RESUMO
Regulatory T cells (Tregs) that express the transcription factor Foxp3 have a critical role in limiting inflammatory processes and tissue damage. Whether Tregs are functional in maintaining epithelial barriers and in control of tight junction expression has not yet been explored. In this study, we investigated the effect of Treg deficiency on the airway epithelial barrier in an experimental murine model in which diphtheria toxin was repeatedly injected in Foxp3-diphtheria toxin receptor (DTR) mice to deplete Tregs. This resulted in spontaneous peribronchial inflammation and led to a systemic and local increase of IL-4, IL-5, CCL3, IFN-γ, and IL-10 and a local (lung) increase of IL-6 and IL-33 and decreased amphiregulin levels. Moreover, Treg depletion increased airway permeability and decreased epithelial tight junction (protein and mRNA) expression. CTLA4-Ig treatment of Treg-depleted mice almost completely prevented barrier dysfunction together with suppression of lung inflammation and cytokine secretion. Treatment with anti-IL-4 partly reversed the effects of Treg depletion on tight junction expression, whereas neutralization of IL-6 of IFN-γ had either no effect or only a limited effect. We conclude that Tregs are essential to protect the epithelial barrier at the level of tight junctions by restricting spontaneous T cell activation and uncontrolled secretion of cytokines, in particular IL-4, in the bronchi.
Assuntos
Toxina Diftérica , Linfócitos T Reguladores , Abatacepte/farmacologia , Anfirregulina/metabolismo , Animais , Citocinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-33/metabolismo , Interleucina-5/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
BACKGROUND: Increased epithelial permeability has been reported in allergic rhinitis, with histamine and type-2 inflammation being responsible for tight junction dysfunction. The impact of an epithelial barrier defect on allergic sensitization and mast cell (MC) degranulation remains speculative. METHODS: Transepithelial passage of allergens was evaluated on primary human nasal epithelial cell cultures. Active sensitization was attempted by repeated intranasal ovalbumin (OVA) applications in Naïve mice. In a passive sensitization model, mice were injected with IgE to Dermatophagoides pteronyssinus (rDer p)2 and then exposed intranasally to the allergen. Chitosan was used to disrupt nasal epithelial integrity in vitro and in vivo. RESULTS: Chitosan strongly reduced transepithelial electrical resistance and facilitated transepithelial allergen passage in cultured primary nasal epithelial cells. In vivo, intranasal chitosan affected occludin expression and facilitated allergen passage. After epithelial barrier disruption, intranasal OVA application induced higher OVA-specific IgG1 and total IgE in serum, and increased eosinophilia and interleukin-5 in bronchoalveolar lavage (BAL) compared to sham-OVA mice. Chitosan exposure, prior to rDer p2 allergen challenge in passively sensitized mice, resulted in increased ß-hexosaminidase levels in serum and BAL compared to sham-rDer p2 mice. Intranasal treatment with the synthetic glucocorticoid fluticasone propionate prevented chitosan-induced barrier dysfunction, allergic sensitization, and MC degranulation. CONCLUSION: Epithelial barrier dysfunction facilitates transepithelial allergen passage, allergic sensitization, and allergen-induced MC degranulation even in the absence of inflammatory environment. These results emphasize the crucial role of an intact epithelial barrier in prevention of allergy.
Assuntos
Mastócitos , Rinite Alérgica , Alérgenos , Animais , Degranulação Celular , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , OvalbuminaRESUMO
BACKGROUND: Allergic rhinitis (AR) is characterized by mucosal inflammation, driven by activated immune cells. Mast cells and TH2 cells might decrease epithelial barrier integrity in AR, maintaining a leaky epithelial barrier. OBJECTIVE: We sought to investigate the role of histamine and TH2 cells in driving epithelial barrier dysfunction in AR. METHODS: Air-liquid interface cultures of primary nasal epithelial cells were used to measure transepithelial electrical resistance, paracellular flux of fluorescein isothiocyanate-dextran 4 kDa, and mRNA expression of tight junctions. Nasal secretions were collected from healthy control subjects, AR patients, and idiopathic rhinitis patients and were tested in vitro. In addition, the effect of activated TH1 and TH2 cells, mast cells, and neurons was tested in vitro. The effect of IL-4, IL-13, IFN-γ, and TNF-α on mucosal permeability was tested in vivo. RESULTS: Histamine as well as nasal secretions of AR but not idiopathic rhinitis patients rapidly decreased epithelial barrier integrity in vitro. Pretreatment with histamine receptor-1 antagonist, azelastine prevented the early effect of nasal secretions of AR patients on epithelial integrity. Supernatant of activated TH1 and TH2 cells impaired epithelial integrity, while treatment with anti-TNF-α or anti-IL-4Rα monoclonal antibodies restored the TH1- and TH2-induced epithelial barrier dysfunction, respectively. IL-4, IFN-γ, and TNF-α enhanced mucosal permeability in mice. Antagonizing IL-4 prevented mucosal barrier disruption and tight junction downregulation in a mouse model of house dust mite allergic airway inflammation. CONCLUSIONS: Our data indicate a key role for allergic inflammatory mediators in modulating nasal epithelial barrier integrity in the pathophysiology in AR.
