High Monocytes Research Articles

Human coronary blood single-cell sequencing combined with exosome proteomic profiling reveals the molecular mechanism of recurrent in-stent restenosis (R-ISR)

Abstract Background Percutaneous coronary intervention (PCI) is recognized as the core treatment for symptomatic myocardial ischemia and acute coronary syndrome (ACS). However, the complex pathogenesis of recurrent in-stent restenosis (R-ISR) after stent implantation is still insufficiently understood. Purpose This study reports the first single-cell RNA sequencing and proteomic profiling to characterize pathogenesis of R-ISR in humans. Methods To reveal the pathophysiology and molecular mechanism of R-ISR, we performed single-cell RNA sequencing to identify RISR-induced changes in transcriptional landscape in coronary blood mononuclear cell composition. Besides, proteomic profiling of human coronary plasma-derived exosomes was used to further screen different proteins. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were performed to identify exosomes. In vitro cell biology experiments were used to validate the discoveries of bioinformatics analysis. Results Compared with normal subjects, the percentage of FCGR3B high monocytes, memory T cells and plasmacytoid dendritic cells (DCs) was increased in R-ISR. Furthermore, we observed a marked increase in the expression of JUN, FOS, CXCL8, S100A8 enriched in the IL-17 signaling pathway that is known to play a crucial role in activating inflammation, mediating immune response, and even maintaining inflammation to chronicity. According to in vitro experiments, the co-cultured system demonstrated that FCGR3B high monocytes isolated from RISR patients activated the expression of inflammatory factors in endothelial cells (ECs), such as IL-1β, IL-6 and TNF-α. Inflammatory activated endothelial cells could release a large amount of exosomes, proteomic profiling of exosomes isolated from ECs observed a significant upregulation of MYLK expression, which could promote proliferation and migration of vascular smooth muscle cells (VSMCs) by activating VEGFR-2 signaling pathway. Conclusions Our study reveals a novel subset of FCGR3B high monocytes in R-ISR patients that could activate ECs inflammation via IL-17 signaling pathway. Activated ECs release exosomes rich in MYLK, which promote proliferation and migration of VSMCs, ultimately leading to repeated restenosis.

A novel VCP modulator, KUS121, attenuates atherosclerosis progression by maintaining intracellular ATP and mitigating ER stress in endothelial cells

Abstract Back ground: Endoplasmic reticulum (ER) stress signaling pathways have pivotal roles in atherosclerosis progression. Recently, we have developed Kyoto University Substance (KUS) 121, which selectively inhibits ATPase activities of valosin-containing protein (VCP) and consequently saves intracellular ATP consumption and mitigates ER stress. KUS121 are known to have protective effects on several disease models including ischemic heart disease and heart failure. However, the efficacy of KUS121 against atherosclerosis was still unclear. Purpose To elucidate the effect of KUS121 on atherosclerosis and its mechanisms. Methods and Results The daily treatment of KUS121 reduced atherosclerosis progression by approximately 40% and macrophage burden in the plaques were also significantly decreased in ApoE knockout mice on high fat diet. Interestingly, we found that C/EBP homologous protein (CHOP), an established ER stress marker, was mainly expressed in plaque endothelium. Therefore, we assessed the action of KUS 121 in endothelial cells using the EA.hy926 cell line. Consequently, it was demonstrated that KUS121 attenuated ER stress-induced apoptosis and downregulated the IRE1α-associated inflammatory pathways. Consistent with these in vitro findings, KUS121 treatment also significantly reduced endothelial apoptosis assessed by TUNEL staining and inflammation examined by immunostaining of NF-κB and ICAM1 in atherosclerotic plaques. Moreover, KUS121 treatment attenuated the recruitment of inflammatory Ly6C high monocytes assessed by bead-labeling technique, which could be due to the reduction of endothelial ICAM1 expression. Furthermore, we also demonstrated that maintenance of intracellular ATP levels mitigates ER stress and prevents pathological states in endothelial cells. Conclusion KUS121 can be a new therapeutic option for atherosclerotic diseases by maintaining intracellular ATP levels and attenuating ER stress-induced apoptosis and inflammation in plaque endothelium.

Single-Cell Analysis Reveals That CD47 mRNA Expression Correlates with Immune Cell Activation, Antiviral Isgs, and Cytotoxicity.

Immune cells are reported to upregulate CD47 during infection, however, the role of CD47 in innate and adaptive immune cells remains unclear. To bridge this knowledge gap, we analysed our single cell (sc)-RNA dataset along with other publicly available sc-RNA datasets from healthy controls, people with HIV-1 (PWH) and COVID-19 patients. We characterized each immune cell based on low, intermediate, and high expression of CD47 . Our analyses revealed that CD47 high pDCs and monocytes exhibited relatively higher expression of IFN-α regulatory genes, antiviral interferon-stimulated genes (ISGs) and MHC-I associated genes compared to CD47 inter. and CD47 low cells. Furthermore, CD47 high NK and CD8+ T cells showed higher expression of antiviral ISGs, as well as genes encoding for cytotoxic markers like granzyme B, perforin, granulysin, interferon gamma and NKG7. Additionally, CD47 high CD8+ T cells expressed higher levels of PD-1 and LAG-3 genes. Lastly, we found that CD47 high B cells had enriched expression of genes involved in cell activation and humoral responses. Overall, our analyses revealed that innate and adaptive immune cells expressing elevated activation and functional gene signatures also express higher CD47 levels.

