Pub Date : 2024-09-14DOI: 10.1101/2024.09.09.612126
Jie Liu, Fengyang Lei, Bin Yan, Naiwen Cui, Jyoti Sharma, Victor Correa, Lara Roach, Savvas Kikolaou, Kristen Pitts, James Chodosh, Daniel E Maidana, Demetrios G Vavvas, Huidan Zhang, David Weitz, Raul Mostoslavsky, Eleftherios I Paschalis
The identification of specific markers for microglia has been a long-standing challenge. Recently, markers such as P2ry12, TMEM119, and Fcrls have been proposed as microglia-specific and widely used to explore microglial functions within various central nervous system (CNS) contexts. The specificity of these markers was based on the assumption that circulating monocytes retain their distinct signatures even after infiltrating the CNS. However, recent findings reveal that infiltrating monocytes can adopt microglia-like characteristics while maintaining a pro-inflammatory profile upon permanent engraftment in the CNS.In this study, we utilize bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry to demonstrate that engrafted monocytes acquire expression of established microglia markers P2ry12, TMEM119, Fcrls and the pan-myeloid marker Iba1, which has been commonly mischaracterized as microglia-specific. These changes are accompanied by alterations in chromatin accessibility and shifts in chromatin binding motifs that are indicative of microglial identity. Moreover, we show that engrafted monocytes dynamically regulate the expression of CX3CR1, CCR2, Ly6C, and transcription factors PU.1, CTCF, RUNX, AP-1, CEBP, and IRF2, all of which are crucial for shaping microglial identity. This study is the first to illustrate that engrafted monocytes in the retina undergo both epigenetic and transcriptional changes, enabling them to express microglia-like signatures. These findings highlight the need for future research to account for these changes when assessing the roles of monocytes and microglia in CNS pathology.
{"title":"Epigenetic adaptation drives monocyte differentiation into microglia-like cells upon engraftment into the retina","authors":"Jie Liu, Fengyang Lei, Bin Yan, Naiwen Cui, Jyoti Sharma, Victor Correa, Lara Roach, Savvas Kikolaou, Kristen Pitts, James Chodosh, Daniel E Maidana, Demetrios G Vavvas, Huidan Zhang, David Weitz, Raul Mostoslavsky, Eleftherios I Paschalis","doi":"10.1101/2024.09.09.612126","DOIUrl":"https://doi.org/10.1101/2024.09.09.612126","url":null,"abstract":"The identification of specific markers for microglia has been a long-standing challenge. Recently, markers such as P2ry12, TMEM119, and Fcrls have been proposed as microglia-specific and widely used to explore microglial functions within various central nervous system (CNS) contexts. The specificity of these markers was based on the assumption that circulating monocytes retain their distinct signatures even after infiltrating the CNS. However, recent findings reveal that infiltrating monocytes can adopt microglia-like characteristics while maintaining a pro-inflammatory profile upon permanent engraftment in the CNS.In this study, we utilize bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry to demonstrate that engrafted monocytes acquire expression of established microglia markers P2ry12, TMEM119, Fcrls and the pan-myeloid marker Iba1, which has been commonly mischaracterized as microglia-specific. These changes are accompanied by alterations in chromatin accessibility and shifts in chromatin binding motifs that are indicative of microglial identity. Moreover, we show that engrafted monocytes dynamically regulate the expression of CX3CR1, CCR2, Ly6C, and transcription factors PU.1, CTCF, RUNX, AP-1, CEBP, and IRF2, all of which are crucial for shaping microglial identity. This study is the first to illustrate that engrafted monocytes in the retina undergo both epigenetic and transcriptional changes, enabling them to express microglia-like signatures. These findings highlight the need for future research to account for these changes when assessing the roles of monocytes and microglia in CNS pathology.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.10.612251
Greta R Webb, Kerry L Hilligan, Samuel I Old, Shiau-Choot Tang, Olivier Lamiable, F Ronchese
Type 2 dendritic cells (DC2s) are essential for TH2 differentiation, but the signaling pathways involved in allergen sensing, DC activation and instruction of CD4+ T cell priming remain unclear. Previous transcriptomic analyses demonstrated a type-I interferon (IFN-I) signature in skin cDC2s following immunization with non-viable larvae of Nippostrongylus brasiliensis (Nb), house dust mite (HDM), and Schistosoma egg antigen (SEA). Blocking IFN-I signaling with anti-IFNAR1 (aIFNAR1) led to reduced TH2 cytokine responses to these antigens, however, the phenotype of cytokine-producing CD4+ T cells was not further defined. Here we show that conditional loss of IFNAR1 signaling in CD11c+ DCs significantly impaired effector TH2 and TFH CD4+ T cell responses to Nb. In vivo proliferation experiments demonstrated reduced numbers of highly divided CD4+ T cells in IFNAR1deltaCD11c mice compared to IFNAR1WT, with the highly divided population comprising both TH2 and TFH. Characterization of the cDC2 compartment by flow cytometry and bulk RNAseq demonstrated lower numbers of Nb+ cDC2s in the skin-draining LN and a reduced expression of Il15 and Il15Ra in IFNAR1dletaCD11c mice compared to IFNAR1WT, while expression of costimulatory molecules including CD80, CD86, Cd40 and Pdcd1lg2 (PD-L2) was not impaired. Therefore, IFN-I conditioning of skin cDC2s is necessary for their effective priming of CD4+ T cell responses to allergens, providing evidence for a role of tissue cytokines in driving cDC2 activation in a TH2 context.
