RESUMEN
Regulatory B (Breg) cells can dampen inflammation, autoreactivity, and transplant rejection. We investigated the frequencies, phenotypes, and function of Breg cells in granulomatosis with polyangiitis (GPA) to gain further knowledge as to whether there are numerical alterations or limitations of their ability to regulate T-cell function. Frequencies and phenotypes of CD24hiCD27+ and CD24hiCD38hi B-cells in the blood were determined with flow cytometry in 37 GPA patients (22 in remission and 15 with active disease) and 31 healthy controls (HC). A co-culture model was used to study the capacity of Breg cells to regulate T-cell activation and proliferation in cells from 10 GPA patients in remission and 12 HC. T-cell cytokine production in vitro and levels in plasma were determined with enzyme-linked immunosorbent assay. Frequencies of CD24hiCD27+ B-cells were reduced both during active disease and remission compared with HC (P = 0.005 and P = 0.010, respectively), whereas CD24hiCD38hi B-cells did not differ. Patient CD24hiCD27+ B-cells exhibited decreased expression of CD25 but increased expression of PD-L1 and PD-L2 during remission. B-cells from GPA patients regulated T-cell proliferation but failed to regulate interferon (IFN)-γ production (median T-cells alone 222 ng/ml vs. T-cells + B-cells 207 ng/ml, P = 0.426). IFN-γ was also elevated in patient plasma samples (P = 0.016). In conclusion, GPA patients exhibit altered numbers and phenotypes of CD24hiCD27+ B-cells. This is accompanied by a disability to control T-cell production of Th1-type cytokines during remission, which might be of fundamental importance for the granulomatous inflammation that characterizes the chronic phase of this disease.
Asunto(s)
Linfocitos B Reguladores , Granulomatosis con Poliangitis , Humanos , Linfocitos B Reguladores/metabolismo , Citocinas/metabolismo , Interferón gamma , InflamaciónRESUMEN
INTRODUCTION AND OBJECTIVE: Interleukin (IL)-32 is a pro-inflammatory cytokine not previously studied in relation to abdominal aortic aneurysm (AAA). The aim of this study was to elucidate the expression and localization of IL-32 in AAA. METHODS: Expression and localization of IL-32 in human aortic tissue was studied with immunohistochemical analysis and Western blot (AAA: n = 5; controls: n = 4). ELISA was used to measure IL-32 in human plasma samples (AAA: n = 140; controls: n = 37) and in media from cultured peripheral blood mononuclear cells (PBMCs) from 3 healthy donors. IL-32 mRNA in PBMCs, endothelial cells, aortic smooth muscle cells (SMCs), and aortic tissue samples of AAA (n = 16) and control aortas (n = 9) was measured with qPCR. RESULTS: IL-32 was predominantly expressed in SMCs and T-cell-rich areas. Highest mRNA expression was observed in the intima/media layer of the AAA. A weaker protein expression was detected in non-aneurysmal aortas. Expression of IL-32 was confirmed in isolated T cells, macrophages, endothelial cells, and SMCs, where expression was also inducible by cytokines such as interferon-γ. There was no difference in IL-32 expression in plasma between patients and controls. CONCLUSION: IL-32 signaling is altered locally in AAA and could potentially play an important role in aneurysm development. Further studies using animal models would be helpful to study its potential role in AAA disease.
