Through Toll-like Receptors Research Articles

SARS-CoV-2 Envelope Protein Tolerizes Macrophage Response to Secondary Inflammatory Stimuli

Abstract Exposure of innate immune cells to bacteria or viruses may result in epigenetic rewiring that either enhances or weakens innate immune responses to a secondary challenge. One primary mechanism of innate immune cell function is through toll-like receptors (TLRs) that sense a variety of bacterial and viral molecules and cause immune activation. SARS-CoV-2 Envelope protein (E protein) has been shown to mediate innate immune activation, through engagement of TLR2. In this study, we determined the secretome of chemokines, cytokines and growth factors from monocytes in response to E protein stimulation and compared this response with other viral antigens and TLR agonists. Next, we stimulated monocytes with E protein followed by secondary stimulation with lipopolysaccharide (LPS) on monocyte-derived macrophages (MDM) 1 week later. Reduced expression of CCL3, CCL4, CCL5, CXCL10, TNF-A, and IL-12 upon secondary LPS or autologous E protein stimulation were statistically reflecting a more tolerant MDM state that is imprinted after E protein exposure. Finally, we demonstrated that immunizing neonatal mice with SARS-CoV-2 E protein induced proinflammatory cytokine secretion and lung tissue inflammation pathology and demonstrated a long-term impact on immune cell function and secondary response to LPS. Thus, E protein induces macrophage proinflammatory networks that lead to tissue inflammation, but also induce a more tolerant state after return to a resting state that could impact the ability to respond to secondary infection. Further understanding of the molecular pathways that drive this immune activation and tolerance could identify treatment targets to restore immunity after SARS-CoV-2 infection.

Effect of NETs/COX-2 pathway on immune microenvironment and metastasis in gastric cancer.

Neutrophil extracellular traps (NETs) are crucial in the progression of several cancers. The formation of NETs is closely related to reactive oxygen species (ROS), and the granule proteins involved in nucleosome depolymerization under the action of ROS together with the loosened DNA compose the basic structure of NETs. This study aims to investigate the specific mechanisms of NETs promoting gastric cancer metastasis in order to perfect the existing immunotherapy strategies. In this study, the cells and tumor tissues of gastric cancer were detected by immunological experiments, real-time polymerase chain reaction and cytology experiments. Besides, bioinformatics analysis was used to analyze the correlation between cyclooxygenase-2 (COX-2) and the immune microenvironment of gastric cancer, as well as its effect on immunotherapy. Examination of clinical specimens showed that NETs were deposited in tumor tissues of patients with gastric cancer and their expression was significantly correlated with tumor staging. Bioinformatics analysis showed that COX-2 was involved in gastric cancer progression and was associated with immune cell infiltration as well as immunotherapy. In vitro experiments, we demonstrated that NETs could activate COX-2 through Toll-like receptor 2 (TLR2) and thus enhance the metastatic ability of gastric cancer cells. In addition, in a liver metastasis model of nude mice we also demonstrated the critical role of NETs and COX-2 in the distant metastasis of gastric cancer. NETs can promote gastric cancer metastasis by initiating COX-2 through TLR2, and COX-2 may become a target for gastric cancer immunotherapy.

Toll-like receptor agonists enhance HIV-specific T cell response mediated by plasmacytoid dendritic cells in diverse HIV-1 disease progression phenotypes