{"title":"IFN-I signaling in type 2 conventional dendritic cells supports TH2 and T follicular helper differentiation after allergen immunization","authors":"Greta R Webb, Kerry L Hilligan, Samuel I Old, Shiau-Choot Tang, Olivier Lamiable, F Ronchese","doi":"10.1101/2024.09.10.612251","DOIUrl":"https://doi.org/10.1101/2024.09.10.612251","url":null,"abstract":"Type 2 dendritic cells (DC2s) are essential for TH2 differentiation, but the signaling pathways involved in allergen sensing, DC activation and instruction of CD4+ T cell priming remain unclear. Previous transcriptomic analyses demonstrated a type-I interferon (IFN-I) signature in skin cDC2s following immunization with non-viable larvae of Nippostrongylus brasiliensis (Nb), house dust mite (HDM), and Schistosoma egg antigen (SEA). Blocking IFN-I signaling with anti-IFNAR1 (aIFNAR1) led to reduced TH2 cytokine responses to these antigens, however, the phenotype of cytokine-producing CD4+ T cells was not further defined. Here we show that conditional loss of IFNAR1 signaling in CD11c+ DCs significantly impaired effector TH2 and TFH CD4+ T cell responses to Nb. In vivo proliferation experiments demonstrated reduced numbers of highly divided CD4+ T cells in IFNAR1deltaCD11c mice compared to IFNAR1WT, with the highly divided population comprising both TH2 and TFH. Characterization of the cDC2 compartment by flow cytometry and bulk RNAseq demonstrated lower numbers of Nb+ cDC2s in the skin-draining LN and a reduced expression of Il15 and Il15Ra in IFNAR1dletaCD11c mice compared to IFNAR1WT, while expression of costimulatory molecules including CD80, CD86, Cd40 and Pdcd1lg2 (PD-L2) was not impaired. Therefore, IFN-I conditioning of skin cDC2s is necessary for their effective priming of CD4+ T cell responses to allergens, providing evidence for a role of tissue cytokines in driving cDC2 activation in a TH2 context.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.10.612362
Heather C Denroche, Victoria Ng, Jane Velghe, Imelda Suen, Liam Stanley, Dominika Nackiewicz, Mitsu Komba, Sam Chen, Galina Soukhatcheva, Lei Dai, C Bruce Verchere
Islet amyloid contributes to beta cell failure in type 2 diabetes through several mechanisms, one being the potent induction of local islet inflammation through activating inflammatory pathways in islet macrophages. We performed an unbiased phenotypic investigation of islet macrophages in the early stage of islet amyloid formation using single cell RNA sequencing of resident islet macrophages in mice with and without the amyloidogenic form of human islet amyloid polypeptide (hIAPP). This revealed that MHC Class II antigen presentation genes were strongly down-regulated in islet macrophages during islet amyloid formation. As islet amyloid has recently been reported in pancreases of people with type 1 diabetes, we sought to investigate the impact of islet amyloid in the NOD mouse model of type 1 diabetes. Both overexpression and physiological expression of hIAPP delayed diabetes in NOD mice relative to littermate controls, corresponding with decreased markers of antigen presentation and activation, as well as decreased immune cell infiltration in islets. Adoptive transfer studies showed that systemic autoimmune function remained intact and beta cells from hIAPP transgenic mice did not evade immune recognition by diabetogenic T cells, collectively indicating the protection from diabetes was mediated by localized disruption of antigen presentation in the pancreas. Consistent with this, incubation of dendritic cells with IAPP aggregates decreased MHC Class II surface expression and diminished antigen-specific T cell activation in vitro, through a phagocytosis-dependent mechanism. Collectively our data show that despite the well-established pro-inflammatory response of macrophages to IAPP aggregates, the uptake of IAPP aggregates during early amyloid formation also disrupts MHC Class II antigen presentation and slows beta cell autoimmunity.
胰岛淀粉样蛋白通过多种机制导致2型糖尿病患者的β细胞衰竭，其中一种机制是通过激活胰岛巨噬细胞的炎症通路强力诱导局部胰岛炎症。我们利用单细胞 RNA 测序技术，对胰岛巨噬细胞在胰岛淀粉样蛋白形成早期阶段的表型进行了无偏见的研究。结果发现，在胰岛淀粉样蛋白形成过程中，胰岛巨噬细胞中的 MHC II 类抗原呈递基因被强烈下调。由于最近有报道称 1 型糖尿病患者的胰腺中存在胰岛淀粉样蛋白，我们试图研究胰岛淀粉样蛋白对 1 型糖尿病 NOD 小鼠模型的影响。与同卵对照组相比，hIAPP 的过表达和生理性表达都能延缓 NOD 小鼠的糖尿病，这与抗原呈递和激活标记物的减少以及胰岛免疫细胞浸润的减少有关。接受性转移研究表明，全身自身免疫功能保持完好，来自 hIAPP 转基因小鼠的 beta 细胞并没有逃避致糖尿病 T 细胞的免疫识别，这共同表明糖尿病保护是由胰腺中抗原呈递的局部破坏介导的。与此相一致的是，用 IAPP 聚集物培养树突状细胞会降低 MHC II 类表面表达，并通过吞噬依赖机制降低体外抗原特异性 T 细胞的活化。总之，我们的数据表明，尽管巨噬细胞对 IAPP 聚集物的促炎反应已得到证实，但在早期淀粉样蛋白形成过程中对 IAPP 聚集物的摄取也会破坏 MHC II 类抗原呈递并减缓β细胞自身免疫。
{"title":"Islet amyloid disrupts MHC Class II antigen presentation and protects NOD mice from autoimmune diabetes.","authors":"Heather C Denroche, Victoria Ng, Jane Velghe, Imelda Suen, Liam Stanley, Dominika Nackiewicz, Mitsu Komba, Sam Chen, Galina Soukhatcheva, Lei Dai, C Bruce Verchere","doi":"10.1101/2024.09.10.612362","DOIUrl":"https://doi.org/10.1101/2024.09.10.612362","url":null,"abstract":"Islet amyloid contributes to beta cell failure in type 2 diabetes through several mechanisms, one being the potent induction of local islet inflammation through activating inflammatory pathways in islet macrophages. We performed an unbiased phenotypic investigation of islet macrophages in the early stage of islet amyloid formation using single cell RNA sequencing of resident islet macrophages in mice with and without the amyloidogenic form of human islet amyloid polypeptide (hIAPP). This revealed that MHC Class II antigen presentation genes were strongly down-regulated in islet macrophages during islet amyloid