RESUMO
Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MCTC) and epithelial MCs expressing tryptase without chymase (MCT). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine-mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MCT and MCTC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38highCD117high MCs that is markedly expanded during T2 inflammation. These CD38highCD117high MCs exhibit an intermediate phenotype relative to the expanded MCT and MCTC subsets. CD38highCD117high MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MCT-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MCTC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.
Assuntos
Mucosa Nasal/patologia , Pólipos Nasais/imunologia , Rinite/imunologia , Sinusite/imunologia , Adulto , Idoso , Proliferação de Células , Endoscopia , Feminino , Citometria de Fluxo , Humanos , Masculino , Mastócitos , Pessoa de Meia-Idade , Mucosa Nasal/citologia , Mucosa Nasal/imunologia , Mucosa Nasal/cirurgia , Pólipos Nasais/patologia , Procedimentos Cirúrgicos Nasais , RNA-Seq , Rinite/patologia , Rinite/cirurgia , Análise de Célula Única , Sinusite/patologia , Sinusite/cirurgia , Adulto JovemRESUMO
BACKGROUND: The cause of severe nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but upstream drivers of local antibody production in nasal polyps are undetermined. OBJECTIVE: We sought to identify upstream drivers and phenotypic properties of local antibody-expressing cells in nasal polyps from subjects with AERD. METHODS: Sinus tissue was obtained from subjects with AERD, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps, and controls without CRS. Tissue antibody levels were quantified via ELISA and immunohistochemistry and were correlated with disease severity. Antibody-expressing cells were profiled with single-cell RNA sequencing, flow cytometry, and immunofluorescence, with IL-5Rα function determined through IL-5 stimulation and subsequent RNA sequencing and quantitative PCR. RESULTS: Tissue IgE and IgG4 levels were elevated in AERD compared with in controls (P < .01 for IgE and P < .001 for IgG4 vs CRSwNP). Subjects with AERD whose nasal polyps recurred rapidly had higher IgE levels than did subjects with AERD, with slower regrowth (P = .005). Single-cell RNA sequencing revealed increased IL5RA, IGHG4, and IGHE in antibody-expressing cells from patients with AERD compared with antibody-expressing cells from patients with CRSwNP. There were more IL-5Rα+ plasma cells in the polyp tissue from those with AERD than in polyp tissue from those with CRSwNP (P = .026). IL-5 stimulation of plasma cells in vitro induced changes in a distinct set of transcripts. CONCLUSIONS: Our study identifies an increase in antibody-expressing cells in AERD defined by transcript enrichment of IL5RA and IGHG4 or IGHE, with confirmed surface expression of IL-5Rα and functional IL-5 signaling. Tissue IgE and IgG4 levels are elevated in AERD, and higher IgE levels are associated with faster nasal polyp regrowth. Our findings suggest a role for IL-5Rα+ antibody-expressing cells in facilitating local antibody production and severe nasal polyps in AERD.
Assuntos
Aspirina/efeitos adversos , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Pólipos Nasais/metabolismo , Sinusite/metabolismo , Adulto , Idoso , Anticorpos/metabolismo , Feminino , Humanos , Interleucina-5/metabolismo , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/induzido quimicamente , Plasmócitos/efeitos dos fármacos , Plasmócitos/metabolismo , Análise de Sequência de RNA/métodos , Sinusite/induzido quimicamente , Adulto JovemRESUMO
Cysteinyl leukotrienes (cysLTs) facilitate eosinophilic mucosal type 2 immunopathology, especially in aspirin-exacerbated respiratory disease (AERD), by incompletely understood mechanisms. We now demonstrate that platelets, activated through the type 2 cysLT receptor (CysLT2R), cause IL-33-dependent immunopathology through a rapidly inducible mechanism requiring the actions of high mobility box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE). Leukotriene C4 (LTC4) induces surface HMGB1 expression by mouse platelets in a CysLT2R-dependent manner. Blockade of RAGE and neutralization of HMGB1 prevent LTC4-induced platelet activation. Challenges of AERD-like Ptges-/- mice with inhaled lysine aspirin (Lys-ASA) elicit LTC4 synthesis and cause rapid intrapulmonary recruitment of platelets with adherent granulocytes, along with platelet- and CysLT2R-mediated increases in lung IL-33, IL-5, IL-13, and bronchoalveolar lavage fluid HMGB1. The intrapulmonary administration of exogenous LTC4 mimics these effects. Platelet depletion, HMGB1 neutralization, and pharmacologic blockade of RAGE eliminate all manifestations of Lys-ASA challenges, including increase in IL-33, mast cell activation, and changes in airway resistance. Thus, CysLT2R signaling on platelets prominently utilizes RAGE/HMGB1 as a link to downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of AERD. Antagonists of HMGB1 or RAGE may be useful to treat AERD and other disorders associated with type 2 immunopathology.