Abstract 3054: Proinflammatory Phenotypes And Role Of Ly-6c Low Monocytes In Atherosclerosis With Hypertriglyceridemia

Background: Monocytes play pivotal roles in atherosclerosis. Ly-6C low monocytes, also known as patrolling monocytes, become foamy in the circulation of mice with hyperlipidemia. However, the role of Ly-6C low monocytes in atherosclerosis with hypertriglyceridemia (HTG) remains to be determined. Aim: To examine the phenotypes and role of Ly-6C low monocytes in atherosclerosis in HTG. Method: Ldlr -/- ApoC3Tg (transgenic expression of human ApoC3) mice fed a western high-fat diet (WD) were used to examine the effects of HTG on monocyte lipid accumulation (foamy monocyte formation), phenotypes, and contributions to atherosclerosis. Results: Plasma triglycerides in Ldlr -/- ApoC3Tg mice were 5.1 times higher than in Ldlr -/- controls at 6 weeks on WD, correlating with more than twice the increase in aortic atherosclerosis (P<0.01). Consistently, Ly-6C low compared to Ly-6C high monocytes in Ldlr -/- mice had increased lipid accumulation and exhibited elevated levels of TNFα, IL-6, IL-1β, Plin2, and PPAR-γ. In Ldlr -/- ApoC3Tg mice, Ly-6C low (also CD11c + ) monocytes demonstrated even greater lipid accumulation and adhesion to VCAM-1 and had further elevations in levels of TNFα, IL-6, IL-1β, Plin2, and PPAR-γ than CD11c + (Ly-6C low ) monocytes in Ldlr -/- controls, indicating an enhanced inflammatory response and lipid accumulation under HTG. Furthermore, compared to Ldlr -/- control mice, Ldlr -/- ApoC3Tg mice had an increase in the level of SIRPα (a ligand of the “don’t-eat me” signal CD47) on Ly-6C low monocytes, indicative of impairment in efferocytosis and clearance of apoptotic cells. Additionally, nuclear c-Rel mRNA and protein levels were upregulated in Ly-6C low monocytes of Ldlr -/- ApoC3Tg mice compared to Ldlr -/- controls, suggesting activation of the NF-κB pathway in Ly-6C low monocytes by HTG. Finally, depletion of Ly-6C low monocytes reduced atherosclerosis by 45% (P<0.05) in Ldlr -/- ApoC3Tg mice, indicating a role of Ly-6C low monocytes in atherosclerosis in HTG. Conclusions: HTG accelerates atherogenesis in Ldlr -/- mice, possibly by increasing lipid accumulation and inflammatory phenotypic changes in monocytes, primarily CD11c + Ly-6C low monocytes.

Abstract 7644: Prognostication of genomic characteristics of residual breast cancer after neoadjuvant chemotherapy

Abstract Residual cancer burden (RCB) based on pathologic characteristics is the most powerful prognostic factor in breast cancer (BC) which received neoadjuvant chemotherapy (NAC) followed by surgery. According to RCB classification, class III predicted short survival duration regardless of BC subtypes according to hormone receptor (HR) and human epidermal growth factor receptor-2 (HER-2) state. However, up to 20-40% of BCs with RCB class III did not experience BC recurrence and had long term survival without disease recurrence. In our study, we evaluated genomic characteristics of BC which had high RCB class. We collected 96 fresh frozen BC specimens which had undergone surgery after NAC between 2010 and 2018 from Samsung Medical Center Biobank. We performed whole genome or exome sequencing, RNA sequencing and high-plex single-cell spatial transcriptomics. Of 96 BCs, 65 BCs had experienced BC recurrence and 31 BCs have not been BC recurrence. Of 65 BCs with dismal prognosis, 12 were HR+HER2-, four in HR+HER2+, 39 in triple negative breast cancer (TNBC) and 10 in HR-HER2+ BC. Of 31 BCs without recurrence, 11 were HR+HER2-, two in HR+HER2+, 14 in TNBC and four in HR-HER2+ BC. In HR+HER2- BC, ten of 12 BCs with dismal prognosis were categorized into basal like intrinsic subtype and two were into luminal B subtype compared that six of luminal A and five of luminal B subtype categorization in good prognosis BC group. Age associated mutation signature were enriched in HR+HER2- BC with good prognosis (p=0.027). Tumor microenvironment (TME) analysis using Kassandra suggested that high monocytes were associated with BC with dismal prognosis (p=0.00031). In single-cell spatial transcriptome analysis, T-cell abundance in the tumoral niche is associated with dismal prognosis in HR+HER2- BC (p=0.039). In TNBC, almost BCs (88.7%) were categorized into basal-like intrinsic subtype regardless of their prognosis. TME analysis presented that immune cells including macrophages and lymphocytes with CD4 and CD8 T cells were significantly enriched in TNBC with good prognosis (ps <0.05, respectively). In HER2+ BC, intrinsic subtypes were heterogeneous in both groups. Focal amplification in HER2+BC patient harbored DNA segments from different chromosomes more frequently than in those with good prognosis (p=0.00091), which was prominent in oncogenic extrachromosomal DNA amplifications compared to other oncogenic amplifications. In TME analysis, there were no specific cell enrichments according to BC prognosis but sing-cell spatial transcriptome analysis revealed that higher T-cell abundance in the tumoral niche was associated with HR+HER2+ BC with good prognosis (p=0.047). In conclusion, each BC subtype had their own genomic characteristics to determine their prognosis. Our study also suggested that TME characteristics was common prognostic factor for survival outcome of BC with residual tumor after NAC. Citation Format: Ji-Yeon Kim, Sabin Park, Sepil Ahn, Eun Seop Seo, Soyeon Kim, Mark Gregory, Emily Killingbeck, Eun Yoon Cho, Jeong Eon Lee, Jin Seok Ahn, Yeon Hee Park, Woong-Yang Park, Hoon Kim, Semin Lee, Young-Hyuck Im. Prognostication of genomic characteristics of residual breast cancer after neoadjuvant chemotherapy [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 7644.

