RESUMO
BACKGROUND: Interleukin (IL)-6 trans-signalling (IL-6TS) is emerging as a pathogenic mechanism in chronic respiratory diseases; however, the drivers of IL-6TS in the airways and the phenotypic characteristic of patients with increased IL-6TS pathway activation remain poorly understood. OBJECTIVE: Our aim was to identify and characterise COPD patients with increased airway IL-6TS and to elucidate the biological drivers of IL-6TS pathway activation. METHODS: We used an IL-6TS-specific sputum biomarker profile (soluble IL-6 receptor (sIL-6R), IL-6, IL-1ß, IL-8, macrophage inflammatory protein-1ß) to stratify sputum data from patients with COPD (n=74; Biomarkers to Target Antibiotic and Systemic Corticosteroid Therapy in COPD Exacerbation (BEAT-COPD)) by hierarchical clustering. The IL-6TS signature was related to clinical characteristics and sputum microbiome profiles. The induction of neutrophil extracellular trap formation (NETosis) and IL-6TS by Haemophilus influenzae were studied in human neutrophils. RESULTS: Hierarchical clustering revealed an IL-6TS-high subset (n=24) of COPD patients, who shared phenotypic traits with an IL-6TS-high subset previously identified in asthma. The subset was characterised by increased sputum cell counts (p=0.0001), persistent sputum neutrophilia (p=0.0004), reduced quality of life (Chronic Respiratory Questionnaire total score; p=0.008), and increased levels of pro-inflammatory mediators and matrix metalloproteinases in sputum. IL-6TS-high COPD patients showed an increase in Proteobacteria, with Haemophilus as the dominating genus. NETosis induced by H. influenzae was identified as a potential mechanism for increased sIL-6R levels. This was supported by a significant positive correlation between sIL-6R and NETosis markers in bronchoalveolar lavage fluid from COPD patients. CONCLUSION: IL-6TS pathway activation due to chronic colonisation with Haemophilus may be an important disease driver in a subset of COPD patients.
Assuntos
Armadilhas Extracelulares , Infecções por Haemophilus , Doença Pulmonar Obstrutiva Crônica , Infecções por Haemophilus/complicações , Humanos , Interleucina-6 , Qualidade de Vida , EscarroRESUMO
Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.
Assuntos
Toxina da Cólera/toxicidade , Gangliosídeo G(M1)/fisiologia , Animais , Células Cultivadas , Gangliosídeo G(M1)/metabolismo , Glicosilação , Células HL-60 , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Ratos , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
OBJECTIVES: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. METHODS: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. RESULTS: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. CONCLUSIONS: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.
Assuntos
Citrulinação/genética , Endopeptidases/genética , Armadilhas Extracelulares/genética , Lúpus Eritematoso Sistêmico/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Epitopos/imunologia , Predisposição Genética para Doença/genética , Humanos , Lúpus Eritematoso Sistêmico/sangue , Camundongos , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Polimorfismo Genético , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Regulação para Cima/genéticaRESUMO
Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov ) 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency.
Assuntos
Imunoterapia/métodos , Leucemia Mieloide/imunologia , Leucemia Mieloide/terapia , Linfócitos T Reguladores/imunologia , Doença Aguda , Adolescente , Adulto , Idoso , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Histamina/imunologia , Histamina/uso terapêutico , Humanos , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Prognóstico , Modelos de Riscos Proporcionais , Indução de Remissão , Linfócitos T Reguladores/metabolismo , Telômero/genética , Adulto JovemRESUMO
Polymorphonuclear neutrophils (PMNs) are innate effector cells with pivotal roles in pathogen recognition, phagocytosis, and eradication. However, their role in the development of subsequent immune responses is incompletely understood. This study aimed to identify mechanisms of relevance to the cross talk between human neutrophils and NK cells and its potential role in promoting adaptive immunity. TLR-stimulated PMNs were found to release soluble mediators to attract and activate NK cells in vitro. PMN-conditioned NK cells displayed enhanced cytotoxicity and cytokine production, and responded vigorously to ensuing stimulation with exogenous and endogenous IL-12. The neutrophil-induced activation of NK cells was prevented by caspase-1 inhibitors and by natural antagonists to IL-1 and IL-18, suggesting a role for the NOD-like receptor family pyrin domain containing-3 inflammasome. In addition, PMN-conditioned NK cells triggered the maturation of monocyte-derived dendritic cells, which promoted T cell proliferation and IFN-γ production. These data imply that neutrophils attract NK cells to sites of infection to convert these cells into an active state, which drives adaptive immune responses via maturation of dendritic cells. Our results add to a growing body of evidence that suggests a sophisticated role for neutrophils in orchestrating the immune response to pathogens.
