RESUMEN
PURPOSE: Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). METHODS: Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant's anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. RESULTS: Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. CONCLUSIONS: Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones Asintomáticas , COVID-19 , Inmunidad Celular , Inmunidad Humoral , SARS-CoV-2 , Humanos , COVID-19/inmunología , SARS-CoV-2/inmunología , Masculino , Femenino , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Adulto Joven , Adulto , Vacunas contra la COVID-19/inmunología , Estudios de Cohortes , Estudios Longitudinales , Vacunación , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Reino Unido/epidemiología , Adolescente , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to "tune" CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.
Asunto(s)
Linfocitos T CD4-Positivos , Virus de la Influenza A , Mutación , Receptores de Antígenos de Linfocitos T , Animales , Femenino , Humanos , Ratones , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Virus de la Influenza A/inmunología , Virus de la Influenza A/genética , Gripe Humana/inmunología , Gripe Humana/virología , Gripe Humana/prevención & control , Activación de Linfocitos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell-mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell-targeted immunotherapy in mice, we find that CD4+ Foxp3- conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3- Tconv cells within tumors adopt a Treg cell-like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10-dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell-targeted therapies.
Asunto(s)
Neoplasias , Linfocitos T Reguladores , Ratones , Humanos , Animales , Interleucina-10/metabolismo , Neoplasias/terapia , Neoplasias/metabolismo , Inmunoterapia , Factores de Transcripción Forkhead/metabolismoRESUMEN
Improving the anti-tumour T cell response as a consequence of immunotherapy can result in eradication of tumour burden, however, the majority of patients fail with current treatment regimens and so novel immunotherapies with greater efficacy and improved tolerability are needed. The phosphoinositide-3-kinase (PI3K) family members that are directly involved in cell signalling comprise PI3Kα, PI3Kß, PI3Kδ and PI3Kγ, with the latter two isoforms expressed primarily by leukocytes. The survival and optimal function of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) is dependent on PI3Kδ, whereas tumour-associated macrophages (TAMs), use PI3Kγ. Blocking these signalling isoforms can boost development of effective anti-cancer immune responses and result in control of tumour burden. The dependence on different PI3K isoforms in immune cells makes targeting this pathway an attractive approach for tumour immunotherapy. Herein, we discuss how inhibiting specific PI3K isoforms in pro-tumoural Tregs, MDSCS and TAMs can unleash a powerful anti-tumour immune response, driven by CD8+ T cells, capable of controlling tumour burden and consider how the immune response to therapy needs careful investigation, to identify both the correlates of successful treatment and those that impede the generation of robust anti-tumour responses. Furthermore, we review how combination immunotherapy approaches with both PI3K inhibitors and subsequent immune checkpoint blockade can potentiate the efficacy of monotherapy. Finally, we discuss the recent advances in the use of PI3K isoform-specific inhibitors as an immunotherapy for solid tumours in clinical trials.
Asunto(s)
Neoplasias , Fosfatidilinositol 3-Quinasas , Linfocitos T CD8-positivos , Humanos , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/uso terapéuticoRESUMEN
T cells specific for SARS-CoV-2 are thought to protect against infection and development of COVID-19, but direct evidence for this is lacking. Here, we associated whole-blood-based measurement of SARS-CoV-2-specific interferon-γ-positive T cell responses with positive COVID-19 diagnostic (PCR and/or lateral flow) test results up to 6 months post-blood sampling. Amongst 148 participants donating venous blood samples, SARS-CoV-2-specific T cell response magnitude is significantly greater in those who remain protected versus those who become infected (P < 0.0001); relatively low magnitude T cell response results in a 43.2% risk of infection, whereas high magnitude reduces this risk to 5.4%. These findings are recapitulated in a further 299 participants testing a scalable capillary blood-based assay that could facilitate the acquisition of population-scale T cell immunity data (14.9% and 4.4%, respectively). Hence, measurement of SARS-CoV-2-specific T cells can prognosticate infection risk and should be assessed when monitoring individual and population immunity status.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Humanos , Interferón gamma , Reacción en Cadena de la Polimerasa , Linfocitos TRESUMEN
BACKGROUND: Recent studies have demonstrated that blocking the PI3Kδ signalling enzyme (by administering a small molecule inhibitor, PI-3065) can potently improve the anti-tumour T-cell response through direct inhibition of Tregs. This treatment also has a negative impact on MDSC numbers but the primary mechanism driving this effect has remained unclear. METHODS: The 4T1 breast cancer mouse model was used in combination with PI-3065 to gain insights into the effect of PI3Kδ inhibition on MDSCs. RESULTS: PI-3065 treatment resulted in a concomitant reduction in MDSC expansion and tumour size. However, targeting Tregs independent of PI-3065 was also associated with reduced tumour volume and MDSC numbers. Surgical removal of tumours resulted in a rapid and significant decline in MDSC numbers, whilst ex vivo studies using cells from PI-3065-treated mice demonstrated no direct effect of the inhibitor on MDSC activity. CONCLUSIONS: Our data suggest that MDSCs are not inhibited directly by PI-3065 treatment but that their reduced recruitment and immunosuppression within the tumour microenvironment is an indirect consequence of PI3Kδ-inhibition-driven tumour control. This indicates that PI3Kδ inhibition drives tumour immunity by breaking down multiple immunosuppressive pathways through both direct mechanisms (on Treg) and indirect mechanisms, secondary to tumour control (on MDSCs).