Abstract PR-009: Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer using human multicellular in vitro models

Abstract Effective treatment of high grade serous ovarian cancer (HGSOC) still represents a clinical challenge. This is partially due to poor understanding of the complexity of this disease and the highly immunosuppressive tumor microenvironment (TME) that makes HGSOC resistant to most of the newly discovered therapies. The lack of relevant and physiological human in vitro models represents a major issue as the development of new treatments are mostly based on mouse models that do not fully recapitulate the human disease. In this study, we developed 3-dimensional (3D) human multicellular in vitro models using as a template and validation, data collected in a previous study that ‘deconstructs’ the metastatic TME of HGSOC. Our human 3D in vitro pentaculture (five cell types) model is composed of patient-derived primary adipocytes, fibroblasts and mesothelial cells isolated from macroscopic normal omentum – the primary site of metastasis in HGSOC – of women undergoing a gynaeco-oncology surgery, combined with different high grade serous malignant cell lines and monocytes isolated from healthy individuals. We found that the peripheral blood monocytes were able to differentiate and polarize to macrophages in our model in a similar way that observed in human HGSOC omental metastasis. Interestingly, our pentaculture models self-rearrange at the cellular level in a different way depending on the malignant cell line used in the culture. RNAseq analysis of the pentacultures showed that the different cultures clustered according to the malignant cell line used suggesting that malignant cells have a major influence on the transcriptome of the TME. Moreover, we demonstrated that our pentacultures partially replicated human biopsies: some of the macrophage and fibroblast clusters identified in a previous scRNAseq experiment on human HGSOC biopsies were also found in the pentacultures. We then confirmed the presence of some these macrophages clusters at the protein level. Finally, these pentacultures reproduce some of the actions of macrophage modifying drugs that have previously been shown in vivo. In conclusion, the study of the immunosuppressive TME of HGSOC in these human 3D in vitro models allows us to identify the contribution of different cell types to tumor growth and spread and provides us a semi-high throughput platform for therapeutic screening. Citation Format: Beatrice Malacrida, Samar Elorbany, Eleni Maniati, Rachel Bryan-Ravenscroft, Sophie L.P. Skingsley, Florian Laforets, Ranjit Manchanda, Frances R. Balkwill. Reconstructing the metastatic tumor microenvironment of high grade serous ovarian cancer using human multicellular in vitro models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-009.

A Murine Model of Maternal Micronutrient Deficiencies and Gut Inflammatory Host-microbe Interactions in the Offspring

Background & AimsMicronutrient deficiency (MND i.e., lack of vitamins and minerals) during pregnancy is a major public health concern. Historically, studies have considered micronutrients in isolation; however, MNDs rarely occur alone. The impact of co-occurring MNDs on public health, mainly in shaping mucosal colonization by pathobionts from Enterobacteriaceae family, remains undetermined due to lack of relevant animal models. MethodsTo establish a maternal murine model of multiple MND (MMND), we customized a diet deficient in vitamins (A, B12 and B9) and minerals (iron and zinc) that most commonly affect children and women of reproductive age. Thereafter, mucosal adherence by Enterobacteriaceae, the associated inflammatory markers and proteomic profile of intestines were determined in the offspring of MMND mothers (hereafter, low micronutrient/LM pups) via bacterial plating, flow cytometry and mass spectrometry, respectively. For human validation, Enterobacteriaceae abundance, assessed via 16s sequencing of 3-month-old infant fecal samples (n= 100), was correlated with micronutrient metabolites using Spearman’s correlation in meconium of children from the CHILD birth cohort. ResultsWe developed an MMND model and reported an increase in colonic abundance of Enterobacteriaceae in LM pups at weaning. Findings from CHILD cohort confirmed a negative correlation between Enterobacteriaceae and micronutrient availability. Furthermore, pro-inflammatory cytokines and increased infiltration of lymphocyte antigen 6 complex high monocytes and M1-like macrophages were evident in the colons of LM pups. Mechanistically, mitochondrial dysfunction marked by reduced expression of nicotinamide adenine dinucleotide (NAD)H dehydrogenase and increased expression of NAD phosphate oxidase (Nox) 1 contributed to the Enterobacteriaceae bloom. ConclusionThis study establishes an early life MMND link to intestinal pathobiont colonization and mucosal inflammation via damaged mitochondria in the offspring.

Validation of a hematology analyzer in donkey medicine

Asinina de Miranda is a protected donkey sub-species from the Mirandês plateau in northeastern of Portugal. Donkeys are animals that have substantially lost their place as working animals in modern society, this had led to a decrease in their population numbers. A need to preserve native species has led to the foundation of organizations like Associação para o Estudo e Proteção do Gado Asinino (AEPGA) and the development of studies regarding breed welfare, such as hematology. The IDEXX ProCyte Dx is a veterinary hematology analyzer validated for several species, but not for donkeys. The aim of this study was to validate the ProCyte Dx for Asinina de Miranda donkeys. The validation requires a controlled study of precision, carryover, linearity and comparison between the equipment and the manually obtained values for the leukocyte differential count and hematocrit. Results indicated coefficient of variation was good (below 5 %) for both the intra-assay and the inter-assay precision, except for basophils. Carryover was 0 % for all the parameters except platelets (5.88 %). Linearity showed a very high Pearson correlation coefficient, above 0.99, for erythrocytes, hematocrit, hemoglobin, leucocytes, neutrophils, lymphocytes, monocytes, eosinophils, platelets and plateletcrit. Comparison demonstrated excellent agreement for hematocrit (rs=0.96) and good Spearman rank correlation for neutrophils (rs=0.84) and lymphocytes (rs=0.90). Accuracy for total leukocyte count and platelets could not be determined. In conclusion, the ProCyte Dx seems appropriate to be used in Asinina de Miranda hematology.

