Monocyte Activation Research Articles

The Effect of Orally Administered Multi-Strain Probiotic Formulation (Lactobacillus, Bifidobacterium) on the Phagocytic Activity and Oxidative Metabolism of Peripheral Blood Granulocytes and Monocytes in Lambs

Probiotic feed additives have attracted considerable research interest in recent years because the effectiveness of probiotics can differ across microbial strains and the supplemented macroorganisms. The present study was conducted on 16 lambs divided equally into two groups (C—control and E—experimental). The examined lambs were aged 11 days at the beginning of the experiment and 40 days at the end of the experiment. The diet of group E lambs was supplemented with a multi-strain probiotic formulation (Lactobacillus plantarum AMT14, Lactobacillus plantarum AMT4, Lactobacillus rhamnosus AMT15, and Bifidobacterium animalis AMT30), whereas group C lambs did not receive the probiotic additive. At the beginning of the experiment (day 0) and on experimental days 15 and 30, blood was sampled from the jugular vein to determine and compare: phagocytic activity (Phagotest) and oxidative metabolism (Phagoburst) of peripheral blood granulocytes and monocytes by flow cytometry. An analysis of the phagocytic activity of granulocytes and monocytes revealed significantly higher levels of phagocytic activity (expressed as the percentage of phagocytic cells and mean fluorescence intensity) in lambs that were administered the multi-strain probiotic formulation compared with lambs in the control group. The probiotic feed additive also exerted a positive effect on the oxidative metabolism of both granulocytes and monocytes (expressed as the percentage of oxidative metabolism and mean fluorescence intensity) after stimulation with Escherichia coli bacteria and with PMA (4-phorbol-12-β-myristate-13-acetate). These findings suggest that the tested probiotic formulation may have a positive effect on the immune status of lambs.

High levels of soluble P-selectin, neutrophil extracellular traps, and myeloperoxidase as risk factor of deep vein thrombosis in malignancy patients receiving platinum-based chemotherapy

Backgrounds Venous Thromboembolism (VTE) is a disease entity comprising Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE). VTE events increase the mortality rate of patients with cancer receiving platinum-based chemotherapy. Soluble P-Selectin, Neutrophil Extracellular Traps (NET), and myeloperoxidase (MPO) are risk factors associated with DVT in malignancy patients receiving platinum-based chemotherapy. The purpose of this study was to determine the role of soluble P-selectin, NET, and MPO as risk factors for DVT in patients with malignancy receiving platinum-based chemotherapy. Patients and Methods This study used a case-control design (matched pair case-control study) based on age and gender. The case group consisted of subjects with DVT, whereas the control group consisted of subjects without DVT. The subjects were 31 in each case and control groups. Soluble P-selectin, NET, and MPO levels were measured in each group. Results The mean age of case group was 50.26±12.15 years meanwhile in control group was 52.81±11.64 years. In the case group, 71% of the subjects were female, whereas 51.6% of the control group were male. Most subjects, either in the case group (71%) or the control group (71%), used carboplatin. In the case group, cervix malignancy was the most common malignancy (32.3%), whereas in the control group, it was nasopharyngeal malignancy (25.8%). High soluble P-selectin level was a risk factor for DVT (OR 3.38, CI 1.180 – 9.780, p=0.02). A high NET level was also a risk factor for DVT (OR 2.88, CI 1.026-8.074, p=0.04). The high MPO levels in this study could not be proven as a risk factor. Conclusions Soluble P-selectin and NET are risk factors that play a role in the pathophysiology of DVT through the pathomechanism of immunothrombosis induced by endothelial injury and activation of monocytes and neutrophils due to the use of platinum-based chemotherapy.

Myeloperoxidase induces monocyte migration and activation after acute myocardial infarction.

