RESUMO
BACKGROUND: The aberrant recognition of self-nucleic acids by the innate immune system contributes to the pathology of several autoimmune diseases. Although microbial DNA and, in certain instances, self-DNA that is released from damaged cells are primarily recognized by Toll-like receptor 9 (TLR9), recent evidence suggests that other cytosolic sequence-nonspecific DNA sensors contribute to DNA recognition. In this study, we focused on the sensing of microbial and host DNA in type 1 diabetes (T1D) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocytes from pediatric patients with T1D and from healthy donors were stimulated with microbial DNA (CpG) or with self-DNA (DNA contained within neutrophil extracellular traps, NETs). The production of cytokines was measured by flow cytometry and multiplex bead assays. The internalization of microbial DNA and its colocalization with STING was detected by image cytometry. Furthermore, the involvement of the TBK1 kinase was investigated by detecting its phosphorylation with phospho-flow cytometry or by using a TBK1 inhibition assay. RESULTS: We observed a prominent proinflammatory response in T1D PBMCs, especially pDCs and monocytes, to microbial DNA in comparison to that in controls. We further confirmed that monocytes could bind and internalize DNA and respond by releasing proinflammatory cytokines in a more pronounced manner in T1D patients than those in controls. Surprisingly, this cytokine production was not affected by TLR9 blockade, suggesting the involvement of intracellular receptors in DNA recognition. We further identified TBK1 and STING as two crucial molecules in the DNA-sensing pathway that were involved in CpG-DNA sensing by T1D cells. A similar DNA-sensing pathway that was dependent on intracellular DNA sensors and the STING-TBK1 interaction was employed in response to NETs, which were used to model self-DNA. CONCLUSIONS: Here, we show that there were significant differences in DNA sensing in T1D patients compared to that in controls. We demonstrate that monocytes from T1D patients are able to sense microbial- and self-DNA, leading to proinflammatory cytokine secretion through the adaptor protein STING and the TBK1 kinase.
Assuntos
DNA/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Monócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Adolescente , Estudos de Casos e Controles , Criança , Ilhas de CpG/genética , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/genética , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Tolerogenic dendritic cells (tolDCs) may offer an interesting intervention strategy to re-establish Ag-specific tolerance in autoimmune diseases, including type 1 diabetes (T1D). T1D results from selective destruction of insulin-producing ß cells leading to hyperglycemia that, in turn, specifically affects a patient's immune system. In this study, we prepared monocyte-derived tolDCs modulated by dexamethasone and vitamin D2 from 31 T1D patients with optimal glycemic control and 60 T1D patients with suboptimal glycemic control and assessed their tolerogenic properties in correlation with metabolic state of patients. tolDCs differentiated from both groups of patients acquired a regulatory phenotype and an anti-inflammatory profile. Interestingly, tolDCs from well-controlled patients expressed higher levels of inhibitory molecules IL-T3 and PD-L1. Additionally, glutamic acid decarboxylase (GAD)65-loaded tolDCs from well-controlled patients decreased significantly primary Th1/Th17 responses, induced stable GAD65-specific T cell hyporesponsiveness, and suppressed markedly control DC-induced GAD65-specific T cell activation compared with poorly controlled patients. The ability of tolDCs from poorly controlled patients to induce durable GAD65-specific T cell hyporesponsiveness was reversed once the control of glycemia improved. In both groups of patients, tolDCs were able to induce regulatory T cells from autologous naive CD4+ T cells. However, regulatory T cells from well-controlled patients had better suppressive abilities. The functionality of tolDCs was confirmed in the adoptive transfer model of NOD-SCID mice where tolDCs delayed diabetes onset. These results suggest that metabolic control of T1D affects the functional characteristics of tolDCs and subsequent effector T cell responses. Metabolic control may be relevant for refining inclusion criteria of clinical trials in the settings of T1D.
Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The profile of the antitumor immune response is an important factor determining patient clinical outcome. However, the influence of the tissue contexture on the composition of the tumor microenvironments of virally induced tumors is not clearly understood. Therefore, we analyzed the immune landscape of two HPV-associated malignancies: oropharyngeal squamous cell carcinoma (OPSCC) and squamous cell carcinoma of uterine cervix (CESC). We employed multiplex immunohistochemistry and immunofluorescence to evaluate the density and spatial distribution of immune cells in retrospective cohorts of OPSCC and CESC patients. This approach was complemented by transcriptomic analysis of purified primary tumor cells and in silico analysis of publicly available RNA sequencing data. Transcriptomic analysis showed similar immune profiles in OPSCC and CESC samples. Interestingly, immunostaining of OPSCC tissues revealed high densities of immune cells in both tumor stroma and tumor epithelium, whereas CESC samples were mainly characterized by the lack of immune cells in the tumor epithelium. However, in contrast to other immune cell populations, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were abundant in both segments of CESC samples and CESC-derived tumor cells expressed markedly higher levels of the PMN-MDSC chemoattractants CXCL1, CXCL5, and CXCL6 than OPSCC tumor cells. Taken together, despite their having the same etiologic agent, the immune infiltration pattern significantly differs between OPSCC and CESC, with a noticeable shift toward prominent MDSC infiltration in the latter. Our data thus present a rationale for a diverse approach to targeted therapy in patients with HPV-associated tumors of different tissue origins.
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At odds with other solid tumors, epithelial ovarian cancer (EOC) is poorly sensitive to immune checkpoint inhibitors (ICIs), largely reflecting active immunosuppression despite CD8+ T cell infiltration at baseline. Accumulating evidence indicates that both conventional chemotherapeutics and targeted anticancer agents commonly used in the clinical management of EOC not only mediate a cytostatic and cytotoxic activity against malignant cells, but also drive therapeutically relevant immunostimulatory or immunosuppressive effects. Here, we discuss such an immunomodulatory activity, with a specific focus on molecular and cellular pathways that can be harnessed to develop superior combinatorial regimens for clinical EOC care.
Assuntos
Antineoplásicos , Neoplasias Ovarianas , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Feminino , Humanos , Imunomodulação , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
In genetically predisposed individuals, ingestion of wheat gliadin provokes a T-cell-mediated enteropathy, celiac disease. Gliadin fragments were previously reported to induce phenotypic maturation and Th1 cytokine production by human dendritic cells (DCs) and to boost their capacity to stimulate allogeneic T cells. Here, we monitor the effects of gliadin on migratory capacities of DCs. Using transwell assays, we show that gliadin peptic digest stimulates migration of human DCs and their chemotactic responsiveness to the lymph node-homing chemokines CCL19 and CCL21. The gliadin-induced migration is accompanied by extensive alterations of the cytoskeletal organization, with dissolution of adhesion structures, podosomes, as well as up-regulation of the CC chemokine receptor (CCR) 7 on cell surface and induction of cyclooxygenase (COX)-2 enzyme that mediates prostaglandin E2 (PGE2) production. Blocking experiments confirmed that gliadin-induced migration is independent of the TLR4 signalling. Moreover, we showed that the α-gliadin-derived 31-43 peptide is an active migration-inducing component of the digest. The migration promoted by gliadin fragments or the 31-43 peptide required activation of p38 mitogen-activated protein kinase (MAPK). As revealed using p38 MAPK inhibitor SB203580, this was responsible for DC cytoskeletal transition, CCR7 up-regulation and PGE2 production in particular. Taken together, this study provides a new insight into pathogenic features of gliadin fragments by demonstrating their ability to promote DC migration, which is a prerequisite for efficient priming of naive T cells, contributing to celiac disease pathology.
Assuntos
Quimiotaxia/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Gliadina/farmacologia , Fragmentos de Peptídeos/farmacologia , Quimiocina CCL19/farmacologia , Quimiocina CCL21/farmacologia , Ciclo-Oxigenase 2/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/enzimologia , Dinoprostona/biossíntese , Ativação Enzimática/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Receptores CCR7/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses.
