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Lymphocyte activation involves a transition from quiescence and associated catabolic metabolism to a metabolic state with noted similarities to cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and cell division 1-3 . Following antigen receptor ligation, lymphocytes require spatiotemporally distinct "second signals". These include costimulatory receptor or cytokine signaling, which engage discrete programs that often involve remodeling of organelles and increased nutrient uptake or synthesis to meet changing biochemical demands 4-6 . One such signaling molecule, IL-4, is a highly pleiotropic cytokine that was first identified as a B cell co-mitogen over 30 years ago 7 . However, how IL-4 signaling mechanistically supports B cell proliferation is incompletely understood. Here, using single cell RNA sequencing we find that the cholesterol biosynthetic program is transcriptionally upregulated following IL-4 signaling during the early B cell response to influenza virus infection, and is required for B cell activation in vivo . By limiting lipid availability in vitro , we determine cholesterol to be essential for B cells to expand their endoplasmic reticulum, progress through cell cycle, and proliferate. In sum, we demonstrate that the well-known ability of IL-4 to act as a B cell growth factor is through a previously unknown rewiring of specific lipid anabolic programs, relieving sensitivity of cells to environmental nutrient availability.
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Single-cell RNA sequencing has been widely used to investigate cell state transitions and gene dynamics of biological processes. Current strategies to infer the sequential dynamics of genes in a process typically rely on constructing cell pseudotime through cell trajectory inference. However, the presence of concurrent gene processes in the same group of cells and technical noise can obscure the true progression of the processes studied. To address this challenge, we present GeneTrajectory, an approach that identifies trajectories of genes rather than trajectories of cells. Specifically, optimal transport distances are calculated between gene distributions across the cell-cell graph to extract gene programs and define their gene pseudotemporal order. Here we demonstrate that GeneTrajectory accurately extracts progressive gene dynamics in myeloid lineage maturation. Moreover, we show that GeneTrajectory deconvolves key gene programs underlying mouse skin hair follicle dermal condensate differentiation that could not be resolved by cell trajectory approaches. GeneTrajectory facilitates the discovery of gene programs that control the changes and activities of biological processes.
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Neutrophils are important innate immune cells with plasticity, heterogenicity, and functional ambivalency. While bone marrow is often regarded as the primary source of neutrophil production, the roles of extramedullary production in regulating neutrophil plasticity and heterogenicity in autoimmune diseases remain poorly understood. Here, we report that the lack of wingless-type MMTV integration site family member 5 (WNT5) unleashes anti-inflammatory protection against colitis in mice, accompanied by reduced colonic CD8+ T cell activation and enhanced splenic extramedullary myelopoiesis. In addition, colitis upregulates WNT5 expression in splenic stromal cells. The ablation of WNT5 leads to increased splenic production of hematopoietic niche factors, as well as elevated numbers of splenic neutrophils with heightened CD8+ T cell suppressive capability, in part due to elevated CD101 expression and attenuated pro-inflammatory activities. Thus, our study reveals a mechanism by which neutrophil plasticity and heterogenicity are regulated in colitis through WNT5 and highlights the role of splenic neutrophil production in shaping inflammatory outcomes.
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Colitis , Neutrófilos , Animales , Ratones , Mielopoyesis , Colitis/inducido químicamente , Médula ÓseaRESUMEN
Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.
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Inflamación , Interleucina-10 , Esfingolípidos , Animales , Humanos , Ratones , Ceramidas/química , Ceramidas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Homeostasis , Inmunidad Innata , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-10/deficiencia , Interleucina-10/genética , Interleucina-10/metabolismo , Proteínas Proto-Oncogénicas c-rel , Esfingolípidos/metabolismoRESUMEN
Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vß2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vß chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vß scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vß2+ Jurkat cells and Vß2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vß2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.
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Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Humanos , Receptores de Antígenos de Linfocitos T/genética , Células Jurkat , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos/genética , Células ClonalesRESUMEN
BACKGROUND: Stimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma. METHODS: We employed a Simon's two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects. RESULTS: Thirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses. CONCLUSIONS: Higher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03502330.
