RESUMEN
Background: Seasonal variations have been reported for immune markers. However, the relative contributions of sunlight and vitamin D variability on such seasonal changes are unknown. Objective: This double-blind, randomized, placebo-controlled trial tested whether daily 400 IU vitamin D3 supplementation affected short-term (12 weeks) and long-term (43 weeks) natural regulatory T cell (nTreg) populations in healthy participants. Design: 62 subjects were randomized equally to vitamin D versus placebo in March and assessed at baseline, April (4w), June (12w), September (25w) and January (43w). Circulating nTregs, ex vivo proliferation, IL-10 and IFN-γ productions were measured. Vitamin D metabolites and sunlight exposure were also assessed. Results: Mean serum 25-hydroxyvitamin D (25(OH)D) increased from 35.8(SD 3.0) to 65.3(2.6) nmol/L in April and remained above 75 nmol/L with vitamin D supplementation, whereas it increased from 36.4(3.2) to 49.8(3.5) nmol/L in June to fall back to 39.6(3.5) nmol/L in January with placebo. Immune markers varied similarly between groups according to the season, but independently of 25(OH)D. For nTregs, the mean (%CD3+CD4+CD127lo cells (SEM)) nadir observed in March (2.9(0.1)%) peaked in September at 4.0(0.2)%. Mean T cell proliferation peaked in June (33156(1813) CPM) returning to the nadir in January (17965(978) CPM), while IL-10 peaked in June and reached its nadir in September (median (IQR) of 262(283) to (121(194) pg/ml, respectively). Vitamin D attenuated the seasonal increase in IFN-γ by ~28% with mean ng/ml (SEM) for placebo vs vitamin D, respectively, for April 12.5(1.4) vs 10.0(1.2) (p=0.02); June 13.9(1.3) vs 10.2(1.7) (p=0.02) and January 7.4(1.1) vs 6.0(1.1) (p=0.04). Conclusions: Daily low dose Vitamin D intake did not affect the nTregs population. There were seasonal variation in nTregs, proliferative response and cytokines, suggesting that environmental changes influence immune response, but the mechanism seems independent of vitamin D status. Vitamin D attenuated the seasonal change in T cell-produced IFN-γ, suggesting a decrease in effector response which could be associated with inflammation. Clinical Trial Registration: https://www.isrctn.com, identifier (ISRCTN 73114576).
Asunto(s)
Proliferación Celular/efectos de los fármacos , Colecalciferol/administración & dosificación , Colecalciferol/inmunología , Interferón gamma/análisis , Estaciones del Año , Linfocitos T Reguladores/inmunología , Adulto , Colecalciferol/sangre , Colecalciferol/farmacología , Suplementos Dietéticos , Método Doble Ciego , Femenino , Humanos , Inflamación/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-10/análisis , Interleucina-10/inmunología , Masculino , Persona de Mediana Edad , Luz Solar , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/fisiologíaRESUMEN
Non-immune cells are increasingly recognized as important in regulating immunity, but the role of red blood cells (RBC) remains relatively unexplored, despite their abundance in the circulation and a cell surface rich in potential ligands. Here, we determine whether RBC influence the activation state of human B cells. Separation of RBC from peripheral blood mononuclear cells increased B-cell expression of HLA-DR/DP/DQ, whilst reconstitution reduced the levels of B-cell activation markers HLA-DR/DP/DQ, CD86, CD69 and CD40, as well as decreasing proliferative responses and IgM secretion. Inhibition of B cells required contact with RBC and was abrogated by either removal of sialic acids from RBC or blocking the corresponding lectin receptor CD22 on B cells. Chronic lymphocytic leukaemia B cells express low levels of CD22 and were less susceptible to inhibition by RBC, which may contribute to their activated phenotype. Taken together, the results identify a novel mechanism that may suppress inappropriate responsiveness of healthy B cells whilst circulating in the bloodstream.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Linfocitos B/inmunología , Eritrocitos/inmunología , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos CD40/metabolismo , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Inmunoglobulina M/metabolismo , Lectinas Tipo C/metabolismo , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Ácidos Siálicos/metabolismo , Regulación hacia ArribaRESUMEN