Asunto(s)
Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/metabolismo , Interleucinas/metabolismo , Adulto , Anciano , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Estudios de Casos y Controles , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Humanos , Interleucinas/genética , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Regulación hacia Arriba , Adulto JovenRESUMEN
Dendritic cells (DCs) are mediators between innate and adaptive immunity and vital in initiating and modulating antigen-specific immune responses. The most important site for induction of tolerance is the gut mucosa, where TGF-ß, retinoic acid, and aryl hydrocarbon receptors collaborate in DCs to induce a tolerogenic phenotype. To mimic this, a novel combination of compounds - the synthetic aryl hydrocarbon receptor (AhR) agonist IGN-512 together with TGF-ß and retinoic acid - was developed to create a platform technology for induction of tolerogenic DCs intended for treatment of several conditions caused by unwanted immune activation. These in vitro-generated cells, designated ItolDCs, are phenotypically characterized by their low expression of co-stimulatory and activating molecules along with high expression of tolerance-associated markers such as ILT3, CD103, and LAP, and a weak pro-inflammatory cytokine profile. When co-cultured with T cells and/or B cells, ItolDC-cultures contain higher frequencies of CD25+Foxp3+ regulatory T cells (Tregs), CD49b+LAG3+ 'type 1 regulatory (Tr1) T cells, and IL-10-producing B cells and are less T cell stimulatory compared to cultures with matured DCs. Factor VIII (FVIII) and tetanus toxoid (TT) were used as model antigens to study ItolDC antigen-loading. ItolDCs can take up FVIII, process, and present FVIII peptides on HLA-DR. By loading both ItolDCs and mDCs with TT, antigen-specific T cell proliferation was observed. Cryo-preserved ItolDCs showed a stable tolerogenic phenotype that was maintained after stimulation with LPS, CD40L, or a pro-inflammatory cocktail. Moreover, exposure to other immune cells did not negatively impact ItolDCs' expression of tolerogenic markers. In summary, a novel protocol was developed supporting the generation of a stable population of human DCs in vitro that exhibited a tolerogenic phenotype with an ability to increase proportions of induced regulatory T and B cells in mixed cultures. This protocol has the potential to constitute the base of a tolDC platform for inducing antigen-specific tolerance in disorders caused by undesired antigen-specific immune cell activation.
Asunto(s)
Tolerancia Inmunológica , Factor de Crecimiento Transformador beta , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Tretinoina/metabolismo , Células Dendríticas , Membrana MucosaRESUMEN
Neutrophil extracellular traps (NETs) and mitochondrial DNA (mtDNA) are inflammatory mediators involved in the development of type 1 diabetes (T1D). Pancreas-infiltrating neutrophils can release NETs, contributing to the inflammatory process. Levels of NETs are increased in serum from patients with T1D and mtDNA is increased in adult T1D patients. Our aim was to investigate extracellular DNA (NETs, mtDNA and nuclear DNA) in children with newly diagnosed T1D and in children at high risk of the disease. We also elucidated if extracellular DNA short after diagnosis could predict loss of endogenous insulin production. Samples were analysed for mtDNA and nuclear DNA using droplet digital PCR and NETs were assessed by a NET-remnants ELISA. In addition, in vitro assays for induction and degradation of NETs, as well as analyses of neutrophil elastase, HLA genotypes, levels of c-peptide, IL-1beta, IFN and autoantibodies (GADA, IA-2A, IAA and ZnT8A) were performed. In serum from children 10 days after T1D onset there was an increase in NETs (p=0.007), mtDNA (p<0.001) and nuclear DNA (p<0.001) compared to healthy children. The elevated levels were found only in younger children. In addition, mtDNA increased in consecutive samples short after onset (p=0.017). However, levels of extracellular DNA short after onset did not reflect future loss of endogenous insulin production. T1D serum induced NETs in vitro and did not deviate in the ability to degrade NETs. HLA genotypes and autoantibodies, except for ZnT8A, were not associated with extracellular DNA in T1D children. Serum from children with high risk of T1D showed fluctuating levels of extracellular DNA, sometimes increased compared to healthy children. Therefore, extracellular DNA in serum from autoantibody positive high-risk children does not seem to be a suitable biomarker candidate for prediction of T1D. In conclusion, we found increased levels of extracellular DNA in children with newly diagnosed T1D, which might be explained by an ongoing systemic inflammation.