Myocardial infarction (MI) is a significant contributor to morbidity and mortality worldwide. Many individuals who survive the acute event continue to experience heart failure (HF), with inflammatory and healing processes post-MI playing a pivotal role. Polymorphonuclear neutrophils (PMN) and monocytes infiltrate the infarcted area, where PMN release high amounts of the heme enzyme myeloperoxidase (MPO). MPO has numerous inflammatory properties and MPO plasma levels are correlated with prognosis and severity of MI. While studies have focused on MPO inhibition and controlling PMN infiltration into the infarcted tissue, less is known on MPO's role in monocyte function. Here, we combined human data with mouse and cell studies to examine the role of MPO on monocyte activation and migration. We revealed a correlation between plasma MPO levels and monocyte activation in a patient study. Using a mouse model of MI, we demonstrated that MPO deficiency led to an increase in splenic monocytes and a decrease in cardiac monocytes compared to wildtype mice (WT). In vitro studies further showed that MPO induces monocyte migration, with upregulation of the chemokine receptor CCR2 and upregulation of inflammatory pathways identified as underlying mechanisms. Taken together, we identify MPO as a pro-inflammatory mediator of splenic monocyte recruitment and activation post-MI and provide mechanistic insight for novel therapeutic strategies after ischemic injury.

Cytomegalovirus Immunoglobulin G Levels and Subclinical Arterial Disease among People Living with HIV in Botswana: A Cross-Sectional Study

Cytomegalovirus (CMV) has been linked with increased cardiovascular risk and monocyte activation in people living with HIV (PLWH). This cross-sectional study aimed to compare CMV immunoglobulin G (IgG) levels between combined antiretroviral therapy (cART)-treated PLWH versus ART-naïve PLWH and those without HIV, and to investigate their associations with biomarkers of endothelial injury and carotid atherosclerosis, in Gaborone, Botswana. All participants were between 30 and 50 years old. Carotid intimal media thickness (cIMT) and biomarkers of endothelial injury and monocyte activation were also assessed. The association between quantitative CMV IgG and cardiovascular disease risk was assessed in multivariate logistic regression analysis. The results showed that the mean CMV IgG level among ART-naïve participants was significantly higher than both the cART group and controls. However, CMV IgG levels did not differ significantly between the controls and cART groups. Among PLWH, CMV IgG levels were associated with ICAM-1 levels and cIMT. Increases in CMV IgG among ART-naïve participants were significantly associated with increases in log VCAM-1. In conclusion, CMV IgG levels are elevated among PLWH in sub-Saharan Africa, and higher levels are associated with biomarkers of endothelial injury and cIMT. Future research should investigate the long-term impact of elevated CMV IgG among PLWH.

CETP inhibition enhances monocyte activation and bacterial clearance and reduces streptococcus pneumonia-associated mortality in mice.

Sepsis is a leading cause of mortality worldwide, and pneumonia is the most common cause of sepsis in humans. Low levels of high-density lipoprotein cholesterol (HDL-C) levels are associated with an increased risk of death from sepsis, and increasing levels of HDL-C by inhibition of cholesteryl ester transfer protein (CETP) decreases mortality from intraabdominal polymicrobial sepsis in APOE*3-Leiden.CETP mice. Here, we show that treatment with the CETP inhibitor (CETPi) anacetrapib reduced mortality from Streptococcus pneumoniae-induced sepsis in APOE*3-Leiden.CETP and APOA1.CETP mice. Mechanistically, CETP inhibition reduced the host proinflammatory response via attenuation of proinflammatory cytokine transcription and release. This effect was dependent on the presence of HDL, leading to attenuation of immune-mediated organ damage. In addition, CETP inhibition promoted monocyte activation in the blood prior to the onset of sepsis, resulting in accelerated macrophage recruitment to the lung and liver. In vitro experiments demonstrated that CETP inhibition significantly promoted the activation of proinflammatory signaling in peripheral blood mononuclear cells and THP1 cells in the absence of HDL; this may represent a mechanism responsible for improved bacterial clearance during sepsis. These findings provide evidence that CETP inhibition represents a potential approach to reduce mortality from pneumosepsis.

Discovery and validation of molecular patterns and immune characteristics in the peripheral blood of ischemic stroke patients.