In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Eritrocitos/metabolismo , Manosa/metabolismo , Fagocitos/metabolismo , Polisacáridos/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitología , Eritrocitos/parasitología , Citometría de Flujo/métodos , Hemólisis , Humanos , Ligandos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Glicoproteínas de Membrana/metabolismo , Fagocitosis , Plasmodium falciparum/fisiología , Unión Proteica , Receptores Inmunológicos/metabolismoAsunto(s)
Antígeno CTLA-4/sangre , Antígeno CTLA-4/química , Interferón-alfa/sangre , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Biomarcadores/sangre , Antígeno CTLA-4/genética , Estudios de Cohortes , Haplotipos , Humanos , Interleucina-10/sangre , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas , SolubilidadRESUMEN
The factors and signals driving T cell activation and polarisation during immune responses have been studied mainly at the level of cells and chemical mediators. Here we describe a physical driver of these processes in the form of physiological-strength electric fields (EFs). EFs are generated at sites where epithelium is disrupted (e.g. wounded skin/bronchial epithelia) and where T cells frequently are present. Using live-cell imaging, we show human primary T cells migrate directionally to the cathode in low strength (50/150 mV/mm) EFs. Strikingly, we show for the first time that EFs significantly downregulate T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. These EF-induced functional changes were accompanied by a significant dampening of CD4+ T cell polarisation. Expression of critical markers of the Th17 lineage, RORγt and IL-17, and the Th17 polarisation mediator phospho-STAT3 were reduced significantly, while STAT1, ERK and c-Jun phosphorylation were comparatively unaffected suggesting STAT3 modulation by EFs as one mechanism driving effects. Overall, we identify electrical signals as important contributors to the co-ordination and regulation of human T cell functions, paving the way for a new research area into effects of naturally occurring and clinically-applied EFs in conditions where control of T cell activity is paramount.
Asunto(s)
Campos Electromagnéticos , Activación de Linfocitos/efectos de la radiación , Subgrupos de Linfocitos T/efectos de la radiación , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de la radiación , División Celular/efectos de la radiación , Movimiento Celular , Polaridad Celular/efectos de la radiación , Células Cultivadas , Citocinas/biosíntesis , Electrodos , Endotoxinas/farmacología , Humanos , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Fosforilación , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Células Th17/inmunología , Células Th17/efectos de la radiaciónRESUMEN
Platelet destruction in immune thrombocytopenia is caused by autoreactive antibody and T-cell responses, most commonly directed against platelet glycoprotein IIb/IIIa. Loss of self-tolerance in the disease is also associated with deficient activity of regulatory T cells. Having previously mapped seven major epitopes on platelet glycoprotein IIIa that are recognized by helper T cells from patients with immune thrombocytopenia, the aim was to test whether peptide therapy with any of these sequences, alone or in combination, could inhibit responses to the antigen in humanized mice expressing HLA-DR15. None of the individual peptides, delivered by a putative tolerogenic regimen, consistently suppressed the antibody response to subsequent immunization with human platelet glycoprotein IIb/IIIa. However, the combination of glycoprotein IIIa peptides aa6-20 and aa711-725, which contain the predominant helper epitopes in patients and elicited the strongest trends to suppress when used individually, did abrogate this response. The peptide combination also blunted, but did not reverse, the ongoing antibody response when given after immunization. Suppression of antibody was associated with reduced splenocyte T-cell responsiveness to the antigen, and with the induction of a regulatory T-cell population that is more responsive to the peptides than to purified platelet glycoprotein IIb/IIIa. Overall, these data demonstrate that combinations of peptides containing helper epitopes, such as platelet glycoprotein IIIa aa6-20 and aa711-725, can promote in vivo suppression of responses to the major antigen implicated in immune thrombocytopenia. The approach offers a promising therapeutic option to boost T-cell regulation, which should be taken forward to clinical trials.