A Novel Model Combining Circulating Absolute Monocyte Counts and a Four-Gene Monocyte Signature in Leukapheresis Identifies Lymphoma Patients at Very High Risk of Progression after Tisagenlecleucel or Axicabtagene Ciloleucel Therapy

Introduction CD19-directed chimeric antigen receptor (CAR) T can induce durable remissions in a fraction of relapsed/refractory large B-cell lymphomas (R/R LBCL), but 60% of patients (pts) still relapse. Biological mechanisms explaining lack of responses are emerging but they are largely unsuccessful in predicting disease response at the patient level. Aim To address the potential role of lymphocyte features before CAR T manufacturing, we performed transcriptomic and functional evaluations of leukapheresis products (LK) in 95 R/R LBCL pts enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel (Tisa-cel) and axicabtagene ciloleucel (Axi-cel). Methods RNA of sorted CD3+ cells from LK was digitally quantified using the nCounter CAR T Characterization Panel (NanoString). Flow cytometry (FCM) was used for CAR T enumeration and monocyte analysis. Disease response at day 90 was assessed by computed tomography (CT) and positron emission tomography-CT (PET/CT) according to Lugano criteria. Statistical analyses were performed by GraphPad Prism v.9.00 and R v.4.1.2. Results We identified a 4-gene transcriptional signature (including MS4A4A, CD86, CD163 and SIGLEC5) able to divide LBCL pts into two groups, [namely Expressors (EXP) and poor-Expressors (poor-EXP)], characterized by significantly different survival probabilities. EXP have shorter PFS (P<0.0001: median PFS for EXP was 61 days and not reached for poor-EXP) (Fig. 1) and are less likely to respond (P<0.01). As the identified genes are highly expressed in monocytes but not in T cells, we sought to define whether monocytes contained in LK have a role in determining post CAR T outcome. In line with the fact that EXP had higher frequencies of monocytes in LK by FCM, we confirmed elevated absolute monocyte counts (AMC) both in LK (P<0.01) and in peripheral blood (PB) (P<0.05) of EXP. We then checked if monocyte levels could influence response and survival. When compared to responders, non-responders (NR) had higher LK AMC (P<0.01), higher percentages of monocytes (P<0.01) but also decreased percentages of lymphocytes (P<0.001), a parameter that was also associated with reduced PFS [HR: 0.97 (CI 0.96 - 0.99) P=0.01]. Consistent with the fact that circulating monocytes significantly correlated with those in LK (r= 0.6621, P<0.0001), NR also had significantly higher AMC (P<0.001) and AMC levels in PB negatively correlated with PFS [HR: 1.8 (CI 1.2 - 2.9) P=0.01]. Additionally, pts with serum C-reactive protein (CRP) and ferritin levels higher than the upper normal limit, displayed significantly higher AMC (P<0.001 for CRP; P<0.05 for ferritin). Accordingly, univariate analysis indicated that high ferritin, CRP and LDH levels at the time of LK were associated with the lack of response, but only high serum ferritin levels correlated with shorter PFS [HR: 2.86 (CI 1.25-6.55) P=0.013]. We next investigated if the combination of the monocyte signature and high ferritin levels and/or high circulating monocytes could better identify a group of pts at high risk of progression, compared to the expression of the 4 genes alone. Remarkably, albeit high ferritin levels and monocyte counts are overall associated with significantly reduced survival, the shortest PFS was achieved in EXP with monocyte counts above the median (P=0.0234: median PFS for EXP with high monocytes was 32.5 days and for EXP with low monocytes 209 days) (Fig. 2), thus supporting the hypothesis that CAR T treatment outcome is influenced by the presence of elevated levels of monocytes expressing the 4 genes. Concatenated monocyte FCM data from 30 pts analyzed with FlowSOM, followed by a consensus clustering algorithm combined with t-SNE (t-stochastic neighbor embedding), indicated that EXP had higher frequencies of CD14hi monocytes co-expressing MS4A4A, CD86, CD163 and SIGLEC5 at the protein level than poor-EXP (P<0.05), and accordingly, the presence of this population was also associated with lack of response (P<0.05). Conclusions Our data suggest that the poor outcome of some LBCL pts receiving CAR T is due to the presence of high monocytes levels expressing a four-gene signature in an inflammatory microenvironment. Additionally, the pre-manufacturing combined analysis of the gene signature and of PB AMC, could be used to plan trials evaluating CAR T cells versus other newly approved therapies such as bispecific antibodies.

Chronic TNF in the Aging Microenvironment Exacerbates TET2-loss-of-Function Myeloid Expansion

