RESUMEN
It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.
Asunto(s)
Movimiento Celular , Forma del Núcleo Celular , Neutrófilos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Cromosomas/química , Cromosomas/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Conformación de Ácido Nucleico , Diferenciación Celular/genética , Inflamación/genética , Elementos de Facilitación Genéticos , Linaje de la Célula/genéticaRESUMEN
Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.
Asunto(s)
Diferenciación Celular/inmunología , Macrófagos/inmunología , Melanoma Experimental/inmunología , Monocitos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Animales , Antígenos Ly/inmunología , Separación Celular , Inmunoprecipitación de Cromatina , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/citología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/deficiencia , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVE: Alcohol-associated liver disease is accompanied by microbial dysbiosis, increased intestinal permeability and hepatic exposure to translocated microbial products that contribute to disease progression. A key strategy to generate immune protection against invading pathogens is the secretion of IgA in the gut. Intestinal IgA levels depend on the polymeric immunoglobulin receptor (pIgR), which transports IgA across the epithelial barrier into the intestinal lumen and hepatic canaliculi. Here, we aimed to address the function of pIgR during ethanol-induced liver disease. DESIGN: pIgR and IgA were assessed in livers from patients with alcohol-associated hepatitis and controls. Wild-type and pIgR-deficient (pIgR-/- ) littermates were subjected to the chronic-binge (NIAAA model) and Lieber-DeCarli feeding model for 8 weeks. Hepatic pIgR re-expression was established in pIgR-/- mice using adeno-associated virus serotype 8 (AAV8)-mediated pIgR expression in hepatocytes. RESULTS: Livers of patients with alcohol-associated hepatitis demonstrated an increased colocalisation of pIgR and IgA within canaliculi and apical poles of hepatocytes. pIgR-deficient mice developed increased liver injury, steatosis and inflammation after ethanol feeding compared with wild-type littermates. Furthermore, mice lacking pIgR demonstrated increased plasma lipopolysaccharide levels and more hepatic bacteria, indicating elevated bacterial translocation. Treatment with non-absorbable antibiotics prevented ethanol-induced liver disease in pIgR-/- mice. Injection of AAV8 expressing pIgR into pIgR-/- mice prior to ethanol feeding increased intestinal IgA levels and ameliorated ethanol-induced steatohepatitis compared with pIgR-/- mice injected with control-AAV8 by reducing bacterial translocation. CONCLUSION: Our results highlight that dysfunctional hepatic pIgR enhances alcohol-associated liver disease due to impaired antimicrobial defence by IgA in the gut.
Asunto(s)
Hígado Graso , Hepatitis , Hepatopatías Alcohólicas , Receptores de Inmunoglobulina Polimérica , Ratones , Animales , Etanol/metabolismo , Receptores de Inmunoglobulina Polimérica/metabolismo , Traslocación Bacteriana , Hígado/metabolismo , Hepatopatías Alcohólicas/prevención & control , Hepatopatías Alcohólicas/metabolismo , Hígado Graso/metabolismo , Hepatitis/metabolismo , Inmunoglobulina A , Ratones Endogámicos C57BLRESUMEN
Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the ß2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates ß2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and ß2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ ß2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted, kindlin-3-knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity ß2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ ß2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity ß2-integrin.