Stroke is a disease with high morbidity, disability, and mortality. Immune factors play a crucial role in the occurrence of ischemic stroke (IS), but their exact mechanism is not clear. This study aims to identify possible immunological mechanisms by recognizing immune-related biomarkers and evaluating the infiltration pattern of immune cells. We downloaded datasets of IS patients from GEO, applied R language to discover differentially expressed genes, and elucidated their biological functions using GO, KEGG analysis, and GSEA analysis. The hub genes were then obtained using two machine learning algorithms (least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE)) and the immune cell infiltration pattern was revealed by CIBERSORT. Gene-drug target networks and mRNA-miRNA-lncRNA regulatory networks were constructed using Cytoscape. Finally, we used RT-qPCR to validate the hub genes and applied logistic regression methods to build diagnostic models validated with ROC curves. We screened 188 differentially expressed genes whose functional analysis was enriched to multiple immune-related pathways. Six hub genes (ANTXR2, BAZ2B, C5AR1, PDK4, PPIH, and STK3) were identified using LASSO and SVM-RFE. ANTXR2, BAZ2B, C5AR1, PDK4, and STK3 were positively correlated with neutrophils and gamma delta T cells, and negatively correlated with T follicular helper cells and CD8, while PPIH showed the exact opposite trend. Immune infiltration indicated increased activity of monocytes, macrophages M0, neutrophils, and mast cells, and decreased infiltration of T follicular helper cells and CD8 in the IS group. The ceRNA network consisted of 306 miRNA-mRNA interacting pairs and 285 miRNA-lncRNA interacting pairs. RT-qPCR results indicated that the expression levels of BAZ2B, C5AR1, PDK4, and STK3 were significantly increased in patients with IS. Finally, we developed a diagnostic model based on these four genes. The AUC value of the model was verified to be 0.999 in the training set and 0.940 in the validation set. Our research explored the immune-related gene expression modules and provided a specific basis for further study of immunomodulatory therapy of IS.

H3K4me3-Mediated FOXJ2/SLAMF8 Axis Aggravates Thrombosis and Inflammation in β2GPI/Anti-β2GPI-Treated Monocytes.

Antiphospholipid syndrome (APS) is characterized by thrombus formation, poor pregnancy outcomes, and a proinflammatory response. H3K4me3-related monocytes activation are key regulators of APS pathogenesis. Therefore, H3K4me3 CUT&Tag and ATAC-seq are performed to examine the epigenetic profiles. The results indicate that the H3K4me3 signal and chromatin accessibility at the FOXJ2 promoter are enhanced in an in vitro monocyte model by stimulation with β2GPI/anti-β2GPI, which mimics APS, and decreases after OICR-9429 administration. Furthermore, FOXJ2 is highly expressed in patients with primary APS (PAPS) and is the highest in patients with triple-positive antiphospholipid antibodies (aPLs). Mechanistically, FOXJ2 directly binds to the SLAMF8 promoter and activates SLAMF8 transcription. SLAMF8 further interacts with TREM1 to stimulate TLR4/NF-κB signaling and prohibit autophagy. Knockdown of FOXJ2, SLAMF8, or TREM1 blocks TLR4/NF-κB and provokes autophagy, subsequently inhibiting the release of inflammatory and thrombotic indicators. A mouse model of vascular APS is established via β2GPI intraperitoneal injection, and the results suggest that OICR-9429 administration attenuates the inflammatory response and thrombus formation by inactivating FOXJ2/SLAMF8/TREM1 signaling. These findings highlight the overexpression of H3K4me3-mediated FOXJ2 in APS, which consequently accelerates APS pathogenesis by triggering inflammation and thrombosis via boosting the SLAMF8/TREM1 axis. Therefore, OICR-9429 is a promising candidate drug for APS therapy.

Pathophysiological basis of hepatopulmonary syndrome

Circulatory changes with increased blood flow and vasodilatation/vasoconstriction imbalance are an integral consequence of liver cirrhosis and portal hypertension and can affect the pulmonary circulation with the development of vascular disorders, with hepatopulmonary syndrome (HPS) being the most common. HPS is a serious pulmonary complication of progressive liver disease, resulting in a poor clinical prognosis. Vascular tone decrease, monocytic infiltration of pulmonary vessels, formation of intrapulmonary arteriovenous shunts, dysfunction of alveolar type II cells, destruction of the endothelial glycocalyx are important in the pathogenesis of HPS. Abnormalities of pulmonary capillaries lead to hypoxemia caused by a violation of the ventilation/perfusion ratio, diffusion disorders, and the development of arteriovenous anastomoses. Infiltration of the pulmonary vessels by monocytes is one of the key factors of HPS. This migration is facilitated by the intestinal microbiota translocation into the portal bloodstream with increased expression of proinflammatory cytokines (tumor necrosis factor α, interleu­kins 1, 6), leading to the activation of monocytes. Monocytes located in the pulmonary circulation promote the vasodilation through the activation of inducible nitric oxide (NO) synthase and thus NO production. This is also associated with endothelial dysfunction due to a decreased hepatic secretion of bone morphogenetic protein 9 and increased endothelin 1, endothelial overexpression of endothelin B receptors, and increased endothelial NO production. Proangiogenic factors such as vascular endothelial growth factor, platelet-derived growth factor, and placental growth factor play an important role in the proliferation of pulmonary capillaries. Circulation of tumor necrosis factor α, bile acids and monocyte infiltration in the pulmonary circulation lead to increased apoptosis of alveolar type II cells and decreased surfactant synthesis. Chronic inflammation in HPS disrupts the continuity of the endothelial glycocalyx layer. This article provides an overview of the current knowledge on the pathogenesis of HPS, summarizes many features of the disease based on the literature research in MEDLINE database on the PubMed platform.