Introduction: Age-associated TET2 somatic mutations impart an intrinsic hematopoietic stem cell (HSC) advantage and contribute to the phenomenon of clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutantCHIP have a higher risk of developing myeloid neoplasms and other age-related conditions, including heart, lung, liver, kidney and infectious disease, and have increased risk of all-cause mortality. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing TNF favours TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSC and progeny with TET2-mutations in vivo. Methods: All human and mouse studies met ethics approval. C57Bl/6 background wildtype (WT), TNF-α knockout ( TNF-/-), Tet2 hematopoietic knockout (Vav1-iCre-mediated; Tet2-/-) and floxed control mice ( Tet2f/f) mice were obtained from the Jackson Laboratory. Young (6-mo [n=9]) and old (18-22 mo [n=9]) WT mice, and old TNF-/- mice (n=7) were subjected to nonirradiative myeloablation with busulfan, and retro-orbital injections of 8x10 6 cells/ml T-cell-depleted bone marrow (BM), equally harvested from 4-mo-old WT CD45.1 (n=5) and Tet2-/- CD45.2 donor mice (n=5). Flow cytometry was used to confirm and monitor engraftment. Mice were harvested 8 weeks post-transplant for analysis of HSC, progenitor, monocyte and neutrophil populations, and cytokine analyses. Consenting human research participants diagnosed with rheumatoid arthritis (RA) were recruited from the Greater Hamilton Area (Ontario, Canada) from 2016-2018. Blood draws occurred prior to any immunomodulatory treatment (baseline), and at 3- and 6-months following treatment with Adalimumab (Humira®), an anti-TNF agent. CHIP status was determined with our successful 48-gene, targeted, Ion-Torrent based sequencing approach to isolated genomic DNA from PBMC. Confirmation of calls and increased sensitivity to detect clones with VAF < 0.02 employed our established single molecule molecular inversion probes (smMIP)-targeted genomic capture technique and high-depth (47,500X avg. coverage) paired-end sequencing, employing high-stringency filters for error-suppression. Statistical analyses were performed in GraphPad Prism V9.2 or R 4.1.2. Results: 1. Mixed BM chimeric mice of WT and TNF -/- genotypes reconstituted with WT CD45.1 + and Tet2 -/- CD45.2 + HSC showed that age-associated increases in TNF significantly increased by 2- to 2.5-fold the proportions of HSC in old recipient mice, with myeloid lineage skewing (2- to 2.5-fold more granulocyte-monocyte, monocyte-dendritic and common monocyte progenitor cells in BM, and dramatic expansion of Tet2-/- monocytes and neutrophils in blood). This aberrant myelomonocytic advantage was mitigated in old TNF -/- recipient mice, suggesting that TNF signalling in the BM is essential for Tet2-mutant myeloid expansion. 2. Age-associated TNF predisposed Tet2-/- HSC to the development of Ly6C high inflammatory monocytes, including increased intracellular and serum TNF levels, further exacerbating an inflammatory environment in favor of Tet2-mutantexpansion. 3. Examination of human RA patients (n=7; avg. age 57y) with serial PBMC sampling revealed one patient with CHIP driven by CBL and PPM1D variants, with a 32% reduction in clone size between 3 and 6 months of anti-TNF therapy (adalimumab). Two additional variants were detected at baseline in this RA patient, TP53 R196* (VAF = 1.34%) and ASXL1 P689L (VAF = 3.13%), but these were not detected at 3- and 6-months post-treatment even with high-depth, error suppression sequencing. Two additional patients had detectible low-level CH clones prior to anti-TNF treatment - one with a STAG2 R1033Q variant at baseline (VAF = 1.65%) and the other with a TET2 baseline E798K variant (VAF = 1.68%) - but not detected during anti-TNF treatment. Conclusions: We present novel findings that TNF in the aging BM environment has a causal role in driving TET2-mutant CHIP in vivo, and the first examination of the impact of TNF blockade on CH dynamics in humans, suggesting promise for reducing clonal burden. Larger, prospective studies are required to confirm these findings and to determine if inflammatory cytokine blockade may improve CHIP-comorbid conditions and myeloid cancer risk.

Umbilical Cord Blood-Derived Monocytes as A Reliable Source of Functional Macrophages for Biomedical Research.

Macrophages are multifunctional immune cells widely used in immunological research. While autologous macrophages have been widely used in several biomedical applications, allogeneic macrophages have also demonstrated similar or even superior therapeutic potential. The umbilical cord blood (UCB) is a well-described source of abundant allogenic monocytes and macrophages that is easy to collect and can be processed without invasive methods. Current monocyte isolation procedures frequently result in heterogenous cell products, with limited yields, activated cells, and high cost. This study outlines a simple isolation method that results in high yields and pure monocytes with the potential to differentiate into functional macrophages. In the experimental study, we describe a simple and efficient protocol to isolate highpurity monocytes. After collection of human UCB samples, we used a gradient-based procedure composed of three consecutive gradient steps: i. Hydroxyethyl starch-based erythrocytes sedimentation, followed by ii. Mononuclear cells (MNCs) isolation by Ficoll-Hypaque gradient, and iii. Separation of monocytes from lymphocytes by a slight hyperosmolar Percoll gradient (0.573 g/ml). Then the differentiation potential of isolated monocytes to pro- and antiinflammatory macrophages were evaluated in the presence of granulocyte colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF), respectively. The macrophages were functionally characterized as well. A high yield of monocytes after isolation (25 to 50 million) with a high purity (>95%) could be obtained from every 100-150 ml UCB. Isolated monocytes were defined based on their phenotype and surface markers expression pattern. Moreover, they possess the ability to differentiate into pro- or anti-inflammatory macrophages with specific phenotypes, gene/surface protein markers, cytokine secretion patterns, T-cell interactions, and phagocytosis activity. Here we describe a simple and reproducible procedure for isolation of pure monocytes from UCB, which could be utilized to provide functional macrophages as a reliable and feasible source of allogenic macrophages for biomedical research.