Assuntos
Citocinas/imunologia , Histamina/imunologia , Mucosa Nasal/imunologia , Rinite Alérgica/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/patologia , Rinite Alérgica/patologia , Células Th1/patologia , Células Th2/patologiaRESUMO
Blocking of costimulatory CD28/B7 and CD40/CD40L interactions is an experimental approach to immune suppression and tolerance induction. We previously reported that administration of a combination of CTLA-4Ig and MR1 (anti-CD40L mAb) for blockade of these interactions induces tolerance in a fully mismatched allogeneic splenocyte transfer model in mice. We now used this model to study whether regulatory T cells (Tregs) contribute to immune suppression and why both pathways have to be blocked simultaneously. Mice were injected with allogeneic splenocytes, CD4(+) T cells, or CD8(+) T cells and treated with MR1 mAb and different doses of CTLA-4Ig. The graft-versus-host reaction of CD4(+) T cells, but not of CD8(+) T cells, was inhibited by MR1. CTLA-4Ig was needed to cover CD8(+) T cells but had only a weak effect on CD4(+) T cells. Consequently, only the combination provided full protection when splenocytes were transferred. Importantly, MR1 and low-dose CTLA-4Ig treatment resulted in a relative increase in Tregs, and immune suppressive efficacy was abolished in the absence of Tregs. High-dose CTLA-4Ig treatment, in contrast, prevented Treg expansion and activity, and in combination with MR1 completely inhibited CD4(+) and CD8(+) T cell activation in a Treg-independent manner. In conclusion, MR1 and CTLA-4Ig act synergistically as they target different T cell populations. The contribution of Tregs to immune suppression by costimulation blockade depends on the concentration of CTLA-4Ig and thus on the degree of available CD28 costimulation.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-1/antagonistas & inibidores , Antígeno B7-1/imunologia , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/imunologia , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/imunologia , Ligante de CD40/antagonistas & inibidores , Ligante de CD40/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Terapia de Imunossupressão , CamundongosRESUMO
BACKGROUND: Asthma is characterized by a heterogeneous inflammatory profile and can be subdivided into T(h)2-high and T(h)2-low airway inflammation. Profiling of a broader panel of airway cytokines in large unselected patient cohorts is lacking. METHODS: Patients (n = 205) were defined as being "cytokine-low/high" if sputum mRNA expression of a particular cytokine was outside the respective 10th/90th percentile range of the control group (n = 80). Unsupervised hierarchical clustering was used to determine clusters based on sputum cytokine profiles. RESULTS: Half of patients (n = 108; 52.6%) had a classical T(h)2-high ("IL-4-, IL-5- and/or IL-13-high") sputum cytokine profile. Unsupervised cluster analysis revealed 5 clusters. Patients with an "IL-4- and/or IL-13-high" pattern surprisingly did not cluster but were equally distributed among the 5 clusters. Patients with an "IL-5-, IL-17A-/F- and IL-25- high" profile were restricted to cluster 1 (n = 24) with increased sputum eosinophil as well as neutrophil counts and poor lung function parameters at baseline and 2 years later. Four other clusters were identified: "IL-5-high or IL-10-high" (n = 16), "IL-6-high" (n = 8), "IL-22-high" (n = 25). Cluster 5 (n = 132) consists of patients without "cytokine-high" pattern or patients with only high IL-4 and/or IL-13. CONCLUSION: We identified 5 unique asthma molecular phenotypes by biological clustering. Type 2 cytokines cluster with non-type 2 cytokines in 4 out of 5 clusters. Unsupervised analysis thus not supports a priori type 2 versus non-type 2 molecular phenotypes. www.clinicaltrials.gov NCT01224938. Registered 18 October 2010.