Assuntos
Células Dendríticas/imunologia , Citometria de Fluxo/métodos , Ativação Linfocitária , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Apoptose , Células Cultivadas , Humanos , FagocitoseRESUMO
Myeloid-derived suppressor cells (MDSC) represent a heterogeneous group of immature myeloid cells with immunoregulatory function in cancer and autoimmune diseases. In humans, two subsets of MDSC were determined based on the characteristic surface markers, monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC). Expansion of MDSC has been reported in some murine models and patients with autoimmune diseases and their immune-suppressive properties were characterized. However, the exact role of MDSC in the pathogenesis of autoimmune diseases is more complex and/or controversial. In type 1 diabetes mellitus (T1D), the increased frequency of MDSC was found in the blood of T1D patients but their suppressor capacity was diminished. In our study, we assessed the role of M-MDSC in the pathogenesis of T1D and showed for the first time the increased frequency of M-MDSC not only in the blood of T1D patients but also in their at-risk relatives compared to healthy donors. T1D patients with inadequate long term metabolic control showed an elevation of M-MDSC compared to patients with better disease control. Furthermore, we described the positive correlation between the percentage of M-MDSC and Th17 cells and IFN-γ producing T cells in T1D patients and their at-risk relatives. Finally, we found that the ability of M-MDSC to suppress autologous T cells is efficient only at the high MDSC: T cells ratio and dependent on cell-cell-contact and TGF-ß production. Our data show that the engagement of MDSC in the pathogenesis of T1D is evident, yet not entirely explored and more experiments are required to clarify whether MDSC are beneficial or harmful in T1D.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Células Supressoras Mieloides/imunologia , Adolescente , Contagem de Linfócito CD4 , Criança , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Interferon gama/metabolismo , Masculino , Linfócitos T Reguladores/imunologia , Células Th17/imunologiaRESUMO
Dendritic cells (DCs) are key regulators of immune responses that operate at the interface between innate and adaptive immunity, and defects in DC functions contribute to the pathogenesis of a variety of disorders. For instance, cancer evolves in the context of limited DC activity, and some autoimmune diseases are initiated by DC-dependent antigen presentation. Thus, correcting aberrant DC functions stands out as a promising therapeutic paradigm for a variety of diseases, as demonstrated by an abundant preclinical and clinical literature accumulating over the past two decades. However, the therapeutic potential of DC-targeting approaches remains to be fully exploited in the clinic. Here, we discuss the unique features of DCs that underlie the high therapeutic potential of DC-targeting strategies and critically analyze the obstacles that have prevented the full realization of this promising paradigm.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Tolerância Imunológica , Imunidade , Animais , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/terapia , Autoimunidade , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Comunicação Celular , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Plasticidade Celular/genética , Plasticidade Celular/imunologia , Suscetibilidade a Doenças , Humanos , Imunoterapia , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
Diabetes mellitus is characterized by long standing hyperglycemia leading to numerous life-threatening complications. For type 1 diabetes mellitus, resulting from selective destruction of insulin producing cells by exaggerated immune reaction, the only effective therapy remains exogenous insulin administration. Despite accurate compliance to treatment of certain patients, transient episodes of hyperglycemia cannot be completely eliminated by this symptomatic treatment. Novel immunotherapeutic approaches based on tolerogenic dendritic cells, T regulatory cells and mesenchymal stem cells (MSCs) have been tested in clinical trials, endeavoring to directly modulate the autoimmune destruction process in pancreas. However, hyperglycemia itself affects the immune system and the final efficacy of cell-based immunotherapies could be affected by the different glycemic control of enrolled patients. The present review explores the impact of hyperglycemia on immune cells while providing greater insight into the molecular mechanisms of high glucose action and subsequent metabolic reprogramming of different immune cells. Furthermore, over-production of mitochondrial reactive oxygen species, formation of advanced glycation end products as a consequence of hyperglycemia and their downstream signalization in immune cells are also discussed. Since hyperglycemia in patients with type 1 diabetes mellitus might have an impact on immune-interventional treatment, the maintenance of a tight glucose control seems to be beneficial in patients considered for cell-based therapy.