MONOCYTE DERIVED CYTOKINES INDUCE ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: Due to aldosterone excess and its pleotropic effects patients with primary aldosteronism (PA) are exposed to high rates of cardiovascular events. Endothelial dysfunction, as a part of vascular disease, is one of the earliest changes related to high cardiovascular risk and monocytes are the main immune cells participating in the development of vascular wall damage. Therefore, we ought to compare endothelial function between PA patients and healthy controls. Next, we wanted to estimate, if monocytes are able to induce endothelial dysfunction solely by their secretome. Design and method: 13 healthy controls and 42 PA patients received flow mediated dilation measurements (FMD). Later, monocytes were isolated from 8 PA patients and 6 healthy individuals and placed in cell culture. 24 hours later supernatants were collected for cytokine measurements using Human Cytokine 17-plex Assay (Bio-rad). Effects of monocyte derived cytokines on endothelial function were assessed using wire Myograph (Danish Myo Technology). Pre-contraction was induced by norepinephrine (1μM) (Sigma) followed by cumulative concentration response curve to carbachol (0.3μM-300μM) (Sigma). Results: FMD was significantly lower among PA patients (6.3(3.8-8.1)% vs 10.5 ± 3%, p < 0.05). Furthermore, monocytes from patients with PA secreted significantly higher concentrations of following cytokines and growth factors than healthy controls: G-CSF, IFN-gamma, TNF-alfa, IL-6, IL-10, IL-8, IL-17, and MCP-1. Especially concentrations of IL-6 (388 (351-580)pg/ml vs 36(4-268)pg/ml, p < 0.05) and TNF-alfa (95(48-135)pg/ml vs 17(5-41)pg/ml, p < 0.05) were much higher among PA patients. Incubation of mouse thoracic aorta with this monocyte secretome for 24 hours revealed stronger vascular contraction than using supernatant from healthy controls. In pre-constricted murine aortas, maximal dilation of 87.9 ± 11% was reached using aortas incubated in PA monocyte secretome whereas a dilation of 90.9 ± 9.1% was reached in controls. Indeed, carbachol EC50 for aortas incubated in monocyte secretome derived from healthy controls was 1.0mkM and 1.9mkM for PA patients. Conclusions: PA patients demonstrate endothelial dysfunction measured by FMD. Moreover, monocytes isolated from these patients are characterized by high secretion of pro-inflammatory cytokines which induce endothelial dysfunction, one of mechanisms triggering vascular dysfunction among these patients.

Abstract 307: Targeted Activation Of The Tnfa Pathway Accelerates Atherosclerosis Regression In Mice

Aggressive lipid lowering in mice with established atherosclerosis induces plaque regression and is characterized by reduced monocyte recruitment and increased expression of the motility receptor C-C chemokine receptor 7 (CCR7) to promote macrophage egress from plaques to nearby lymph nodes. Notably, genetic deletion of receptors critical for the recruitment of inflammatory Ly6C high monocytes is necessary to facilitate plaque regression. Our group previously showed that deletion of macrophage LDL receptor-related protein 1 (LRP1) in mice with established atherosclerosis accelerates plaque regression after lipid lowering and is associated with increased inflammation and macrophage CCR7-mediated egress. We tested the hypothesis that targeted activation of inflammation following lipid lowering in atherosclerotic mice accelerates atherosclerosis regression. LC-MS/MS proteomics performed on murine macrophages revealed that the loss of LRP1 upregulated the inflammatory protein tumor necrosis factor alpha (TNFα) 1.48-fold (P<0.05). Apoe -/- mice were fed a Western diet for 12 weeks to establish atherosclerosis and then subjected to bone marrow transplantation (BMT) using wildtype ( Apoe +/+ ) mice as donors to restore plasma APOE and reduce lipids. A cohort was sacrificed two weeks after BMT and used as baseline (N=11). The remaining mice (N=12/group) received intraperitoneal injections of recombinant murine TNFα (0.075mg/kg/day) or saline for 6 weeks on chow diet. After 6 weeks on chow diet, total plasma cholesterol levels ranged 82 -120 mg/dL with no significant differences between treatment cohorts. TNFα-treated mice had elevated spleen-to-body weight ratios compared to saline treated controls (P<0.0001), confirming successful activation of the immune system. Following 6 weeks of lipid lowering, TNFα treatment reduced total plaque area and lipid content 17.0% (P<0.05) and 34.4% (P<0.05), respectively, compared to saline treated controls (P<0.05).Herein, we show that the administration of TNFα, to stimulate inflammation after lipid lowering, significantly reduces plaque size and plaque lipid content in mice with established atherosclerosis.

Longitudinal Immune Cell Profiling in Patients With Early Systemic Lupus Erythematosus.

To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time. We employed mass cytometry with 3 different panels of 38-39 markers (an immunophenotyping panel, a Tcell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning-driven clustering, flow self-organizing maps, and dimensional reduction with t-distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed. Levels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki-67+ proliferating subsets (ICOS+Ki-67+ CD8 T cells, Ki-67+ regulatory T cells, CD19intermediate Ki-67high plasmablasts, and PU.1high Ki-67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE. Two major helper T cell subsets and unique Ki-67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

Detecting monocyte trafficking in an animal model of glioblastoma using R2* and quantitative susceptibility mapping.

The role of tumor-associated macrophages (TAMs) in glioblastoma (GBM) disease progression has received increasing attention. Recent advances have shown that TAMs can be re-programmed to exert a pro-inflammatory, anti-tumor effect to control GBMs. However, imaging methods capable of differentiating tumor progression from immunotherapy treatment effects have been lacking, making timely assessment of treatment response difficult. We showed that tracking monocytes using iron oxide nanoparticle (USPIO) with MRI can be a sensitive imaging method to detect therapy response directed at the innate immune system. We implanted syngeneic mouse glioma stem cells into C57/BL6 mice and treated the animals with either niacin (a stimulator of innate immunity) or vehicle. Animals were imaged using an anatomical MRI sequence, R2* mapping, and quantitative susceptibility mapping (QSM) before and after USPIO injection. Compared to vehicles, niacin-treated animals showed significantly higher susceptibility and R2*, representing USPIO and monocyte infiltration into the tumor. We observed a significant reduction in tumor size in the niacin-treated group 7days later. We validated our MRI results with flow cytometry and immunofluoresence, which showed that niacin decreased pro-inflammatory Ly6C high monocytes in the blood but increased CD16/32 pro-inflammatory macrophages within the tumor, consistent with migration of these pro-inflammatory innate immune cells from the blood to the tumor. MRI with USPIO injection can detect therapeutic responses of innate immune stimulating agents before changes in tumor size have occurred, providing a potential complementary imaging technique to monitor cancer immunotherapies. We show that iron oxide nanoparticles (USPIOs) can be used to label innate immune cells and detect the trafficking of pro-inflammatory monocytes into the glioblastoma. This preceded changes in tumor size, making it a more sensitive imaging technique.