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Anticuerpos Monoclonales , Melanoma , Humanos , Animales , Ratones , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Nivolumab/uso terapéutico , Melanoma/patología , Proteínas Tirosina Quinasas ReceptorasRESUMEN
Accurate cell marker identification in single-cell RNA-seq data is crucial for understanding cellular diversity and function. An ideal marker is highly specific in identifying cells that are similar in terms of function and state. Current marker identification methods, commonly based on clustering and differential expression, capture general cell-type markers but often miss markers for subtypes or functional cell subsets, with their performance largely dependent on clustering quality. Moreover, cluster-independent approaches tend to favor genes that lack the specificity required to characterize regions within the transcriptomic space at multiple scales. Here we introduce Localized Marker Detector (LMD), a novel tool to identify "localized genes" - genes with expression profiles specific to certain groups of highly similar cells - thereby characterizing cellular diversity in a multi-resolution and fine-grained manner. LMD's strategy involves building a cell-cell affinity graph, diffusing the gene expression value across the cell graph, and assigning a score to each gene based on its diffusion dynamics. We show that LMD exhibits superior accuracy in recovering known cell-type markers in the Tabula Muris bone marrow dataset relative to other methods for marker identification. Notably, markers favored by LMD exhibit localized expression, whereas markers prioritized by other clustering-free algorithms are often dispersed in the transcriptomic space. We further group the markers suggested by LMD into functional gene modules to improve the separation of cell types and subtypes in a more fine-grained manner. These modules also identify other sources of variation, such as cell cycle status. In conclusion, LMD is a novel algorithm that can identify fine-grained markers for cell subtypes or functional states without relying on clustering or differential expression analysis. LMD exploits the complex interactions among cells and reveals cellular diversity at high resolution.
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Recent advances in sample preparation and sequencing technology have made it possible to profile the transcriptomes of individual cells using single-cell RNA sequencing (scRNA-Seq). Compared to bulk RNA-Seq data, single-cell data often contain a higher percentage of zero reads, mainly due to lower sequencing depth per cell, which affects mostly measurements of low-expression genes. However, discrepancies between platforms are observed regardless of expression level. Using four paired datasets with multiple samples each, we investigated technical and biological factors that can contribute to this expression shift. Using two separate machine learning models we found that, in addition to expression level, RNA integrity, gene or UTR3 length, and the number of transcripts potentially also influence the occurrence of zeros. These findings could enable the development of novel analytical methods for cross-platform expression shift correction. We also identified genes and biological pathways in our diverse datasets that consistently showed differences when assessed at the single cell versus bulk level to assist in interpreting analysis across transcriptomic platforms. At the gene level, 25 genes (0.12%) were found in all datasets as discordant, but at the pathway level, 7 pathways (2.02%) showed shared enrichment in discordant genes.
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Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.
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Células Endoteliales , Hígado , Animales , Humanos , Ratones , Células Endoteliales/metabolismo , Hepatocitos/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/citología , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fibrosis/metabolismoRESUMEN
BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.
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Neoplasias , Factor A de Crecimiento Endotelial Vascular , Humanos , Animales , Ratones , Microambiente Tumoral , Oncología Médica , Modelos Animales de EnfermedadRESUMEN
Unchecked chronic inflammation is the underlying cause of many diseases, ranging from inflammatory bowel disease to obesity and neurodegeneration. Given the deleterious nature of unregulated inflammation, it is not surprising that cells have acquired a diverse arsenal of tactics to limit inflammation. IL-10 is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types; however, the exact mechanism by which IL-10 signaling subdues inflammation remains unclear. Here, we find that IL-10 signaling constrains sphingolipid metabolism. Specifically, we find increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10-deficient macrophages. Genetic deletion of CerS2, the enzyme responsible for VLC ceramide production, limited exacerbated inflammatory gene expression associated with IL-10 deficiency both in vitro and in vivo , indicating that "metabolic correction" is able to reduce inflammation in the absence of IL-10. Surprisingly, accumulation of saturated VLC ceramides was regulated by flux through the de novo mono-unsaturated fatty acid (MUFA) synthesis pathway, where addition of exogenous MUFAs could limit both saturated VLC ceramide production and inflammatory gene expression in the absence of IL-10 signaling. Together, these studies mechanistically define how IL-10 signaling manipulates fatty acid metabolism as part of its molecular anti-inflammatory strategy and could lead to novel and inexpensive approaches to regulate aberrant inflammation.
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Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin ß3 resulting in increased tumor necrosis factor [TNF]-α signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin ß3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.
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Aterosclerosis , Placa Aterosclerótica , Humanos , Ratones , Animales , Anciano , Placa Aterosclerótica/metabolismo , Médula Ósea/metabolismo , Integrina beta3/metabolismo , Aterosclerosis/genética , Miocitos del Músculo Liso , Músculo Liso/metabolismoRESUMEN
The incidence of cutaneous T-cell lymphoma (CTCL) increases with age, and blood involvement portends a worse prognosis. To advance our understanding of the development of CTCL and identify potential therapeutic targets, we performed integrative analyses of paired single-cell RNA and T-cell receptor (TCR) sequencing of peripheral blood CD4+ T cells from patients with CTCL to reveal disease-unifying features. The malignant CD4+ T cells of CTCL showed highly diverse transcriptomic profiles across patients, with most displaying a mature Th2 differentiation and T-cell exhaustion phenotype. TCR-CDR3 peptide prediction analysis suggested limited diversity between CTCL samples, consistent with a role for a common antigenic stimulus. Potential of heat diffusion for affinity-based trajectory embedding transition analysis identified putative precancerous circulating populations characterized by an intermediate stage of gene expression and mutation level between the normal CD4+ T cells and malignant CTCL cells. We further revealed the therapeutic potential of targeting CD82 and JAK that endow the malignant CTCL cells with survival and proliferation advantages.
