Pro-inflammatory Myeloid Cells Research Articles

Bruton Tyrosine Kinase Inhibition Decreases Inflammation and Differentially Impacts Phagocytosis and Cellular Metabolism in Mouse- and Human-derived Myeloid Cells.

Bruton tyrosine kinase (BTK) is a kinase expressed by various immune cells and is often activated under proinflammatory states. Although the majority of BTK-related research has historically focused on B cells, understanding the role of BTK in non-B cell populations is critical given myeloid cells also express BTK at comparable levels. In this study, we investigated and compared how BTK inhibition in human and murine myeloid cells alters cell phenotype and function. All experiments were performed using two BTK inhibitors (evobrutinib and tolebrutinib) that are currently in late-stage clinical trials for the treatment of multiple sclerosis. Assays were performed to assess the impact of BTK inhibition on cytokine and microRNA expression, phagocytic capacity, and cellular metabolism. In all cells, both evobrutinib and tolebrutinib significantly decreased phosphorylated BTK and LPS-induced cytokine release. BTK inhibition also significantly decreased the oxygen consumption rate and extracellular acidification rate in myeloid cells, and significantly decreased phagocytosis in murine-derived cells, but not human macrophages. To further elucidate the mechanism, we also investigated the expression of microRNAs known to impact the function of myeloid cells. BTK inhibition resulted in an altered microRNA expression profile (i.e., decreased miR-155-5p and increased miR-223-3p), which is consistent with a decreased proinflammatory myeloid cell phenotype. In summary, these results provide further insights into the mechanism of action of BTK inhibitors in the context of immune-related diseases, while also highlighting important species-specific and cell-specific differences that should be considered when interpreting and comparing results between preclinical and human studies.

Abstract 4886: An in-depth single-cell map of inflammatory breast cancer reveals wide-scaled restructuring associated to an immunosuppressive and inflammation-like microenvironment

Abstract Inflammatory Breast Cancer (IBC) has increasingly been recognised as a distinct form of breast cancer, that is characterised by its distinctive clinical presentation, as well as its fast onset and poor prognosis. Despite previous efforts, the underlying molecular heterogeneity of IBC remains poorly understood. We used single-cell transcriptomics to dissect the transcriptional landscape of IBC at single-cell resolution, and elucidate differences to non-inflammatory breast cancer (non-IBC), of which we created and curated a subtype-matched comparison dataset. Our analysis revealed a previously underappreciated role of the microenvironment in driving the immune suppressive and angiogenic phenotype of IBC. Furthermore, we identified key transcriptional signatures associated with IBC aggressiveness, including dysregulated pathways across all major cell compartments. Within each compartment, we used a combination of approaches to assign functional roles to subsets of cells, including expression of marker genes, gene programmes and projection onto thoroughly annotated reference-spaces. We thus identify cell populations present in significantly different proportions between IBC and non-IBC. Interestingly, among the populations enriched in IBC we identified iCAFs, pro-inflammatory M1-like myeloid cells as well as regulatory T cells. In non-IBC on the other hand, enriched cell types include myCAFs, M2-like myeloid cells and cytotoxic T cells.In addition to differences in the cell composition, we also find differences in the expression of certain markers and gene programmes. A pseudo-time analysis of T cells reveals a shift towards an exhausted T cell phenotype in IBC, in line with a lower expression of HLA genes in its epithelial compartment. In agreement with the known angiogenic phenotype, we also see an enrichment in mitogenic signatures in IBC-associated endothelial cells. In summary, our data describe an immunosuppressive environment, highlighted by the presence of cell types with an inflammation-like phenotype, signs of immune evasion and exhaustion and angiogenesis. Our comprehensive and unique single-cell transcriptomic dataset of this rare form of breast cancer provides valuable insights into the complex cellular composition of IBC, offering a foundation for further functional exploration of involved pathways, and finally development of new targeted therapy approaches. Our study contributes to a deeper understanding of IBC biology and pathology, enabling future identification of direly needed biomarkers and therapeutic approaches for this hard-to-treat breast cancer type. Citation Format: Paul Schwerd-Kleine, Tasneem Cheytan, Roberto Würth, Ewgenija Gutjahr, Laura Michel, Verena Thewes, Peter Lichter, Andreas Schneeweiss, Andreas Trumpp, Martin Sprick. An in-depth single-cell map of inflammatory breast cancer reveals wide-scaled restructuring associated to an immunosuppressive and inflammation-like microenvironment [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 4886.