The Effects of Water Temperature and Simulated Angling on the Physiological Stress Response of Largemouth Bass

AbstractThe Largemouth Bass Micropterus salmoides, a popular sport fish, is subjected to multiple sublethal stressors during angling, including high water temperature, exercise, handling, live‐well retention, and weigh‐in procedures. Combined effects of ambient and live‐well temperatures on the stress response and recovery from angling‐induced exercise have not been tested in conditions similar to those encountered in tournaments. Therefore, we assessed the effects of ambient temperature (17, 25, and 33°C) and live‐well temperature differential (−4, 0, and +4°C) on the physiological stress response of Largemouth Bass (mean length = 331 mm) at rest, following a simulated angling stressor, and throughout 8 h of recovery in live wells. Stress variables were measured in whole blood (hematocrit, hemoglobin, pH, partial pressure of oxygen [pO2], partial pressure of carbon dioxide [pCO2], Na+, K+, Ca2+, Cl−, and leukocytes) and plasma (cortisol, glucose, lactate, and osmolality). Fish acclimated to 17°C showed the greatest cortisol response after the chasing stressor; however, higher levels of glucose, lactate, pCO2, K+, and monocyte percentage were found at 33°C, and blood pH, Cl−, and lymphocyte percentage were lower at 33°C than at 17°C. When live‐well temperature was manipulated, cortisol levels were highest in fish subjected to the coldest conditions (acclimated to 17°C and retained in 13°C and 17°C live wells) and the warmest condition (acclimated to 33°C and retained in 37°C live wells). However, all fish subjected to the colder extremes survived, whereas 100% mortality occurred in the warmest condition. Besides cortisol, indicators of stress were less pronounced at colder temperatures. Glucose, lactate, and notably K+ concentrations were highest in 37°C live wells, and blood pH, Ca2+, Na+, and Cl− were lowest. Low blood lymphocytes and high monocytes at the warmest conditions indicate reduced immunocompetence or inflammation. Mortality at high temperature may result from exhaustion of aerobic and anaerobic energy sources, failure to recover from metabolic acidosis, and an inability to regain ionic balance.

Abstract 6179: Increased circulating PD-L1 expressing CD14 high CD16 negative classical monocytes correlate with poor prognosis in cancer patients treated with PD-1 blockade therapies

Abstract Background: Immune checkpoint inhibitors (ICIs), like PD-1 and PD-L1 blockade therapies, are currently considered as one of the most significant breakthroughs in potent cancer immunotherapy. However, many patients are non-responders or develop resistance following an initial response to ICIs. ICIs have no accurate predictive biomarkers. Previously, we showed that increased expression of PD-L1 molecules on CD14+ monocytes was significantly correlated with shorter overall survival (OS) for patients with several cancer types on PD-1 blockade therapies. As monocytes can be further classified into subsets (classical, intermediate, non-classical), we herein investigated the prognostic factors in peripheral blood mononuclear cell (PBMC) subsets and assessed the clinical significance for the PD-L1-expressing subsets of each of these cells including classical, intermediate and non-classical monocyte subsets in patients with various cancer types, along with their clinical implications in PD-1 blockade therapies. Material and Methods: We evaluated 44 patients (20 with gastric cancer, 17 with non-small cell lung carcinoma, and 7 with esophageal cancer) undergoing PD-L1 blockade therapy (pembrolizumab or nivolumab) for PD-L1 expression levels in classical (CD14high, CD16-), intermediate (CD14high, CD16+), and non-classical (CD14low, CD16+) monocytes measured via flow cytometry before ICI treatment. The percentages of PD-L1+ cells in respective monocyte subsets were compared with respect to different clinicopathological conditions and the association with survival time was assessed. Results: The number of PD-1 positive cells in CD14+ monocytes was very low (almost <1%). Also, the monocyte subset expressing PD-L1 had a high percentage of classical monocytes, whereas non-classical monocytes expressed less PD-L1. Higher levels of classical monocytes were correlated with shorter OS (P = 0.044), whereas higher levels of non-classical monocytes were correlated with longer OS (P = 0.027). Focusing on non-small cell lung cancer population (N = 17), higher levels of PD-L1-expressing CD14+ monocytes were statistically correlated with shorter OS (P = 0.025). Furthermore, analysis of a subset of monocytes showed that the higher PD-L1-expressing classical monocytes correlated with the shorter OS, suggesting that they predict poor prognosis (P = 0.0095). Conclusion: Circulating PD-L1-expressing classical monocytes in the peripheral bloods were associated with poor prognosis in patients treated with PD-1 blockade therapies. Among the monocyte subsets, classical monocyte, especially PD-L1-expressing classical monocytes can be a potential biomarker for prognosis in these patients. Citation Format: Ryotaro Ohkuma, Katsuaki Ieguchi, Makoto Watanabe, Tsubasa Goshima, Rie Onoue, Kazuyuki Hamada, Yutaro Kubota, Atsushi Horiike, Toshiaki Tsurui, Risako Suzuki, Nana Iriguchi, Tomoyuki Ishiguro, Yuya Hirasawa, Hirotsugu Ariizumi, Kiyoshi Yoshimura, Mayumi Tsuji, Yuji Kiuchi, Shinichi Kobayashi, Takuya Tsunoda, Satoshi Wada. Increased circulating PD-L1 expressing CD14 high CD16 negative classical monocytes correlate with poor prognosis in cancer patients treated with PD-1 blockade therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6179.

