RESUMO
Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.
Assuntos
Fator 1-alfa Nuclear de Hepatócito , Hipersensibilidade , Fator 1 de Ligação ao Facilitador Linfoide , Células-Tronco Multipotentes , Fator 1 de Transcrição de Linfócitos T , Células Th2 , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Células Th2/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Hipersensibilidade/imunologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Linfopoietina do Estroma do Timo , Animais , Células Cultivadas , CamundongosRESUMO
Invariant natural killer T cells (iNKT cells) are lipid-sensing innate T cells that are restricted by the antigen-presenting molecule CD1d and express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, as well as their targets in adipose tissue, are unknown. Here we found that iNKT cells in adipose tissue had a unique transcriptional program and produced interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lacked PLZF but expressed the transcription factor E4BP4, which controlled their IL-10 production. The adipose iNKT cells were a tissue-resident population that induced an anti-inflammatory phenotype in macrophages and, through the production of IL-2, controlled the number, proliferation and suppressor function of regulatory T cells (Treg cells) in adipose tissue. Thus, iNKT cells in adipose tissue are unique regulators of immunological homeostasis in this tissue.
Assuntos
Tecido Adiposo/imunologia , Fatores de Transcrição Kruppel-Like/biossíntese , Macrófagos/imunologia , Células T Matadoras Naturais/metabolismo , Linfócitos T Reguladores/metabolismo , Tecido Adiposo/citologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Processos de Crescimento Celular/imunologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Homeostase/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Macrófagos/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Organismos Livres de Patógenos Específicos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Invariant natural killer T cells (iNKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled gene expression in iNKT cells during ontogeny and in peripheral subsets as part of the Immunological Genome Project. High-resolution comparative transcriptional analyses defined developmental and subset-specific programs of gene expression by iNKT cells. In addition, we found that iNKT cells shared an extensive transcriptional program with NK cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Notably, the program shared by NK cells and iNKT cells also operated constitutively in γδ T cells and in adaptive T cells after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.
Assuntos
Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Transcriptoma , Imunidade Adaptativa/genética , Animais , Diferenciação Celular , Linhagem da Célula , Genoma Humano/imunologia , Humanos , Imunidade Inata/genética , Memória Imunológica/genética , Camundongos , Análise em Microsséries , Timo/crescimento & desenvolvimentoRESUMO
Macrophages expressing group V phospholipase A2 (Pla2g5) release the free fatty acid (FFA) linoleic acid (LA), potentiating lung type 2 inflammation. Although Pla2g5 and LA increase in viral infections, their role remains obscure. We generated Pla2g5flox/flox mice, deleted Pla2g5 by using the Cx3cr1cre transgene, and activated bone marrow-derived macrophages (BM-Macs) with poly:IC, a synthetic double-stranded RNA that triggers a viral-like immune response, known Pla2g5-dependent stimuli (IL-4, LPS + IFNγ, IL-33 + IL-4 + GM-CSF) and poly:IC + LA followed by lipidomic and transcriptomic analysis. Poly:IC-activated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs had downregulation of major bioactive lipids and critical enzymes producing those bioactive lipids. In addition, AKT phosphorylation was lower in poly:IC-stimulated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs, which was not restored by adding LA to poly:IC-stimulated BM-Macs. Consistently, Pla2g5flox/flox;Cx3cr1cre/+ mice had diminished poly:IC-induced lung inflammation, including inflammatory macrophage proliferation, while challenging Pla2g5flox/flox;Cx3cr1cre/+ mice with poly:IC + LA partially restored lung inflammation and inflammatory macrophage proliferation. Finally, mice lacking FFA receptor-1 (Ffar1)-null mice had reduced poly:IC-induced lung cell recruitment and tissue macrophage proliferation, not corrected by LA. Thus, Pla2g5 contributes to poly:IC-induced lung inflammation by regulating inflammatory macrophage proliferation and LA/Ffar1-mediated lung cell recruitment and tissue macrophage proliferation.