Asunto(s)
Formación de Anticuerpos/inmunología , Antígenos HLA/inmunología , Inmunoterapia/métodos , Fragmentos de Péptidos/administración & dosificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Formación de Anticuerpos/efectos de los fármacos , Epítopos/inmunología , Humanos , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Macrophages are key in orchestrating immune responses to micro-environmental stimuli, sensed by a complex set of surface receptors. The human cell line THP-1 has a monocytic phenotype, including the ability to differentiate into macrophages, providing a tractable, standardised surrogate for human monocyte-derived macrophages. Here we assessed the expression of 49 surface markers including Fc, complement, C-type lectin and scavenger receptors; TIMs; Siglecs; and co-stimulatory molecules by flow cytometry on both THP-1 monocytes and macrophages and following macrophage activation with seven standard conditioning/polarizing stimuli. Of the 34 surface markers detected on macrophages, 18 altered expression levels on activation. From these, expression of 9 surface markers were consistently altered by all conditioning regimens, while 9 were specific to individual polarizing stimuli. This study provides a resource for the study of macrophages and highlights that macrophage polarization states share much in common and the differences do not easily fit a simple classification system.
Asunto(s)
Diferenciación Celular/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Células THP-1/inmunología , Biomarcadores/sangre , Línea Celular , Proteínas del Sistema Complemento/inmunología , Humanos , Lectinas Tipo C/inmunología , Activación de Macrófagos/inmunologíaRESUMEN
DISCLOSURES: Vickers: University of Aberdeen: Patents & Royalties: About to apply for patent. Barker: University of Aberdeen: Employment, Patents & Royalties: About to apply for patent. Cao: University of Aberdeen: Patents & Royalties: About to apply for patent.
RESUMEN
BACKGROUND: The inhibitory CTLA-4 molecule is a crucial regulator of immune responses and a target for therapeutic intervention in both autoimmunity and cancer. In particular, CTLA-4 is important in controlling antigen-specific immunity, including responses to autoantigens associated with autoimmune disease. Here, we investigate cytokine responses to a range of lupus-associated autoantigens and assess whether the alternatively spliced isoform of CTLA-4, soluble CTLA-4 (sCTLA-4), contributes to immune regulation of autoantigen-specific immunity in systemic lupus erythematosus (SLE). METHODS: The cell culture supernatant production of sCTLA-4 as well as the cytokines IL-10, IFN-γ, and IL-17 from peripheral blood mononuclear cells (PBMC) from lupus patients and age- and sex-matched healthy volunteer donors were measured in response to previously identified histone and small nuclear ribonucleoprotein (snRNP) autoantigen-derived peptides (H391-105, H471-93, and U170K131-151) by ELISA. We also examined the functional contribution of sCTLA-4 to immune regulation in the context of these autoantigenic peptides following blockade of sCTLA-4 with a selective anti-sCTLA-4 monoclonal antibody, JMW-3B3. RESULTS: We identified responses to autoantigenic peptides, which revealed qualitative differences in cytokine (IL-10, IL-17, and IFN-γ) profiles between SLE patients and healthy donors. PBMC from healthy donors responded to each of the lupus peptides by secreting IFN-γ and IL-17, but PBMC from SLE patients produced IL-10. Although we did not observe differences in the levels of serum or PBMC culture supernatant sCTLA-4 in either cohort, blockade of sCTLA-4 in PBMC cultures responding to antigen enhanced the cytokine profiles associated with each group. CONCLUSION: The results show that lupus autoantigen-derived peptides display varied immunogenicity in lupus versus healthy volunteer donors, while sCTLA-4 acts to regulate the T-cell activity independently of response profile.
Asunto(s)
Antígeno CTLA-4/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Autoantígenos/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4+ T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4+ Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.
Asunto(s)
Eosinófilos/metabolismo , Mycobacterium tuberculosis/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Presentación de Antígeno , Antígenos Dermatofagoides/inmunología , Antígenos Dermatofagoides/metabolismo , Antígenos de Plantas/inmunología , Antígenos de Plantas/metabolismo , Proteínas de Artrópodos/inmunología , Proteínas de Artrópodos/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Citocinas/metabolismo , Eosinófilos/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Activación de Linfocitos , Phleum , Pyroglyphidae , Tuberculina/inmunología , Tuberculina/metabolismoRESUMEN
Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte-derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live-cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking â¼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.