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Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Hiperglicemia/imunologia , Imunoterapia Adotiva/métodos , Células-Tronco Mesenquimais/imunologia , Mitocôndrias/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Reprogramação Celular , Ensaios Clínicos como Assunto , Células Dendríticas/imunologia , Células Dendríticas/transplante , Humanos , Tolerância Imunológica/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais , Monitorização Fisiológica , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Reguladores/transplanteRESUMO
Tolerogenic dendritic cells (tolDCs) are explored as a promising standalone or combination therapy in type 1 diabetes (T1D). The therapeutic application of tolDCs, including in human trials, has been tested also in other autoimmune diseases, however, T1D displays some unique features. In addition, unlike in several disease-induced animal models of autoimmune diseases, the prevalent animal model for T1D, the NOD mouse, develops diabetes spontaneously. This review compares evidence of various tolDCs approaches obtained from animal (mainly NOD) models of T1D with a focus on parameters of this cell-based therapy such as protocols of tolDC preparation, antigen-specific vs. unspecific approaches, doses of tolDCs and/or autoantigens, application schemes, application routes, the migration of tolDCs as well as their preventive, early pre-onset intervention or curative effects. This review also discusses perspectives of tolDC therapy and areas of preclinical research that are in need of better clarification in animal models in a quest for effective and optimal tolDC therapies of T1D in humans.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/transplante , Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos NODRESUMO
Inflammasomes are large protein complexes involved in the maturation of IL-1ß, a cytokine associated with the pathophysiology of type 1 diabetes (T1D). The data presented in this article focused on the role of inflammasomes in DNA recognition in T1D patients. This data extend knowledge on DNA sensing in T1D patients and relate to our research paper "Monocytes contribute to DNA sensing through the TBK1 signaling pathway in type 1 diabetes patients" Zentsova et al., 2009. To examine inflammasome involvement, we blocked the known mechanism of inflammasome activation - potassium efflux via various approaches: 1) high concentration of KCl; 2) Glybenclamide, which selectively blocks the ATP sensitive K+ channel; 3) KN-62, an inhibitor of P2X7 receptor, which activates K+ channel after ATP binding. Moreover, we used an inhibitor which blocks Nod-like receptor family containing pyrin domain 3 (NLRP3) inflammasome. In T1D patients, we show that secretion of cytokines IL-1ß, TNFα, IL-6 and IFNα after microbial DNA stimulation is promoted by potassium efflux and is not dependent on P2X7 receptor signaling. Surprisingly, the microbial DNA induced IL-1ß release was independent of NLRP3.
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Radiotherapy (RT) plays an important role in the management of cancer patients. RT is used in more than 50% of patients during the course of their disease in a curative or palliative setting. In the past decades it became apparent that the abscopal effect induced by RT might be dependent on the activation of immune system, and that the induction of immunogenic cancer cell death and production of danger-associated molecular patterns from dying cells play a major role in the radiotherapy-mediated anti-tumor efficacy. Therefore, the combination of RT and immunotherapy is of a particular interest that is reflected in designing clinical trials to treat patients with various malignancies. The use of cytokines as immunoadjuvants in combination with RT has been explored over the last decades as one of the immunotherapeutic combinations to enhance the clinical response to anti-cancer treatment. Here we review mainly the data on the efficacy of IFN-α, IL-2, IL-2-based immunocytokines, GM-CSF, and TNF-α used in combinations with various radiotherapeutic techniques in clinical trials. Moreover, we discuss the potential of IL-15 and its analogs and IL-12 cytokines in combination with RT based on the efficacy in preclinical mouse tumor models.
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High hydrostatic pressure (HHP) has been known to affect biological systems for >100â¯years. In this review, we describe the technology of HHP and its effect macromolecules and physiology of eukaryotic cells. We discuss the use of HHP in cancer immunotherapy to kill tumor cells for generation of whole cell and dendritic cell-based vaccines. We further summarize the current use and perspectives of HHP application in biomedicine, specifically in orthopedic surgery and for the viral, microbial and protozoan inactivation to develop vaccines against infectious diseases.