Assuntos
Ácido Linoleico , Pneumonia , Animais , Camundongos , Proliferação de Células , Interleucina-4/metabolismo , Ácido Linoleico/metabolismo , Pulmão , MacrófagosRESUMO
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread rapidly throughout the world since being identified in South Africa in November 2021. Few studies have assessed primary series and booster vaccine effectiveness against Omicron among US healthcare workers. METHODS: We conducted a test-negative case-control design to estimate BNT162b2 and mRNA1273 primary vaccination and booster effectiveness against SARS-CoV-2 infection and symptomatic coronavirus disease 2019 during an Omicron surge among employees of the University of Pennsylvania Health System. The study period was between 1 July 2021 and 5 April 2022. We defined the Delta period as 1 July to 12 December 2021 and the Omicron period as beginning 12 December 21. RESULTS: Our sample included 14 520 tests (2776 [19%] positive)-7422 (506 [7%] positive) during Delta and 7098 (2270 [32%] positive) during Omicron. Benchmarked against Delta, the vaccine effectiveness of 2 vaccine doses was lower during Omicron, with no significant protection against infection. Booster doses added significant protection, although they also showed reduced effectiveness during Omicron. Compared with findings in employees who had received 2 vaccine doses, 3 doses of BNT162b2 had a relative effectiveness of 50% (95% confidence interval, 42%-56%) during Omicron, relative to 78% (63%-87%) during Delta; 3 doses of mRNA1273 had a relative effectiveness of 56% (45%-65%) during Omicron, relative to 96% (82%-99%) during Delta. Restricting the sample to symptomatic tests yielded similar results to our primary analysis. After initial waning in BNT162b2 booster protection against infection, it remained largely stable for ≥16 weeks after vaccination. CONCLUSIONS: Our findings provide a strong rationale for boosters among healthcare workers in the Omicron era.
Assuntos
COVID-19 , Vacinas , Humanos , Vacina BNT162 , SARS-CoV-2 , COVID-19/prevenção & controle , Pessoal de Saúde , Vacina de mRNA-1273 contra 2019-nCoV , RNA , RNA MensageiroRESUMO
Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self antigen. We found that an abundant endogenous lipid, ß-D-glucopyranosylceramide (ß-GlcCer), was a potent iNKT cell self antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, ß-GlcCer accumulated during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of ß-GlcCer by the invariant T cell antigen receptor translates innate danger signals into iNKT cell activation.
Assuntos
Autoantígenos/imunologia , Infecções Bacterianas/imunologia , Glicoesfingolipídeos/imunologia , Células T Matadoras Naturais/imunologia , Animais , Autoimunidade/imunologia , Linhagem Celular , Glicoesfingolipídeos/metabolismo , Humanos , Ativação Linfocitária/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
BACKGROUND: Intestinal microenvironmental perturbations may increase food allergy risk. We hypothesize that children with clinical food allergy, those with food sensitization, and healthy children can be differentiated by intestinal metabolites in the first years of life. METHODS: In this ancillary analysis of the Vitamin D Antenatal Asthma Reduction Trial (VDAART), we performed untargeted metabolomic profiling in 824 stool samples collected at ages 3-6 months, 1 year and 3 years. Subjects included 23 with clinical food allergy at age 3 and/or 6 years, 151 with food sensitization but no clinical food allergy, and 220 controls. We identified modules of correlated, functionally related metabolites and sought associations of metabolite modules and individual metabolites with food allergy/sensitization using regression models. RESULTS: Several modules of functionally related intestinal metabolites were reduced among subjects with food allergy, including bile acids at ages 3-6 months and 1 year, amino acids at age 3-6 months, steroid hormones at 1 year, and sphingolipids at age 3 years. One module primarily containing diacylglycerols was increased in those with food allergy at age 3-6 months. Fecal caffeine metabolites at age 3-6 months, likely derived from breast milk, were increased in those with food allergy and/or sensitization (beta = 5.9, 95% CI 1.0-10.8, p = .02) and were inversely correlated with fecal bile acids and bilirubin metabolites, though maternal plasma caffeine levels were not associated with food allergy and/or sensitization. CONCLUSIONS: Several classes of bioactive fecal metabolites are associated with food allergy and/or sensitization including bile acids, steroid hormones, sphingolipids, and caffeine metabolites.
