV-1-specific proliferation of lymphocytes and production of IFNγ, TNFα, and IL-4. Downregulation of the expression of endosome Toll-like receptors 8-9 was detected after booster vaccination. This is the first thorough study of the immunity generated by a commercial vaccine against BoHV-1 in cattle.

Dulaglutide improves muscle function by attenuating inflammation through OPA-1-TLR-9 signaling in aged mice.

Dulaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is widely used to treat diabetes. However, its effects on muscle wasting due to aging are poorly understood. In the current study, we investigated the therapeutic potential and underlying mechanism of dulaglutide in muscle wasting in aged mice. Dulaglutide improved muscle mass and strength in aged mice. Histological analysis revealed that the cross-sectional area of the tibialis anterior (TA) in the dulaglutide-treated group was thicker than that in the vehicle group. Moreover, dulaglutide increased the shift toward middle and large-sized fibers in both young and aged mice compared to the vehicle. Dulaglutide increased myofiber type I and type IIa in young (18.5% and 8.2%) and aged (1.8% and 19.7%) mice, respectively, compared to the vehicle group. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, decreased but increased by dulaglutide in aged mice. The expression of atrophic factors such as myostatin, atrogin-1, and muscle RING-finger protein-1 was decreased in aged mice, whereas that of the myogenic factor, MyoD, was increased in both young and aged mice following dulaglutide treatment. In aged mice, optic atrophy-1 (OPA-1) protein was decreased, whereas Toll-like receptor-9 (TLR-9) and its targeting inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were elevated in the TA and quadriceps (QD) muscles. In contrast, dulaglutide administration reversed this expression pattern, thereby significantly attenuating the expression of inflammatory cytokines in aged mice. These data suggest that dulaglutide may exert beneficial effects in the treatment of muscle wasting due to aging.

Talaromyces marneffei promotes M2-like polarization of human macrophages by downregulating SOCS3 expression and activating the TLR9 pathway.

Little is known about how Talaromyces marneffei, a thermally dimorphic fungus that causes substantial morbidity and mortality in Southeast Asia, evades the human immune system. Polarization of macrophages into fungal-inhibiting M1-like and fungal-promoting M2-like types has been shown to play an important role in the innate immune response against fungal pathogens. This mechanism has not been defined for T. marneffei. Here, we demonstrated that T. marneffei promotes its survival in human macrophages by inducing them toward M2-like polarization. Our investigations of the mechanism revealed that T. marneffei infection led to SOCS3 protein degradation by inducing tyrosine phosphorylation, thereby relieving the inhibitory effect of SOCS3 on p-STAT6, a key factor for M2-like polarization. Our SOCS3-overexpression experiments showed that SOCS3 is a positive regulator of M1-like polarization and plays an important role in limiting M2-like polarization. Furthermore, we found that inhibition of the TLR9 pathway partially blocked T. marneffei-induced M2-like polarization and significantly enhanced the killing activity of macrophages against T. marneffei. Collectively, these results reveal a novel mechanism by which T. marneffei evades the immune response of human macrophages.

IRF8 Impacts Self-Renewal of Hematopoietic Stem Cells by Regulating TLR9 Signaling Pathway of Innate Immune Cells.

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

TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice.

Mice reconstituted with a human immune system (humanized mice) provide a robust model to study human immunology, vaccinology, and human infectious diseases. However, the development and function of B cells in humanized mice is impaired. B cells from humanized mice are immature and are impaired in IgM to IgG isotype switch in response to infection or vaccination. In the present study we report that Toll-like receptor 9 (TLR9) agonist CpG-B combined with CD40-targeting vaccination triggered human B cell immunoglobin class-switch from IgM+ to IgG+ B cells in humanized mice. Human B cells from mice vaccinated with CpG-B as adjuvant were more mature in phenotype and produced significant levels of both total IgG and antigen-specific IgG. We found that CpG-B treatment activated human pDCs (plasmacytoid dendritic cells) in vivo to induce interferon-alpha (IFN-α)expression in humanized mice. Pre-depletion of human pDC in vivo abrogated the adjuvant effect of CpG-B. Our results indicate that TLR9 and CD40-targeting vaccination triggers human B cell maturation and immunoglobulin class-switch in a pDC-dependent manner in humanized mice. The findings also shed light on induction of human IgG antibodies in humanized mouse models.