Assuntos
Asma/imunologia , Asma/patologia , Citocinas/imunologia , Escarro/imunologia , Células Th2/imunologia , Adolescente , Adulto , Idoso , Bélgica/epidemiologia , Biomarcadores , Análise por Conglomerados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: Tight junction (TJ) defects have recently been associated with asthma and chronic rhinosinusitis. The expression, function, and regulation of nasal epithelial TJs remain unknown in patients with allergic rhinitis (AR). OBJECTIVE: We investigated the expression, function, and regulation of TJs in the nasal epithelium of patients with house dust mite (HDM)-induced AR and in an HDM-induced murine model of allergic airway disease. METHODS: Air-liquid interface cultures of primary nasal epithelial cells of control subjects and patients with HDM-induced AR were used for measuring transepithelial resistance and passage to fluorescein isothiocyanate-dextran 4 kDa (FD4). Ex vivo transtissue resistance and FD4 permeability of nasal mucosal explants were measured. TJ expression was evaluated by using real-time quantitative PCR and immunofluorescence. In addition, the effects of IL-4, IFN-γ, and fluticasone propionate (FP) on nasal epithelial cells were investigated in vitro. An HDM murine model was used to study the effects of allergic inflammation and FP treatment on transmucosal passage of FD4 in vivo. RESULTS: A decreased resistance in vitro and ex vivo was found in patients with HDM-induced AR, with increased FD4 permeability and reduced occludin and zonula occludens-1 expression. AR symptoms correlated inversely with resistance in patients with HDM-induced AR. In vitro IL-4 decreased transepithelial resistance and increased FD4 permeability, whereas IFN-γ had no effect. FP prevented IL-4-induced barrier dysfunction in vitro. In an HDM murine model FP prevented the allergen-induced increased mucosal permeability. CONCLUSION: We found impaired nasal epithelial barrier function in patients with HDM-induced AR, with lower occludin and zonula occludens-1 expression. IL-4 disrupted epithelial integrity in vitro, and FP restored barrier function. Better understanding of nasal barrier regulation might lead to a better understanding and treatment of AR.
Assuntos
Mucosa Nasal/metabolismo , Ocludina/metabolismo , Pyroglyphidae/imunologia , Rinite Alérgica Perene/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Adulto , Animais , Anti-Inflamatórios/uso terapêutico , Biomarcadores/metabolismo , Estudos de Casos e Controles , Dextranos/metabolismo , Feminino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluticasona/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Permeabilidade , Reação em Cadeia da Polimerase em Tempo Real , Rinite Alérgica Perene/tratamento farmacológico , Rinite Alérgica Perene/imunologiaRESUMO
Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA-4 on effector T (Teff) cells, its immune-modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA-4Ig at day 7 and 9 after immunization, when myelin-reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL-17 and IFN-γ. Importantly, CTLA-4Ig treatment resulted in a transient reduction of Ki67 and CTLA-4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.
Assuntos
Autoimunidade , Antígenos B7/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Antígenos B7/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Progressão da Doença , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Expressão Gênica , Interferon gama/biossíntese , Interleucina-17/biossíntese , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Camundongos , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.
Assuntos
Antígenos B7/metabolismo , Antígenos CD28/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Abatacepte , Animais , Antígenos B7/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Antígenos de Histocompatibilidade Classe I/farmacologia , Imunoconjugados/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Camundongos , Antígenos de Histocompatibilidade Menor , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
RATIONALE: Airway hyperreactivity (AHR) is a key feature of bronchial asthma, and inhalation of irritants may facilitate development of nonallergic AHR. Swimmers exposed to hypochlorite (ClO(-))-containing water show a higher risk of developing AHR. We developed a mouse model in which instillation of ClO(-) before ovalbumin (OVA) induces AHR without bronchial inflammatory cells. OBJECTIVES: To investigate the mechanisms of ClO(-)-OVA-induced nonallergic AHR. METHODS: The involvement of the transient receptor potential ankyrin (TRPA)1 channel was checked in vivo by the use of TRPA1(-/-) mice and in vitro by Ca(2+) imaging experiments. The role of substance P (SP) was investigated by pretreating animals with the receptor antagonist RP67580, by replacing ClO(-) with SP in vivo, and by immunofluorescent staining of large airways of exposed mice. The role of mast cells was evaluated by exposing mast cell-deficient Kit(Wh)/Kit(Wsh) mice to ClO(-)-OVA with or without mast cell reconstitution. MEASUREMENTS AND MAIN RESULTS: ClO(-)-OVA did not induce AHR in TRPA1(-/-) mice, and ClO(-) generates a Ca(2+) influx in TRPA1-transfected cells. Pretreatment with RP67580 reduces ClO(-)-OVA-induced AHR, although no increased SP expression was shown in the airways. SP-OVA exposure resulted in the same AHR as induced by ClO(-)-OVA. Kit(Wsh)/Kit(Wsh) mice did not develop AHR in response to ClO(-)-OVA unless they were reconstituted with bone marrow-derived mast cells. CONCLUSIONS: Induction of AHR by exposure to ClO(-)-OVA depends on a neuroimmune interaction that involves TRPA1-dependent stimulation of sensory neurons and mast cell activation.
Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Ácido Hipocloroso/efeitos adversos , Irritantes/efeitos adversos , Mastócitos/imunologia , Canais de Potencial de Receptor Transitório/imunologia , Animais , Hiper-Reatividade Brônquica/etiologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neuroimunomodulação , Nociceptores/imunologia , Ovalbumina/efeitos adversos , Substância P/metabolismo , Canal de Cátion TRPA1RESUMO
Interleukin-15 (IL-15) is a pro-inflammatory cytokine thought to contribute to the inflammation in inflammatory bowel diseases (IBD). The specific receptor chain IL-15Rα can be expressed as a transmembranous signalling receptor, or can be cleaved by a disintegrin and metalloprotease domain 17 (ADAM17) into a neutralizing, soluble receptor (sIL-15Rα). The aim of this study is to evaluate the expression of IL-15Rα in ulcerative colitis (UC) and Crohn's disease (CD) patients before and after infliximab (IFX) therapy. Gene expression of IL-15Rα, IL-15 and ADAM17 was measured at the mRNA level by quantitative reverse transcription-PCR in mucosal biopsies harvested before and after first IFX therapy. Concentrations of sIL-15Rα were measured in sera of patients by ELISA and IL-15Rα protein was localized in the gut by immunohistochemistry and immunofluorescence. Mucosal expression of IL-15Rα is increased in UC and CD patients compared with controls and it remains elevated after IFX therapy in both responder and non-responder patients. The concentration of sIL-15Rα in serum is also increased in UC patients when compared with controls and does not differ between responders and non-responders either before or after IFX. CD patients have levels of sIL-15Rα comparable to healthy controls before and after therapy. In mucosal tissues, IL-15Rα(+) cells closely resemble activated memory B cells with a pre-plasmablastic phenotype. To conclude, IBD patients have an increased expression of IL-15Rα mRNA in the mucosa. Expression is localized in B cells, suggesting that IL-15 regulates B-cell functions during bowel inflammation. No change in release of sIL-15Rα is observed in patients treated with IFX.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Subunidade alfa de Receptor de Interleucina-15/biossíntese , Subunidade alfa de Receptor de Interleucina-15/imunologia , Adulto , Anti-Inflamatórios não Esteroides/uso terapêutico , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Inflamação/tratamento farmacológico , Infliximab , Subunidade alfa de Receptor de Interleucina-15/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.
Assuntos
Asma/imunologia , Brônquios/imunologia , Edema/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Proteínas da Gravidez/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Fator de Crescimento Placentário , Reação em Cadeia da Polimerase em Tempo RealRESUMO
[This corrects the article DOI: 10.3389/fimmu.2022.849155.].
RESUMO
Rationale: Non-allergic asthma is driven by multiple endotypes of which neutrophilic and pauci-granulocytic asthma have been best established. However, it is still puzzling what drives inflammation and airway hyperreactivity (AHR) in these patients and how it can be treated effectively. Recently, a potential role of the innate immune system and especially the innate lymphoid cells (ILC) has been proposed. Objective: In this study, we investigated the effects of LPS inhalation on airway inflammation and AHR as a potential model for elucidating the pathogenesis of non-allergic asthma. Methods: Wild-type (BALB/c), SCID, IL-17A-/-, and Rag2-/- γC-/- mice were endonasally exposed to lipopolysaccharide (LPS, 2 µg) on four consecutive days. Twenty-four hours after the last exposure, AHR to methacholine was assessed. Cytokine levels and ILC subpopulations were determined in lung tissue. Cellular differential analysis was performed in BAL fluid. Main Results: In this study, we developed a murine model for non-allergic neutrophilic asthma. We found that repeated endonasal applications of low-dose LPS in BALB/c mice led to AHR, BAL neutrophilia, and a significant increase in lung ILC3 as well as a significant increase in lung chemokines KC and MIP-2 and cytokines IL-1ß, IL-17A, IL-22, and TNF. The adoptive transfer of ILC in Rag2-/- γC-/- mice showed that ILC played a causal role in the induction of AHR in this model. Antagonising IL-1ß, but not IL-17A or neutrophils, resulted in a partial reduction in LPS-induced AHR. Conclusion: In conclusion, we report here a murine model for neutrophilic asthma where ILC are required to induce airway hyperreactivity.