Plasmacytoid dendritic cells (pDCs) sense viral and bacterial products through Toll-like receptor (TLR)-7 and-9 and translate this sensing into Interferon-α (IFN-α) production and T-cell activation. The understanding of the mechanisms involved in pDCs stimulation may contribute to HIV-cure immunotherapeutic strategies. The objective of the present study was to characterize the immunomodulatory effects of TLR agonist stimulations in several HIV-1 disease progression phenotypes and in non HIV-1 infected donors. pDCs, CD4 and CD8 T-cells were isolated from 450ml of whole blood from non HIV-1 infected donors, immune responders (IR), immune non responders (INR), viremic (VIR) and elite controller (EC) participants. pDCs were stimulated overnight with AT-2, CpG-A, CpG-C and GS-9620 or no stimuli. After that, pDCs were co-cultured with autologous CD4 or CD8 T-cells and with/without HIV-1 (Gag peptide pool) or SEB (Staphylococcal Enterotoxin B). Cytokine array, gene expression and deep immunophenotyping were assayed. pDCs showed an increase of activation markers levels, interferon related genes, HIV-1 restriction factors and cytokines levels after TLR stimulation in the different HIV-disease progression phenotypes. This pDC activation was prominent with CpG-C and GS-9620 and induced an increase of HIV-specific T-cell response even in VIR and INR comparable with EC. This HIV-1 specific T-cell response was associated with the upregulation of HIV-1 restriction factors and IFN-α production by pDC. These results shed light on the mechanisms associated with TLR-specific pDCs stimulation associated with the induction of a T-cell mediated antiviral response which is essential for HIV-1 eradication strategies. This work was supported by Gilead fellowship program, the Instituto de Salud Carlos III (Fondo Europeo de Desarrollo Regional, FEDER, "a way to make Europe") and the Red Temática de Investigación Cooperativa en SIDA and by the Spanish National Research Council (CSIC).

Macrophage metabolic rewiring improves heme-suppressed efferocytosis and tissue damage in sickle cell disease

AbstractSickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory condition, which is contributed by heme-activated proinflammatory macrophages. Although previous studies addressed heme ability to stimulate macrophage inflammatory skewing through Toll-like receptor4 (TLR4)/reactive oxygen species signaling, how heme alters cell functional properties remains unexplored. Macrophage-mediated immune cell recruitment and apoptotic cell (AC) clearance are relevant in the context of SCD, in which tissue damage, cell apoptosis, and inflammation occur owing to vaso-occlusive episodes, hypoxia, and ischemic injury. Here we show that heme strongly alters macrophage functional response to AC damage by exacerbating immune cell recruitment and impairing cell efferocytic capacity. In SCD, heme-driven excessive leukocyte influx and defective efferocytosis contribute to exacerbated tissue damage and sustained inflammation. Mechanistically, these events depend on heme-mediated activation of TLR4 signaling and suppression of the transcription factor proliferator-activated receptor γ (PPARγ) and its coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). These changes reduce efferocytic receptor expression and promote mitochondrial remodeling, resulting in a coordinated functional and metabolic reprogramming of macrophages. Overall, this results in limited AC engulfment, impaired metabolic shift to mitochondrial fatty acid β-oxidation, and, ultimately, reduced secretion of the antiinflammatory cytokines interleukin-4 (IL-4) and IL-10, with consequent inhibition of continual efferocytosis, resolution of inflammation, and tissue repair. We further demonstrate that impaired phagocytic capacity is recapitulated by macrophage exposure to plasma of patients with SCD and improved by hemopexin-mediated heme scavenging, PPARγ agonists, or IL-4 exposure through functional and metabolic macrophage rewiring. Our data indicate that therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PGC1α/PPARγ modulation significantly ameliorates tissue damage associated with SCD pathophysiology.

A DNA/DMXAA/Metal-Organic Framework Activator of Innate Immunity for Boosting Anticancer Immunity.

Immunotherapy has achieved revolutionary success in clinics, but it remains challenging for treating hepatocellular carcinoma (HCC) characterized by high vascularization. Here, it is reported that metal-organic framework-801 (MOF-801) can be employed as a stimulator of interferon genes (STING) through Toll-like receptor 4 (TLR4) not just as a drug delivery carrier. Notably, cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) and 5, 6-dimethylxanthenone-4-acetic acid (DMXAA) STING agonist with vascular disrupting function coordinates with MOF-801 to self-assemble into a nanoparticle (MOF-CpG-DMXAA) that effectively delivers CpG ODNs and DMXAA to cells for synergistically improving the tumor microenvironment by reprogramming tumor-associated macrophages (TAMs), promoting dendritic cells (DCs) maturation, as well as destroying tumor blood vessels. In HCC-bearing mouse models, it is demonstrated that MOF-CpG-DMXAA triggers systemic immune activation and stimulates robust tumoricidal immunity, resulting in a superior immunotherapeutic efficiency in orthotopic and recurrent HCC.

I329L: A Dual Action Viral Antagonist of TLR Activation Encoded by the African Swine Fever Virus (ASFV).