Functional Immunostimulating DNA Materials: The Rising Stars for Cancer Immunotherapy.

Cancer immunotherapy has risen as a promising method in clinical practice for cancer treatment and DNA-based immune intervention materials, along with DNA nanotechnology, have obtained increasing importance in this field. In this review, various immunostimulating DNA materials are introduced and the mechanisms via which they exerted an immune effect are explained. Then, representative examples in which DNA is used as the leading component for anticancer applications through immune stimulation are provided and their efficacy is evaluated. Finally, the challenges for those materials in clinical applications are discussed and suggestions for possible further research directions are also put forward.

Toll-like Receptor 9 Agonists as Adjuvants for Nanoparticle-Based Nicotine Vaccine.

Nicotine vaccine was considered a promising therapy against smoking addiction. The level of immune response that a nicotine vaccine can induce is pivotal to its efficacy. In this study, Toll-like receptor 9 agonists, namely, CpG ODN 1555 and CpG ODN 1826, were incorporated into a nanoparticle-based nicotine vaccine (NanoNicVac) to enhance its immunogenicity. The results showed that NanoNicVac containing either CpG ODN 1555 or CpG ODN 1826 could be rapidly internalized by dendritic cells. In mice trials, it was found that NanoNicVac with CpG ODN 1555 and CpG ODN 1826 induced 3.3- and 3.2-fold higher anti-nicotine antibody titer than that by the native NanoNicVac after two injections, respectively. Instead of enhancing the immunogenicity of the vaccine, however, mixtures of the two CpG ODNs were observed to exert an immune-suppressing effect on NanoNicVac. Finally, the histopathological examination on major organs of the mice immunized with the NanoNicVacs proved that NanoNicVac with either CpG ODN 1555 or CpG ODN 1826 as adjuvants did not cause detectable toxicity to the mice.

Helicobacter pylori-Induced TLR9 Activation and Injury Are Associated With the Virulence-Associated Adhesin HopQ.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma. The H. pylori cancer-associated cag pathogenicity island (cag-PAI) encodes a type IV secretion system (T4SS), which translocates microbial DNA and activates TLR9; however, most cag-PAI+-infected persons do not develop cancer and cag-PAI-independent regulators of pathogenesis, including strain-specific adhesins, remain understudied. We defined the relationships between H. pylori HopQ adhesin allelic type, gastric injury, and TLR9 activation. Type I hopQ alleles were significantly associated with magnitude of injury, cag-T4SS function, and TLR9 activation. Genetic deletion of hopQ significantly decreased H. pylori-induced TLR9 activation, implicating this adhesin in H. pylori-mediated disease.

Molecular and Structural Basis of DNA Sensors in Antiviral Innate Immunity.

DNA viruses are a source of great morbidity and mortality throughout the world by causing many diseases; thus, we need substantial knowledge regarding viral pathogenesis and the host's antiviral immune responses to devise better preventive and therapeutic strategies. The innate immune system utilizes numerous germ-line encoded receptors called pattern-recognition receptors (PRRs) to detect various pathogen-associated molecular patterns (PAMPs) such as viral nucleic acids, ultimately resulting in antiviral immune responses in the form of proinflammatory cytokines and type I interferons. The immune-stimulatory role of DNA is known for a long time; however, DNA sensing ability of the innate immune system was unraveled only recently. At present, multiple DNA sensors have been proposed, and most of them use STING as a key adaptor protein to exert antiviral immune responses. In this review, we aim to provide molecular and structural underpinnings on endosomal DNA sensor Toll-like receptor 9 (TLR9) and multiple cytosolic DNA sensors including cyclic GMP-AMP synthase (cGAS), interferon-gamma inducible 16 (IFI16), absent in melanoma 2 (AIM2), and DNA-dependent activator of IRFs (DAI) to provide new insights on their signaling mechanisms and physiological relevance. We have also addressed less well-understood DNA sensors such as DEAD-box helicase DDX41, RNA polymerase III (RNA pol III), DNA-dependent protein kinase (DNA-PK), and meiotic recombination 11 homolog A (MRE11). By comprehensive understanding of molecular and structural aspects of DNA-sensing antiviral innate immune signaling pathways, potential new targets for viral and autoimmune diseases can be identified.