formation. As islet amyloid has recently been reported in pancreases of people with type 1 diabetes, we sought to investigate the impact of islet amyloid in the NOD mouse model of type 1 diabetes. Both overexpression and physiological expression of hIAPP delayed diabetes in NOD mice relative to littermate controls, corresponding with decreased markers of antigen presentation and activation, as well as decreased immune cell infiltration in islets. Adoptive transfer studies showed that systemic autoimmune function remained intact and beta cells from hIAPP transgenic mice did not evade immune recognition by diabetogenic T cells, collectively indicating the protection from diabetes was mediated by localized disruption of antigen presentation in the pancreas. Consistent with this, incubation of dendritic cells with IAPP aggregates decreased MHC Class II surface expression and diminished antigen-specific T cell activation in vitro, through a phagocytosis-dependent mechanism. Collectively our data show that despite the well-established pro-inflammatory response of macrophages to IAPP aggregates, the uptake of IAPP aggregates during early amyloid formation also disrupts MHC Class II antigen presentation and slows beta cell autoimmunity.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252136","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.11.611691
Julia Castillo Gonzalez, Pablo Vargas Rodriguez, Ignacio Serrano Martinez, Irene Forte-Lago, Melanie Price, Lara Buscemi, Lorenz Hirt, Elena Gonzalez-Rey
Ischemic stroke is the second leading cause of death globally. Neuroinflammation, blood-brain barrier (BBB) disruption, and immune dysregulation are key features of the pathogenesis and clinical outcomes of brain ischemia. A comprehensive understanding of the interconnected mechanisms and endogenous mediators that regulate these processes is essential for the development of effective therapeutic strategies. In this context, the present study investigates the role of cortistatin, a neuropeptide broadly distributed within the central nervous and immune systems. Given its anti-inflammatory, immunomodulatory, and neuroprotective properties, cortistatin positions as a promising endogenous factor in the pathogenesis of ischemic stroke. To evaluate its potential effects, we employed the widely recognized preclinical model of stroke, middle cerebral artery occlusion (MCAO), in young (3 months) and middle-aged (6 months) cortistatin-deficient mice and compared them with wild-type mice, assessing both short-term (48 hours) and long-term (7 days) outcomes. Our findings indicate that cortistatin deficiency in both young and middle-aged mice results in increased susceptibility to stroke and a significantly worsened prognosis, characterized by severe neurological deficits, altered microglial activity, impaired astrocytic scar formation, BBB disruption, dysregulated angiogenesis, and exacerbated immune infiltration and peripheral immune responses. Notably, the administration of cortistatin reversed these adverse outcomes, underscoring the critical role of cortistatin in regulating the complex interactions between the nervous and immune systems. Furthermore, cortistatin treatment for one week markedly improved key processes involved in stroke recovery, including neuronal repopulation, myelin repair, and the restoration of BBB integrity. The multifaceted therapeutic effects of cortistatin across various pathological stages and phenotypes suggest that multimodal therapies could represent a novel and more effective approach for treating ischemic stroke, offering advantages over current interventions that typically target only a single aspect of stroke pathology. In conclusion, our findings emphasize the significance of identifying the endogenous and therapeutic roles of neuro-immune mediators such as cortistatin in ischemic stroke
{"title":"Lack of cortistatin worsens neurological outcome and exacerbates aging-related glial and vascular dysfunction in stroke","authors":"Julia Castillo Gonzalez, Pablo Vargas Rodriguez, Ignacio Serrano Martinez, Irene Forte-Lago, Melanie Price, Lara Buscemi, Lorenz Hirt, Elena Gonzalez-Rey","doi":"10.1101/2024.09.11.611691","DOIUrl":"https://doi.org/10.1101/2024.09.11.611691","url":null,"abstract":"Ischemic stroke is the second leading cause of death globally. Neuroinflammation, blood-brain barrier (BBB) disruption, and immune dysregulation are key features of the pathogenesis and clinical outcomes of brain ischemia. A comprehensive understanding of the interconnected mechanisms and endogenous mediators that regulate these processes is essential for the development of effective therapeutic strategies. In this context, the present study investigates the role of cortistatin, a neuropeptide broadly distributed within the central nervous and immune systems. Given its anti-inflammatory, immunomodulatory, and neuroprotective properties, cortistatin positions as a promising endogenous factor in the pathogenesis of ischemic stroke. To evaluate its potential effects, we employed the widely recognized preclinical model of stroke, middle cerebral artery occlusion (MCAO), in young (3 months) and middle-aged (6 months) cortistatin-deficient mice and compared them with wild-type mice, assessing both short-term (48 hours) and long-term (7 days) outcomes. Our findings indicate that cortistatin deficiency in both young and middle-aged mice results in increased susceptibility to stroke and a significantly worsened prognosis, characterized by severe neurological