Assuntos
Biotecnologia/métodos , Pressão Hidrostática , Imunoterapia/métodos , Neoplasias/terapia , Procedimentos Ortopédicos/métodos , Biotecnologia/instrumentação , Células Dendríticas/imunologia , Células Eucarióticas , Humanos , Neoplasias/patologia , Vacinas/imunologia , Inativação de VírusRESUMO
Tolerogenic DCs (tolDCs) are being researched as a promising intervention strategy also in autoimmune diseases including type 1 diabetes (T1D). T1D is a T-cell-mediated, organ-specific disease with several well-defined and rather specific autoantigens, i.e., proinsulin, insulin, glutamic acid decarboxylase 65 (GAD65), that have been used in animal as well as human intervention trials in attempts to achieve a more efficient, specific immunotherapy. In this study, we have tested tolerogenic DCs for their effectiveness to prevent adoptive transfer of diabetes by diabetogenic splenocytes into non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) recipients. While i.p. application of tolDCs prepared from bone marrow of prediabetic NOD mice by vitamin D2 and dexamethasone significantly reduced diabetes transfer into the NOD-SCID females, this effect was completely abolished when tolDCs were loaded with the mouse recombinant GAD65, but also with a control protein-ovalbumin (OVA). The effect was not dependent on the presence of serum in the tolDC culture. Similar results were observed in NOD mice. Removal of possible bystander antigen-presenting cells within the diabetogenic splenocytes by negative magnetic sorting of T cells did not alter this surprising effect. Tolerogenic DCs loaded with an immunodominant mouse GAD65 peptide also displayed diminished diabetes-preventive effect. Tolerogenic DCs were characterized by surface maturation markers (CD40, CD80, CD86, MHC II) and the lipopolysaccharide stability test. Data from alloreactive T cell proliferation and cytokine induction assays (IFN-γ) did not reveal the differences observed in the diabetes incidence. Migration of tolDCs, tolDCs-GAD65 and tolDCs-OVA to spleen, mesenteric- and pancreatic lymph nodes displayed similar, mucosal pattern with highest accumulation in pancreatic lymph nodes present up to 9 days after the i.p. APPLICATION: These data document that mechanisms by which tolDCs operate in vivo require much better understanding for improving efficacy of this promising cell therapy, especially in the presence of an antigen, e.g., GAD65.
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Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Tolerância Imunológica/imunologia , Transferência Adotiva , Animais , Autoantígenos/imunologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Cell-based tolerogenic therapy is a promising approach for the treatment of autoimmune diseases and transplant rejection. Regulatory T cells and tolerogenic dendritic cells have been particularly explored in the treatment of various autoimmune disorders in experimental models of disease. Although some of these cells have already been tested in a limited number of clinical trials, there is still a need for preclinical research on tolerogenic cells in animal models of autoimmunity. This review will focus on the relevance of data obtained from studies in experimental animal models for the use of tolerogenic cell-based therapy in humans. Also, perspectives for further improvement of tolerogenic cell preparation towards enhanced suppressive activity and stability of the cells will be discussed.
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Artrite Reumatoide/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Diabetes Mellitus Tipo 1/terapia , Modelos Animais de Doenças , Esclerose Múltipla/terapia , Animais , Artrite Reumatoide/imunologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunossupressores/administração & dosagem , Esclerose Múltipla/imunologia , Compostos Orgânicos/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Initial events and effector mechanisms of most inflammatory and autoimmune diseases remain largely unknown. Dysfunction of the innate and adaptive immune systems associated with mucosae (the major interface between the organism and its environment, e.g., microbiota, food) can conceivably cause impairment of mucosal barrier function and development of localized or systemic inflammatory and autoimmune processes. Animal models help in elucidating the etiology and pathogenetic mechanisms of human diseases, such as the inflammatory bowel diseases, Crohn's disease and ulcerative colitis, severe chronic diseases affecting the gut. To study the role of innate immunity and gut microbiota in intestinal inflammation, colitis was induced by dextran sulfate sodium (DSS) in mice with severe combined immunodeficiency (SCID). Conventionally reared (microflora-colonized) SCID mice displayed severe inflammation like that seen in immunocompetent Balb/c mice, whereas only minor changes appeared in the intestinal mucosa of DSS-fed gnotobiotic germ-free SCID mice. The presence of microflora facilitates the inflammation in DSS-induced colitis that develops in immunodeficient SCID mice, that is, in the absence of T and B lymphocytes. Celiac disease, a chronic autoimmune small bowel disorder, afflicts genetically susceptible individuals with wheat gluten intolerance. We showed that, in contrast with any other food proteins, wheat gliadin and its peptic fragments activate mouse macrophages and human monocytes to produce proinflammatory cytokines through the nuclear factor-kappaB signaling pathway. Activation of innate immunity cells by food proteins or components from gut microbiota thus could participate in the impairment of intestinal mucosa and the development of intestinal and/or systemic inflammation.