Discrepant antitumor efficacies of three CpG oligodeoxynucleotide classes in monotherapy and co-therapy with PD-1 blockade.

Unmethylated CpG oligodeoxynucleotides (ODNs) activate plasmacytoid dendritic cells (pDCs) and B cells to induce humoral and cellular immunity, and are under development for the treatment of multiple cancers. However, the specific differences in antitumor effects among the three CpG ODN classes when administered as a monotherapy or in co-therapy with the anti-PD-1 antibody are unclear. We compared the immunostimulatory effects in vitro and antitumor effects in a CT26 subcutaneous mouse tumor model among the three CpG ODN classes. We found that CpG-A slightly suppressed tumor growth but possessed no synergistic antitumor effects with the anti-PD-1 antibody. CpG-B at low doses significantly inhibited tumor growth and possessed synergistic antitumor effects with the anti-PD-1 antibody. A high dose of CpG-C was required to achieve antitumor effects comparable to those of CpG-B, which was consistent with the immunostimulatory effects in B-cell proliferation and TLR9-NF-κB activation. Importantly, CpG-C in combination with anti-PD-1 antibody inhibited tumor growth more quickly and effectively than CpG-B because CpG-B significantly upregulated PD-L1 expression on multiple host immune cells to promote tumor immune escape. Moreover, co-therapy increased the infiltration of effector memory T cells. In summary, CpG-B and CpG-C with different optimal concentrations possessed strong antitumor effects, while CpG-C was more rapid and effective for co-therapy with the anti-PD-1 antibody.

In situ immunization of a TLR9 agonist virus-like particle enhances anti-PD1 therapy.

BACKGROUND: CMP-001 is a novel Toll-like receptor-9 agonist that consists of an unmethylated CpG-A motif-rich G10 oligodeoxynucleotide (ODN) encapsulated in virus-like particles. In situ vaccination of CMP-001 is believed to activate local tumor-associated plasmacytoid dendritic cells (pDCs) leading to type I interferon secretion and tumor antigen presentation to T cells and systemic antitumor T cell responses. This study is designed to investigate if CMP-001 would enhance head and neck squamous cell carcinoma (HNSCC) tumor response to anti-programmed cell death protein-1 (anti-PD-1) therapy in a human papilloma virus-positive (HPV+) tumor mouse model. METHODS: Immune cell activation in response to CMP-001±anti-Qß was performed using co-cultures of peripheral blood mononuclear cells and HPV+/HPV- HNSCC cells and then analyzed by flow cytometry. In situ vaccination with CMP-001 alone and in combination with anti-PD-1 was investigated in C57BL/6 mice-bearing mEERL HNSCC tumors and analyzed for anti-Qß development, antitumor response, survival and immune cell recruitment. The role of antitumor immune response due to CMP-001+anti-PD-1 treatment was investigated by the depletion of natural killer (NK), CD4+ T, and CD8+ T cells. RESULTS: Results showed that the activity of CMP-001 on immune cell (pDCs, monocytes, CD4+/CD8+ T cells and NK cells) activation depends on the presence of anti-Qß. A 2-week 'priming' period after subcutaneous administration of CMP-001 was required for robust anti-Qß development in mice. In situ vaccination of CMP-001 was superior to unencapsulated G10 CpG-A ODN at suppressing both injected and uninjected (distant) tumors. In situ vaccination of CMP-001 in combination with anti-PD-1 therapy induced durable tumor regression at injected and distant tumors and significantly prolonged mouse survival compared with anti-PD-1 therapy alone. The antitumor effect of CMP-001+anti-PD-1 was accompanied by increased interferon gamma (IFNγ)+ CD4+/CD8+ T cells compared with control-treated mice. The therapeutic and abscopal effect of CMP-001+ anti-PD-1 therapy was completely abrogated by CD8+ T cell depletion. CONCLUSIONS: These results demonstrate that in situ vaccination with CMP-001 can induce both local and abscopal antitumor immune responses. Additionally, the antitumor efficacy of CMP-001 combined with α-PD-1 therapy warrants further study as a novel immunotherapeutic strategy for the treatment of HNSCC.