Abstract 6938: Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant phenotypes and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was hidden in the bulk tumor, and the extended granularity of NF-1 glioma tumor microenvironment (TME) remained still unexplored. Here, we collected glioma samples from 46 NF-1 patients including 22 high-grade (HGG) and 24 low-grade (LGG) tumors, and we analyzed their gene expression by single nuclei RNA sequencing. A total of 239,044 single cells were classified into tumor and non-tumor components by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, and clustering). We defined the pattern of intra-tumor heterogeneity of NF-1 glioma cells using non-negative matrix factorization and derived 7 malignant meta-programs (MPs) that we respectively defined as Neuronal-like, EMT, Astrocyte-like, Dividing Radial Glia-like, Ependymal-like, Immune, and Glycolytic/Hypoxic-like. These MPs recapitulated normal brain cell subtypes, thereby reflecting broad cell plasticity. The non-tumor cell compartment (121,364 cells, 51%) was dissected for the characterization of the cell types that populate the TME of NF-1 glioma. We identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma ecomodules were highlighted by comparing the relative composition of the TME across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that could mediate a potential anti-tumor response in low-grade NF-1 glioma (LGG immune high). Conversely, regulatory T cell infiltration and effector T cell exhaustion induced immune suppression in a low-grade glioma immune dysfunctional ecomodule. The absence of lymphocytes characterized a large set of cold tumors, mostly including high-grade glioma. Together, the complex interplay of tumor cell states with different TME compartments elucidated the existence of separate ecomodules in NF-1 glioma, with the LGG immune high TME associated with Neuronal-like and the LGG immune dysfunctional with Ependymal-like tumor cells. The Ependymal-like state also exhibits maximal association with brain-specific normal cells, including oligodendrocytes, neurons and astrocytes, whereas the HGG are enriched with Dividing Radial Glia- and Glycolytic/Hypoxic-like tumor cell states. The elucidation of different ecomodules provides novel insights for the application of targeted therapies in NF-1 glioma patients. Citation Format: Luciano Garofano, Fulvio D'Angelo, Michael Oh, Michele Ceccarelli, Franck Bielle, Marc Sanson, Anna Lasorella, Antonio Iavarone. Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1 [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 6938.

Single-cell RNA sequencing reveals myeloid and T cell co-stimulation mediated by IL-7 anti-cancer immunotherapy

BackgroundImmune checkpoint inhibitors unleash inhibitory signals on T cells conferred by tumors and surrounding stromal cells. Despite the clinical efficacy of checkpoint inhibitors, the lack of target expression and persistence of immunosuppressive cells limit the pervasive effectiveness of the therapy. These limitations may be overcome by alternative approaches that co-stimulate T cells and the immune microenvironment.MethodsWe analyzed single-cell RNA sequencing data from multiple human cancers and a mouse tumor transplant model to discover the pleiotropic expression of the Interleukin 7 (IL-7) receptor on T cells, macrophages, and dendritic cells.ResultsOur experiment on the mouse model demonstrated that recombinant IL-7 therapy induces tumor regression, expansion of effector CD8 T cells, and pro-inflammatory activation of macrophages. Moreover, spatial transcriptomic data support immunostimulatory interactions between macrophages and T cells.ConclusionThese results indicate that IL-7 therapy induces anti-tumor immunity by activating T cells and pro-inflammatory myeloid cells, which may have diverse therapeutic applicability.

COVID-19 immune signatures in Uganda persist in HIV co-infection and diverge by pandemic phase

Little is known about the pathobiology of SARS-CoV-2 infection in sub-Saharan Africa, where severe COVID-19 fatality rates are among the highest in the world and the immunological landscape is unique. In a prospective cohort study of 306 adults encompassing the entire clinical spectrum of SARS-CoV-2 infection in Uganda, we profile the peripheral blood proteome and transcriptome to characterize the immunopathology of COVID-19 across multiple phases of the pandemic. Beyond the prognostic importance of myeloid cell-driven immune activation and lymphopenia, we show that multifaceted impairment of host protein synthesis and redox imbalance define core biological signatures of severe COVID-19, with central roles for IL-7, IL-15, and lymphotoxin-α in COVID-19 respiratory failure. While prognostic signatures are generally consistent in SARS-CoV-2/HIV-coinfection, type I interferon responses uniquely scale with COVID-19 severity in persons living with HIV. Throughout the pandemic, COVID-19 severity peaked during phases dominated by A.23/A.23.1 and Delta B.1.617.2/AY variants. Independent of clinical severity, Delta phase COVID-19 is distinguished by exaggerated pro-inflammatory myeloid cell and inflammasome activation, NK and CD8+ T cell depletion, and impaired host protein synthesis. Combining these analyses with a contemporary Ugandan cohort of adults hospitalized with influenza and other severe acute respiratory infections, we show that activation of epidermal and platelet-derived growth factor pathways are distinct features of COVID-19, deepening translational understanding of mechanisms potentially underlying SARS-CoV-2-associated pulmonary fibrosis. Collectively, our findings provide biological rationale for use of broad and targeted immunotherapies for severe COVID-19 in sub-Saharan Africa, illustrate the relevance of local viral and host factors to SARS-CoV-2 immunopathology, and highlight underemphasized yet therapeutically exploitable immune pathways driving COVID-19 severity.