Assuntos
Asma , Interleucina-17 , Animais , Citocinas , Modelos Animais de Doenças , Humanos , Imunidade Inata , Inflamação , Interleucina-17/farmacologia , Lipopolissacarídeos/farmacologia , Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCIDRESUMO
Although the concept of "global airway allergy" has become widely accepted during recent years, nasobronchial interaction and its mechanisms remain incompletely understood. The experimental study of the effect of nasal allergen deposition on lower airway pathology is hampered by the difficulty of avoiding lower airway penetration of the allergens. In ovalbumin-sensitized mice with experimental airway allergy, nasal allergen provocations were performed after complete anatomical separation of upper and lower airways by means of a tracheotomy. A canula was inserted in the trachea, and the trachea was ligated, thus inhibiting any passage of allergens from upper to lower airways. Mice showed bronchial hyperresponsiveness to methacholine as early as 4 hours after nasal allergen provocation in the absence of recruitment of inflammatory cells. An increased substance P (SP) concentration in the bronchial lumen was found, as well as an increased number of SP-positive pulmonary nerves. Treatment with a neurokinin (NK) 1 receptor antagonist abolished the allergen-induced bronchial hyperresponsiveness. Moreover, endobronchial administration of SP caused NK1 receptor-dependent bronchial hyperresponsiveness in mice with airway allergy. Nasal allergen provocation rapidly induces bronchial hyperresponsiveness via pulmonary up-regulation of SP and activation of NK1 receptors.
Assuntos
Alérgenos/imunologia , Hiper-Reatividade Brônquica/imunologia , Testes de Provocação Nasal , Substância P/metabolismo , Administração por Inalação , Animais , Asma/imunologia , Asma/patologia , Asma/fisiopatologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/patologia , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/patologia , Imunização , Contagem de Leucócitos , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Antagonistas dos Receptores de Neurocinina-1 , Ovalbumina/imunologia , Receptores da Neurocinina-1/metabolismo , Substância P/administração & dosagem , Substância P/farmacologia , Triptofano/análogos & derivados , Triptofano/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL-6. HP has been demonstrated to modulate the immune response and to have anti-inflammatory activities. To analyze HP's effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp(-/-)) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). Hp(-/-)mice suffered from a more severe disease that was associated with increased expression of IL-17A, IL-6, and IFN-gamma mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL-17+ CD4+ and IFN-gamma+ CD4+ cells persisted in the CNS of Hp(-/-) mice. Absence of HP affected the priming and differentiation of T cells after MOG(35-55) immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp(-/-) T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.
Assuntos
Doenças Autoimunes/metabolismo , Haptoglobinas/deficiência , Inflamação/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/imunologia , Haptoglobinas/genética , Haptoglobinas/metabolismo , Imunização , Imunoglobulina G/sangue , Inflamação/genética , Inflamação/imunologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-17/genética , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas da Mielina , Glicoproteína Associada a Mielina/química , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Baço/metabolismo , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Células Th2/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Mouse models of asthma suffer from the necessity to prime the animals by injections before respiratory exposure. Our aim was to develop a mouse model that mimics the progression of human allergic disease upon low-dose inhaled allergen exposure. METHODS: Mice were primed intraperitoneally to ovalbumin (OVA) before they were exposed repeatedly to aerosols of either OVA, ryegrass (Lolium perenne) pollen extract, or both concomitantly. The sensitization to ryegrass pollen proteins was evaluated by measurement of specific serum antibody, by the respiratory response to a challenge with ryegrass pollen extract and by lung cytokine production after challenge. RESULTS: Inhalation of ryegrass pollen extract alone did not result in sensitization. Sensitization to inhaled ryegrass pollen proteins, however, did occur in mice that had been sensitized to OVA by intraperitoneal injections and were then exposed to inhaled ryegrass pollen extract and OVA simultaneously. T and B cell priming was ascertained by ryegrass pollen-specific IgG1 and IgE antibody production and by induction of airway inflammation and of Th2 cytokine mRNA transcripts in the lungs upon airway challenge with ryegrass pollen extract. A progressive spread of the IgE/IgG1 response to different ryegrass pollen proteins could be visualized in immunoblots by comparing antibody patterns at day 56 and 86. CONCLUSIONS: Low-dose inhalatory allergen exposure results in sensitization when airways are exposed at the same time to another allergen to which the animals are already sensitized. This model can help to unravel the mechanisms that underlie the development and progression of respiratory allergic diseases.