Assuntos
Asma Induzida por Aspirina/imunologia , Plaquetas/imunologia , Proteína HMGB1/metabolismo , Pulmão/imunologia , Mastócitos/imunologia , Receptores de Leucotrienos/metabolismo , Animais , Células Cultivadas , Humanos , Interleucina-33/metabolismo , Leucotrieno C4/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prostaglandina-E Sintases/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptores de Leucotrienos/genética , Transdução de SinaisRESUMO
Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases1. Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway2-4. Allergic inflammation can develop from persistent activation5 of type 2 immunity6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps7. Basal cell hyperplasia is a hallmark of severe disease7-9, but it is not known how these progenitor cells2,10,11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing12, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic13, epigenetic14,15 and extrinsic factors11,16,17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.
Assuntos
Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Células-Tronco/imunologia , Células-Tronco/patologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Epigênese Genética , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Interleucina-13/imunologia , Interleucina-4/imunologia , Subunidade alfa de Receptor de Interleucina-4/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-4/imunologia , Pessoa de Meia-Idade , Pólipos Nasais/imunologia , Pólipos Nasais/patologia , Rinite/imunologia , Rinite/patologia , Análise de Sequência de RNA , Análise de Célula Única , Sinusite/imunologia , Sinusite/patologia , Transcrição Gênica , Transcriptoma , Adulto JovemRESUMO
BACKGROUND: Mast cells are present in the airways of patients who have severe asthma despite glucocorticoid treatment; these cells are associated with disease characteristics including poor quality of life and inadequate asthma control. Stem cell factor and its receptor, KIT, are central to mast-cell homeostasis. We conducted a proof-of-principle trial to evaluate the effect of imatinib, a KIT inhibitor, on airway hyperresponsiveness, a physiological marker of severe asthma, as well as on airway mast-cell numbers and activation in patients with severe asthma. METHODS: We conducted a randomized, double-blind, placebo-controlled, 24-week trial of imatinib in patients with poorly controlled severe asthma who had airway hyperresponsiveness despite receiving maximal medical therapy. The primary end point was the change in airway hyperresponsiveness, measured as the concentration of methacholine required to decrease the forced expiratory volume in 1 second by 20% (PC20). Patients also underwent bronchoscopy. RESULTS: Among the 62 patients who underwent randomization, imatinib treatment reduced airway hyperresponsiveness to a greater extent than did placebo. At 6 months, the methacholine PC20 increased by a mean (±SD) of 1.73±0.60 doubling doses in the imatinib group, as compared with 1.07±0.60 doubling doses in the placebo group (P=0.048). Imatinib also reduced levels of serum tryptase, a marker of mast-cell activation, to a greater extent than did placebo (decrease of 2.02±2.32 vs. 0.56±1.39 ng per milliliter, P=0.02). Airway mast-cell counts declined in both groups. Muscle cramps and hypophosphatemia were more common in the imatinib group than in the placebo group. CONCLUSIONS: In patients with severe asthma, imatinib decreased airway hyperresponsiveness, mast-cell counts, and tryptase release. These results suggest that KIT-dependent processes and mast cells contribute to the pathobiologic basis of severe asthma. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01097694 .).