Nonpreferential but Detrimental Accumulation of Macrophages With Clonal Hematopoiesis-Driver Mutations in Cardiovascular Tissues-Brief Report.

Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired genetic risk factor for both leukemia and cardiovascular disease. It results in proinflammatory myeloid cells in the bone marrow and blood; however, how these cells behave in the cardiovascular tissue remains unclear. Our study aimed at investigating whether CHIP-mutated macrophages accumulate preferentially in cardiovascular tissues and examining the transcriptome of tissue macrophages from DNMT3A (DNA methyltransferase 3 alpha) or TET2 (Tet methylcytosine dioxygenase 2) mutation carriers. We recruited patients undergoing carotid endarterectomy or heart surgeries to screen for CHIP mutation carriers using targeted genomic sequencing. Myeloid and lymphoid cells were isolated from blood and cardiovascular tissue collected during surgeries using flow cytometry. DNA and RNA extracted from these sorted cells were subjected to variant allele frequency measurement using droplet digital polymerase chain reaction and transcriptomic profiling using bulk RNA sequencing, respectively. Using droplet digital polymerase chain reaction, we detected similar variant allele frequency of CHIP in monocytes from blood and macrophages from atheromas and heart tissues, even among heart macrophages with and without CCR2 (C-C motif chemokine receptor 2) expression. Bulk RNA sequencing revealed a proinflammatory gene profile of myeloid cells from DNMT3A or TET2 mutation carriers compared with those from noncarriers. Quantitatively, CHIP-mutated myeloid cells did not preferentially accumulate in cardiovascular tissues, but qualitatively, they expressed a more disease-prone phenotype.

MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS

Abstract BACKGROUND & AIMS After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients with and without CDP using single cell analyses. METHODS Endoscopic samples from the pouch and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8). RESULTS We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases. CONCLUSIONS CDP demonstrates altered immune and non-immune cell populations/states in the pouch and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones. Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p<0.01. Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.

P048 Identification of IL36RN missense mutations in Crohn’s disease patients as a potentially new drug target for treating rare patients with over-activation of the IL36 signaling cascade

Abstract Background The interleukin 36 (IL-36) family includes the agonists IL-36α, IL-36β, IL-36γ, which activate the NF-κB pathway and the natural antagonist IL-36RA, which inhibits IL-36 signaling. While missense mutations in the gene encoding IL-36RA (IL36RN) are associated with severe skin inflammation, their role in Crohn’s disease (CD) is currently unknown. Therefore, we here investigated if IL36RN mutations contribute to inflammation in CD and whether IL-36 signaling might represent a potential drug target for personalized therapy. Methods Mutations in IL36RN were identified by whole exome sequencing (WES) followed by targeted Sanger sequencing. Peripheral blood mononuclear cells (PBMCs) of affected patients were analyzed by mass cytometry and serum cytokines were measured by ELISA. Identified IL36RN mutations were characterized by overexpression experiments and functional assays. Clinical and molecular responses of one IL-36RN mutant patient to combined treatment with spesolimab and certolizumab pegol was assessed by fecal calprotectin, endoscopy, RNA sequencing and mass cytometry at week 0 and 12 of treatment. Results In a cohort of 47 ulcerative colitis patients, 177 CD patients and 34 healthy controls, we identified 3 CD patients with heterozygous missense mutations in IL36RN (IL-36RA S113L, IL-36RA P76L and IL-36RA L133I). In the patient carrying the IL-36RA S113L variant, we detected elevated serum levels of IL-18 and IL-23, as well as an increased frequency of TH17 cells in PBMCs, suggesting an overactivation of the IL-36 pathway. Overexpression experiments showed that IL-36RA S113L causes decreased expression of IL-36RA, resulting in increased IL-36 signaling. The patient was therefore treated with 1200 mg of the IL-36R blocking antibody spesolimab every 4 weeks and 200 mg of certolizumab pegol biweekly on the basis of an Out Of Scope request. This approach resulted in reduced intestinal inflammation and a reduction in the frequency of pro-inflammatory myeloid cells in PBMCs. Conclusion In this study, we show that pathogenic IL36RN mutations are present in a subset of CD patients and that blocking IL-36 signaling may represent a personalized therapeutic approach for this rare subset of patients in whom conventional biologic therapies have failed. Disclaimer Boehringer Ingelheim was given the opportunity to review the abstract for medical and scientific accuracy as well as intellectual property considerations in relation to the potential mention of BI substances. Boehringer Ingelheim had no role in the design, analysis or interpretation of the results in this study. Spesolimab was made available on the basis of an out of scope request. Boehringer Ingelheim was given the opportunity to review this abstract as a courtesy.

Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis

ObjectiveWheat has become a main staple globally. We studied the effect of defined pro-inflammatory dietary proteins, wheat amylase trypsin inhibitors (ATI), activating intestinal myeloid cells via toll-like receptor 4, in...

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomic analysis of lungs from JHU083-treated Mtb-infected mice reveals citrulline accumulation, suggesting elevated nitric oxide (NO) synthesis, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. JHU083-treated macrophages also produce more NO potentiating their antibacterial activity. When tested in an immunocompromised mouse model of Mtb infection, JHU083 loses its therapeutic efficacy suggesting the drug’s host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

EPCO-35. IDENTIFICATION OF DIVERSE IMMUNE PHENOTYPES IN NF-1 GLIOMA BY SINGLE NUCLEI GENE EXPRESSION ANALYSIS

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the high molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant clones and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was mostly hidden in the bulk tumor mass, and the extended granularity of NF-1 glioma immune microenvironment remained still unexplored. Here, to address this challenge, we collected glioma samples from 53 NF-1 patients including 27 high-grade and 26 low-grade tumors, and we analyzed their gene expression at the highest resolution through single nuclei RNA sequencing. A total of 288,372 single cells have been classified by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, pathway-based deconvolution and integrative non-negative matrix factorization). The non-tumor cell compartment (124,082 cells, 43%) has been dissected for the deep characterization of each cell type that populate the microenvironment of NF-1 glioma. We unraveled the complexity of the immune infiltration and identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma niches have been revealed by comparing the relative composition of the immune microenvironment across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that can mediate a potential anti-tumor response in low-grade NF-1 glioma. Regulatory T cell infiltration and effector T cell exhaustion, instead, induced immune suppression in another tumor niche. Conversely, the complete absence of lymphocytes characterized a large set of cold tumors, mostly high-grade glioma. The elucidation of the complex immune interplay in the different tumor niches provides novel insights for the application of immunotherapies in NF-1 glioma patients.

Haematopoietic loss of KDM6A impairs cardiac recovery post myocardial infarction via pro-inflammatory myeloid cells

Abstract Background Ischaemic heart failure (IHF) remains a leading cause of death worldwide. Clonal haematopoiesis has emerged as a major risk factor for IHF and patients with CH have worse outcomes after acute myocardial infarction (AMI). CH is characterized by clonal expansion of a haematopoietic stem cell that carries a leukaemogenic mutation. Recently, acquired loss-of-function mutations in the histone demethylase KDM6A were identified as CH-drivers in IHF patients. KDM6A is frequently mutated in human cancer and acts as a tumour suppressor in acute myeloid leukaemia. However, the role of KDM6A in the regulation of cardiac damage after MI is not understood. Purpose Investigate the effect of leukocyte specific loss of KDM6A in the development of IHF after AMI in mice and humans. Methods/Results A mouse model of haematopoietic specific loss of Kdm6a (VaviCre-Kdm6aflox) combined with a model of myocardial injury was employed to investigate the role of Kdm6a in leukocytes in post-MI cardiac recovery. Firstly, following 28 days (D) post-MI, mice lacking Kdm6a in haematopoietic cells (KDM6A KO) showed significantly worse cardiac ejection fraction compared to controls (controls 43.98 ± 2.55 % vs KDM6A KO 31.86 ± 3.23 % recovery from baseline, P=0.009). This was accompanied by an increase in cardiac scar size at D28 post-MI (controls 22.46±5.25% vs KDM6A KO 56.83±11.1 % area, P<0.0001). Moreover, KDM6A KO mice showed peripheral blood monocytosis (controls 33793±5644 vs KDM6A KO 76227±14035 monocytes/ml blood, P =0.008) at D3 post-MI, which was accompanied by an increased influx of CD11b+ myeloid cells in the injured heart (7804±1023 vs 11444±1436 CD11b+ cells/mg tissue, P=0.05), as well as by an increased expression of pro-inflammatory marker TNF-α (controls 1.01±0.14 vs KDM6A KO 2.16±0.45 fold change, P=0.04) in the ischaemic myocardium. These data were corroborated by single-cell RNA sequencing (scRNA-seq) profiling of CD45+ cardiac leukocytes 3D post-MI. Here, transcriptomically distinct subsets of macrophages/monocytes showed upregulation of pro-inflammatory markers such as Il1-β, Nlrp3, Cxcl2, and Nfkb1 (P < 0.05, Bonferroni correction). Finally, scRNA-seq of circulating blood cells from IHF patients carrying KDM6A CH-driver mutations showed elevated levels of pro-inflammatory genes such as IL32, IL7R and CCL5 in CD14+ classical monocytes (P < 0.05, Bonferroni correction) compared to non-carriers. Conclusions In summary, this study shows that loss of KDM6A in myeloid cells amplifies acute cardiac inflammation and impairs recovery of left ventricular function after AMI accompanied by increased pro-inflammatory myeloid cells in the heart. Further interrogation of genes identified in the aforementioned murine and human sequencing data and subsequent in vitro mechanistic studies will be essential to identify the mechanism by which loss of KDM6A results in altered leukocyte function and worse HF prognosis post-MI.