Asunto(s)
Núcleo Celular/genética , ADN Mitocondrial/sangre , ADN/sangre , Diabetes Mellitus Tipo 1/sangre , Trampas Extracelulares/metabolismo , Adolescente , Factores de Edad , Autoanticuerpos/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Humanos , Estudios Longitudinales , Masculino , Medición de Riesgo , Factores de Riesgo , Regulación hacia ArribaRESUMEN
How do you wash cells? Three out of four of our colleagues use experimental procedures during everyday lab-bench work that can severely impair data interpretation depending on how cells are handled. We show here that a subpopulation (2-3%) of human leukocytes immediately induce a yet unclassified lytic cell death, concomitant with discharge of chromatin entities and cell elimination, when placed in protein-free solutions (i.e., PBS and HBSS). DNA release was not restricted to hematopoietic cells but occurred also in HEK293T cells. Albumin, fetal bovine serum, polyethylene glycol, and Pluronic F-68 supplements prevented chromatin discharge. Expelled chromatin was devoid of surrounding membranes but maintained its original nuclear shape, although â¼10 times enlarged. These structures differed from DNA appearance after osmotic or detergent-induced cell lysis. Besides sounding a cautionary note to the neutrophil extracellular trap (NET) research community, in which â¼50% of all published studies used protein-free media for NET-formation, our study also provides a rapid tool for analysis of chromatin organization.
RESUMEN
BACKGROUND: Small vessel vasculitis (SVV) is a group of systemic autoimmune diseases that are mediated by neutrophil extracellular traps (NETs) in response to cathelicidin LL37, an aging molecular marker, which could be induced by telomere dysfunction. Therefore, in this study, we evaluated the hypothesis that telomere dysfunction in neutrophils may promote SVV via an LL37-NETs-dependent mechanism. METHODS: We contrasted the release of neutrophil NETs from mice with telomere dysfunction, mice with DNA damage and wide-type mice. Neutrophil telomere length, the expression of LL37, and the formation of NETs were measured in SVV patients and healthy controls (HCs). The co-expression of γH2AX, LL37, and NETs were detected in SVV patients to evaluate the association of the immune aging of neutrophils and pro-inflammatory conditions. LL37 inhibitor was used to verify its key role in NETs release in SVV patients and DNA damage mice. RESULTS: We found that NETs were over-induced by telomere dysfunction and DNA damage in mice, which may be associated with a marked increase in LL37. For patients with SVV, telomeres in neutrophils were significantly shortened, which was also associated with higher levels of LL37 and NETs. Inhibition of LL37 reduced the NETs released from neutrophils. CONCLUSIONS: Taken together, the results of these studies suggest that dysfunction of telomeres may promote SVV through the mechanism of LL37-dependent NETs. Thus, targeting the LL37-NETs may be a novel therapy for SVV.
RESUMEN
Neutrophils operate as part of the innate defence in the skin and may eliminate the Borrelia spirochaete via phagocytosis, oxidative bursts, and hydrolytic enzymes. However, their importance in Lyme neuroborreliosis (LNB) is unclear. Neutrophil extracellular trap (NET) formation, which is associated with the production of reactive oxygen species, involves the extrusion of the neutrophil DNA to form traps that incapacitate bacteria and immobilise viruses. Meanwhile, NET formation has recently been studied in pneumococcal meningitis, the role of NETs in other central nervous system (CNS) infections has previously not been studied. Here, cerebrospinal fluid (CSF) samples from clinically well-characterised children (N = 111) and adults (N = 64) with LNB and other CNS infections were analysed for NETs (DNA/myeloperoxidase complexes) and elastase activity. NETs were detected more frequently in the children than the adults (p = 0.01). NET presence was associated with higher CSF levels of CXCL1 (p < 0.001), CXCL6 (p = 0.007), CXCL8 (p = 0.003), CXCL10 (p < 0.001), MMP-9 (p = 0.002), TNF (p = 0.02), IL-6 (p < 0.001), and IL-17A (p = 0.03). NETs were associated with fever (p = 0.002) and correlated with polynuclear pleocytosis (rs = 0.53, p < 0.0001). We show that neutrophil activation and active NET formation occur in the CSF samples of children and adults with CNS infections, mainly caused by Borrelia and neurotropic viruses. The role of NETs in the early phase of viral/bacterial CNS infections warrants further investigation.