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Doenças Autoimunes/etiologia , Imunidade Inata , Inflamação/etiologia , Animais , Doenças Autoimunes/imunologia , Doença Celíaca/etiologia , Modelos Animais de Doenças , Humanos , Imunidade nas Mucosas , Inflamação/imunologia , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/imunologia , CamundongosRESUMO
Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1ß and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1ß and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1ß production in celiac PBMC show that PDWGF-induced de novo pro-IL-1ß synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1ß. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1ß release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3(-/-) and ASC(-/-) knockout mice. Moreover, treatment of human PBMC as well as MyD88(-/-) and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-ß (TRIF)(-/-) BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1ß in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD.
Assuntos
Proteínas de Transporte/imunologia , Gliadina/imunologia , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Adulto , Animais , Proteínas de Transporte/genética , Doença Celíaca , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Gliadina/química , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-1beta/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pepsina A , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
BACKGROUND AND AIMS: The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy. METHODOLOGY/PRINCIPAL FINDINGS: Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by ß-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes. CONCLUSIONS: Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity.
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Apresentação de Antígeno/imunologia , Hipersensibilidade Alimentar/imunologia , Ovalbumina/química , Linfócitos T Reguladores/imunologia , Animais , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/sangue , Temperatura Alta , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Camundongos , Ovalbumina/imunologia , Estrutura Secundária de ProteínaRESUMO
To elucidate the role of innate immune responses in celiac disease, we investigated the effect of gliadin on blood monocytes from patients with celiac disease. Gliadin induced substantial TNF-alpha and IL-8 production by monocytes from patients with active celiac disease, lower levels by monocytes from patients with inactive celiac disease, and even lower levels by monocytes from healthy donors. In healthy donor monocytes gliadin induced IL-8 from monocytes expressing HLA-DQ2 and increased monocyte expression of the costimulatory molecules CD80 and CD86, the dendritic cell marker CD83, and the activation marker CD40. Gliadin also increased DNA binding activity of NF-kappaB p50 and p65 subunits in monocytes from celiac patients, and NF-kappaB inhibitors reduced both DNA binding activity and cytokine production. Thus, gliadin activation of HLA-DQ2(+) monocytes leading to chemokine and proinflammatory cytokine production may contribute to the host innate immune response in celiac disease.
Assuntos
Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Citocinas/biossíntese , Gliadina/metabolismo , Monócitos/metabolismo , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Citometria de Fluxo , Antígenos HLA-DQ/metabolismo , Humanos , Imunoglobulinas/metabolismo , Interleucina-8/biossíntese , Ativação de Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos , Fator de Necrose Tumoral alfa/biossíntese , Antígeno CD83RESUMO
Celiac disease is a chronic inflammatory disease developing in genetically predisposed individuals. Ingested gliadin, the triggering agent of the disease, can cross the epithelial barrier and elicit a harmful T cell-mediated immune response. Dendritic cells (DC) are supposed to play a pivotal role in shaping the immune response. The direction of the immune response toward immunity or tolerance depends on the stage of maturation and the functional properties of the DC. DC become fully functional APC upon maturation by various stimuli. We investigated the effect of a peptic digest of gliadin on the maturation of human monocyte-derived DC. Stimulation of cells with gliadin, in contrast with other tested food proteins, led to enhanced expression of maturation markers (CD80, CD83, CD86, and HLA-DR molecules) and increased secretion of chemokines and cytokines (mainly of IL-6, IL-8, IL-10, TNF-alpha, growth-related oncogene, MCP-1, MCP-2, macrophage-derived chemokine, and RANTES). Maturation was accompanied by a greater capacity to stimulate proliferation of allogeneic T cells and significantly reduced endocytic activity. Furthermore, gliadin-induced phosphorylation of members of three MAPK families (ERK1/2, JNK, and p38 MAPK) was demonstrated. The largest contribution of p38 MAPK was confirmed using its inhibitor SB203580, which markedly down-regulated the gliadin-triggered up-regulation of maturation markers and cytokine production. Gliadin treatment also resulted in increased NF-kappaB/DNA binding activity of p50 and p65 subunits. Taken together, gliadin peptides can contribute to overcoming the stage of unresponsiveness of immature DC by inducing phenotypic and functional DC maturation, resulting in more efficient processing and presentation of gliadin peptides to specific T lymphocytes.