Tacrolimus alleviates pulmonary fibrosis progression through inhibiting the activation and interaction of ILC2 and monocytes

Idiopathic pulmonary fibrosis (IPF) is a heterogeneous group of lung diseases with different etiologies and characterized by progressive fibrosis. This disease usually causes pulmonary structural remodeling and decreased pulmonary function. The median survival of IPF patients is 2–5 years. Predominantly accumulation of type II innate immune cells accelerates fibrosis progression by secreting multiple pro-fibrotic cytokines. Group 2 innate lymphoid cells (ILC2) and monocytes/macrophages play key roles in innate immunity and aggravate the formation of pro-fibrotic environment. As a potent immunosuppressant, tacrolimus has shown efficacy in alleviating the progression of pulmonary fibrosis. In this study, we found that tacrolimus is capable of suppressing ILC2 activation, monocyte differentiation and the interaction of these two cells. This effect further reduced activation of monocyte-derived macrophages (Mo-M), thus resulting in a decline of myofibroblast activation and collagen deposition. The combination of tacrolimus and nintedanib was more effective than either drug alone. This study will reveal the specific process of tacrolimus alleviating pulmonary fibrosis by regulating type II immunity, and explore the potential feasibility of tacrolimus combined with nintedanib in the treatment of pulmonary fibrosis. This project will provide new ideas for clinical optimization of anti-pulmonary fibrosis drug strategies.

Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes.

Inflammasomes are multi-protein complexes, which are formed in response to tissue injury, infections, and metabolic stress. However, aberrant inflammasome activation has been linked to several inflammatory diseases. Anthocyanins have been reported to attenuate NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, but the influence of grape/blueberry anthocyanins and especially their gut-derived metabolites on NLRP3 inflammasome activation in human monocytes remains unclear. Therefore, human leukemic monocytes (THP-1 cells, Tohoku Hospital Pediatrics-1 cells) were preincubated with different concentrations of grape/blueberry anthocyanins, homovanillyl alcohol, or 2,4,6-trihydroxybenzaldehyde (THBA) before the NLRP3 inflammasome was activated by lipopolysaccharide and/or nigericin. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, as well as ASC and NLRP3 protein expression, were determined using flow cytometry. Caspase-1 activity was measured in cultured cells, and pro-inflammatory cytokine secretion was determined using enzyme-linked immunosorbent assays. Anthocyanins and their metabolites had no effect on ASC or NLRP3 protein expression. However, THBA significantly inhibited ASC speck formation in primed and unprimed THP-1 monocytes, while caspase-1 activity was significantly declined by grape/blueberry anthocyanins. Furthermore, reduced inflammasome activation resulted in lower pro-inflammatory cytokine secretion. In conclusion, our results show for the first time that grape/blueberry anthocyanins and their gut-derived metabolites exert anti-inflammatory effects by attenuating NLRP3 inflammasome activation in THP-1 monocytes.