Asunto(s)
Antígenos CD18/metabolismo , Rodamiento de Leucocito/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Infiltración Neutrófila/fisiología , Neutrófilos/metabolismo , Animales , Membrana Celular/metabolismo , Células HL-60 , Humanos , Ratones , Transporte de Proteínas/fisiologíaRESUMEN
AIMS/HYPOTHESIS: We aimed to characterise and quantify the expression of HLA class II (HLA-II) in human pancreatic tissue sections and to analyse its induction in human islets. METHODS: We immunostained human pancreatic tissue sections from non-diabetic (n = 5), autoantibody positive (Aab+; n = 5), and type 1 diabetic (n = 5) donors, obtained from the Network of Pancreatic Organ Donors (nPOD), with HLA-II, CD68 and insulin. Each tissue section was acquired with a widefield slide scanner and then analysed with QuPath software. In total, we analysed 7415 islets that contained 338,480 cells. Widefield microscopy was further complemented by high resolution imaging of 301 randomly selected islets, acquired using a Zeiss laser scanning confocal (LSM880) to confirm our findings. Selected beta cells were acquired in enhanced resolution using LSM880 with an Airyscan detector. Further, we cultured healthy isolated human islets and reaggregated human islet microtissues with varying concentrations of proinflammatory cytokines (IFN-γ, TNF-α and IL-1ß). After proinflammatory cytokine culture, islet function was measured by glucose-stimulated insulin secretion, and HLA-I and HLA-II expression was subsequently evaluated with immunostaining or RNA sequencing. RESULTS: Insulin-containing islets (ICIs) of donors with type 1 diabetes had a higher percentage of HLA-II positive area (24.31%) compared with type 1 diabetic insulin-deficient islets (IDIs, 0.67%), non-diabetic (3.80%), and Aab+ (2.31%) donors. In ICIs of type 1 diabetic donors, 45.89% of the total insulin signal co-localised with HLA-II, and 27.65% of the islet beta cells expressed both HLA-II and insulin, while in non-diabetic and Aab+ donors 0.96% and 0.59% of the islet beta cells, respectively, expressed both markers. In the beta cells of donors with type 1 diabetes, HLA-II was mostly present in the cell cytoplasm, co-localising with insulin. In the experiments with human isolated islets and reaggregated human islets, we observed changes in insulin secretion upon stimulation with proinflammatory cytokines, as well as higher expression of HLA-II and HLA-I when compared with controls cultured with media, and an upregulation of HLA-I and HLA-II RNA transcripts. CONCLUSIONS/INTERPRETATION: After a long-standing controversy, we provide definitive evidence that HLA-II can be expressed by pancreatic beta cells from patients with type 1 diabetes. Furthermore, this upregulation can be induced in vitro in healthy isolated human islets or reaggregated human islets by treatment with proinflammatory cytokines. Our findings support a role for HLA-II in type 1 diabetes pathogenesis since HLA-II expressing beta cells can potentially become a direct target of autoreactive CD4+ lymphocytes.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Células Secretoras de Insulina/metabolismo , Adolescente , Adulto , Autoanticuerpos/sangre , Células Cultivadas , Niño , Femenino , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Donantes de Tejidos , Regulación hacia Arriba , Adulto JovenRESUMEN
We defined the effect of the anti-inflammatory cytokines IL4 and IL10 on an in vitro model of human T1D. After preincubation with IL4 or IL10, human islet microtissues were co-cultured with PBMC and proinflammatory cytokines for a few hours or for multiple days to assess acute and chronic effects. This resulted in an immune attack with infiltration of T cells into the islet, a loss of beta cell endocrine function, and an upregulation of HLA-I on the beta cells. HLA-I upregulation was associated with infiltration of T cells and both HLA-I expression and infiltration were associated with impaired insulin secretion. Preincubation with IL4 or IL10 did not preserve beta cell function but decreased infiltration of T cells. Our data support the hypothesis that a loss of beta cell endocrine function mediates an increase in beta cell specific antigen presentation. IL4 and IL10 failed to preserve beta cell endocrine function.
Asunto(s)
Diabetes Mellitus Tipo 1 , Interleucina-10 , Citocinas , Humanos , Interleucina-4/farmacología , Leucocitos Mononucleares/metabolismoRESUMEN
BACKGROUND: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4+ T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (TH1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4+ T cells with an atheroprotective, regulatory T cell (Treg) phenotype in healthy individuals. Yet, the function of apoB-reactive Tregs and their relationship with pathogenic TH1 cells remain unknown. METHODS: To interrogate the function of autoreactive CD4+ T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993 (apoB+) at the single-cell level. RESULTS: We found that apoB+ T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a Treg-like transcriptome, although only 21% of all apoB+ T cells expressed the Treg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB+ T cells formed several clusters with mixed TH signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of TH1, T helper cell type 2 (TH2), and T helper cell type 17 (TH17), and of follicular-helper T cells. ApoB+ T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic TH1/TH17-like cells with proinflammatory properties and only a residual Treg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed TH1/TH17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB+ Tregs in lineage tracing of hyperlipidemic Apoe-/- mice. In adoptive transfer experiments, converting apoB+ Tregs failed to protect from atherosclerosis. CONCLUSIONS: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive Tregs as a novel cellular target in atherosclerosis.