Assuntos
Cafeína , Hipersensibilidade Alimentar , Criança , Humanos , Feminino , Gravidez , Pré-Escolar , Lactente , Hipersensibilidade Alimentar/diagnóstico , Metabolômica , Alérgenos , Leite Humano , EsfingolipídeosRESUMO
BACKGROUND: Ensuring equitable care remains a critical issue for healthcare systems. Nationwide evidence highlights the persistence of healthcare disparities and the need for research-informed approaches for reducing them at the local level. OBJECTIVE: To characterize key contributors in racial/ethnic disparities in emergency department (ED) throughput times. DESIGN: We conducted a sequential mixed methods analysis to understand variations in ED care throughput times for patients eventually admitted to an emergency department at a single academic medical center from November 2017 to May 2018 (n=3152). We detailed patient progression from ED arrival to decision to admit and compared racial/ethnic differences in time intervals from electronic medical record time-stamp data. We then estimated the relationships between race/ethnicity and ED throughput times, adjusting for several patient-level variables and ED-level covariates. These quantitative analyses informed our qualitative study design, which included observations and semi-structured interviews with patients and physicians. KEY RESULTS: Non-Hispanic Black as compared to non-Hispanic White patients waited significantly longer during the time interval from arrival to the physician's decision to admit, even after adjustment for several ED-level and patient demographic, clinical, and socioeconomic variables (Beta (average minutes) (SE): 16.35 (5.8); p value=.005). Qualitative findings suggest that the manner in which providers communicate, advocate, and prioritize patients may contribute to such disparities. When the race/ethnicity of provider and patient differed, providers were more likely to interrupt patients, ignore their requests, and make less eye contact. Conversely, if the race/ethnicity of provider and patient were similar, providers exhibited a greater level of advocacy, such as tracking down patient labs or consultants. Physicians with no significant ED throughput disparities articulated objective criteria such as triage scores for prioritizing patients. CONCLUSIONS: Our findings suggest the importance of (1) understanding how our communication style and care may differ by race/ethnicity; and (2) taking advantage of structured processes designed to equalize care.
Assuntos
Serviços Médicos de Emergência , Etnicidade , Serviço Hospitalar de Emergência , Disparidades em Assistência à Saúde , Hospitalização , Humanos , Estados UnidosRESUMO
Vaccination remains key to reducing the risk of COVID-19-related severe illness and death. Because of historic medical exclusion and barriers to access, Black communities have had lower rates of COVID-19 vaccination than White communities. We describe the efforts of an academic medical institution to implement community-based COVID-19 vaccine clinics in medically underserved neighborhoods in Philadelphia, Pennsylvania. Over a 13-month period (April 2021-April 2022), the initiative delivered 9038 vaccine doses to community members, a majority of whom (57%) identified as Black. (Am J Public Health. 2022;112(12):1721-1725. https://doi.org/10.2105/AJPH.2022.307030).
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , Área Carente de Assistência Médica , COVID-19/epidemiologia , COVID-19/prevenção & controle , Philadelphia/epidemiologia , VacinaçãoRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic continues to surge in the United States and globally. OBJECTIVE: To describe the epidemiology of COVID-19-related critical illness, including trends in outcomes and care delivery. DESIGN: Single-health system, multihospital retrospective cohort study. SETTING: 5 hospitals within the University of Pennsylvania Health System. PATIENTS: Adults with COVID-19-related critical illness who were admitted to an intensive care unit (ICU) with acute respiratory failure or shock during the initial surge of the pandemic. MEASUREMENTS: The primary exposure for outcomes and care delivery trend analyses was longitudinal time during the pandemic. The primary outcome was all-cause 28-day in-hospital mortality. Secondary outcomes were all-cause death at any time, receipt of mechanical ventilation (MV), and readmissions. RESULTS: Among 468 patients with COVID-19-related critical illness, 319 (68.2%) were treated with MV and 121 (25.9%) with vasopressors. Outcomes were notable for an all-cause 28-day in-hospital mortality rate of 29.9%, a median ICU stay of 8 days (interquartile range [IQR], 3 to 17 days), a median hospital stay of 13 days (IQR, 7 to 25 days), and an all-cause 30-day readmission rate (among nonhospice survivors) of 10.8%. Mortality decreased over time, from 43.5% (95% CI, 31.3% to 53.8%) to 19.2% (CI, 11.6% to 26.7%) between the first and last 15-day periods in the core adjusted model, whereas patient acuity and other factors did not change. LIMITATIONS: Single-health system study; use of, or highly dynamic trends in, other clinical interventions were not evaluated, nor were complications. CONCLUSION: Among patients with COVID-19-related critical illness admitted to ICUs of a learning health system in the United States, mortality seemed to decrease over time despite stable patient characteristics. Further studies are necessary to confirm this result and to investigate causal mechanisms. PRIMARY FUNDING SOURCE: Agency for Healthcare Research and Quality.