The African Swine Fever Virus (ASFV) is an economically important, large DNA virus which causes a highly contagious and frequently fatal disease in domestic pigs. Due to the acute nature of the infection and the complexity of the protective porcine anti-ASFV response, there is no accepted vaccine in use. As resistance to ASFV is known to correlate with a robust IFN response, the virus is predicted to have evolved strategies to inhibit innate immunity by modulating the IFN response. The deletion of virus host evasion gene(s) inhibiting IFN is a logical solution to develop an attenuated virus vaccine. One such candidate, the ASFV ORF I329L gene, is highly conserved in pathogenic and non-pathogenic virus isolates and in this study we confirm and extend the conclusion that it has evolved for the inhibition of innate immunity initiated through Toll-like receptors (TLRs). Specifically, the ASFV I329L extracellular (ECD) and intracellular (ICD) domains inhibit TLR signalling by two entirely different mechanisms. Bioinformatics modelling suggests that the ECD inhibits several TLR signalling pathways through a short sequence homologous to the conserved TLR dimerization domain, here termed the putative dimerization domain (PDD). Remarkably, both full length and PDD constructs of I329L were demonstrated to inhibit activation, not only of TLR3, but also TLR4, TLR5, TLR8 and TLR9. Additionally, the demonstration of a weak association of I329L with TLR3 is consistent with the formation of a non-signalling I329L-TLR3 heterodimer, perhaps mediated through the PDD of I329L. Finally, the ICD associates with TRIF, thereby impacting on both TLR3 and TLR4 signalling. Thus, I329L offers potential as a general inhibitor of TLR responses and is a rational candidate for construction and testing of an I329L deletion mutant vaccine.

Neutrophil Extracellular Traps and Their Possible Implications in Ocular Herpes Infection.

Neutrophil extracellular traps (NETs) are net-like structures released from neutrophils. NETs predominantly contain cell-free deoxyribonucleic acid (DNA) decorated with histones and neutrophil granule proteins. Numerous extrinsic and intrinsic stimuli can induce the formation of NETs such as pathogens, cytokines, immune complexes, microcrystals, antibodies, and other physiological stimuli. The mechanism of NETosis induction can either be ROS-dependent or independent based on the catalase producing activity of the pathogen. NADPH is the source of ROS production, which in turn depends on the upregulation of Ca2+ production in the cytoplasm. ROS-independent induction of NETosis is regulated through toll-like receptors (TLRs). Besides capturing and eliminating pathogens, NETs also aggravate the inflammatory response and thus act as a double-edged sword. Currently, there are growing reports of NETosis induction during bacterial and fungal ocular infections leading to different pathologies, but there is no direct report suggesting its role during herpes simplex virus (HSV) infection. There are innumerable independent reports showing that the major effectors of NETosis are also directly affected by HSV infection, and thus, there is a strong possibility that HSV interacts with these facilitators that can either result in virally mediated modulation of NETosis or NETosis-mediated suppression of ocular HSV infection. This review focuses on the mechanism of NETs formation during different ocular pathologies, with its prime focus on highlighting their potential implications during HSV ocular infections and acting as prospective targets for the treatment of ocular diseases.

Selenomonas sputigena Interactions with Gingival Epithelial Cells That Promote Inflammation.

Increased prevalence and abundance of Selenomonas sputigena have been associated with periodontitis, a chronic inflammatory disease of tooth-supporting tissues, for more than 50 years. Over the past decade, molecular surveys of periodontal disease using 16S and shotgun metagenomic sequencing approaches have confirmed the disease association of classically recognized periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia while highlighting previously underappreciated organisms such as Filifactor alocis and S. sputigena. Despite abundant clinical association between S. sputigena and periodontal disease, we have little to no understanding of its pathogenic potential, and virulence mechanisms have not been studied. In this study, we sought to characterize the response of gingival epithelial cells to infection with S. sputigena. Here, we show that S. sputigena attaches to gingival keratinocytes and induces expression and secretion of cytokines and chemokines associated with inflammation and leukocyte recruitment. We demonstrate that S. sputigena induces signaling through Toll-like receptor 2 (TLR2) and TLR4 but evades activation of TLR5. Cytokines released from S. sputigena-infected keratinocytes induced monocyte and neutrophil chemotaxis. These results show that S. sputigena-host interactions have the potential to contribute to bacterially driven inflammation and tissue destruction, the hallmark of periodontitis. Characterization of previously unstudied pathogens may provide novel approaches to develop therapeutics to treat or prevent periodontal disease.