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Linfoma Cutáneo de Células T , Neoplasias Cutáneas , Humanos , Transcriptoma , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Linfoma Cutáneo de Células T/patología , Linfocitos T CD4-Positivos/metabolismo , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) is being studied in multiple tumor types. However, little is known about clonal cell expansion in vitro and persistence of the ACT product in vivo. We performed single-cell RNA and T-Cell Receptor (TCR) sequencing on serial blood and tumor samples from a patient undergoing ACT, who did not respond. We found that clonal expansion varied during preparation of the ACT product, and only one expanded clone was preserved in the ACT product. The TCR of the preserved clone which persisted and remained activated for five months was previously reported as specific for cytomegalovirus and had upregulation of granzyme family genes and genes associated with effector functions (HLA-DQB1, LAT, HLA-DQA1, and KLRD1). Clones that contracted during TIL preparation had features of exhaustion and apoptosis. At disease progression, all previously detected clonotypes were detected. New clonotypes appearing in blood or tumor at disease progression were enriched for genes associated with cytotoxicity or stemness (FGFBP2, GNLY, GZMH, GZMK, IL7R, SELL and KLF2), and these might be harnessed for alternative cellular therapy or cytokine therapy. In-depth single-cell analyses of serial samples from additional ACT-treated patients is warranted, and viral- versus tumor-specificity should be carefully analyzed.
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Melanoma , Humanos , Melanoma/genética , Linfocitos Infiltrantes de Tumor/patología , Receptores de Antígenos de Linfocitos T/genética , Análisis de la Célula Individual , Insuficiencia del Tratamiento , Progresión de la Enfermedad , Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia AdoptivaRESUMEN
BACKGROUND: The pituitary gland regulates essential physiological processes such as growth, pubertal onset, stress response, metabolism, reproduction, and lactation. While sex biases in these functions and hormone production have been described, the underlying identity, temporal deployment, and cell-type specificity of sex-biased pituitary gene regulatory networks are not fully understood. METHODS: To capture sex differences in pituitary gene regulation dynamics during postnatal development, we performed 3' untranslated region sequencing and small RNA sequencing to ascertain gene and microRNA expression, respectively, across five postnatal ages (postnatal days 12, 22, 27, 32, 37) that span the pubertal transition in female and male C57BL/6J mouse pituitaries (n = 5-6 biological replicates for each sex at each age). RESULTS: We observed over 900 instances of sex-biased gene expression and 17 sex-biased microRNAs, with the majority of sex differences occurring with puberty. Using miRNA-gene target interaction databases, we identified 18 sex-biased genes that were putative targets of 5 sex-biased microRNAs. In addition, by combining our bulk RNA-seq with publicly available male and female mouse pituitary single-nuclei RNA-seq data, we obtained evidence that cell-type proportion sex differences exist prior to puberty and persist post-puberty for three major hormone-producing cell types: somatotropes, lactotropes, and gonadotropes. Finally, we identified sex-biased genes in these three pituitary cell types after accounting for cell-type proportion differences between sexes. CONCLUSION: Our study reveals the identity and postnatal developmental trajectory of sex-biased gene expression in the mouse pituitary. This work also highlights the importance of considering sex biases in cell-type composition when understanding sex differences in the processes regulated by the pituitary gland.
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MicroARNs , Hipófisis , Regiones no Traducidas 3' , Animales , Femenino , Expresión Génica , Hormonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Hipófisis/metabolismoRESUMEN
γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.
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Desmetilasa de ARN, Homólogo 5 de AlkB , Linfocitos Intraepiteliales , ARN Mensajero , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Animales , Linfocitos Intraepiteliales/enzimología , Linfocitos Intraepiteliales/inmunología , Proteína Jagged-1/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Receptor Notch2/metabolismo , Transducción de Señal/genéticaRESUMEN
Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.