Asunto(s)
Apolipoproteína B-100/inmunología , Aterosclerosis/inmunología , Autoinmunidad , Linfocitos T Reguladores/inmunología , Animales , Apolipoproteína B-100/genética , Aterosclerosis/genética , Ratones , Ratones Noqueados para ApoE , Linfocitos T Reguladores/patologíaRESUMEN
RATIONALE: Macrophages are essential regulators of atherosclerosis. They secrete cytokines, process lipoproteins and cholesterol, and take up apoptotic cells. Multiple subsets of plaque macrophages exist and their differential roles are emerging. OBJECTIVE: Here, we explore macrophage heterogeneity in atherosclerosis plaques using transgenic fluorescent mice in which subsets of macrophages are labeled by GFP (green fluorescent protein), YFP (yellow fluorescent protein), neither, or both. The objective was to define migration patterns of the visible subsets and relate them to their phenotypes and transcriptomes. METHODS AND RESULTS: Apoe-/-Cx3cr1GFPCd11cYFP mice have 4 groups of macrophages in their aortas. The 3 visible subsets show varying movement characteristics. GFP and GFP+YFP+ macrophages extend and retract dendritic processes, dancing on the spot with little net movement while YFP macrophages have a more rounded shape and migrate along the arteries. RNA sequencing of sorted cells revealed significant differences in the gene expression patterns of the 4 subsets defined by GFP and YFP expression, especially concerning chemokine and cytokine expression, matrix remodeling, and cell shape dynamics. Gene set enrichment analysis showed that GFP+ cells have similar transcriptomes to cells found in arteries with tertiary lymphoid organs and regressing plaques while YFP+ cells were associated with progressing and stable plaques. CONCLUSIONS: The combination of quantitative intravital imaging with deep transcriptomes identified 4 subsets of vascular macrophages in atherosclerosis that have unique transcriptomic profiles. Our data link vascular macrophage transcriptomes to their in vivo migratory function. Future work on the functional significance of the change in gene expression and motility characteristics will be needed to fully understand how these subsets contribute to disease progression.
Asunto(s)
Aterosclerosis/patología , Movimiento Celular/fisiología , Macrófagos/patología , Macrófagos/fisiología , Placa Aterosclerótica/patología , Animales , Aterosclerosis/genética , Proteínas Bacterianas/análisis , Femenino , Proteínas Fluorescentes Verdes/análisis , Proteínas Luminiscentes/análisis , Macrófagos/química , Masculino , Ratones , Ratones Transgénicos , Placa Aterosclerótica/genéticaRESUMEN
IL-6 is a pro-inflammatory cytokine upregulated in some autoimmune diseases. The role of IL-6 in the development of type 1 diabetes (T1D) is unclear. Clinical studies are investigating whether tocilizumab (anti-IL-6 receptor) can help preserve beta cell function in patients recently diagnosed with T1D. However, in some rodent models and isolated human islets, IL-6 has been found to have a protective role for beta cells by reducing oxidative stress. Hence, we systematically investigated local tissue expression of IL-6 in human pancreas from non-diabetic, auto-antibody positive donors and donors with T1D and T2D. IL-6 was constitutively expressed by beta and alpha cells regardless of the disease state. However, expression of IL-6 was highly reduced in insulin-deficient islets of donors with T1D, and the expression was then mostly restricted to alpha cells. Our findings suggest that the implication of IL-6 in T1D pathogenesis might be more complex than previously assumed.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Glucagón/inmunología , Células Secretoras de Insulina/inmunología , Interleucina-6/inmunología , Adolescente , Adulto , Anciano , Diabetes Mellitus Tipo 2/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
RATIONALE: Nonclassical mouse monocyte (CX3CR1high, Ly-6Clow) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Because of technical challenges, it is currently not established how patrolling occurs in large arteries. OBJECTIVE: This study was undertaken to elucidate the molecular, migratory, and functional phenotypes of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic, and atherosclerotic conditions. METHODS AND RESULTS: Applying a new method for stable, long-term 2-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP (green fluorescent protein) mice, we show that nonclassical monocytes patrol inside healthy carotid arteries at a velocity of 36 µm/min, 3× faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wild-type mice a Western diet or by applying topical TLR7/8 (Toll-like receptor) agonists. A similar increase is seen in CX3CR1+/GFP/apoE-/- mice on chow diet, with a further 2- to 3-fold increase on Western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 (lymphocyte-associated antigen 1) and α4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1-/- apoE-/- mice leads to pronounced endothelial apoptosis. CONCLUSIONS: Arterial patrolling is a prominent new feature of nonclassical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in a CX3CR1-independent fashion and plays a potential role in endothelial protection.