Enhanced cellular uptake of CpG DNA by α-helical antimicrobial peptide Kn2-7: Effects on macrophage responsiveness to CpG DNA.

DNA containing unmethylated cytosine-guanine motifs (CpG DNA) initiates innate immune responses, including the secretion of cytokines from macrophages. Some antimicrobial peptides modulate the responses to CpG DNA, although the molecular mechanisms of this process remain unclear. This study examined the effects of four α-helical antimicrobial peptides on the immune responses induced by CpG DNA. The antimicrobial peptide FIKRIARLLRKIF, known as Kn2-7, increased the CpG DNA-dependent secretion of interleukin-10 (IL-10) and tumor necrosis factor-α from mouse macrophage-like RAW264.7 cells. Kn2-7 enhanced the cellular uptake of CpG DNA; this effect was decreased by the substitution of arginine residues with alanine residues, and increased by the substitution of lysine residues with arginine residues. The degree to which these peptides enhanced the cellular uptake of CpG DNA correlated well with their ability to increase CpG DNA-dependent IL-10 secretion. In contrast, Kn2-7 synthesized with d-amino acids did not increase CpG DNA-dependent IL-10 secretion, although the ability of the D-form of Kn2-7 to enhance the cellular uptake of CpG DNA was not diminished relative to that of Kn2-7. These results indicate that enhanced cellular uptake of CpG DNA is necessary but insufficient to augment CpG DNA-dependent immune responses.

The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice.

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

Overcoming Genetically Based Resistance Mechanisms to PD-1 Blockade.

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti-PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti-PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy. SIGNIFICANCE: The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy.This article is highlighted in the In This Issue feature, p. 1079.

Induction of mucosal immunity against pathogens by using recombinant baculoviral vectors: Mechanisms, advantages, and limitations.

Over 90% of pathogens of medical importance invade the organism through mucosal surfaces, which makes it urgent to develop safe and effective mucosal vaccines and mucosal immunization protocols. Besides, parenteral immunization does not provide adequate protective immunity in mucosal surfaces. Effective mucosal vaccination could protect local and systemic compartments and favor herd immunity. Although various mucosal adjuvants and Ag-delivery systems have been developed, none has filled the gap to control diseases caused by complex mucosal pathogens. Among the strategies to counteract them, recombinant virions from the baculovirus Autographa californica multiple nucleopolyhedrovirus (rAcMNPV) are useful vectors, given their safety and efficacy to produce mucosal and systemic immunity in animal infection models. Here, we review the immunogenic properties of rAcMNPV virions from the perspectives of mucosal immunology and vaccinology. Some features, which are analyzed and extrapolated from studies with different particulate antigens, include size, shape, surface molecule organization, and danger signals, all needed to break the tolerogenic responses of the mucosal immune tissues. Also, we present a condensed discussion on the immunity provided by rAcMNPV virions against influenza virus and human papillomavirus in animal models. Through the text, we highlight the advantages and limitations of this experimental immunization platform.

Synthetic High-density Lipoprotein Nanodiscs for Personalized Immunotherapy Against Gliomas.