Assuntos
COVID-19/mortalidade , COVID-19/terapia , Estado Terminal/mortalidade , Estado Terminal/terapia , Pneumonia Viral/mortalidade , Pneumonia Viral/terapia , Choque/mortalidade , Choque/terapia , APACHE , Centros Médicos Acadêmicos , Idoso , Feminino , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Pandemias , Readmissão do Paciente/estatística & dados numéricos , Pennsylvania/epidemiologia , Pneumonia Viral/virologia , Respiração Artificial/estatística & dados numéricos , Estudos Retrospectivos , SARS-CoV-2 , Choque/virologia , Taxa de SobrevidaRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic challenges hospital leaders to make time-sensitive, critical decisions about clinical operations and resource allocations. OBJECTIVE: To estimate the timing of surges in clinical demand and the best- and worst-case scenarios of local COVID-19-induced strain on hospital capacity, and thus inform clinical operations and staffing demands and identify when hospital capacity would be saturated. DESIGN: Monte Carlo simulation instantiation of a susceptible, infected, removed (SIR) model with a 1-day cycle. SETTING: 3 hospitals in an academic health system. PATIENTS: All people living in the greater Philadelphia region. MEASUREMENTS: The COVID-19 Hospital Impact Model (CHIME) (http://penn-chime.phl.io) SIR model was used to estimate the time from 23 March 2020 until hospital capacity would probably be exceeded, and the intensity of the surge, including for intensive care unit (ICU) beds and ventilators. RESULTS: Using patients with COVID-19 alone, CHIME estimated that it would be 31 to 53 days before demand exceeds existing hospital capacity. In best- and worst-case scenarios of surges in the number of patients with COVID-19, the needed total capacity for hospital beds would reach 3131 to 12 650 across the 3 hospitals, including 338 to 1608 ICU beds and 118 to 599 ventilators. LIMITATIONS: Model parameters were taken directly or derived from published data across heterogeneous populations and practice environments and from the health system's historical data. CHIME does not incorporate more transition states to model infection severity, social networks to model transmission dynamics, or geographic information to account for spatial patterns of human interaction. CONCLUSION: Publicly available and designed for hospital operations leaders, this modeling tool can inform preparations for capacity strain during the early days of a pandemic. PRIMARY FUNDING SOURCE: University of Pennsylvania Health System and the Palliative and Advanced Illness Research Center.
Assuntos
Betacoronavirus , Infecções por Coronavirus/terapia , Tomada de Decisões , Unidades de Terapia Intensiva/organização & administração , Modelos Organizacionais , Pandemias , Pneumonia Viral/terapia , COVID-19 , Infecções por Coronavirus/epidemiologia , Humanos , Pneumonia Viral/epidemiologia , SARS-CoV-2 , Estados Unidos/epidemiologiaRESUMO
Glycolipid antigens recognized by αß T-cell receptors (TCRs) drive the activation of invariant natural killer T (iNKT) cells, a specialized subset of innate T lymphocytes. Glycolipids with α-linked anomeric carbohydrates have been identified as potent microbial lipid antigens for iNKT cells, and their unusual α-anomeric linkage has been thought to define a "foreign" lipid antigen motif. However, mammals use endogenous lipids to select iNKT cells, and there is compelling evidence for iNKT cell responses in various types of sterile inflammation. The nature of endogenous or environmental lipid antigens encountered by iNKT cells is not well defined. Here, we sought to identify lipid antigens in cow's milk, a prominent part of the human diet. We developed a method to directly capture lipid antigens within CD1d-lipid-TCR complexes, while excluding CD1d bound to nonantigenic lipids, followed by direct biochemical analysis of the lipid antigens trapped at the TCR-CD1d interface. The specific antigens captured by this "TCR trap" method were identified as α-linked monohexosylceramides by mass spectrometry fragmentation patterns that distinguished α- from ß-anomeric monohexosylceramides. These data provide direct biochemical evidence for α-linked lipid antigens from a common dietary source.
Assuntos
Antígenos CD1d/imunologia , Galactosilceramidas/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Bioensaio/métodos , Dieta , Humanos , Camundongos , Leite/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-γ, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα paired with unique Vß7 or Vß8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity.