Asunto(s)
Arterias/metabolismo , Aterosclerosis/metabolismo , Dieta Occidental/efectos adversos , Endotelio Vascular/metabolismo , Monocitos/metabolismo , Receptores de Quimiocina/deficiencia , Animales , Arterias/patología , Aterosclerosis/patología , Receptor 1 de Quimiocinas CX3C , Endotelio Vascular/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
RATIONALE: CD4 T cells are involved in the pathogenesis of atherosclerosis, but atherosclerosis-specific CD4 T cells have not been described. Moreover, the chemokine(s) that regulates T-cell trafficking to the atherosclerotic lesions is also unknown. OBJECTIVE: In Apoe(-/-) mice with mature atherosclerotic lesions (5 months of high fat diet), we find that most aortic T cells express CCR5 and interferon-γ with a unique combination of cell surface markers (CD4(+)CD25(-)CD44(hi)CD62L(lo)) and transcription factors (FoxP3(+)T-bet(+)). We call these cells CCR5Teff. We investigated the role of CCR5 in regulating T-cell homing to the atherosclerotic aorta and the functionality of the CCR5Teff cells. METHODS AND RESULTS: CCR5Teff cells are exclusively found in the aorta and para-aortic lymph nodes of Apoe(-/-) mice. They do not suppress T-cell proliferation in vitro and are less potent than regulatory T cells at inhibiting cytokine secretion. Blocking or knocking out CCR5 or its ligand CCL5 significantly blocks T-cell homing to atherosclerotic aortas. Transcriptomic analysis shows that CCR5Teff cells are more similar to effector T cells than to regulatory T cells. They secrete interferon-γ, interleukin-2, interleukin-10, and tumor necrosis factor. Adoptive transfer of these CCR5Teff cells significantly increases atherosclerosis. CONCLUSIONS: CCR5 is specifically needed for CD4 T-cell homing to the atherosclerotic plaques. CCR5(+)CD4 T cells express an unusual combination of transcription factors, FoxP3 and T-bet. Although CCR5Teff express FoxP3, we showed that they are not regulatory and adoptive transfer of these cells exacerbates atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Receptores CCR5/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Linfocitos T CD4-Positivos/fisiología , Proliferación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucinas/metabolismo , Selectina L/genética , Selectina L/metabolismo , Ratones , Receptores CCR5/genética , Proteínas de Dominio T Box/genéticaRESUMEN
BACKGROUND: Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice. METHODS: We generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. RESULTS: DARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues. CONCLUSIONS: Immunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.
Asunto(s)
Sistema del Grupo Sanguíneo Duffy , Células Endoteliales , Endotelio Vascular , Regulación de la Expresión Génica , Ratones , Receptores de Superficie Celular , Animales , Ratones/genética , Ratones/metabolismo , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Venas/metabolismoRESUMEN
Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4+ T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-Ab by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC50), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model (Apoe-/- mice on Western diet). Immunization with each of the three peptides (1 time in complete Freund's adjuvant subcuntaneously and 4 time in incomplete Freund's adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4+ T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3+ regulatory CD4+ T cells and more than tripled the number of CCR5+FoxP3+ cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection.NEW & NOTEWORTHY Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility complex class II-restricted ApoB peptides, which reduce atherosclerosis and induce IL-10-producing CD4+ T cells and chemokine (C-C motif) receptor 5 expression on regulatory T cells, suggesting that immunization with ApoB peptides inhibits atherosclerosis by inducing anti-inflammatory cytokines.