Past history of obesity triggers persistent epigenetic changes in innate immunity and exacerbates neuroinflammation.

Age-related macular degeneration is a prevalent neuroinflammatory condition and a major cause of blindness driven by genetic and environmental factors such as obesity. In diseases of aging, modifiable factors can be compounded over the life span. We report that diet-induced obesity earlier in life triggers persistent reprogramming of the innate immune system, lasting long after normalization of metabolic abnormalities. Stearic acid, acting through Toll-like receptor 4 (TLR4), is sufficient to remodel chromatin landscapes and selectively enhance accessibility at binding sites for activator protein-1 (AP-1). Myeloid cells show less oxidative phosphorylation and shift to glycolysis, ultimately leading to proinflammatory cytokine transcription, aggravation of pathological retinal angiogenesis, and neuronal degeneration associated with loss of visual function. Thus, a past history of obesity reprograms mononuclear phagocytes and predisposes to neuroinflammation.

Ruxolitinib does not completely abrogate the functional capabilities of TLR4/9 ligand-activated NK cells.

Natural killer (NK) cells are lymphocytes from the innate immune system part of the first defense barrier against infected and transformed cells, representing 5%-15% of peripheral blood lymphocytes. The cytotoxic capacity of NK cells is controlled by a balance between inhibitory and activating NK receptors expressed on their surface, which recognize and interact with the ligands on stressed cells. The cytokines involved in NK cell activation, proliferation, survival, and cytotoxicity are signaled mainly through the Janus kinase and signal transducer and activator of transcription proteins (JAK/STAT) pathway. NK cells are also activated in response to pathogens through Toll-like receptors (TLRs) expressed on their surface. Ruxolitinib is a specific JAK1/2 inhibitor approved for treating myelofibrosis and for steroid-refractory acute and chronic graft-versus-host disease (SR-GvHD). Purified NK cells from healthy donors were stimulated with two TOLL-like receptor ligands, LPS and CpG, in the presence of different concentrations of Ruxolitinib. This study showed the effects of ruxolitinib on TLR4 and TLR9 ligand-activated NK cells from healthy donors. Ruxolitinib did not completely inhibit STAT3 phosphorylation and had a moderate effect on NK cell cytokine activation via the TLR pathway. Only the highest doses of ruxolitinib led to a decrease in the pro-inflammatory cytokines tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-1β. The cytotoxic capacity of stimulated NK cells versus K562, SEM, and MV-4-11 cell lines was reduced by increasing doses of ruxolitinib, but it was not completely abolished and we observed no major changes in degranulation capacity. Phenotypic changes were observed in activated NK cells in the presence of ruxolitinib. In a small cohort of pediatric patients treated with ruxolitinib for SR-GvHD, we observed no decrease in NK cell counts; however, further prospective studies with larger cohorts are necessary to confirm this finding. In summary, our results showed that the functional capabilities and phenotype of NK cells activated through TLR4/9 agonists were not completely abolished by the inhibition of the JAK-STAT pathway by ruxolitinib.

Microglia regulate neuronal and behavioural functions under physiological and pathological conditions.

Microglia are immune cells in the central nervous system that engulf unnecessary synapses during development. In vivo imaging has substantially improved in recent years, besides the development of tools for manipulating microglia and neurons. These techniques reveal the novel functions of microglia. Microglia regulate neuronal activity to prevent synchronization. This neuron-microglia interaction is mediated by adenosine triphosphate-P2Y12 and adenosine-adenosine A1 receptor signalling in the striatum. Moreover, microglia release inflammation-related molecules that suppress neuronal activity, thus leading to lipopolysaccharide-induced aversion. Prostaglandin E2 (PGE2)-PGE receptor 1 signalling in the striatum underlies this behavioural alteration. Chronic stress activates microglia through toll-like receptor (TLR) 2 and TLR4 to release pro-inflammatory cytokines in the medial prefrontal cortex, thereby causing social avoidance. Microglia play multiple functions under physiological conditions, as well as pathological and psychological stress.