deficits, altered microglial activity, impaired astrocytic scar formation, BBB disruption, dysregulated angiogenesis, and exacerbated immune infiltration and peripheral immune responses. Notably, the administration of cortistatin reversed these adverse outcomes, underscoring the critical role of cortistatin in regulating the complex interactions between the nervous and immune systems. Furthermore, cortistatin treatment for one week markedly improved key processes involved in stroke recovery, including neuronal repopulation, myelin repair, and the restoration of BBB integrity. The multifaceted therapeutic effects of cortistatin across various pathological stages and phenotypes suggest that multimodal therapies could represent a novel and more effective approach for treating ischemic stroke, offering advantages over current interventions that typically target only a single aspect of stroke pathology. In conclusion, our findings emphasize the significance of identifying the endogenous and therapeutic roles of neuro-immune mediators such as cortistatin in ischemic stroke","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.10.612119
Qiuyu Gong, Mehul Sharma, Emma L. Kuan, Marla C. Glass, Aishwarya Chander, Mansi Singh, Lucas T. Graybuck, Zachary J. Thomson, Christian M. LaFrance, Samir Rachid Zaim, Tao Peng, Lauren Y. Okada, Palak C. Genge, Katherine E. Henderson, Elisabeth M. Dornisch, Erik D. Layton, Peter J. Wittig, Alexander T. Heubeck, Nelson M. Mukuka, Julian Reading, Charles R. Roll, Veronica Hernandez, Vaishnavi Parthasarathy, Tyanna J. Stuckey, Blessing Musgrove, Elliott Swanson, Cara Lord, Morgan D.A. Weiss, Cole G. Phalen, Regina R. Mettey, Kevin J. Lee, John B. Johanneson, Erin K. Kawelo, Jessica Garber, Upaasana Krishnan, Megan Smithmeyer, E. John Wherry, Laura Vella, Sarah E. Henrickson, Mackenzie S. Kopp, Adam K. Savage, Lynne A. Becker, Paul Meijer, Ernest M. Coffey, Jorg J. Goronzy, Cate Speake, Thomas F. Bumol, Ananda W. Goldrath, Troy R. Torgerson, Xiao-jun Li, Peter J. Skene, Jane H. Buckner, Claire E. Gustafson
The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.
{"title":"Longitudinal Multi-omic Immune Profiling Reveals Age-Related Immune Cell Dynamics in Healthy Adults","authors":"Qiuyu Gong, Mehul Sharma, Emma L. Kuan, Marla C. Glass, Aishwarya Chander, Mansi Singh, Lucas T. Graybuck, Zachary J. Thomson, Christian M. LaFrance, Samir Rachid Zaim, Tao Peng, Lauren Y. Okada, Palak C. Genge, Katherine E. Henderson, Elisabeth M. Dornisch, Erik D. Layton, Peter J. Wittig, Alexander T. Heubeck, Nelson M. Mukuka, Julian Reading, Charles R. Roll, Veronica Hernandez, Vaishnavi Parthasarathy, Tyanna J. Stuckey, Blessing Musgrove, Elliott Swanson, Cara Lord, Morgan D.A. Weiss, Cole G. Phalen, Regina R. Mettey, Kevin J. Lee, John B. Johanneson, Erin K. Kawelo, Jessica Garber, Upaasana Krishnan, Megan Smithmeyer, E. John Wherry, Laura Vella, Sarah E. Henrickson, Mackenzie S. Kopp, Adam K. Savage, Lynne A. Becker, Paul Meijer, Ernest M. Coffey, Jorg J. Goronzy, Cate Speake, Thomas F. Bumol, Ananda W. Goldrath, Troy R. Torgerson, Xiao-jun Li, Peter J. Skene, Jane H. Buckner, Claire E. Gustafson","doi":"10.1101/2024.09.10.612119","DOIUrl":"https://doi.org/10.1101/2024.09.10.612119","url":null,"abstract":"The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.11.612497
Ikechukwu B Jacob, Akinkunmi O Lawal, Salma S Mahmoud, Emerson M Kopsack, Erin S Reynolds, Qinghe Meng, HongKuan Fan, Paul T Massa, Saravanan Thangamani, Hongpeng Jia, Guirong Wang
COVID-19 remains a significant threat to public health globally. Infection in some susceptible individuals causes life-threatening acute lung injury (ALI/ARDS) and/or death. Human surfactant protein A (SP-A) is a C-type lectin expressed in the lung and other mucosal tissues, and it plays a critical role in host defense against various pathogens. The human SP-A genes (SFTPA1 and SFTPA2) are highly polymorphic and comprise several common genetic variants, i.e., SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). Here, we elucidated the differential antiviral and immunoregulatory roles of SP-A variants in response to SARS-CoV-2 infection in vivo. Six genetically-modified mouse lines, expressing both hACE2 (SARS-CoV-2 receptor) and individual SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3 (1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were challenged intranasally with 103 PFU SARS-CoV-2 or saline (Sham). Infected KO and 1A0 mice had more weight loss and mortality compared to other mouse lines. Relative to other infected mouse lines, a more severe ALI was observed in KO, 1A0, and 6A2 mice. Reduced viral titers were generally observed in the lungs of infected SP-A mice relative to KO mice. Transcriptomic analysis revealed an upregulation in genes that play central roles in immune responses such as MyD88, Stat3, IL-18, and Jak2 in the lungs of KO and 1A0 mice. However, Mapk1 was significantly downregulated in 6A2 versus 1A0 mice. Analysis of biological pathways identified those involved in lung host defense and innate immunity, including pathogen-induced cytokine, NOD1/2, and Trem1 signaling pathways. Consistent with the transcriptomic data, levels of cytokines and chemokines such as G-CSF, TNF-α, IL-6 and, IL-1β, and IL-10 were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. These findings demonstrate that human SP-A variants differentially modulate SARS-CoV-2-induced lung injury and disease severity by differentially inhibiting viral infectivity and regulating immune-related gene expressions.