The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

556 Maternal Opioid Use Leads to Aberrant Maternal and Fetal Immunity

OBJECTIVES/GOALS: Maternal opioid use disorder (OUD) is linked to poor fetal outcomes. While it has been established that opioids can cross from maternal to fetal circulation, the mechanisms underlying these adverse outcomes remain poorly defined. This study aims to uncover OUD-associated immunological changes in maternal and fetal circulation. METHODS/STUDY POPULATION: To study the effect of maternal OUD on the maternal immune system at delivery, we collected maternal blood samples at delivery from healthy pregnant women (controls) and pregnant women with a diagnosis of severe OUD. To study the impact of maternal OUD on newborn immunity, we also collected umbilical cord blood (UCB) at delivery. Flow cytometry was used to determine the frequency and phenotype of circulating immune cell subsets and responses to stimulation. Isolated monocytes were stimulated with bacterial/viral agonist cocktails, while T and NK cells were stimulated with PMA/ionomycin. The impact of maternal OUD on circulating immune mediators was determined by Luminex and on monocyte activation markers sCD14 and CRP by ELISA. RESULTS/ANTICIPATED RESULTS: In maternal circulation, OUD was associated with a significant decrease in markers of inflammation, cell proliferation, and activation. Frequencies of immune cell subsets were impacted by OUD, shown by an expansion of CD8+ EMRA T cells, marginal-zone B cells, mDCs, non-classical monocytes, and CD16low NK cells. While no differences were seen in T and NK cell responses to stimulation, monocytes and pDCs had significantly lower responses to bacterial and viral agonist stimulation. Analysis of UCB revealed increased levels of pro-apoptotic/T cell exhaustion mediators and pro-inflammatory cytokines, albeit decreased levels of several chemokines and growth factors. The UCB immune landscape is altered with maternal OUD, as demonstrated by a shift from naive to memory CD8+ T cells and a decrease in pDC frequency. DISCUSSION/SIGNIFICANCE: OUD dampens maternal peripheral immunity, possibly contributing to poor placental function or premature/delayed labor. Monocytes and pDCs lack antimicrobial functionality, suggesting increased infection susceptibility with OUD. Finally, these implications extend to the fetal compartment, shown by heightened immune activation in UCB.

Systematic immune cell dysregulation and molecular subtypes revealed by single-cell RNA-seq of subjects with type 1 diabetes

BackgroundType 1 diabetes mellitus (T1DM) is a prototypic endocrine autoimmune disease resulting from an immune-mediated destruction of pancreatic insulin-secreting β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta$$\\end{document} cells. A comprehensive immune cell phenotype evaluation in T1DM has not been performed thus far at the single-cell level.MethodsIn this cross-sectional analysis, we generated a single-cell transcriptomic dataset of peripheral blood mononuclear cells (PBMCs) from 46 manifest T1DM (stage 3) cases and 31 matched controls.ResultsWe surprisingly detected profound alterations in circulatory immune cells (1784 dysregulated genes in 13 immune cell types), far exceeding the count in the comparator systemic autoimmune disease SLE. Genes upregulated in T1DM were involved in WNT signaling, interferon signaling and migration of T/NK cells, antigen presentation by B cells, and monocyte activation. A significant fraction of these differentially expressed genes were also altered in T1DM pancreatic islets. We used the single-cell data to construct a T1DM metagene z-score (TMZ score) that distinguished cases and controls and classified patients into molecular subtypes. This score correlated with known prognostic immune markers of T1DM, as well as with drug response in clinical trials.ConclusionsOur study reveals a surprisingly strong systemic dimension at the level of immune cell network in T1DM, defines disease-relevant molecular subtypes, and has the potential to guide non-invasive test development and patient stratification.

Different Influence Pattern of Conventional and Alternative Sources of Smoking on Adhesion Molecules and Cytokine Secretion in THP-1 Monocytes.

Tobacco is a carcinogen that is closely associated with the occurrence of lung cancer and head and neck squamous cell carcinoma (HNSCC). The consumption of tobacco is also leading to alterations in different immune cell subtypes. However, the impact of different conventional and alternative smoking sources on human monocytes remains elusive. In this study, we investigated the influence of aqueous extracts of different sources of smoking (cigarettes; heated tobacco product IQOS; e-cigarettes with and without nicotine; nicotine pouches) on different monocytic adhesion molecules, chemokine receptors and checkpoint molecule PD-L1 by flow cytometry. Cytokine expression patterns were evaluated using human cytokine arrays and the human monocyte leukemia cell line THP-1 as a model. Data revealed differential effects of the analyzed conventional and alternative smoking devices on monocyte adhesion molecules and cytokine secretion. The examined smoking devices can be assigned to two differential monocyte activation patterns. Monocytes stimulated with aqueous extracts of cigarettes, e-cigarette without nicotine, and heat not burn product IQOS revealed distinct alterations of surface markers and cytokines compared to the monocyte activation pattern in response to aqueous extracts of nicotine, nicotine pouches, and e-cigarette with nicotine. Our data indicate differential immunological consequences of different conventional and alternative smoking sources with and without nicotine. Further comprehensive analysis as well as in vivo investigations on peripheral blood monocyte subsets from smoking individuals using different smoking sources are required to better understand the impact on monocyte characteristics, especially with regard to the development of cancer.