Potential effects of hydroxysafflor yellow A on reducing pulmonary inflammation and fibrosis due to SARS-COV2

Cytokine storm is a condition that is characterized by a massive production of proinflammatory cytokines. Failure in balancing the up-regulation and down-regulation causes excessive production of proinflammatory cytokines in the fight against SARS-CoV2 virus infection, leading to lung damage and acute respiratory distress syndrome; in addition, high levels of IL-6 can activate the clotting pathways and vascular endothelial cells, which can inhibit blood circulation and heart muscle function and cause pulmonary, kidney, and liver fibrosis. Hydroxysafflor Yellow A (HSYA) is a compound that has been shown to reduce tissue lung damage through Toll-Like Receptor (TLR) 4, inhibits phosphorylation of the NF-κB pathway, and plays a role in balancing the up-regulation and down-regulation of inflammatory cytokines. This review of literature discusses the ability of HSYA to reduce inflammation that causes pulmonary cell and tissue damage. HSYA can inhibit the activation of the NF-κB signaling pathway and suppress the binding of the TGF-β1 promoter. This molecular mechanism can reduce lung damage by attenuating the inflammatory response by inhibiting the TLR 4-dependent pathways that can improve the condition of mice affected by pulmonary fibrosis, including inflammation that leads to vascular tissue repair. The molecular mechanism of HSYA can inhibit inflammatory mechanisms in lung injury, vascular tissue damage, and liver and kidney fibrosis. Therefore, this literature review can be used as a reference for in vivo research and clinical trials for further research on the ability to heal patients with cytokine storm that causes cardiovascular tissue damage and lung injury in patients infected with SARS-CoV-19.

Cisplatin Toxicity Is Mediated by Direct Binding to Toll-Like Receptor 4 through a Mechanism That Is Distinct from Metal Allergens.

Cisplatin is an effective chemotherapeutic agent, yet its use is limited by several adverse drug reactions, known as cisplatin-induced toxicities (CITs). We recently demonstrated that cisplatin could elicit proinflammatory responses associated with CITs through Toll-like receptor 4 (TLR4). TLR4 is best recognized for binding bacterial lipopolysaccharide (LPS) via its coreceptor, MD-2. TLR4 is also proposed to directly bind transition metals, such as nickel. Little is known about the nature of the cisplatin-TLR4 interaction. Here, we show that soluble TLR4 was capable of blocking cisplatin-induced, but not LPS-induced, TLR4 activation. Cisplatin and nickel, but not LPS, were able to directly bind soluble TLR4 in a microscale thermophoresis binding assay. Interestingly, TLR4 histidine variants that abolish nickel binding reduced, but did not eliminate, cisplatin-induced TLR4 activation. This was corroborated by binding data that showed cisplatin, but not nickel, could directly bind mouse TLR4 that lacks these histidine residues. Altogether, our findings suggest that TLR4 can directly bind cisplatin in a manner that is enhanced by, but not dependent on, histidine residues that facilitate binding to transition metals. SIGNIFICANCE STATEMENT: This work describes how the xenobiotic cisplatin interacts with Toll-like receptor 4 (TLR4) to initiate proinflammatory signaling that underlies cisplatin toxicities, which are severe adverse outcomes in cisplatin treatment. Here, this study provides a mechanistic bridge between cisplatin extracellular interactions with TLR4 and previous observations that genetic and chemical inhibition of TLR4 mitigates cisplatin-induced toxicity.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

Innate Immune System Activation, Inflammation and Corneal Wound Healing.

Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

Formulated Phospholipids as Non-Canonical TLR4 Agonists.

Immunogenic agents known as adjuvants play a critical role in many vaccine formulations. Adjuvants often signal through Toll-like receptor (TLR) pathways, including formulations in licensed vaccines that target TLR4. While TLR4 is predominantly known for responding to lipopolysaccharide (LPS), a component of Gram-negative bacterial membranes, it has been shown to be a receptor for a number of molecular structures, including phospholipids. Therefore, phospholipid-based pharmaceutical formulations might have off-target effects by signaling through TLR4, confounding interpretation of pharmaceutical bioactivity. In this study we examined the individual components of a clinical stage oil-in-water vaccine adjuvant emulsion (referred to as a stable emulsion or SE) and their ability to signal through murine and human TLR4s. We found that the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) activated TLR4 and elicited many of the same immune phenotypes as canonical TLR4 agonists. This pathway was dependent on the saturation, size, and headgroup of the phospholipid. Interestingly, DMPC effects on human cells were evident but overall appeared less impactful than emulsion oil composition. Considering the prevalence of DMPC and other phospholipids used across the pharmaceutical space, these findings may contextualize off-target innate immune responses that could impact preclinical and clinical development.