Assuntos
Imunidade Adaptativa/imunologia , Comunicação Celular/imunologia , Células Dendríticas/citologia , Células Matadoras Naturais/imunologia , Neutrófilos/imunologia , Linfócitos T/imunologia , Proteínas de Transporte/imunologia , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Células Dendríticas/imunologia , Humanos , Inflamassomos/imunologia , Interferon gama/biossíntese , Subunidade p35 da Interleucina-12/imunologia , Interleucina-18/antagonistas & inibidores , Interleucina-1beta/antagonistas & inibidores , Ativação Linfocitária/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
Polymorphonuclear neutrophils (PMN) are potent inflammatory effector cells essential to host defense, but at the same time they may cause significant tissue damage. Thus, timely induction of neutrophil apoptosis is crucial to avoid tissue damage and induce resolution of inflammation. NK cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and neutrophils. Coculture experiments revealed that human NK cells could trigger caspase-dependent neutrophil apoptosis in vitro. This event was dependent on cell-cell contact, and experiments using blocking Abs indicated that the effect was mediated by the activating NK cell receptor NKp46 and the Fas pathway. CD56-depleted lymphocytes had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis is specific to NK cells. Our findings provide evidence that NK cells may accelerate neutrophil apoptosis, and that this interaction may be involved in the resolution of acute inflammation.
Assuntos
Apoptose , Citotoxicidade Imunológica , Proteína Ligante Fas/metabolismo , Células Matadoras Naturais/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Neutrófilos/imunologia , Caspase 8/metabolismo , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Humanos , Células Matadoras Naturais/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Patients with metastatic melanoma have a historically poor prognosis, but recent advances in treatment options, including targeted therapy and immunotherapy, have drastically improved the outcomes for some of these patients. However, not all patients respond to available treatments, and around 50% of patients with metastatic cutaneous melanoma and almost all patients with metastases of uveal melanoma die of their disease. Thus, there is a need for novel treatment strategies for patients with melanoma that do not benefit from the available therapies. Chimeric antigen receptor-expressing T (CAR-T) cells are largely unexplored in melanoma. Traditionally, CAR-T cells have been produced by transducing blood-derived T cells with a virus expressing CAR. However, tumor-infiltrating lymphocytes (TILs) can also be engineered to express CAR, and such CAR-TILs could be dual-targeting. To this end, tumor samples and autologous TILs from metastasized human uveal and cutaneous melanoma were expanded in vitro and transduced with a lentiviral vector encoding an anti-HER2 CAR construct. When infused into patient-derived xenograft (PDX) mouse models carrying autologous tumors, CAR-TILs were able to eradicate melanoma, even in the absence of antigen presentation by HLA. To advance this concept to the clinic and assess its safety in an immune-competent and human-patient-like setting, we treated four companion dogs with autologous anti-HER2 CAR-TILs. We found that these cells were tolerable and showed signs of anti-tumor activity. Taken together, CAR-TIL therapy is a promising avenue for broadening the tumor-targeting capacity of TILs in patients with checkpoint immunotherapy-resistant melanoma.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/farmacologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Medições Luminescentes/métodos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oniocompostos/imunologia , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rituximab , Células Tumorais CultivadasRESUMO
Bleomycin hydrolase (BLMH) is a well-conserved cysteine protease widely expressed in several mammalian tissues. In skin, which contains high levels of BLMH, this protease is involved in the degradation of citrullinated filaggrin monomers into free amino acids important for skin hydration. Interestingly, the expression and activity of BLMH is reduced in patients with atopic dermatitis (AD) and psoriasis, and BLMH knockout mice acquire tail dermatitis. Apart from its already known function, we have discovered a novel role of BLMH in the regulation of inflammatory chemokines and wound healing. We show that lowered BLMH levels in keratinocytes result in increased release of the pro-inflammatory chemokines CXCL8 and GROα, which are upregulated in skin from AD patients compared to healthy individuals. Conditioned media from keratinocytes expressing low levels of BLMH increased chemotaxis by neutrophils and caused a delayed wound healing in the presence of low-level TNFα. This defective wound healing was improved by blocking the shared receptor of CXCL8 and GROα, namely CXCR2, using a specific receptor antagonist. Collectively, our results present a novel function of BLMH in regulating the secretion of chemokines involved in inflammation and wound healing in human keratinocytes.
Assuntos
Quimiocinas/metabolismo , Cisteína Endopeptidases/metabolismo , Inflamação/metabolismo , Queratinócitos/metabolismo , Cicatrização/fisiologia , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Meios de Cultivo Condicionados , Cisteína Endopeptidases/genética , Proteínas Filagrinas , Humanos , Inflamação/genética , Queratinócitos/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Chronic myelomonocytic leukemia (CMML) is a myeloproliferative and myelodysplastic neoplasm with few treatment options and dismal prognosis. The role of natural killer (NK) cells and other antileukemic lymphocytes in CMML is largely unknown. We aimed to provide insight into the mechanisms of immune evasion in CMML with a focus on immunosuppressive reactive oxygen species (ROS) formed by the myeloid cell NADPH oxidase-2 (NOX2). The dominant population of primary human CMML cells was found to express membrane-bound NOX2 and to release ROS, which, in turn, triggered extensive PARP-1-dependent cell death in cocultured NK cells, CD8+ T effector memory cells, and CD8+ T effector cells. Inhibitors of ROS formation and scavengers of extracellular ROS prevented CMML cell-induced lymphocyte death and facilitated NK cell degranulation toward Ab-coated, primary CMML cells. In patients with CMML, elevation of immature cell counts (CD34+) in blood was associated with reduced expression of several NK cell-activating receptors. We propose that CMML cells may use extracellular ROS as a targetable mechanism of immune escape.