Non-preferential, but detrimental accumulation of macrophages with clonal hematopoiesis-driver mutations in cardiovascular tissues

Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired genetic risk factor of cardiovascular (CV) disease transmitted by myeloid cells. Harboring CHIP is associated with higher risk of coronary artery disease and worse prognosis of congestive heart failure. Studies show that circulating monocytes of CHIP carriers are more pro-inflammatory than those of non-carriers, but to what extend mutant monocytes and their progenies accumulate in the tissue of CV lesions remains unknown. First, we aimed to identify the frequencies of mutant macrophages in human carotid plaque and heart tissues relative to those of mutant monocytes in the patients’ blood. Second, we compared the gene expression profiles of circulating and tissue myeloid cells of CHIP carriers with those from non-carriers. Methods We recruited patients undergoing carotid endarterectomy and aortic valve replacement. Blood and CV tissue (carotid plaque or right atrium appendix) of patients who provided study consents were retrieved during the surgeries and immediately processed for the isolation of lymphocytes and myeloid cells. Whole blood DNA was subjected to CHIP mutation screening. DNA and RNA of eight DNMT3A or TET2 mutation carriers and eight matched non-carriers were isolated from sorted cells. We measured the frequencies of CHIP mutations in each sorted cell type using a droplet-digital PCR method. RNA of the sorted myeloid cells from the carriers and matched non-carriers were subjected to RNA sequencing. Results Our data showed that CHIP-mutant monocytes in the blood is similar to that of CHIP-mutant macrophages in both the plaque (ratio versus monocytes =1.2, p=0.44) and the heart (ratio versus monocytes=0.89, p=0.47), suggesting a comparable ability of mutant and non-mutant monocytes to accumulate in the cardiovascular lesions. Near absence of CHIP clones in lymphocytes (ratio versus monocytes=0.06, p<0.0001) confirmed the myeloid skewing driven by DNMT3A or TET2 mutations. Unexpectedly, we detected similar size of CHIP clones in CCR2-positive and CCR2-negative cardiac macrophages (ratio=0.95, p=0.77), challenging the current paradigm that CCR2-negative cardiac macrophages represent a resident population with minimal monocyte contribution. Transcriptomic profiles of blood and tissue monocytes/macrophages from these carriers were distinct from those of eight matched non-carriers, featuring differentially up-regulated cytokines (eg. IL1B, S100A9) and chemokines (eg. CXCL1, CCL3) in cells from the carriers. Myeloid cells from the carriers up-regulated genes enriching pathways related to immune activation and inflammation. Conclusion Our study manifested the presence of pro-inflammatory CHIP-mutant myeloid cells in human plaque and heart tissues. The equivalent size of CHIP clones in the circulation and tissue suggests a comparable ability of mutant and non-mutant monocytes to accumulate in CV lesions, but inflammation may mediate CHIP-associated CV diseases.

Abstract 15818: Association of Clonal Hematopoiesis of Indeterminate Potential With Incident Heart Failure With Preserved Ejection Fraction

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is the age-related expansion of blood cells with somatic mutations, most commonly in DMNT3A , TET2 , and ASXL1 . CHIP is a risk factor for coronary heart disease as well as heart failure (HF). Experimental HF models suggest a role of CHIP pro-inflammatory myeloid cells on cardiac dysfunction via fibrotic remodeling and hypertrophy. However, whether CHIP associates with incident HF with preserved ejection fraction (HFpEF), HF with reduced ejection fraction (HFrEF), or both is currently unknown. Hypothesis: We hypothesized that CHIP is more strongly related to HFpEF than HFrEF. Methods: We studied a multiethnic sample of postmenopausal women without prevalent HF from the Women’s Health Initiative (WHI). Whole genome sequencing was performed through the NHLBI TOPMed Program, and CHIP was detected based on 74 pre-specified myeloid driver mutations. Participants were followed through March 2020 with physician adjudication of HFrEF and HFpEF. Cox models tested the association of CHIP with incident HFpEF and HFrEF with adjustment for demographic/clinical risk factors and inverse probability weighting to account for the TOPMed sampling scheme. Results: Among 5,214 women, the mean age was 62 years, and 7.8% had CHIP. Over a median follow-up of 15 years, age-adjusted HFpEF incidence rates were 5.86 (95%CI 5.63-6.10) vs. 3.72 (95%CI 3.68-3.77) per 1,000 person-years among CHIP carriers vs. non-carriers. After multivariable adjustment, CHIP was associated with a 42% increased risk of incident HFpEF (HR 1.42, 95%CI 6-91%; P =0.02). In contrast, there was no evidence of association between CHIP and HFrEF ( Table 1 ). HFpEF was more strongly associated with TET2 CHIP (HR 2.50, 95%CI 1.54-4.06; P <0.001) than with DNMT3A or ASXL1 ( Table 1 ). Conclusions: CHIP, especially TET2 CHIP, is associated with incident HFpEF. This finding has potential precision medicine implications for HFpEF prevention and treatment.