Assuntos
Diferenciação Celular , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/fisiologia , Citocinas/genética , Citocinas/imunologia , Citotoxicidade Imunológica/imunologia , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Técnicas de Transferência Nuclear , Especificidade de Órgãos , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Invariant natural killer T (iNKT) cells are innate T cells that express a semi-invariant T cell receptor (TCR) and recognize lipid antigens presented by CD1d molecules. As part of innate immunity, iNKT cells rapidly produce large amounts of cytokines after activation and regulate the function of innate and adaptive immune cells in antimicrobial immunity, tumor rejection and inflammatory diseases. Global transcriptional profiling has advanced our understanding of all aspects of iNKT cell biology. In this review, we discuss transcriptional analyses of iNKT cell development, functional subsets of iNKT cells, and global comparisons of iNKT cells to other innate and adaptive immune cells. Global transcriptional analysis revealed that iNKT cells have a transcriptional profile distinct from NK cells and MHC-restricted T cells, both during thymic development and in the periphery. The transcription factors EGR2 and PLZF (and microRNA like miR-150) are key regulators of the iNKT cell transcriptome during development. PLZF is one of several factors that control the homing and maintenance of organ-specific iNKT cell populations. As in MHC-restricted T cells, specific transcription factors are characteristic of functional subsets of iNKT cells, such as the transcription factor T-bet in the NKT1 subset. Exciting future directions for global transcriptional analyses include iNKT cells in disease models, diverse NKT cells and human studies.
Assuntos
Células T Matadoras Naturais/metabolismo , Transcrição Gênica , Animais , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Humanos , Inflamação/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , TranscriptomaRESUMO
The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.
Assuntos
Proteínas de Bactérias/genética , Parede Celular/metabolismo , Galactanos/metabolismo , Mycobacterium tuberculosis/metabolismo , Peptidoglicano/metabolismo , Ácidos Teicoicos/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Galactanos/genética , Mycobacterium tuberculosis/genética , Peptidoglicano/genética , Ácidos Teicoicos/genéticaRESUMO
Invariant natural killer T (iNKT) cells are a specialized T-cell subset that recognizes lipids as antigens, contributing to immune responses in diverse disease processes. Experimental data suggests that iNKT cells can recognize both microbial and endogenous lipid antigens. Several candidate endogenous lipid antigens have been proposed, although the contextual role of specific antigens during immune responses remains largely unknown. We have previously reported that mammalian glucosylceramides (GlcCers) activate iNKT cells. GlcCers are found in most mammalian tissues, and exist in variable molecular forms that differ mainly in N-acyl fatty acid chain use. In this report, we purified, characterized, and tested the GlcCer fractions from multiple animal species. Although activity was broadly identified in these GlcCer fractions from mammalian sources, we also found activity properties that could not be reconciled by differences in fatty acid chain use. Enzymatic digestion of ß-GlcCer and a chromatographic separation method demonstrated that the activity in the GlcCer fraction was limited to a rare component of this fraction, and was not contained within the bulk of ß-GlcCer molecular species. Our data suggest that a minor lipid species that copurifies with ß-GlcCer in mammals functions as a lipid self antigen for iNKT cells.
Assuntos
Glucosilceramidas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Animais , Bovinos , Cromatografia em Camada Fina , Glucosilceramidase/metabolismo , Glucosilceramidas/química , Humanos , Ativação Linfocitária/imunologia , Espectrometria de Massas , Camundongos , Leite/química , Células T Matadoras Naturais/efeitos dos fármacosRESUMO
Genome-wide association studies (GWAS) and subsequent dense-genotyping of associated loci identified over a hundred single-nucleotide polymorphism (SNP) variants associated with the risk of rheumatoid arthritis (RA), type 1 diabetes (T1D), and celiac disease (CeD). Immunological and genetic studies suggest a role for CD4-positive effector memory T (CD+ TEM) cells in the pathogenesis of these diseases. To elucidate mechanisms of autoimmune disease alleles, we investigated molecular phenotypes in CD4+ effector memory T cells potentially affected by these variants. In a cohort of genotyped healthy individuals, we isolated high purity CD4+ TEM cells from peripheral blood, then assayed relative abundance, proliferation upon T cell receptor (TCR) stimulation, and the transcription of 215 genes within disease loci before and after stimulation. We identified 46 genes regulated by cis-acting expression quantitative trait loci (eQTL), the majority of which we detected in stimulated cells. Eleven of the 46 genes with eQTLs were previously undetected in peripheral blood mononuclear cells. Of 96 risk alleles of RA, T1D, and/or CeD in densely genotyped loci, eleven overlapped cis-eQTLs, of which five alleles completely explained the respective signals. A non-coding variant, rs389862A, increased proliferative response (p=4.75 × 10-8). In addition, baseline expression of seventeen genes in resting cells reliably predicted proliferative response after TCR stimulation. Strikingly, however, there was no evidence that risk alleles modulated CD4+ TEM abundance or proliferation. Our study underscores the power of examining molecular phenotypes in relevant cells and conditions for understanding pathogenic mechanisms of disease variants.