Abstract B014: TLR signaling coordinates SAA-dependent liver metastasis in pancreatic cancer

Abstract Metastasis remains the primary cause of mortality in cancer. This is especially true in pancreatic ductal adenocarcinoma (PDAC) - where the liver is the most common site of metastasis and the 5-year overall survival rate for these patients has remained <3% for the past 20 years. Accumulating evidence shows a critical role for a niche microenvironment in the liver, termed the pro-metastatic niche, that supports metastatic seeding and outgrowth. Our previous work demonstrated that hepatocytes orchestrate the formation of this pro-metastatic niche during early tumorigenesis through the release of Serum Amyloid A1 and 2 (SAA). However, the mechanisms by which SAA directs formation of a pro-metastatic niche in the liver, and the impact of this niche on the fate of disseminating tumor cells (DTCs) seeding the liver, remain ill-defined. To begin to identify the cellular determinants of niche formation, we first analyzed the expression of SAA receptors within the normal liver and found predominant expression of receptors on the resident F4/80+ macrophages. This is in line with current published work as SAA is well characterized to control liver biology through toll-like receptors (TLRs) expressed by liver resident immune cells and hepatic stellate cells. Consistent with this, we also found that SAA is a specific, potent agonist of TLR2 in vitro. In tumor bearing mice we then found increased expression of TLR2 transcripts as well as an accumulation of TLR2+ inflammatory monocytes within the niche. Together, these findings suggested that SAA might form a pro-metastatic niche in the liver withough a TLR2-dependent mechanism. To test this, we next examined the livers of TLR2-/- tumor bearing mice and found a decrease in the expression of surrogate markers of the pro-metastatic niche. We also sought to determine a role for TLR2 in metastatic outgrowth. Using TLR2-/- mice in a model of PDAC with direct metastasis to the liver, we found significantly decreased metastatic burden and outgrowth in the liver. Analysis of micro-metastatic lesions in TLR2-/- tumor bearing mice also revealed that DTCs demonstrated molecular markers of cellular dormancy. Altogether, our findings suggest the evolution of an inflammatory mechanism within the niche wherein SAA may continue to signal through TLR2+ inflammatory monocytes, which then act to coordinate metastatic outgrowth. Future work aims to further elucidate the role of TLR2+ resident macrophages and monocytes in the formation of a pro-metastatic niche in the liver and in regulating metastatic outgrowth. Citation Format: Jesse Lee, Meredith L. Stone, Hana Choi, Gregory L. Beatty. TLR signaling coordinates SAA-dependent liver metastasis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B014.

Abstract 11904: Lipoprotein(a) Integrates Monocyte-Mediated Thrombosis and Inflammation in Atherosclerotic Cardiovascular Disease

Introduction: Lipoprotein (a) [Lp(a)] drives atherosclerotic cardiovascular disease (ASCVD) through multiple mechanisms including cholesterol accumulation, inflammation, and thrombosis. However, the effects of Lp(a) on monocyte activation and monocyte-mediated thrombosis are unknown. Hypothesis: Lp(a) itself can activate monocytes, driving inflammation and thrombosis in parallel. Methods: We employed systems biology approaches consisting of proteomics, transcriptomics, and mass cytometry to define the immune cellular and molecular phenotypes in ASCVD subjects with high and low Lp(a) levels and leveraged cell models to define the mechanisms through which Lp(a) drives monocyte inflammation and thrombosis. We included study participants with CHD who were aged 18 to 80 years with Lp(a) concentrations > 150 nmol/L (cases) and <75 nmol/L (controls). Results: Circulating markers of inflammation (CCL3, CD40) and vascular dysfunction (PAR1, tissue factor [TF]) were elevated in subjects with high Lp(a) levels compared with those with low Lp(a) levels. Additionally, although total monocytes levels and hs-CRP levels were similar between the groups, CD14+ monocytes from ASCVD subjects with an elevated Lp(a) were primed and expressed more TF at baseline and in response to stress. Mechanistically, we found that Lp(a) itself can activate monocytes through Toll-like receptors (TLR) and nuclear factor kappa B (NFκB) signaling, driving both TF expression and TF activity (Figure). Conclusions: Overall, these studies are the first to link Lp(a) to monocyte-mediated inflammation and thrombosis in ASCVD subjects, demonstrating a novel mechanism through TLR2, NFκB, and monocyte TF through which Lp(a) amplifies immunothrombotic risk.