Asunto(s)
Apolipoproteínas B/inmunología , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Genes MHC Clase II/inmunología , Interleucina-10/biosíntesis , Vacunación , Secuencia de Aminoácidos , Animales , Apolipoproteína B-100 , Apolipoproteínas E/genética , Apolipoproteínas E/inmunología , Aterosclerosis/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Inmunoglobulina G/inmunología , Lipoproteínas LDL/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/inmunologíaRESUMEN
SARS-CoV-2-reactive T cells are detected in some healthy unexposed individuals. Human studies indicate these T cells could be elicited by the common cold coronavirus OC43. To directly test this assumption and define the role of OC43-elicited T cells that are cross-reactive with SARS-CoV-2, we develop a model of sequential infections with OC43 followed by SARS-CoV-2 in HLA-B*0702 and HLA-DRB1*0101 Ifnar1-/- transgenic mice. We find that OC43 infection can elicit polyfunctional CD8+ and CD4+ effector T cells that cross-react with SARS-CoV-2 peptides. Furthermore, pre-exposure to OC43 reduces subsequent SARS-CoV-2 infection and disease in the lung for a short-term in HLA-DRB1*0101 Ifnar1-/- transgenic mice, and a longer-term in HLA-B*0702 Ifnar1-/- transgenic mice. Depletion of CD4+ T cells in HLA-DRB1*0101 Ifnar1-/- transgenic mice with prior OC43 exposure results in increased viral burden in the lung but no change in virus-induced lung damage following infection with SARS-CoV-2 (versus CD4+ T cell-sufficient mice), demonstrating that the OC43-elicited SARS-CoV-2 cross-reactive T cell-mediated cross-protection against SARS-CoV-2 is partially dependent on CD4+ T cells. These findings contribute to our understanding of the origin of pre-existing SARS-CoV-2-reactive T cells and their effects on SARS-CoV-2 clinical outcomes, and also carry implications for development of broadly protective betacoronavirus vaccines.
Asunto(s)
COVID-19 , Coronavirus Humano OC43 , Humanos , Ratones , Animales , SARS-CoV-2 , Ratones Transgénicos , Cadenas HLA-DRB1/genética , Linfocitos T CD4-Positivos , Glicoproteína de la Espiga del CoronavirusRESUMEN
Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.
RESUMEN
Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Ebolavirus/genética , Compartimentos de Replicación Viral , Transcripción Genética , Replicación Viral , Nucleotidiltransferasas/genéticaRESUMEN
Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response of an aggressive low TMB squamous cell tumor to ICB could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4 + and CD8 + T cells. We found that, whereas vaccination with CD4 + or CD8 + NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1 + tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4 + /CD8 + T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8 + T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. The concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.
RESUMEN
BACKGROUND: Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab). METHODS: KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses. RESULTS: The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4-6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups. CONCLUSIONS: IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.
Asunto(s)
Neoplasias Hepáticas , Neoplasias Pancreáticas , Animales , Masculino , Ratones , Anticuerpos/uso terapéutico , Electroporación/métodos , Inmunidad , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias PancreáticasRESUMEN
Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response to ICB of an aggressive low-TMB squamous cell tumor could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4+ and CD8+ T cells. We found that, whereas vaccination with CD4+ or CD8+ NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1+ tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4+/CD8+ T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8+ T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. We believe that the concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Vacunación , Epítopos , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivosRESUMEN
Although mucosal-associated invariant T (MAIT) cells provide rapid, innate-like responses, they are not pre-set, and memory-like responses have been described for MAIT cells following infections. The importance of metabolism for controlling these responses, however, is unknown. Here, following pulmonary immunization with a Salmonella vaccine strain, mouse MAIT cells expanded as separate CD127-Klrg1+ and CD127+Klrg1- antigen-adapted populations that differed in terms of their transcriptome, function and localization in lung tissue. These populations remained altered from steady state for months as stable, separate MAIT cell lineages with enhanced effector programmes and divergent metabolism. CD127+ MAIT cells engaged in an energetic, mitochondrial metabolic programme, which was critical for their maintenance and IL-17A synthesis. This programme was supported by high fatty acid uptake and mitochondrial oxidation and relied on highly polarized mitochondria and autophagy. After vaccination, CD127+ MAIT cells protected mice against Streptococcus pneumoniae infection. In contrast, Klrg1+ MAIT cells had dormant but ready-to-respond mitochondria and depended instead on Hif1a-driven glycolysis to survive and produce IFN-γ. They responded antigen independently and participated in protection from influenza virus. These metabolic dependencies may enable tuning of memory-like MAIT cell responses for vaccination and immunotherapies.