Estrogen receptor blockade and radiation therapy cooperate to enhance the response of immunologically cold ER+ breast cancer to immunotherapy

BackgroundMost patients with estrogen receptor positive (ER+) breast cancer do not respond to immune checkpoint inhibition (ICI); the tumor microenvironment (TME) of these cancers is generally immunosuppressive and contains few tumor-infiltrating lymphocytes. Radiation therapy (RT) can increase tumor inflammation and infiltration by lymphocytes but does not improve responses to ICIs in these patients. This may result, in part, from additional effects of RT that suppress anti-tumor immunity, including increased tumor infiltration by myeloid-derived suppressor cells and regulatory T cells. We hypothesized that anti-estrogens, which are a standard of care for ER+ breast cancer, may ameliorate these detrimental effects of RT by reducing the recruitment/ activation of suppressive immune populations in the radiated TME, increasing anti-tumor immunity and responsiveness to ICIs.MethodsTo interrogate the effect of the selective estrogen receptor downregulator, fulvestrant, on the irradiated TME in the absence of confounding growth inhibition by fulvestrant on tumor cells, we used the TC11 murine model of anti-estrogen resistant ER+ breast cancer. Tumors were orthotopically transplanted into immunocompetent syngeneic mice. Once tumors were established, we initiated treatment with fulvestrant or vehicle, followed by external beam RT one week later. We examined the number and activity of tumor infiltrating immune cells using flow cytometry, microscopy, transcript levels, and cytokine profiles. We tested whether fulvestrant improved tumor response and animal survival when added to the combination of RT and ICI.ResultsDespite resistance of TC11 tumors to anti-estrogen therapy alone, fulvestrant slowed tumor regrowth following RT, and significantly altered multiple immune populations in the irradiated TME. Fulvestrant reduced the influx of Ly6C+Ly6G+ cells, increased markers of pro-inflammatory myeloid cells and activated T cells, and augmented the ratio of CD8+: FOXP3+ T cells. In contrast to the minimal effects of ICIs when co-treated with either fulvestrant or RT alone, combinatorial treatment with fulvestrant, RT and ICIs significantly reduced tumor growth and prolonged survival.ConclusionsA combination of RT and fulvestrant can overcome the immunosuppressive TME in a preclinical model of ER+ breast cancer, enhancing the anti-tumor response and increasing the response to ICIs, even when growth of tumor cells is no longer estrogen sensitive.

Investigating the role of "Immature Myeloid Cells" as Drivers of Inflammation and Disease Persistence in Psoriatic Arthritis.

Despite the development of treatments targeting T cell co-stimulation and cytokines TNF, IL-12/23, and IL-17, less than half of patients within clinical trials achieve high levels of clinical response. This fact, as well as the absence of prognostic biomarkers represents major unmet clinical needs that necessitate further investigation of the disease pathophysiology. Myeloid cells are critical components of PsA inflammatory mechanisms, being a highly prevalent immune population in biopsies of PsA target tissues, such as the skin and the synovium. Through their antigen-presenting capacity and their pro-angiogenic and pro-inflammatory properties myeloid cells could contribute to persistent inflammation in PsA leading to treatment-resistant disease. To this end, we have recently shown the expansion of monocytes in the blood of PsA patients compared to healthy subjects. Importantly, we have also identified an immature myeloid cell population in patients with highly active, refractory disease, indicating the presence of an "emergency myelopoiesis" process in PsA. In this research protocol, we aim to decipher the pro-inflammatory "myeloid signature" in patients with active PsA and explore the role of immature myeloid cells in disease pathophysiology and their potential as prognostic biomarkers. To address this, we will isolate and analyse monocytes and immature myeloid cells from PsA patients -before and after a 6-month treatment course- focusing on differences between responders and non-responders. In this context, we will perform a thorough phenotypic and functional analysis of these cells, identify their expression signature in an already established whole blood RNA-seq dataset and investigate their presence in target tissues, such as the skin and synovial fluid. This study will elucidate the role of myeloid cells in disease propagation by further defining the involvement of immature myeloid cells in PsA.