Asunto(s)
Electricidad , Macrófagos/citología , Macrófagos/metabolismo , Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Movimiento Celular , Citocinas/metabolismo , Electrodos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Espacio Intracelular/metabolismo , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Regulación hacia ArribaRESUMEN
Macrophages are antigen presenting cells that can adopt different activation states as directed by microenvironmental stimuli. It is well-recognised how CD4(+) T helper (Th) signals drive macrophage activation, but the ability of differentially activated human macrophages to stimulate the major types of CD4(+) T helper (Th) response by presenting antigen have not been well defined. Previous studies have focussed on murine cells, undifferentiated human monocytes, or macrophage products, and have been limited to non-physiological mitogenic Th responses. The aim was therefore to compare the Th cell polarising abilities of different human macrophage subsets when presenting specific antigen. We demonstrate for the first time that the way macrophages are activated, while naturally presenting antigen, has profound effects on downstream adaptive immune responses. In autologous co-cultures, LPS-activation was the most potent stimulus for antigen-loaded macrophages to drive Th17 polarisation from both unfractionated CD4(+) T-cells and the CD45RO(+) memory population, while IFNγ/LPS activated macrophages preferentially induced a Th1 phenotype. By contrast, IL-4-activated macrophages were ineffective in inducing responses by either Th subset. Although antigen-loaded dendritic cells were superior to macrophages in driving Th1 responses, the Th17 polarising capacity of the two antigen-presenting cell types was equivalent, and was strongly dependent on IL-1ß secretion. Taken together, these results clearly demonstrate for the first time how differentially activated human macrophages present antigen to bias specific, rather than mitogen-driven, Th responses and lead us to propose that they impact adaptive immunity in vivo, particularly in determining Th17 polarisation within inflamed tissues.
Asunto(s)
Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Epítopos/inmunología , Macrófagos/inmunología , Células Th17/inmunología , Técnicas de Cultivo de Célula , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/inmunología , Humanos , Activación de Linfocitos/inmunología , Activación de Macrófagos , Macrófagos/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th17/metabolismoRESUMEN
CTLA-4 is an inhibitory protein that contributes to immune homeostasis and tolerance, a role that has led to its exploitation as a therapeutic in several clinical settings including cancer and autoimmune disease. Development of CTLA-4 therapies focused largely on the full-length receptor isoform but other CTLA-4 isoforms are also expressed, including a secretable form of CTLA-4 (soluble CTLA-4 [sCTLA-4]). The contribution of sCTLA-4 to immune regulation has been less well studied, primarily because it was identified some years after the original description of CTLA-4. Here, we examine how sCTLA-4 might contribute to immune regulation and ask whether it might be a biomarker to inform current CTLA-4 therapies or represent a novel CTLA-4 target for future therapeutics.
Asunto(s)
Antígeno CTLA-4/inmunología , Inmunoterapia , Empalme Alternativo , Animales , Humanos , Isoformas de Proteínas/inmunologíaRESUMEN
Autoreactive CD4⺠helper T cells specific for a range of nucleoprotein-derived autoantigens are an important feature of systemic lupus erythematosus, driving B cell differentiation and autoantibody production and contributing to the inflammatory lesions caused by immune complex deposition. Several peptide epitopes from nucleoprotein antigens have been identified and offer a means selectively to manipulate T cell responses by skewing toward a profile of cytokines that is less pro-inflammatory. Antigen-specific T cell lines and clones can be useful in the study of helper T cell subsets because their life span is prolonged and many individual cells can be generated, allowing particular phenotypes to be studied in detail. Magnetic beads offer a robust and convenient method for the isolation, polarization, and expansion of T cells, which can be adapted for a broad range of applications.
Asunto(s)
Técnicas de Cultivo de Célula , Separación Inmunomagnética/métodos , Linfocitos T Colaboradores-Inductores/citología , Línea Celular , Células Clonales , Humanos , Fenotipo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Proliferación Celular , Factores de Transcripción Forkhead/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Candida albicans/fisiología , Candidiasis/microbiología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Interacciones Huésped-Patógeno/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Enfermedades Renales/inmunología , Enfermedades Renales/virología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/virología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/microbiología , Células Th17/metabolismo , Células Th17/microbiologíaRESUMEN
The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.