Asunto(s)
Células Supresoras de Origen Mieloide , Neoplasias , Animales , Ratones , Linfocitos T Reguladores , Microambiente Tumoral , Proliferación CelularRESUMEN
High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass.
Asunto(s)
Neoplasias , Linfocitos T Reguladores , Humanos , Vénulas , Imagenología Tridimensional , Ganglios LinfáticosRESUMEN
Emerging studies have demonstrated the potential of PI3Kδ blockade as an immunotherapy for solid tumours. In pre-clinical models, we recently demonstrated that anti-LAG3 immune checkpoint blockade vastly potentiated PI3Kδ-based immunotherapy, enabling successful tumour control in all treated mice.
Asunto(s)
Antígenos CD/inmunología , Fosfatidilinositol 3-Quinasa Clase I/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias/tratamiento farmacológico , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia , Ratones , Neoplasias/inmunología , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
BACKGROUND: Despite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors. METHODS: Mice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later. RESULTS: As observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8+ T cells, T cell factor 1 (TCF1)+ T cells and CD69- T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies. CONCLUSIONS: These data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies.
Asunto(s)
Antígenos CD/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Inmunoterapia/métodos , Neoplasias/inmunología , Animales , Femenino , Humanos , Ratones , Microambiente Tumoral , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
The nature of the tumor microenvironment (TME) influences the ability of tumor-specific T cells to control tumor growth. In this study, we performed an unbiased comparison of the TME of regulatory T-cell (Treg)-replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T-cell infiltrates where responding tumors were T-cell rich and ECM poor, whereas the converse was observed in non-responder tumors. For this reason, we hypothesized that the ECM acted as a barrier to successful T-cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T-cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell-like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells but in widescale remodeling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity.
Asunto(s)
Neoplasias/etiología , Células Madre Neoplásicas/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Matriz Extracelular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral/genéticaRESUMEN
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Epítopos/inmunología , Región Variable de Inmunoglobulina/genética , Virus de la Influenza A/inmunología , Adulto , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Aves/virología , Regiones Determinantes de Complementariedad/química , Secuencia Conservada , Epítopos/química , Femenino , Células Germinativas/metabolismo , Antígeno HLA-DR1/inmunología , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Masculino , Persona de Mediana Edad , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Porcinos/virología , Donantes de Tejidos , Proteínas Virales/inmunología , Adulto Joven , Zoonosis/inmunología , Zoonosis/virologíaRESUMEN
Although metastatic disease is responsible for the majority of cancer deaths, tests of novel immunotherapies in mouse tumour models often focus on primary tumours without determining whether these therapies also target metastatic disease. This study examined the impact of depleting Foxp3+ regulatory T cells (Treg), on lung metastases, using a mouse model of breast cancer. After Treg-depletion, generation of an immune response to the primary tumour was a critical determinant for limiting development of metastasis. Indeed, resection of the primary tumour abrogated any effect of Treg-depletion on metastases. In addition, whilst the immune response, generated by the primary tumour, prevented metastases development, it had little impact on controlling established disease. Collectively, the data indicate that metastatic cells in the lung are not controlled by immune responses induced by the primary tumour. These findings indicate that targeting Tregs alone will not suffice for treating lung metastases.
Asunto(s)
Inmunoterapia/métodos , Neoplasias Pulmonares/inmunología , Depleción Linfocítica/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos T Reguladores/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Animales , Femenino , Factores de Transcripción Forkhead/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Ratones , Linfocitos T Reguladores/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
BACKGROUND: The relationship between intestinal epithelial integrity and the development of intestinal disease is of increasing interest. A reduction in mucosal integrity has been associated with ulcerative colitis, Crohn's disease and potentially could have links with colorectal cancer development. The Ussing chamber system can be utilised as a valuable tool for measuring gut integrity. Here we describe step-by-step methodology required to measure intestinal permeability of both mouse and human colonic tissue samples ex vivo, using the latest equipment and software. This system can be modified to accommodate other tissues. METHODS: An Ussing chamber was constructed and adapted to support both mouse and human tissue to measure intestinal permeability, using paracellular flux and electrical measurements. Two mouse models of intestinal inflammation (dextran sodium sulphate treatment and T regulatory cell depletion using C57BL/6-FoxP3DTR mice) were used to validate the system along with human colonic biopsy samples. RESULTS: Distinct regional differences in permeability were consistently identified within mouse and healthy human colon. In particular, mice showed increased permeability in the mid colonic region. In humans the left colon is more permeable than the right. Furthermore, inflammatory conditions induced chemically or due to autoimmunity reduced intestinal integrity, validating the use of the system. CONCLUSIONS: The Ussing chamber has been used for many years to measure barrier function. However, a clear and informative methods paper describing the setup of modern equipment and step-by-step procedure to measure mouse and human intestinal permeability isn't available. The Ussing chamber system methodology we describe provides such detail to guide investigation of gut integrity.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Electrodiagnóstico/instrumentación , Mucosa Intestinal/metabolismo , Animales , Colitis/inducido químicamente , Sulfato de Dextran , Electrodiagnóstico/métodos , Fluorescencia , Humanos , Ratones , Ratones Endogámicos C57BL , PermeabilidadRESUMEN
Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, â¼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.