Intranasal Delivery of TGFα – A Novel Therapeutic Approach for Lesion Resolution in Multiple Sclerosis? (P7-3.001)

<h3>Objective:</h3> We here evaluate the therapeutic potential of tissue-protective TGFα for the resolution of CNS inflammation. <h3>Background:</h3> After acute lesions in the central nervous system (CNS) such as in Multiple Sclerosis (MS), the interaction of microglia, astrocytes and infiltrating immune cells defines the inflammatory microenvironment in the CNS which can either induce resolution of inflammation or lead to its chronification. However, the interaction between microglia, astrocytes and infiltrating immune cells and their respective contribution to the resolution of tissue damage is poorly characterized. In this context, we have recently identified microglia-derived TGFα as a novel driver of recovery in MS and its preclinical animal model experimental autoimmune encephalomyelitis (EAE). Here, we describe its regulation over the course of the disease and investigate its translational potential during acute inflammation in the CNS. <h3>Design/Methods:</h3> We here use CRISPR–Cas9-based genetic perturbation models, high-dimensional flow cytometry, immunohistochemistry and magnetic resonance imaging (MRI), as well as fine-tuned <i>in vitro</i> assays to investigate cellular targets, net effects and therapeutic potential of microglia-derived TGFα for recovery in MS and its preclinical animal model experimental autoimmune encephalomyelitis (EAE). <h3>Results:</h3> During acute CNS inflammation, microglia secrete TGFα in a highly regulated temporospatial manner. We show that microglial TGFα is required for EAE recovery by decreasing the number of infiltrating T cells and pro-inflammatory myeloid cells, as well as reducing oligodendrocyte loss, axonal damage, and demyelination. In a therapeutic approach, intranasally delivered TGFα diminishes the pro-inflammatory profile of astrocytes and CNS infiltrating immune cells, while promoting neuronal survival and lesion resolution, therefore promoting clinical recovery. <h3>Conclusions:</h3> Together, we show that TGFα acts on glial and infiltrating cells to promote the resolution of CNS inflammation. TGFα signaling might represent a novel checkpoint of CNS recovery, which offers therapeutic potential to promote recovery in acute and chronic stages of MS. <b>Disclosure:</b> Mrs. Loesslein has nothing to disclose. Mr. Linnerbauer has nothing to disclose. Dr. Tsaktanis has nothing to disclose. Dr. Quintana has nothing to disclose. Prof. Rothhammer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Prof. Rothhammer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Merck. The institution of Prof. Rothhammer has received research support from German Research Foundation. The institution of Prof. Rothhammer has received research support from European Research Council.

PD02-04 SINGLE CELL TRANSCRIPTIONAL PROFILES OF BENIGN PROSTATIC HYPERPLASIA

You have accessJournal of UrologyCME1 Apr 2023PD02-04 SINGLE CELL TRANSCRIPTIONAL PROFILES OF BENIGN PROSTATIC HYPERPLASIA Rei Unno, Jon Akutagawa, Hanging Song, Heiko Yang, Thomas Chi, and Franklin W. Huang Rei UnnoRei Unno More articles by this author , Jon AkutagawaJon Akutagawa More articles by this author , Hanging SongHanging Song More articles by this author , Heiko YangHeiko Yang More articles by this author , Thomas ChiThomas Chi More articles by this author , and Franklin W. HuangFranklin W. Huang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) affects the majority of aging men and leads to lower urinary tract symptoms. Based on histological characteristics and bulk sequencing data, the pathophysiology and underlying mechanisms of BPH are poorly understood. The aim of this study was to investigate the transcriptional profile of BPH at single cell resolution to improve our understanding of the molecular features of BPH. METHODS: Fifteen BPH specimens were collected from patients undergoing holmium laser enucleation of the prostate. Cells from each specimen were isolated, captured, and sequenced using a bead-based single-cell RNA sequencing platform (Seq-well). Transcriptomic analysis of the gene expression matrices was performed using software including Scanpy and cellxgene. The absence of malignant pathologic diagnoses was independently confirmed. RESULTS: 16,234 cells from 15 BPH specimens were analyzed. By canonical cell type markers, we annotated three major cell types (epithelial, stromal, and immune cells). Using significantly upregulated genes in BPH compared to normal prostates previously derived from a bulk RNA sequencing comparison, the signature score of the bulk sequencing data was high only in the fibroblast populations. After performing a sub-clustering analysis of the 6249 epithelial cells, we identified four luminal cell subclusters showing similar levels of luminal epithelial markers KLK3, KLK2, and NKX3-1, as well as one distinct basal and one club cell population. Analysis of the differentially expressed genes among the four luminal subclusters revealed that luminal subcluster 4 was enriched in a specific signature gene set, including ribosomal genes and KLK4, which has been shown to be abundant in BPH, compared to the other subclusters. Gene ontology analysis using this gene set demonstrated that luminal subcluster 4 was associated with pathways such as oxidative phosphorylation and adipogenesis. Analyses of the BPH immune microenvironment showed a larger proportion of pro-inflammatory cells, such as M1 macrophages, compared to anti-inflammatory cells within the myeloid cell population. CONCLUSIONS: Single cell profiling of BPH identified epithelial cells, stromal cells, and immune cells. We confirmed the upregulation of previously derived BPH-associated genes in the fibroblast population and the enrichment of pro-inflammatory myeloid cells in the immune microenvironment. Within the epithelial cell population, we identified a luminal epithelial subcluster that may represent a cell state associated with BPH. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e70 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rei Unno More articles by this author Jon Akutagawa More articles by this author Hanging Song More articles by this author Heiko Yang More articles by this author Thomas Chi More articles by this author Franklin W. Huang More articles by this author Expand All Advertisement PDF downloadLoading ...