COVID-19 仍是全球公共卫生的重大威胁。一些易感人群感染后会导致危及生命的急性肺损伤（ALI/ARDS）和/或死亡。人类表面活性蛋白 A（SP-A）是一种在肺部和其他粘膜组织中表达的 C 型凝集素，在宿主抵御各种病原体的过程中发挥着关键作用。人类 SP-A 基因（SFTPA1 和 SFTPA2）具有高度的多态性，包括几个常见的基因变体，即 SP-A1（变体 6A2、6A4）和 SP-A2（变体 1A0、1A3）。在这里，我们阐明了SP-A变体在体内应对SARS-CoV-2感染时的不同抗病毒和免疫调节作用。六种基因改造的小鼠品系同时表达 hACE2（SARS-CoV-2 受体）和单个 SP-A 变体：(小鼠（hACE2/6A2 (6A2)、hACE2/6A4 (6A4)、hACE2/1A0 (1A0)和 hACE2/1A3 (1A3)）、一种 SP-A 基因敲除小鼠（hACE2/SP-A KO (KO)）和一种 hACE2/小鼠 SP-A (K18)）经鼻内注射 103 PFU SARS-CoV-2 或生理盐水（Sham）。与其他小鼠品系相比，受感染的 KO 和 1A0 小鼠体重下降和死亡率更高。与其他受感染的小鼠品系相比，KO、1A0 和 6A2 小鼠的 ALI 更严重。与 KO 小鼠相比，在受感染的 SP-A 小鼠肺部普遍观察到病毒滴度降低。转录组分析显示，在 KO 和 1A0 小鼠的肺中，MyD88、Stat3、IL-18 和 Jak2 等在免疫反应中起核心作用的基因上调。然而，与 1A0 小鼠相比，6A2 小鼠的 Mapk1 基因明显下调。生物通路分析确定了参与肺宿主防御和先天性免疫的通路，包括病原体诱导的细胞因子、NOD1/2 和 Trem1 信号通路。与转录组数据一致的是，在死亡率最高的 KO 和 1A0 小鼠的肺部和血清中，细胞因子和趋化因子（如 G-CSF、TNF-α、IL-6 和 IL-1β 及 IL-10）的水平相对较高。这些研究结果表明，人类SP-A变体通过抑制病毒的感染性和调节免疫相关基因的表达，对SARS-CoV-2诱发的肺损伤和疾病严重程度有不同的调节作用。
{"title":"Differential Immunoregulation by Human Surfactant Protein A Variants Determines Severity of SARS-CoV-2-induced Lung Disease","authors":"Ikechukwu B Jacob, Akinkunmi O Lawal, Salma S Mahmoud, Emerson M Kopsack, Erin S Reynolds, Qinghe Meng, HongKuan Fan, Paul T Massa, Saravanan Thangamani, Hongpeng Jia, Guirong Wang","doi":"10.1101/2024.09.11.612497","DOIUrl":"https://doi.org/10.1101/2024.09.11.612497","url":null,"abstract":"COVID-19 remains a significant threat to public health globally. Infection in some susceptible individuals causes life-threatening acute lung injury (ALI/ARDS) and/or death. Human surfactant protein A (SP-A) is a C-type lectin expressed in the lung and other mucosal tissues, and it plays a critical role in host defense against various pathogens. The human SP-A genes (SFTPA1 and SFTPA2) are highly polymorphic and comprise several common genetic variants, i.e., SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). Here, we elucidated the differential antiviral and immunoregulatory roles of SP-A variants in response to SARS-CoV-2 infection in vivo. Six genetically-modified mouse lines, expressing both hACE2 (SARS-CoV-2 receptor) and individual SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3 (1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were challenged intranasally with 103 PFU SARS-CoV-2 or saline (Sham). Infected KO and 1A0 mice had more weight loss and mortality compared to other mouse lines. Relative to other infected mouse lines, a more severe ALI was observed in KO, 1A0, and 6A2 mice. Reduced viral titers were generally observed in the lungs of infected SP-A mice relative to KO mice. Transcriptomic analysis revealed an upregulation in genes that play central roles in immune responses such as MyD88, Stat3, IL-18, and Jak2 in the lungs of KO and 1A0 mice. However, Mapk1 was significantly downregulated in 6A2 versus 1A0 mice. Analysis of biological pathways identified those involved in lung host defense and innate immunity, including pathogen-induced cytokine, NOD1/2, and Trem1 signaling pathways. Consistent with the transcriptomic data, levels of cytokines and chemokines such as G-CSF, TNF-α, IL-6 and, IL-1β, and IL-10 were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. These findings demonstrate that human SP-A variants differentially modulate SARS-CoV-2-induced lung injury and disease severity by differentially inhibiting viral infectivity and regulating immune-related gene expressions.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"8 42 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612051
Wiktor Prus, Frederic Grabowski, Paulina Koza, Zbigniew Korwek, Maciej Czerkies, Marek Kochańczyk, Tomasz Lipniacki
Type III interferons (IFN-λ1–λ4) are known to limit influenza infections in vivo and are non-redundant to type I interferons (IFN-α and IFN-β). Here, we demonstrated in vitro that type III interferons secreted by infected epithelial cells limit infection with influenza A virus (IAV). This effect occurs independently of STAT1 and STAT2 activation. Knockout of the IFN-λ receptor subunit IFNLR1 results in a slight reduction of STAT1/2 phosphorylation during infection with IAV, but leads to a substantially increased fraction of IAV-infected cells and higher levels of viral RNA and protein in the cell population. In contrast, knockout of the IFN-β receptor subunit IFNAR1 results in a pronounced reduction of STAT1/2 phosphorylation but a smaller increase in IAV proliferation. We propose that type III interferons limit the spread of IAV by promoting death of IAV-infected cells and demonstrate that the ratio of dying to infected cells is lower in IFNLR1 KO cells compared to wild-type cells. Overall, our results suggest an additional non-transcriptional role of type III interferons in the control of viral infection.