Indole-3-carbinol attenuates lipopolysaccharide-induced acute respiratory distress syndrome through activation of AhR: role of CCR2+ monocyte activation and recruitment in the regulation of CXCR2+ neutrophils in the lungs.

Indole-3-carbinol (I3C) is found in cruciferous vegetables and used as a dietary supplement. It is known to act as a ligand for aryl hydrocarbon receptor (AhR). In the current study, we investigated the role of AhR and the ability of I3C to attenuate LPS-induced Acute Respiratory Distress Syndrome (ARDS). To that end, we induced ARDS in wild-type C57BL/6 mice, Ccr2gfp/gfp KI/KO mice (mice deficient in the CCR2 receptor), and LyZcreAhRfl/fl mice (mice deficient in the AhR on myeloid linage cells). Additionally, mice were treated with I3C (65 mg/kg) or vehicle to investigate its efficacy to treat ARDS. I3C decreased the neutrophils expressing CXCR2, a receptor associated with neutrophil recruitment in the lungs. In addition, LPS-exposed mice treated with I3C revealed downregulation of CCR2+ monocytes in the lungs and lowered CCL2 (MCP-1) protein levels in serum and bronchoalveolar lavage fluid. Loss of CCR2 on monocytes blocked the recruitment of CXCR2+ neutrophils and decreased the total number of immune cells in the lungs during ARDS. In addition, loss of the AhR on myeloid linage cells ablated I3C-mediated attenuation of CXCR2+ neutrophils and CCR2+ monocytes in the lungs from ARDS animals. Interestingly, scRNASeq showed that in macrophage/monocyte cell clusters of LPS-exposed mice, I3C reduced the expression of CXCL2 and CXCL3, which bind to CXCR2 and are involved in neutrophil recruitment to the disease site. These findings suggest that CCR2+ monocytes are involved in the migration and recruitment of CXCR2+ neutrophils during ARDS, and the AhR ligand, I3C, can suppress ARDS through the regulation of immune cell trafficking.

Inhibition of PCSK9 with evolocumab modulates lipoproteins and monocyte activation in high-risk ASCVD subjects

BackgroundMechanistic studies suggest that proprotein convertase subtilisin/kexin type 9 inhibitors can modulate inflammation. MethodsDouble-blind, placebo-controlled trial randomized 41 ASCVD subjects with type 2 diabetes with microalbuminuria and LDL-C level >70 mg/dL on maximum tolerated statin therapy received subcutaneous evolocumab 420 mg every 4 weeks or matching placebo. The primary outcomes were change in circulating immune cell transcriptional response, lipoproteins and blood viscosity at 2 weeks and 12 weeks. Safety was assessed in all subjects who received at least one dose of assigned treatment and analyses were conducted in the intention-to-treat population. ResultsAll 41 randomized subjects completed the 2-week visit. Six subjects did not receive study medication consistently after the 2-week visit due to COVID-19 pandemic suspension of research activities. The groups were well-matched with respect to age, comorbidities, baseline LDL-C, white blood cell counts, and markers of systemic inflammation. Evolocumab reduced LDL-C by −68.8% (p < 0.0001) and −52.8% (p < 0.0001) at 2 and 12 weeks, respectively. There were no differences in blood viscosity at baseline nor at 2 and 12 weeks. RNA-seq was performed on peripheral blood mononuclear cells with and without TLR4 stimulation ("Stress" transcriptomics). "Stress" transcriptomics unmasked immune cell phenotypic differences between evolocumab and placebo groups at 2 and 12 weeks. ConclusionsThis trial is the first to demonstrate that PCSK9 mAB with evolocumab can modulate circulating immune cell properties and highlights the importance of "stress" profiling of circulating immune cells that more clearly define immune contributions to ASCVD.