Asunto(s)
Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Antígenos de Histocompatibilidad/genética , Fragmentos de Péptidos/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Adulto , Animales , Femenino , Humanos , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/inmunología , Inmunoterapia , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fragmentos de Péptidos/administración & dosificación , Sistema del Grupo Sanguíneo Rh-Hr/química , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Adulto JovenRESUMEN
Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3(hi) -expressing, but not SOCS1(hi) -expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1ß, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.
Asunto(s)
Activación de Macrófagos/inmunología , Macrófagos/inmunología , Nefritis/inmunología , Peritonitis/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Enfermedad Aguda , Animales , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Silenciador del Gen , Humanos , Activación de Macrófagos/genética , Macrófagos/patología , Masculino , FN-kappa B/genética , FN-kappa B/inmunología , Nefritis/genética , Nefritis/patología , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Peritonitis/genética , Peritonitis/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Ratas , Ratas Sprague-Dawley , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Low maternal dietary vitamin E (but not vitamin C) intake during pregnancy has been associated with increased in vitro cord blood mononuclear cell (CBMC) proliferative responses, childhood wheezing and asthma. We investigated whether these associations reflect direct effects of vitamin E by investigating the effects of supplementing CBMC cultures with physiological concentrations of vitamin E. CBMC from seventy neonates were cultured supplemented with either nothing, α-tocopherol or ascorbic acid. Proliferative, IFN-γ, IL-4, IL-10 and TGF-ß responses were measured. In general, vitamin E supplementation was associated with a trend for reduced proliferative responses after stimulation with antigens and house dust mite, and with increased proliferation after stimulation with timothy grass allergen. There was a trend for CBMC cultures to exhibit decreased secretion of IFN-γ, IL-10 and IL-4. Supplementation with vitamin C had no effect on CBMC proliferation, but increased IFN-γ and IL-4 production, and decreased IL-10 production. In conclusion, in vitro vitamin E and C supplementation of CBMC modifies neonatal immune function, but not in a manner predicted by observational epidemiological studies. The observed associations between vitamin E and childhood respiratory disease are complex, and the nature and form of nutritional intervention need to be carefully considered before inclusion in trials.
Asunto(s)
Alérgenos/inmunología , Ácido Ascórbico/administración & dosificación , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Vitamina E/administración & dosificación , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Suplementos Dietéticos , Femenino , Voluntarios Sanos , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Embarazo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , alfa-Tocoferol/administración & dosificaciónRESUMEN
CTLA-4 is a crucial immune regulator that mediates both negative costimulation signals to T cells, and regulatory T (Treg)-cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased Ag-driven proliferation and cytokine (IFN-γ, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to Ag in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antígeno CTLA-4/inmunología , Melanoma Experimental/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/genética , Células Cultivadas , Femenino , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Solubilidad , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
CD22, an inhibitory co-receptor of the BCR, has been identified as a potential candidate gene for the development of autoimmune haemolytic anaemia in mice. In this study, we have examined Cd22(tm1Msn) CD22-deficient mice and identified an increase in RBC turnover and stress erythropoiesis, which might be consistent with haemolysis. We then, however, eliminated CD22 deficiency as the cause of accelerated RBC turnover and established that enhanced RBC turnover occurs independently of B cells and anti-RBC autoanti-bodies. Accelerated RBC turnover in this particular strain of CD22-deficient mice is red cell intrinsic and appears to be the consequence of a defective allele of glucose phosphate isomerase, Gpi1(c). This form of Gpi1 was originally derived from wild mice and results in a substantial reduction in enzyme activity. We have identified the polymorphism that causes impaired catalytic activity in the Gpi1(c) allele, and biochemically confirmed an approximate 75% reduction of GPI1 activity in Cd22(-/-) RBCs. The Cd22(-/-).Gpi1(c) congenic mouse provides a novel animal model of GPI1-deficiency, which is one of the most common causes of chronic non-spherocytic haemolytic anaemia in humans.