{"title":"Type III interferons suppress influenza A virus infection independently of STAT activation by triggering cell death","authors":"Wiktor Prus, Frederic Grabowski, Paulina Koza, Zbigniew Korwek, Maciej Czerkies, Marek Kochańczyk, Tomasz Lipniacki","doi":"10.1101/2024.09.09.612051","DOIUrl":"https://doi.org/10.1101/2024.09.09.612051","url":null,"abstract":"Type III interferons (IFN-λ1–λ4) are known to limit influenza infections in vivo and are non-redundant to type I interferons (IFN-α and IFN-β). Here, we demonstrated in vitro that type III interferons secreted by infected epithelial cells limit infection with influenza A virus (IAV). This effect occurs independently of STAT1 and STAT2 activation. Knockout of the IFN-λ receptor subunit IFNLR1 results in a slight reduction of STAT1/2 phosphorylation during infection with IAV, but leads to a substantially increased fraction of IAV-infected cells and higher levels of viral RNA and protein in the cell population. In contrast, knockout of the IFN-β receptor subunit IFNAR1 results in a pronounced reduction of STAT1/2 phosphorylation but a smaller increase in IAV proliferation. We propose that type III interferons limit the spread of IAV by promoting death of IAV-infected cells and demonstrate that the ratio of dying to infected cells is lower in IFNLR1 KO cells compared to wild-type cells. Overall, our results suggest an additional non-transcriptional role of type III interferons in the control of viral infection.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.08.19.608538
Sarah Staudt, Fabian Nikolka, Markus Perl, Julia Franz, Noemie Leblay, Xiaoli-Kat Yuan, Linda Warmuth, Matthias A Fantes, Aiste Skorpskaite, Teng Fei, Maria Bromberg, Patxi San Martin-Uriz, Juan Roberto Rodriguez-Madoz, Kai Ziegler-Martin, Nazdar Adil-Gholam, Pascal Benz, Phuc Tran Huu, Christoph Stein-Thoeringer, Michael Schmitt, Karin Kleigrewe, Fabian Freitag, Zeno Riester, Justus Weber, Kira Mangold, Hermann Einsele, Felipe Prosper, Wilfried Ellmeier, Dirk Busch, Alexander Visekruna, John Slingerland, Roni Shouval, Karsten Hiller, Marcel van den Brink, Patrick Pausch, Paola Neri, Michael Hudecek, Hendrik Poeck, Maik Luu
Emerging data have highlighted a correlation between microbiome composition and cancer immunotherapy outcome. While commensal bacteria and their metabolites are known to modulate the host environment, contradictory effects and a lack of mechanistic understanding impede the translation of microbiome-based therapies into the clinic. In this study, we demonstrate that abundance of the commensal metabolite pentanoate is predictive for survival of chimeric antigen receptor (CAR) T cell patients in two independent cohorts. Its implementation in the CAR T cell manufacturing workflow overcomes solid tumor microenvironments in immunocompetent cancer models by hijacking the epigenetic-metabolic crosstalk, reducing exhaustion and promoting naive-like differentiation. While synergy of clinically relevant drugs mimicked the phenotype of pentanoate-engineered CAR T cells in vitro, in vivo challenge showed inferior tumor control. Metabolic tracing of 13C-pentanoate revealed citrate generation in the TCA cycle via the acetyl- and succinyl-CoA entry points as a unique feature of the C5 aliphatic chain. Inhibition of the ATP-citrate lyase, which links metabolic output and histone acetylation, led to accumulation of pentanoate-derived citrate from the succinyl-CoA route and decreased functionality of SCFA-engineered CAR T cells. Our data demonstrate that microbial metabolites are incorporated as epigenetic imprints and implementation into CAR T cell production might serve as embodiment of the microbiome-host axis benefits for clinical applications.