Assuntos
Modelos Animais de Doenças , Imunização/métodos , Lolium/imunologia , Pólen/imunologia , Hipersensibilidade Respiratória/imunologia , Resistência das Vias Respiratórias/fisiologia , Animais , Antígenos de Plantas/administração & dosagem , Antígenos de Plantas/imunologia , Western Blotting , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Expressão Gênica/genética , Expressão Gênica/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Inflamação/patologia , Interleucina-13/genética , Interleucina-4/genética , Interleucina-5/genética , Pulmão/metabolismo , Pulmão/patologia , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/imunologia , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/imunologia , Pólen/química , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Hipersensibilidade Respiratória/fisiopatologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante , Transferência Adotiva , Animais , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Reguladores/metabolismo , Quimeras de Transplante/imunologiaRESUMO
INTRODUCTION: Insight in the pathogenesis of intestinal fibrosis is an unmet medical need in inflammatory bowel diseases. Studies in murine models and human organ fibrosis point to a potential role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis. MATERIALS AND METHODS: Dextran sodium sulfate (DSS) in drinking water was used to induce chronic colitis and remodeling in C57Bl/6 wild type (WT), RAG-deficient, RAG-/- common γ chain deficient and anti-CD90.2 monoclonal antibody treated RAG-/- mice. Inflammation was scored by macroscopic and histological examination and fibrosis was evaluated by hydroxyproline quantification and histology. RESULTS: In RAG-/- mice (which have a normal ILC population but no adaptive immunity), chronic intestinal inflammation and fibrosis developed similarly as in WT mice, with a relative increase in ILC2 during repeated DSS exposure. Chronic colitis could also be induced in the absence of ILC (RAG-/- γc-/- or anti-CD90.2 treated RAG-/- mice) with no attenuation of fibrosis. Importantly, clinical recovery based on weight gain after stopping DSS exposure was impaired in ILC-deficient or ILC-depleted mice. CONCLUSION: These data argue against a profibrotic effect of ILC in chronic colitis, but rather suggest that ILC have a protective and recovery-enhancing effect after repeated intestinal injury.
Assuntos
Colite , Imunidade Inata , Animais , Sulfato de Dextrana , Feminino , Linfócitos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Patients with Crohn disease (CD) and ulcerative colitis (UC) suffer from chronic relapsing intestinal inflammation. While many studies focused on adaptive immunity, less is known about the role of innate immune cells in these diseases. Innate lymphoid cells (ILCs) are recently identified cells with a high cytokine-producing capacity at mucosal barriers. The aim was to study the impact of biological treatment on ILC in CD and UC. Patients initiating anti-tumor necrosis factor (TNF), ustekinumab, or vedolizumab treatment were prospectively followed up and peripheral and intestinal ILCs were determined. In the inflamed gut tissue of patients with inflammatory bowel disease, we found an increase of ILC1 and in immature NKp44- ILC3, whereas there was a decrease of mature NKp44+ ILC3 when compared to healthy controls (HCs). Similar but less pronounced changes in ILC1 were observed in blood, whereas circulating NKp44- ILC3 were decreased. Fifteen percent of CD patients had NKp44+ ILC3 in blood and these cells were not detected in blood of HCs or UC patients. Therapy with three different biologicals (ustekinumab targeting the IL-12/23 cytokines, anti-TNF and vedolizumab) partly restored intestinal ILC subset equilibrium with a decrease of ILC1 (except for ustekinumab) and an increase of NKp44+ ILC3. Anti-TNF also mobilized more NKp44+ ILC3 in circulation. As ILC1 are proinflammatory cells and as NKp44+ ILC3 contribute to homeostasis of intestinal mucosa, the observed effects of biologicals on ILCs might contribute to their clinical efficacy.