Assuntos
Leucemia Mielomonocítica Crônica/imunologia , Glicoproteínas de Membrana/imunologia , Células Mieloides/imunologia , NADPH Oxidases/imunologia , Espécies Reativas de Oxigênio/imunologia , Evasão Tumoral/imunologia , Separação Celular , Citometria de Fluxo , Humanos , Imunofenotipagem , Microscopia Confocal , NADPH Oxidase 2RESUMO
In a phase IV trial, 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin 2 (IL-2) for 18 months to prevent leukemic relapse. During cycles, the treatment resulted in expansion of CD56(bright) (CD3(-)/16(-)/56(bright)) and CD16(+) (CD3(-)/16(+)/56(+)) natural killer (NK) cells in the blood along with increased NK cell expression of the natural cytotoxicity receptors (NCRs) NKp30 and NKp46. Multivariate analyses correcting for age and risk group demonstrated that high CD56(bright) NK cell counts and high expression of NKp30 or NKp46 on CD16(+) NK cells independently predicted leukemia-free survival (LFS) and overall survival (OS). Our results suggest that the dynamics of NK cell subsets and their NCR expression may determine the efficiency of relapse-preventive immunotherapy in AML.
RESUMO
Preventing relapse after chemotherapy remains a challenge in acute myeloid leukemia (AML). Eighty-four non-transplanted AML patients in first complete remission received relapse-preventive immunotherapy with histamine dihydrochloride and low-dose interleukin-2 in an international phase IV trial (ClinicalTrials.gov; NCT01347996). Blood samples were drawn during cycles of immunotherapy and analyzed for CD8+ (cytotoxic) T cell phenotypes in blood. During the first cycle of therapy, a re-distribution of cytotoxic T cells was observed comprising a reduction of T effector memory cells and a concomitant increase of T effector cells. The dynamics of T cell subtypes during immunotherapy prognosticated relapse and survival, in particular among older patients and remained significantly predictive of clinical outcome after correction for potential confounders. Presence of CD8+ T cells with specificity for leukemia-associated antigens identified patients with low relapse risk. Our results point to novel aspects of T cell-mediated immunosurveillance in AML and provide conceivable biomarkers in relapse-preventive immunotherapy.
Assuntos
Imunoterapia , Leucemia Mieloide Aguda/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Antineoplásicos/administração & dosagem , Células Cultivadas , Feminino , Citometria de Fluxo , Seguimentos , Histamina/administração & dosagem , Agonistas dos Receptores Histamínicos/administração & dosagem , Humanos , Interleucina-2/administração & dosagem , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Subpopulações de Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Indução de Remissão , Taxa de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Adulto JovemRESUMO
In a phase IV trial, eighty-four patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin-2 (IL-2) to prevent relapse in the post-consolidation phase. Aspects of natural killer (NK) cell biology were analyzed before and during immunotherapy with focus on outcome in older patients. In younger (<60 years old, n = 37) and older patients (>60 years old, n = 47), treatment with HDC/IL-2 resulted in an expansion of CD56(bright) and CD16+ NK cells in blood along with an increased NK cell expression of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. In older patients, a high expression of NKp30 or NKp46 on CD16+ NK cells before and during therapy predicted leukemia-free and overall survival. These results suggest that NK cell functions determine relapse risk and survival in older AML patients and point to biomarkers of efficacy in protocols for remission maintenance.
Assuntos
Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Subpopulações de Linfócitos/imunologia , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno CD56/biossíntese , Antígeno CD56/imunologia , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/imunologia , Histamina/administração & dosagem , Humanos , Interleucina-2/administração & dosagem , Interleucina-2/análogos & derivados , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Quimioterapia de Manutenção/métodos , Masculino , Pessoa de Meia-Idade , Receptor 1 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Recidiva Local de Neoplasia/imunologia , Receptores de IgG/biossíntese , Receptores de IgG/imunologia , Proteínas Recombinantes/administração & dosagem , Indução de Remissão , Adulto JovemRESUMO
NK cells and T cells are commonly dysfunctional in CML, and their status may determine the course of disease. We aimed to define the molecular mechanisms of leukemia-induced immunosuppression with focus on the role of ROS and the PARP-1 pathway of cell death. Malignant granulocytes from patients with BCR-ABL-positive CML expressed the oxygen radical-producing enzyme NOX, produced large amounts of ROS, and triggered extensive cell death in NK cells. Inhibition of PARP-1 maintained NK cell viability in cocultures with suppressive leukemic cells. Under conditions of oxidative stress, PARP-1 inhibition upheld the capacity of NK cells to kill myeloid leukemic cells, in addition to restoring the proliferation and cytokine production of NK cells and cytotoxic T cells. Our findings are suggestive of a novel pathway of relevance to immunosuppression in CML.