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COVID-19 , Inflamasomas , Macrófagos , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19/patología , COVID-19/fisiopatología , COVID-19/virología , Humanos , Inflamasomas/metabolismo , Interleucina-1 , Interleucina-18 , Pulmón/patología , Pulmón/virología , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/virología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neumonía/metabolismo , Neumonía/virología , Piroptosis , Receptores de IgG , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidadRESUMEN
Organ formation requires integrating signals to coordinate proliferation, specify cell fates, and shape tissue. Tracing these events and signals remains a challenge, as intermediate states across many critical transitions are unresolvable over real time and space. Here, we designed a unique computational approach to decompose a non-linear differentiation process into key components to resolve the signals and cell behaviors that drive a rapid transition, using the hair follicle dermal condensate as a model. Combining scRNA sequencing with genetic perturbation, we reveal that proliferative Dkk1+ progenitors transiently amplify to become quiescent dermal condensate cells by the mere spatiotemporal patterning of Wnt/ß-catenin and SHH signaling gradients. Together, they deterministically coordinate a rapid transition from proliferation to quiescence, cell fate specification, and morphogenesis. Moreover, genetically repatterning these gradients reproduces these events autonomously in "slow motion" across more intermediates that resolve the process. This analysis unravels two morphogen gradients that intersect to coordinate events of organogenesis.
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Transducción de Señal , Piel , Diferenciación Celular , Folículo Piloso , Proteínas Hedgehog/genética , Morfogénesis , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Although programmed cell death protein 1 (PD-1) inhibitors have revolutionised treatment for advanced melanoma, not all patients respond. We previously showed that inhibition of the flavoprotein renalase (RNLS) in preclinical melanoma models decreases tumour growth. We hypothesised that RNLS inhibition promotes tumour rejection by effects on the tumour microenvironment (TME). METHODS: We used two distinct murine melanoma models, studied in RNLS knockout (KO) or wild-type (WT) mice. WT mice were treated with the anti-RNLS antibody, m28, with or without anti-PD-1. 10X single-cell RNA-sequencing was used to identify transcriptional differences between treatment groups, and tumour cell content was interrogated by flow cytometry. Samples from patients treated with immunotherapy were examined for RNLS expression by quantitative immunofluorescence. RESULTS: RNLS KO mice injected with wild-type melanoma cells reject their tumours, supporting the importance of RNLS in cells in the TME. This effect was blunted by anti-cluster of differentiation 3. However, MØ-specific RNLS ablation was insufficient to abrogate tumour formation. Anti-RNLS antibody treatment of melanoma-bearing mice resulted in enhanced T cell infiltration and activation and resulted in immune memory on rechallenging mice with injection of melanoma cells. At the single-cell level, treatment with anti-RNLS antibodies resulted in increased tumour density of MØ, neutrophils and lymphocytes and increased expression of IFNγ and granzyme B in natural killer cells and T cells. Intratumoural Forkhead Box P3 + CD4 cells were decreased. In two distinct murine melanoma models, we showed that melanoma-bearing mice treated with anti-RNLS antibodies plus anti-PD-1 had superior tumour shrinkage and survival than with either treatment alone. Importantly, in pretreatment samples from patients treated with PD-1 inhibitors, high RNLS expression was associated with decreased survival (log-rank P = 0.006), independent of other prognostic variables. CONCLUSIONS: RNLS KO results in melanoma tumour regression in a T-cell-dependent fashion. Anti-RNLS antibodies enhance anti-PD-1 activity in two distinct aggressive murine melanoma models resistant to PD-1 inhibitors, supporting the development of anti-RNLS antibodies with PD-1 inhibitors as a novel approach for melanomas poorly responsive to anti-PD-1.
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Inhibidores de Puntos de Control Inmunológico , Melanoma , Animales , Humanos , Inmunoterapia , Melanoma/tratamiento farmacológico , Ratones , Monoaminooxidasa/uso terapéutico , Microambiente TumoralRESUMEN
Coronavirus disease 2019 (COVID-19) is an infectious disease that can present as an uncontrolled, hyperactive immune response, causing severe immunological injury. Existing rodent models do not recapitulate the sustained immunopathology of patients with severe disease. Here we describe a humanized mouse model of COVID-19 that uses adeno-associated virus to deliver human ACE2 to the lungs of humanized MISTRG6 mice. This model recapitulates innate and adaptive human immune responses to severe acute respiratory syndrome coronavirus 2 infection up to 28 days after infection, with key features of chronic COVID-19, including weight loss, persistent viral RNA, lung pathology with fibrosis, a human inflammatory macrophage response, a persistent interferon-stimulated gene signature and T cell lymphopenia. We used this model to study two therapeutics on immunopathology, patient-derived antibodies and steroids and found that the same inflammatory macrophages crucial to containing early infection later drove immunopathology. This model will enable evaluation of COVID-19 disease mechanisms and treatments.