Abstract 6734: Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC

Abstract Background: Stimulator of interferon genes (STING) agonists elicit potent innate immune enhancement and mobilize robust adaptive immune responses. Given the potential risk of immunotoxicity from associated cytokine release due to STING agonist treatment, there is a need to identify mitigation strategies that minimize immune-related toxicity while offering a therapeutic window for STING-mediated anti-tumor effects. This strategy is particularly relevant for myeloid-targeted immunotherapies, as cytokine release syndrome (CRS) can be myeloid cell-mediated. We utilized preclinical models to evaluate the pharmacological impact of pretreatment with agents routinely used for management of CRS in advance of delivering TAK-500, a novel, systemic, immune-cell directed STING agonist antibody drug conjugate (iADC) that selectively targets CCR2+ myeloid cells. Methods: Dexamethasone and anti-IL-6R antibodies were tested at clinically relevant doses 1 hour prior to treatment with TAK-500 or mTAK-500, a murine surrogate, to study immunomodulatory and anti-tumor effects in preclinical models. Induction of innate and adaptive immune responses and cytokine release was evaluated in human, ex vivo, and in tumor-bearing mouse models to characterize potential impact of premedication strategies on drug pharmacology and anti-tumor efficacy. Results: TAK-500 triggered dose-dependent monocyte activation and cytokine secretion ex vivo. Monocyte activation was minimally impacted by dexamethasone or anti-IL-6R pretreatment. Suppression of IL-6 and IFN-ƴ and increase in IL-10 were observed following dexamethasone pretreatment, whereas anti-IL-6R maintained a more proinflammatory milieu. In syngeneic mouse models, pretreatment with dexamethasone and anti-mouse IL-6R had minimal impact on mTAK-500 anti-tumor efficacy. Key pharmacodynamic effects, including innate immune cell activation in periphery and tumor-draining lymph node and intratumoral CD8+ T cell accumulation, were minimally impacted by either pretreatment agent. Serum cytokine analyses demonstrated suppression of IL-6, IFN-ƴ and increases in IL-10 with dexamethasone, but not with anti-IL-6R, similar to ex vivo findings. Conclusion: Premedication of TAK-500 and mTAK-500 with dexamethasone or anti-IL-6R antibodies does not substantially impact STING-mediated innate immune activation, mobilization of adaptive immune responses or anti-tumor efficacy, despite altered cytokine profiles compared to (m)TAK-500 without premedication. Clinically, these premedication strategies have been effective in mitigating immunotherapy-associated CRS with T cell therapeutics, and here resulted in maintained anti-tumor activity when used in combination with TAK-500, suggesting a viable potential strategy for mitigation of STING-induced immunotoxicities in patients. Citation Format: Emily Rosentrater, Michelle L. Ganno, Tiquella Hatten, Henry Mack, Arjun Reddy Bollampalli, Dong Mei Zhang, Alex Parent, Jeffrey Raizer, Richard C. Gregory, Neil Lineberry, Vicky Appleman. Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6734.

Novel chemotype NLRP3 inhibitors that target the CRID3-binding pocket with high potency.

The NLRP3 inflammasome plays a central role in various human diseases. Despite significant interest, most clinical-grade NLRP3 inhibitors are derived from sulfonylurea inhibitor CRID3 (also called MCC950). Here, we describe a novel chemical class of NLRP3-inhibiting compounds (NIC) that exhibit potent and selective NLRP3 inflammasome inhibition in human monocytes and mouse macrophages. BRET assays demonstrate that they physically interact with NLRP3. Structural modeling further reveals they occupy the same binding site of CRID3 but in a critically different conformation. Furthermore, we show that NIC-11 and NIC-12 lack the off-target activity of CRID3 against the enzymatic activity of carbonic anhydrases I and II. NIC-12 selectively reduces circulating IL-1ß levels in the LPS-endotoxemia model in mice and inhibits NLRP3 inflammasome activation in CAPS patient monocytes and mouse macrophages with about tenfold increased potency compared with CRID3. Altogether, this study unveils a new chemical class of highly potent and selective NLRP3-targeted inhibitors with a well-defined molecular mechanism to complement existing CRID3-based NLRP3 inhibitors in pharmacological studies and serve as novel chemical leads for the development of NLRP3-targeted therapies.