Assuntos
Artrite Reumatoide/genética , Doenças Autoimunes/genética , Doença Celíaca/genética , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/genética , Locos de Características Quantitativas/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Proliferação de Células/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The innate immune system recognizes microbial pathogens via pattern recognition receptors. One such receptor, NOD2, via recognition of muramyl dipeptide (MDP), triggers a distinct network of innate immune responses, including the production of interleukin-32 (IL-32), which leads to the differentiation of monocytes into dendritic cells (DC). NOD2 has been implicated in the pathogenesis of human leprosy, yet it is not clear whether Mycobacterium leprae, which has a distinct MDP structure, can activate this pathway. We investigated the effect of MDP structure on the innate immune response, finding that infection of monocytes with M. leprae induces IL-32 and DC differentiation in a NOD2-dependent manner. The presence of the proximal l-Ala instead of Gly in the common configuration of the peptide side chain of M. leprae did not affect recognition by NOD2 or cytokine production. Furthermore, amidation of the d-Glu residue did not alter NOD2 activation. These data provide experimental evidence that NOD2 recognizes naturally occurring structural variants of MDP.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Mycobacterium leprae/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunidade Inata/imunologia , Interleucinas/metabolismo , Hanseníase/imunologia , Hanseníase/metabolismo , Monócitos/metabolismo , Mycobacterium leprae/imunologiaRESUMO
Invariant natural killer T (iNKT) cells are a specialized T cell subset that plays an important role in host defense, orchestrating both innate and adaptive immune effector responses against a variety of microbes. Specific microbial lipids and mammalian self lipids displayed by the antigen-presenting molecule CD1d can activate iNKT cells through their semi-invariant αß T cell receptors (TCRs). iNKT cells also constitutively express receptors for inflammatory cytokines typically secreted by antigen-presenting cells (APCs) after recognition of pathogen-associated molecular patterns (PAMPs), and they can be activated through these cytokine receptors either in combination with TCR signals, or in some cases even in the absence of TCR signaling. During infection, experimental evidence suggests that both TCR-driven and cytokine-driven mechanisms contribute to iNKT cell activation. While the relative contributions of these two signaling mechanisms can vary widely depending on the infectious context, both lipid antigens and PAMPs mediate reciprocal activation of iNKT cells and APCs, leading to downstream activation of multiple other immune cell types to promote pathogen clearance. In this review, we discuss the mechanisms involved in iNKT cell activation during infection, focusing on the central contributions of both lipid antigens and PAMP-induced inflammatory cytokines, and highlight in vivo examples of activation during bacterial, viral, and fungal infections.
Assuntos
Apresentação de Antígeno/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , HumanosRESUMO
Defining and characterizing pathologies of the immune system requires precise and accurate quantification of abundances and functions of cellular subsets via cytometric studies. At this time, data analysis relies on manual gating, which is a major source of variability in large-scale studies. We devised an automated, user-guided method, X-Cyt, which specializes in rapidly and robustly identifying targeted populations of interest in large data sets. We first applied X-Cyt to quantify CD4(+) effector and central memory T cells in 236 samples, demonstrating high concordance with manual analysis (r = 0.91 and 0.95, respectively) and superior performance to other available methods. We then quantified the rare mucosal associated invariant T cell population in 35 samples, achieving manual concordance of 0.98. Finally we characterized the population dynamics of invariant natural killer T (iNKT) cells, a particularly rare peripheral lymphocyte, in 110 individuals by assaying 19 markers. We demonstrated that although iNKT cell numbers and marker expression are highly variable in the population, iNKT abundance correlates with sex and age, and the expression of phenotypic and functional markers correlates closely with CD4 expression.