PURPOSE: Gliomas are brain tumors with dismal prognoses. The standard-of-care treatments for gliomas include surgical resection, radiation, and temozolomide administration; however, they have been ineffective in providing significant increases in median survival. Antigen-specific cancer vaccines and immune checkpoint blockade may provide promising immunotherapeutic approaches for gliomas. EXPERIMENTAL DESIGN: We have developed immunotherapy delivery vehicles based on synthetic high-density lipoprotein (sHDL) loaded with CpG, a Toll-like receptor-9 agonist, and tumor-specific neoantigens to target gliomas and elicit immune-mediated tumor regression. RESULTS: We demonstrate that vaccination with neoantigen peptide-sHDL/CpG cocktail in combination with anti-PD-L1 immune checkpoint blocker elicits robust neoantigen-specific T-cell responses against GL261 cells and eliminated established orthotopic GL261 glioma in 33% of mice. Mice remained tumor free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of immunologic memory. Moreover, in a genetically engineered murine model of orthotopic mutant IDH1 (mIDH1) glioma, sHDL vaccination with mIDH1 neoantigen eliminated glioma in 30% of animals and significantly extended the animal survival, demonstrating the versatility of our approach in multiple glioma models. CONCLUSIONS: Overall, our strategy provides a general roadmap for combination immunotherapy against gliomas and other cancer types.

Resolution of Deep Venous Thrombosis: Proposed Immune Paradigms.

Venous thromboembolism (VTE) is a pathology encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE) associated with high morbidity and mortality. Because patients often present after a thrombus has already formed, the mechanisms that drive DVT resolution are being investigated in search of treatment. Herein, we review the current literature, including the molecular mechanisms of fibrinolysis and collagenolysis, as well as the critical cellular roles of macrophages, neutrophils, and endothelial cells. We propose two general models for the operation of the immune system in the context of venous thrombosis. In early thrombus resolution, neutrophil influx stabilizes the tissue through NETosis. Meanwhile, macrophages and intact neutrophils recognize the extracellular DNA by the TLR9 receptor and induce fibrosis, a complimentary stabilization method. At later stages of resolution, pro-inflammatory macrophages police the thrombus for pathogens, a role supported by both T-cells and mast cells. Once they verify sterility, these macrophages transform into their pro-resolving phenotype. Endothelial cells both coat the stabilized thrombus, a necessary early step, and can undergo an endothelial-mesenchymal transition, which impedes DVT resolution. Several of these interactions hold promise for future therapy.

Elevated EPSTI1 promote B cell hyperactivation through NF-κB signalling in patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by aberrant B cell hyperactivation, whose mechanism is partially understood. METHODS: We performed whole transcriptome sequencing of B cells from three pSS patients and three matched healthy controls (HC). Differentially expression genes (DEGs) were confirmed with B cells from 40 pSS patients and 40 HC by quantitative PCR and western blot. We measured the proliferation potential and immunoglobulins production of siRNA-transfected or plasmid-transfected B cells stimulated with cytosine-phosphate-guanine (CpG) or anti-IgM. We also explored Toll-like receptor 9 (TLR9) signalling to reveal the potential mechanism of B cell hyperactivation in pSS. RESULTS: We identified 77 upregulated and 32 downregulated DEGs in pSS B cells. We confirmed that epithelial stromal interaction (EPST1) expression in pSS B cells was significantly higher than that from HCs. EPSTI1-silencing B cells stimulated with CpG were less proliferated and produced lower level of IgG and IgM comparing with control B cells. EPSTI1-silencing B cells expressed lower level of p-p65 and higher level of IκBα, and B cells with overexpressed EPSTI1 showed higher level of p-p65 and lower level of IκBα. Finally, IκBα degradation inhibitor Dehydrocostus Lactone treatment attenuated p65 phosphorylation promoted by EPSTI1. CONCLUSION: Elevated EPSTI1 expression in pSS B cells promoted TLR9 signalling activation and contributed to the abnormal B cell activation, which was promoted by facilitating p65 phosphorylation and activation of NF-κB signalling via promoting IκBα degradation. EPSTI1 might be implicated in pSS pathogenesis and was a potential therapeutic target of pSS.