Abstract 4603: Single-cell and spatial dissection of the effect of combined PD-L1 and TGFb blockade by bintrafusp alfa from the ICING window-of-opportunity trial in resectable locally advanced HNSCC patients

Abstract Despite high tumor mutational burden, immune checkpoint blockade has shown limited efficacy in head and neck squamous cell carcinoma (HNSCC), highlighting the need for additional therapeutic targets. TGFb signaling has been associated with an immunosuppressive tumor microenvironment as well as a lack of response to anti-PD(L)1 therapy, which led to the development of combined therapeutic approaches. ICING is a prospective (NCT04428047) open label, multicenter, phase II, window-of-opportunity preoperative, single-agent trial. The primary objective was to evaluate the efficacy and biological activity of pre-operative combined PD-L1 and TGFb blockade by bintrafusp alfa in patients with untreated resectable stage III/IV locally advanced HNSCC and its effect on the tumor microenvironment (TME). Tumor samples were harvested at baseline prior to immunotherapy and at surgery after two doses (1200mg; D1 and D15) for pre- and post-treatment comparison. Treatment efficacy was assessed using the pathological response (PathR) with a 10% threshold for response as primary endpoint. Changes in the TME were determined by single-cell RNA sequencing (scRNAseq) and multiplexed imaging. Seven patients were included and six received the full treatment scheme (5 oral cavity and 2 oropharyngeal, all HPV-negative; median age = 61y, range 34y-74y). Four patients had a PathR exceeding 10%, including one with a major PathR above 50%. The TME pre-treatment of this major responder did not differ from the other samples, but this sample was set apart by the presence of proliferative, p16+, HPV-negative tumor cells with mildly higher genomic alterations whilst still classified as low tumor mutational burden. Regardless of treatment response, we report several changes in post-treatment samples: (i) the remodeling of the stromal architecture concomitant with a phenotypic shift in cancer-associated fibroblasts (CAF) with a reduction in activated LRRC15+ CAF and an increase in CD10+IL24+ CAF; (ii) a reduction of TGFb-induced genes in both stromal and cancer cells; (iii) a reduction in both conventional and regulatory CD4+ T cells; (iv) an increase in CD8+ T cell infiltration in previously excluded tumors; (v) an interferon gamma-mediated activation of monocytes, macrophages and dendritic cells expressing CXCL9 and CXCL10. Collectively, our study deciphers the effects of bintrafusp alfa both molecularly and spatially at unprecedented granularity, which is of major importance for the many combined PD(L)1 and TGFb blockade therapeutic approaches currently under clinical development. Citation Format: Caroline Hoffmann, Jan-Timon Werle, Jocelyn Gal, Philemon Sirven, Jerzy Klijanienko, Jeremy Mesple, Benjamin Demaille, Wail Zeitouni, Olivier Choussy, Gregoire Marret, Nathalie Badois, Antoine Dubray-Vautrin, Gilles Dolivet, Lionnel Geoffrois, Maud Kamal, Ivan Bieche, Laure Monard, Clotilde Simon, Christophe Le Tourneau, Helene Salmon. Single-cell and spatial dissection of the effect of combined PD-L1 and TGFb blockade by bintrafusp alfa from the ICING window-of-opportunity trial in resectable locally advanced HNSCC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4603.

InPACT: a computational method for accurate characterization of intronic polyadenylation from RNA sequencing data

Alternative polyadenylation can occur in introns, termed intronic polyadenylation (IPA), has been implicated in diverse biological processes and diseases, as it can produce noncoding transcripts or transcripts with truncated coding regions. However, a reliable method is required to accurately characterize IPA. Here, we propose a computational method called InPACT, which allows for the precise characterization of IPA from conventional RNA-seq data. InPACT successfully identifies numerous previously unannotated IPA transcripts in human cells, many of which are translated, as evidenced by ribosome profiling data. We have demonstrated that InPACT outperforms other methods in terms of IPA identification and quantification. Moreover, InPACT applied to monocyte activation reveals temporally coordinated IPA events. Further application on single-cell RNA-seq data of human fetal bone marrow reveals the expression of several IPA isoforms in a context-specific manner. Therefore, InPACT represents a powerful tool for the accurate characterization of IPA from RNA-seq data.