Stroke-induced hexokinase 2 in circulating monocytes exacerbates vascular inflammation and atheroprogression

BackgroundStroke accelerates inflammatory monocyte recruitment to the endothelium and consequent atheroprogression via high-mobility group box 1-receptor for advanced glycation end products signaling. Notably, Hmgb1 interacts with multiple toll-like receptors (TLRs) and promotes TLR4-mediated proinflammatory myeloid cell activation. Therefore, TLR-associated mechanism(s) within monocytes may play a role in Hmgb1-driven poststroke atheroprogression. ObjectivesWe aimed to elucidate the TLR-associated mechanism(s) within monocytes that contribute to stroke-induced exacerbation of atherosclerotic disease. MethodsA weighted gene coexpression network analysis on the whole blood transcriptomes of stroke model mice identified hexokinase 2 (HK2) as a key gene associated with TLR signaling in ischemic stroke. We conducted a cross-sectional analysis of monocyte HK2 levels in patients with ischemic stroke patients. We performed in vitro and in vivo studies using high-cholesterol diet–fed myeloid-specific Hk2-null ApoE−/− (ApoE−/−;Hk2ΔMφ) mice and ApoE−/−;Hk2fl/fl controls. ResultsWe found markedly higher monocyte HK2 levels in patients with ischemic stroke patients during the acute and subacute phases poststroke. Similarly, stroke model mice displayed a profound increase in monocyte Hk2 levels. Using aortas and aortic valve samples collected from high-cholesterol diet–fed ApoE−/−;Hk2ΔMφ mice and ApoE−/−;Hk2fl/fl controls, we found that stroke-induced monocyte Hk2 upregulation enhanced poststroke atheroprogression and inflammatory monocyte recruitment to the endothelium. Stroke-induced monocyte Hk2 upregulation induced inflammatory monocyte activation, systemic inflammation, and atheroprogression via Il-1β. Mechanistically, we demonstrated that stroke-induced monocyte Hk2 upregulation was dependent upon Hmgb1-driven p38-dependent hypoxia-inducible factor-1α stabilization. ConclusionStroke-induced monocyte Hk2 upregulation is a key mechanism underlying poststroke vascular inflammation and atheroprogression.

NF-κB-Inducing Kinase Governs the Mitochondrial Respiratory Capacity, Differentiation, and Inflammatory Status of Innate Immune Cells.

NF-κB-inducing kinase (NIK), which is essential for the activation of the noncanonical NF-κB pathway, regulates diverse processes in immunity, development, and disease. Although recent studies have elucidated important functions of NIK in adaptive immune cells and cancer cell metabolism, the role of NIK in metabolic-driven inflammatory responses in innate immune cells remains unclear. In this study, we demonstrate that murine NIK-deficient bone marrow-derived macrophages exhibit defects in mitochondrial-dependent metabolism and oxidative phosphorylation, which impair the acquisition of a prorepair, anti-inflammatory phenotype. Subsequently, NIK-deficient mice exhibit skewing of myeloid cells characterized by aberrant eosinophil, monocyte, and macrophage cell populations in the blood, bone marrow, and adipose tissue. Furthermore, NIK-deficient blood monocytes display hyperresponsiveness to bacterial LPS and elevated TNF-α production ex vivo. These findings suggest that NIK governs metabolic rewiring, which is critical for balancing proinflammatory and anti-inflammatory myeloid immune cell function. Overall, our work highlights a previously unrecognized role for NIK as a molecular rheostat that fine-tunes immunometabolism in innate immunity, and suggests that metabolic dysfunction may be an important driver of inflammatory diseases caused by aberrant NIK expression or activity.