Asunto(s)
Aorta Abdominal/fisiopatología , Aneurisma de la Aorta Abdominal , Fosfolípidos , Angiotensinas/metabolismo , Animales , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/fisiopatología , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/metabolismo , Modelos Animales de Enfermedad , Femenino , Lipooxigenasa/genética , Lipooxigenasa/metabolismo , Masculino , Ratones , Ratones Noqueados para ApoE , Fosfolípidos/genética , Fosfolípidos/metabolismoRESUMEN
Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Hemorragia/enzimología , Hemorragia/metabolismo , Fosfolípidos/metabolismo , Trombina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Coagulación Sanguínea , Factores de Coagulación Sanguínea/genética , Plaquetas , Puente Cardiopulmonar/efectos adversos , Proteínas Portadoras , Cisteína Endopeptidasas , Factor IX/genética , Factor VIII/genética , Factor VIIa/metabolismo , Factor X/genética , Hemofilia A , Hemorragia/prevención & control , Hemostasis , Humanos , Ácidos Hidroxieicosatetraenoicos , Lipoproteínas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas de Neoplasias , Resonancia por Plasmón de Superficie , Tromboplastina/antagonistas & inhibidores , Tromboplastina/metabolismoRESUMEN
Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein ß2GP1 (ß2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Hemostasis , Fosfolípidos/metabolismo , Activación Plaquetaria , Adulto , Anciano , Animales , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/enzimología , Coagulación Sanguínea , Membrana Celular/ultraestructura , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Humanos , Ácidos Hidroxieicosatetraenoicos/análisis , Ácidos Hidroxieicosatetraenoicos/metabolismo , Lipooxigenasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Modelos Teóricos , Fosfolípidos/análisis , Trombosis de la Vena/sangre , Trombosis de la Vena/inducido químicamente , Trombosis de la Vena/enzimología , beta 2 Glicoproteína I/metabolismoRESUMEN
Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 µM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Caliciformes/metabolismo , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Interleucina-13/farmacología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Araquidonato 15-Lipooxigenasa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor Nuclear 3-beta del Hepatocito/biosíntesis , Factor Nuclear 3-gamma del Hepatocito/biosíntesis , Humanos , Ácidos Linoleicos/biosíntesis , Mucina 5AC/biosíntesisRESUMEN
Background. The myeloid enzyme 12/15-lipoxygenase (LOX), which generates bioactive oxidized lipids, has been implicated in numerous inflammatory diseases, with several studies demonstrating an improvement in pathology in mice lacking the enzyme. However, the ability of 12/15-LOX to directly regulate B cell function has not been studied. Methods. The influence of 12/15-LOX on B cell phenotype and function, and IgM generation, was compared using wildtype (WT) and 12/15-LOX (Alox15-/-) deficient mice. The proliferative and functional capacity of splenic CD19+ B cells was measured in vitro in response to various toll-like receptor agonists. Results. WT and Alox15-/- displayed comparable responses. However in vivo, splenic B cell numbers were significantly elevated in Alox15-/- mice with a corresponding elevation in titres of total IgM in lung, gut and serum, and lower serum IgM directed against the 12/15-LOX product, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (HETE-PE). Discussion. Myeloid 12/15-LOX can regulate B cell numbers and innate immune antibody levels in vivo, potentially contributing to its ability to regulate inflammatory disease. Furthermore, the alterations seen in 12/15-LOX deficiency likely result from changes in the equilibrium of the immune system that develop from birth. Further studies in disease models are warranted to elucidate the contribution of 12/15-LOX mediated alterations in B cell numbers and innate immune antibody generation to driving inflammation in vivo.
RESUMEN
Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.
Asunto(s)
Plaquetas/metabolismo , Dioxolanos/sangre , Integrinas/sangre , Lípidos/sangre , Fosfatidiletanolaminas/sangre , Calcio/sangre , Ciclooxigenasa 1/sangre , Eicosanoides/sangre , Regulación de la Expresión Génica , Humanos , Integrinas/biosíntesis , Antígeno de Macrófago-1/genética , Neutrófilos/metabolismo , Oxidación-Reducción , Fosfolipasas A2 Citosólicas/sangre , Activación Plaquetaria/genética , Receptor PAR-1/sangre , Receptores de Trombina/sangre , Trombina/metabolismo , Fosfolipasas de Tipo C/sangreRESUMEN
Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.