The Association between Daily Dietary Intake of Riboflavin and Lung Function Impairment Related with Dibutyl Phthalate Exposure and the Possible Mechanism

This study aimed to evaluate the relationship between the daily dietary intake of riboflavin (DDIR) and impaired lung function associated with dibutyl phthalate (DBP) exposure. Data of 4631 adults in this national cross-sectional survey were included. Urinary mono-benzyl phthalate (MBP) was used to evaluate the level of DBP exposure. The ln-transformed urinary creatinine-corrected MBP (ln(MBP/UCr)) level was used in the statistical models. High DDIR was defined as the DDIR ≥1.8 mg per day. The results of lung function impairment and high monocytes were significantly higher in the highest MBP group compared with the lowest MBP group. A significant interaction between ln(MBP/UCr) and DDIR (Pinteraction = 0.029) was detected for the risk of lung function impairment. The risk of lung function impairment (ORquartiles4 vs. 1 1.85, 95% CI, 1.27–2.71; Ptrend = 0.018) and high neutrophils (ORquartiles4 vs. 1 1.45, 95% CI, 1.06–1.97; Ptrend = 0.018) was significantly higher in the highest vs. the lowest quartile of MBP in participants with low/normal DDIR but not in in participants with high DDIR. The results of this study showed that high DDIR was associated with less lung function impairment related with DBP exposure, and the inhibiting of the neutrophil recruitment might be the potential mechanism.

A Potential Target for Diabetic Vascular Damage: High Glucose-Induced Monocyte Extracellular Vesicles Impair Endothelial Cells by Delivering miR-142-5p.

Endothelial dysfunction is a key accessory to diabetic cardiovascular complications, and the regulatory role of the extracellular vesicles (EVs) from the innate immune system is growing. We tested whether EVs derived from high glucose-induced monocytes could shuttle microRNAs and impair endothelial cells. EVs from high glucose- and basal glucose-treated THP-1 cells (HG-THP-1 EVs and BG-THP-1 EVs) were isolated and identified. After coculture with THP-1 EVs, human umbilical vein endothelial cells (HUVECs) were tested by proliferation, migration, reactive oxygen species (ROS) detection assays, and western blot for Nrf2/NLRP3 signaling. MiR-142-5p was predicted by miRNAs databases and further verified by RT–qPCR and dual-luciferase reporter gene assays that inhibit Nrf2 expression. The regulation of miR-142-5p in HUVECs was further evaluated. A type 1 diabetes mellitus (T1DM) mouse model was developed for miR-142-5p inhibition. Aorta tissue was harvested for hematoxylin-eosin staining and immunohistochemistry of interleukin-1β (IL-1β). Compared to BG-THP-1 EVs, HG-THP-1 EVs significantly reduced migration and increased ROS production in HUVECs but did not affect proliferation. HG-THP-1 EVs induced suppression of Nrf2 signaling and NLRP3 signaling activation. RT–qPCR results showed that HG-THP-1 EVs overexpressed miR-142-5p in HUVECs. The transfection of miR-142-5p mimics into HUVECs exhibited consistent regulatory effects on HG-THP-1 EVs, whereas miR-142-5p inhibitors demonstrated protective effects. The miR-142-5p antagomir significantly reduced the IL-1β level in T1DM aortas despite morphological changes. To conclude, miR-142-5p transferred by high glucose-induced monocyte EVs participates in diabetic endothelial damage. The inhibition of miR-142-5p could be a potential adjuvant to diabetic cardiovascular protection.

The clinical relevance of lymphocyte to monocyte ratio in patients with Idiopathic Pulmonary Fibrosis (IPF)

Disease course in Idiopathic Pulmonary Fibrosis (IPF) is highly heterogeneous and markers of disease progression would be helpful. Blood leukocyte count has been studied in cancer patients and a reduced lymphocyte to monocyte ratio (LMR) has been show to predict survival. Thus, we aimed to investigate the role of monocytes count and LMR in three distinct population of patients with IPF: 77 newly-diagnosed IPF, 40 with end-stage IPF and 17 IPF with lung cancer. In newly-diagnosed IPF patients, we observed a negative correlation between forced vital capacity (FVC) at diagnosis and both white blood cells and monocytes count (r = −0.24; p = 0.04 and r = −0.27; p = 0.01; respectively). Moreover, a high monocytes count was independently associated with functional decline (OR: 1.004, 95%CI 1.00–1.01; p = 0.03). In newly-diagnosed IPF, the LMR cut-off at diagnosis was 4.18 with an AUC of 0.67 (95%CI 0.5417–0.7960; p = 0.025), and overall survival was significantly worse in patients with a LMR<4.18 compared to patients with a LMR≥4.18 (HR: 6.88, 95%CI 2.55–18.5; p = 0.027). LMR was significantly lower in IPF patients with lung cancer compared to those newly diagnosed with IPF [2.2 (0.8–4.4), 3.5 (0.8–8.8); p < 0.0001] and those with end-stage disease [3.6 (2–6.5); p < 0.0001]. In conclusion, a LMR<4.18 is associated with significantly shorter survival in newly-diagnosed IPF patients. In addition, LMR is significantly lower in patients with IPF and lung cancer compared to patients with newly-diagnosed IPF. High monocytes count at baseline negatively correlates with FVC and is an independent predictor of disease progression in newly-diagnosed IPF patients.