新出现的数据强调了微生物组组成与癌症免疫疗法结果之间的相关性。众所周知，共生细菌及其代谢产物可调节宿主环境，但相互矛盾的影响和缺乏对机理的了解阻碍了将基于微生物的疗法应用于临床。在这项研究中，我们证明了共生代谢产物戊酸盐的丰度可预测两个独立队列中嵌合抗原受体（CAR）T细胞患者的存活率。在 CAR T 细胞制造工作流程中使用这种物质，可以通过劫持表观遗传学-代谢串扰、减少衰竭和促进天真样分化来克服免疫功能正常癌症模型中的实体肿瘤微环境。虽然临床相关药物的协同作用在体外模拟了戊酸盐工程 CAR T 细胞的表型，但体内挑战显示肿瘤控制能力较差。对13C-戊酸盐的代谢追踪显示，在TCA循环中通过乙酰和琥珀酰-CoA入口点生成柠檬酸盐是C5脂肪链的一个独特特征。ATP 柠檬酸裂解酶将代谢输出和组蛋白乙酰化联系在一起，抑制该酶会导致从琥珀酰-CoA途径获得的戊酸柠檬酸积累，并降低 SCFA 工程 CAR T 细胞的功能。我们的数据表明，微生物代谢产物被纳入表观遗传学印记，将其应用于 CAR T 细胞的生产可体现微生物组-宿主轴对临床应用的益处。
{"title":"Microbial metabolite-guided CAR T cell engineering enhances anti-tumor immunity via epigenetic-metabolic crosstalk","authors":"Sarah Staudt, Fabian Nikolka, Markus Perl, Julia Franz, Noemie Leblay, Xiaoli-Kat Yuan, Linda Warmuth, Matthias A Fantes, Aiste Skorpskaite, Teng Fei, Maria Bromberg, Patxi San Martin-Uriz, Juan Roberto Rodriguez-Madoz, Kai Ziegler-Martin, Nazdar Adil-Gholam, Pascal Benz, Phuc Tran Huu, Christoph Stein-Thoeringer, Michael Schmitt, Karin Kleigrewe, Fabian Freitag, Zeno Riester, Justus Weber, Kira Mangold, Hermann Einsele, Felipe Prosper, Wilfried Ellmeier, Dirk Busch, Alexander Visekruna, John Slingerland, Roni Shouval, Karsten Hiller, Marcel van den Brink, Patrick Pausch, Paola Neri, Michael Hudecek, Hendrik Poeck, Maik Luu","doi":"10.1101/2024.08.19.608538","DOIUrl":"https://doi.org/10.1101/2024.08.19.608538","url":null,"abstract":"Emerging data have highlighted a correlation between microbiome composition and cancer immunotherapy outcome. While commensal bacteria and their metabolites are known to modulate the host environment, contradictory effects and a lack of mechanistic understanding impede the translation of microbiome-based therapies into the clinic. In this study, we demonstrate that abundance of the commensal metabolite pentanoate is predictive for survival of chimeric antigen receptor (CAR) T cell patients in two independent cohorts. Its implementation in the CAR T cell manufacturing workflow overcomes solid tumor microenvironments in immunocompetent cancer models by hijacking the epigenetic-metabolic crosstalk, reducing exhaustion and promoting naive-like differentiation. While synergy of clinically relevant drugs mimicked the phenotype of pentanoate-engineered CAR T cells in vitro, in vivo challenge showed inferior tumor control. Metabolic tracing of 13C-pentanoate revealed citrate generation in the TCA cycle via the acetyl- and succinyl-CoA entry points as a unique feature of the C5 aliphatic chain. Inhibition of the ATP-citrate lyase, which links metabolic output and histone acetylation, led to accumulation of pentanoate-derived citrate from the succinyl-CoA route and decreased functionality of SCFA-engineered CAR T cells. Our data demonstrate that microbial metabolites are incorporated as epigenetic imprints and implementation into CAR T cell production might serve as embodiment of the microbiome-host axis benefits for clinical applications.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"266 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211179","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612060
John Sidney, AReum Kim, Rory Dylan de Vries, Bjoern Peters, Philip S. Meade, Florian Krammer, Alba Grifoni, Alessandro Sette
Frequent recent spillovers of subtype H5N1 clade 2.3.4.4b highly pathogenic avian influenza (HPAI) virus into poultry and mammals, especially dairy cattle, including several human cases, increased concerns over a possible future pandemic. Here, we performed an analysis of epitope data curated in the Immune Epitope Database (IEDB). We found that the patterns of immunodominance of seasonal influenza viruses circulating in humans and H5N1 are similar. We further conclude that a significant fraction of the T cell epitopes is conserved at a level associated with cross-reactivity between avian and seasonal sequences, and we further experimentally demonstrate extensive cross-reactivity in the most dominant T cell epitopes curated in the IEDB. Based on these observations, and the overall similarity of the neuraminidase (NA) N1 subtype encoded in both HPAI and seasonal H1N1 influenza virus as well as cross-reactive group 1 HA stalk-reactive antibodies, we expect that a degree of pre-existing immunity is present in the general human population that could blunt the severity of human H5N1 infections.