Asunto(s)
Infecciones del Sistema Nervioso Central/inmunología , Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/líquido cefalorraquídeo , Infecciones del Sistema Nervioso Central/metabolismo , Quimiocinas CXC/líquido cefalorraquídeo , Quimiocinas CXC/metabolismo , Niño , Preescolar , Trampas Extracelulares/fisiología , Femenino , Humanos , Neuroborreliosis de Lyme/líquido cefalorraquídeo , Neuroborreliosis de Lyme/inmunología , Neuroborreliosis de Lyme/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Neutrófilos/patologíaRESUMEN
In mice, B1 and marginal zone (MZ) B-cells play an important role in prevention of autoimmunity through production of regulatory cytokines and natural antibodies. There is limited knowledge about the human counterparts of these cells. We therefore investigated functions of MZ-like B-cells and the frequency of circulating MZ-like and B1-like B-cells in healthy controls (HC), as well as in patients with autoimmune vasculitis to learn more about the role of these cells in autoimmune disease. After stimulation with CpG oligodeoxynucleotides (ODN) of class B in vitro, MZ-like B-cells were the main producers of IgM whereas switched memory B-cells primarily produced IgG and IgA. TNF and IL-10 were produced by both MZ-like and switched memory B-cells. Neither antibody nor TNF/IL-10 production by the B-cell subsets differed between patients and HC. Patients with autoimmune vasculitis, irrespective of disease activity, had lower percentage and absolute numbers of circulating MZ-like B-cells, and lower absolute numbers of B1-like B-cells. The percentage of B1-like B-cells was reduced during active disease. These findings remained significant when the analysis was confined to active treatment-naïve patients (disease onset).Our results suggest that human innate-like B-cells might have a physiological role in prevention of autoimmunity.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Inmunoglobulina M/inmunología , Memoria Inmunológica , Vasculitis/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades Autoinmunes/patología , Linfocitos B/patología , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Interleucina-10/inmunología , Masculino , Persona de Mediana Edad , Oligodesoxirribonucleótidos/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Vasculitis/patologíaRESUMEN
Although neutrophil extracellular traps (NETs) have been highlighted in several systemic inflammatory diseases, their clinical correlates and potential pathological role remain obscure. Herein, we describe a dramatic onset of systemic lupus erythematosus (SLE) with clear-cut pathogenic implications for neutrophils and NET formation in a young woman with cardiac (Libman-Sacks endocarditis) and central nervous system (psychosis and seizures) involvement. Despite extensive search, circulating antiphospholipid autoantibodies, a hallmark of Libman-Sacks endocarditis, could not be detected. Instead, we observed active NET formation in the tissue of the mitral valve, as well as in the circulation. Levels of NET remnants were significantly higher in serially obtained sera from the patient compared with sex-matched blood donors (p = .0011), and showed a non-significant but substantial correlation with blood neutrophil counts (r = 0.65, p = .16). The specific neutrophil elastase activity measured in serum seemed to be modulated by the provided immunosuppressive treatment. In addition, we found anti-Ro60/SSA antibodies in the cerebrospinal fluid of the patient but not NET remnants or increased elastase activity. This case illustrates that different disease mechanisms mediated via autoantibodies can occur simultaneously in SLE. NET formation with release of cytotoxic NET remnants is a candidate player in the pathogenesis of this non-canonical form of Libman-Sacks endocarditis occurring in the absence of traditional antiphospholipid autoantibodies. The case description includes longitudinal results with clinical follow-up data and a discussion of the potential roles of NETs in SLE.