Chemical inhibition of TRAF6-TAK1 axis as therapeutic strategy of endotoxin-induced liver disease

The liver is exposed to gut-derived bacterial endotoxin via portal circulation, and recognizes it through toll-like receptor 4 (TLR4). Endotoxin lipopolysaccharide (LPS) stimulates the self-ubiquitination of ubiquitin ligase TRAF6, which is linked to scaffold with protein kinase TAK1 for auto-phosphorylation and subsequent activation. TAK1 activity is a signal transducer in the activating pathways of transcription factors NF-κB and AP-1 for production of various cytokines. Here, we hypothesized that TRAF6-TAK1 axis would be implicated in endotoxin-induced liver disease. Following exposure to endotoxin LPS, TLR4-mediated phosphorylation of TAK1 and transcription of cell-death cytokine TNF-α were triggered in Kupffer cells but not in hepatocytes as well as TNF receptor-mediated and caspase-3-executed apoptosis was occurred in D-galactosamine (GalN)-sensitized hepatocytes under co-culture with Kupffer cells. Treatment with pyridinylmethylene benzothiophene (PMBT) improved endotoxin LPS-induced hepatocyte apoptosis in GalN-sensitized C57BL/6 mice via suppressing NF-κB- and AP-1-regulated expression of TNF-α in Kupffer cells, and rescued the mice from hepatic damage-associated bleeding and death. As a mechanism, PMBT directly inhibited Lys 63-linked ubiquitination of TRAF6, and mitigated scaffold assembly between TRAF6 and the TAK1-activator adaptors TAB1 and TAB2 complex in Kupffer cells. Thereby, PMBT interrupted TRAF6 ubiquitination-induced activation of TAK1 activity in the TLR4-mediated signal cascade leading to TNF-α production. However, PMBT did not directly affect the apoptotic activity of TNF-α on GalN-sensitized hepatocytes. Finally, we propose chemical inhibition of TRAF6-TAK1 axis in Kupffer cells as a strategy for treating liver disease due to gut-derived endotoxin or Gram-negative bacterial infection.

Inhibition of MyD88 attenuates angiotensin II-induced hypertensive kidney disease via regulating renal inflammation

BackgroundKidney damage is a frequent event in the course of hypertension. Recent researches highlighted a critical role of non-hemodynamic activities of angiotensin II (Ang II) in hypertension-associated kidney fibrosis and inflammation. These activities are mediated through toll-like receptors (TLRs) but the mechanisms by which Ang II links TLRs to downstream inflammatory and fibrogenic responses is not fully known. In this study, we investigated the role of TLR adapter protein called myeloid differentiation primary-response protein-88 (MyD88) as the potential link. MethodsC57BL/6 mice were administered Ang II by micro-osmotic pump infusion for 4 weeks to develop nephropathy. Mice were treated with small-molecule MyD88 inhibitor LM8. In vitro, MyD88 was blocked using siRNA or LM8 in Ang II-challenged renal tubular epithelial cells. ResultsWe show that MyD88 is mainly located in tubular epithelial cells and Ang II increases the interaction between TLR4 and MyD88. This interaction activates MAPKs and nuclear factor-κB (NF-κB), leading to increased production of inflammatory and fibrogenic factors. Inhibition of MyD88 by siRNA or selective inhibitor LM8 supresses MyD88-TLR4 interaction, NF-κB activation, and elaboration of inflammatory cytokines and fibrosis-associated factors. These protective actions resulted in decreased renal pathological changes and preserved renal function in LM8-treated hypertensive mice, without affecting hypertension. ConclusionThese results demonstrate that Ang II induces inflammation and fibrosis in renal tubular epithelial cells through MyD88 and present MyD88 as a potential point of intervention for hypertension-associated kidney disease.