最近，亚型 H5N1 2.3.4.4b 支系高致病性禽流感（HPAI）病毒频繁扩散到家禽和哺乳动物（尤其是奶牛）中，其中包括几例人类病例，这增加了人们对未来可能发生大流行的担忧。在此，我们对免疫表位数据库（IEDB）中收集的表位数据进行了分析。我们发现，在人类中流行的季节性流感病毒和 H5N1 病毒的免疫优势模式相似。我们还进一步得出结论，T 细胞表位中有相当一部分在禽类和季节性序列之间的交叉反应水平上是保守的，我们还进一步通过实验证明了 IEDB 中最主要的 T 细胞表位存在广泛的交叉反应。基于这些观察结果，以及高致病性禽流感和季节性 H1N1 流感病毒中编码的神经氨酸酶（NA）N1 亚型的总体相似性和交叉反应性 1 组 HA 柄反应抗体，我们预计普通人群中存在一定程度的原有免疫力，可减轻人类 H5N1 感染的严重程度。
{"title":"Targets of influenza Human T cell response are mostly conserved in H5N1","authors":"John Sidney, AReum Kim, Rory Dylan de Vries, Bjoern Peters, Philip S. Meade, Florian Krammer, Alba Grifoni, Alessandro Sette","doi":"10.1101/2024.09.09.612060","DOIUrl":"https://doi.org/10.1101/2024.09.09.612060","url":null,"abstract":"Frequent recent spillovers of subtype H5N1 clade 2.3.4.4b highly pathogenic avian influenza (HPAI) virus into poultry and mammals, especially dairy cattle, including several human cases, increased concerns over a possible future pandemic. Here, we performed an analysis of epitope data curated in the Immune Epitope Database (IEDB). We found that the patterns of immunodominance of seasonal influenza viruses circulating in humans and H5N1 are similar. We further conclude that a significant fraction of the T cell epitopes is conserved at a level associated with cross-reactivity between avian and seasonal sequences, and we further experimentally demonstrate extensive cross-reactivity in the most dominant T cell epitopes curated in the IEDB. Based on these observations, and the overall similarity of the neuraminidase (NA) N1 subtype encoded in both HPAI and seasonal H1N1 influenza virus as well as cross-reactive group 1 HA stalk-reactive antibodies, we expect that a degree of pre-existing immunity is present in the general human population that could blunt the severity of human H5N1 infections.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"46 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.08.611875
Fatemeh Gholizadeh, Mehri Hajiaghayi, Jennifer S. Choi, Samuel R. Little, Niloufar Rahbari, Kelly Brotto, Eric Han, Steve C.C. Shih, Peter J. Darlington
Memory T helper (Th) cells, generated after immunogenic challenge, are crucial in directing the adaptive immune response. Muscarinic ACh receptor (mAChR) subtypes expressed by immune cells can be stimulated with acetylcholine or muscarinic-selective drug oxotremorine-M. Cholinergic signaling can influence immune cells, but it is not known how cholinergic stimuli regulate memory Th cells. This study focused on the role of mAChRs, specifically the M3 muscarinic ACh receptor (M3R), in the cytokine profile and NF-κB p65 activity of primary human memory Th cells. Memory Th cells (CD3+CD4+CD45RA-CD45RO+) were isolated from healthy participants' peripheral blood. Cell culture was performed with anti-CD3/anti-CD28/anti-CD2 reagent, oxotremorine-M (M1R-M5R agonist), atropine (M1R-M5R antagonist), and J104129 (M3R-selective antagonist). MR1-MR5 genes CHRM1-CHRM5 were measured with RT-qPCR. Protein expression of M3R and phosphorylated NF-κB p65 were quantified by Western blot. The secretion of IFN-γ, IL-17A, and IL-4 was assessed by ELISA and intracellular cytokine staining flow cytometry. CHRM3, encoding M3R, was knocked out using CRISPR-Cas9 gene targeting. Memory Th cells expressed all five mAChR subtypes. Oxotremorine-M increased IFN-γ and IL-17A while reducing IL-4 in an atropine-sensitive manner. Stimulation of mAChRs in cells with CHRM3-knockout or M3R blockade prevented increases in IFN-γ and IL-17A but continued to inhibit IL-4. mAChR stimulation enhanced NF-κB p65 activity without affecting cell proliferation, viability, or M3R expression. This investigation demonstrates that muscarinic signaling increases the pro-inflammatory profile of memory Th cells, including NF-κB p65, IFN-γ, and IL-17A, with a reduction in IL-4. Focusing on M3R blockers could modulate adaptive immune responses and alleviate immune-related conditions.
{"title":"Modulatory Effects of M3 Muscarinic Acetylcholine Receptor on Inflammatory Profiles of Human Memory T Helper Cells","authors":"Fatemeh Gholizadeh, Mehri Hajiaghayi, Jennifer S. Choi, Samuel R. Little, Niloufar Rahbari, Kelly Brotto, Eric Han, Steve C.C. Shih, Peter J. Darlington","doi":"10.1101/2024.09.08.611875","DOIUrl":"https://doi.org/10.1101/2024.09.08.611875","url":null,"abstract":"Memory T helper (Th) cells, generated after immunogenic challenge, are crucial in directing the adaptive immune response. Muscarinic ACh receptor (mAChR) subtypes expressed by immune cells can be stimulated with acetylcholine or muscarinic-selective drug oxotremorine-M. Cholinergic signaling can influence immune cells, but it is not known how cholinergic stimuli regulate memory Th cells. This study focused on the role of mAChRs, specifically the M3 muscarinic ACh receptor (M3R), in the cytokine profile and NF-κB p65 activity of primary human memory Th cells.\u0000Memory Th cells (CD3+CD4+CD45RA-CD45RO+) were isolated from healthy participants' peripheral blood. Cell culture was performed with anti-CD3/anti-CD28/anti-CD2 reagent, oxotremorine-M (M1R-M5R agonist), atropine (M1R-M5R antagonist), and J104129 (M3R-selective antagonist). MR1-MR5 genes CHRM1-CHRM5 were measured with RT-qPCR. Protein expression of M3R and phosphorylated NF-κB p65 were quantified by Western blot. The secretion of IFN-γ, IL-17A, and IL-4 was assessed by ELISA and intracellular cytokine staining flow cytometry. CHRM3, encoding M3R, was knocked out using CRISPR-Cas9 gene targeting.\u0000Memory Th cells expressed all five mAChR subtypes. Oxotremorine-M increased IFN-γ and IL-17A while reducing IL-4 in an atropine-sensitive manner. Stimulation of mAChRs in cells with CHRM3-knockout or M3R blockade prevented increases in IFN-γ and IL-17A but continued to inhibit IL-4. mAChR stimulation enhanced NF-κB p65 activity without affecting cell proliferation, viability, or M3R expression.\u0000This investigation demonstrates that muscarinic signaling increases the pro-inflammatory profile of memory Th cells, including NF-κB p65, IFN-γ, and IL-17A, with a reduction in IL-4. Focusing on M3R blockers could modulate adaptive immune responses and alleviate immune-related conditions.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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