Assuntos
Asma/tratamento farmacológico , Mesilato de Imatinib/uso terapêutico , Mastócitos/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Testes de Provocação Brônquica , Contagem de Células , Método Duplo-Cego , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Mesilato de Imatinib/efeitos adversos , Masculino , Cloreto de Metacolina , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/efeitos adversos , Qualidade de Vida , Triptases/sangue , Triptases/metabolismoRESUMO
The C-type lectin receptor Dectin-2 can trigger the leukotriene C4 synthase-dependent generation of cysteinyl leukotrienes and the caspase-associated recruitment domain 9- and NF-κB-dependent generation of cytokines, such as IL-23, IL-6, and TNF-α, to promote Th2 and Th17 immunity, respectively. Dectin-2 activation also elicits the type 2 cytokine IL-33, but the mechanism by which Dectin-2 induces these diverse innate mediators is poorly understood. In this study, we identify a common upstream requirement for PI3Kδ activity for the generation of each Dectin-2-dependent mediator elicited by the house dust mite species, Dermatophagoides farinae, using both pharmacologic inhibition and small interfering RNA knockdown of PI3Kδ in bone marrow-derived dendritic cells. PI3Kδ activity depends on spleen tyrosine kinase (Syk) and regulates the activity of protein kinase Cδ, indicating that PI3Kδ is a proximal Syk-dependent signaling intermediate. Inhibition of PI3Kδ also reduces cysteinyl leukotrienes and cytokines elicited by Dectin-2 cross-linking, confirming the importance of this molecule in Dectin-2 signaling. Using an adoptive transfer model, we demonstrate that inhibition of PI3Kδ profoundly reduces the capacity of bone marrow-derived dendritic cells to sensitize recipient mice for Th2 and Th17 pulmonary inflammation in response to D. farinae Furthermore, administration of a PI3Kδ inhibitor during the sensitization of wild-type mice prevents the generation of D. farinae-induced pulmonary inflammation. These results demonstrate that PI3Kδ regulates Dectin-2 signaling and its dendritic cell function.
Assuntos
Células Dendríticas/imunologia , Hipersensibilidade/imunologia , Lectinas Tipo C/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Th17/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Dermatophagoides farinae/imunologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
BACKGROUND: Prostaglandin (PG) D2 is the dominant COX product of mast cells and is an effector of aspirin-induced respiratory reactions in patients with aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: We evaluated the role of the innate cytokine thymic stromal lymphopoietin (TSLP) acting on mast cells to generate PGD2 and facilitate tissue eosinophilia and nasal polyposis in patients with AERD. METHODS: Urinary eicosanoid levels were measured in aspirin-tolerant control subjects and patients with AERD. Nasal polyp specimens from patients with AERD and chronic rhinosinusitis were analyzed by using quantitative PCR, Western blotting, and immunohistochemistry. Human cord blood-and peripheral blood-derived mast cells were stimulated with TSLP in vitro to assess PGD2 generation. RESULTS: Urinary levels of a stable PGD2 metabolite (uPGD-M) were 2-fold higher in patients with AERD relative to those in control subjects and increased further during aspirin-induced reactions. Peak uPGD-M levels during aspirin reactions correlated with reductions in blood eosinophil counts and lung function and increases in nasal congestion. Mast cells sorted from nasal polyps expressed PGD2 synthase (hematopoietic PGD2 synthase) mRNA at higher levels than did eosinophils from the same tissue. Whole nasal polyp TSLP mRNA expression correlated strongly with mRNA encoding hematopoietic PGD2 synthase (r = .75), the mast cell-specific marker carboxypeptidase A3 (r = .74), and uPGD-M (r = 0.74). Levels of the cleaved active form of TSLP were increased in nasal polyps from patients with AERD relative to those in aspirin-tolerant control subjects. Recombinant TSLP induced PGD2 generation by cultured human mast cells. CONCLUSIONS: Our study demonstrates that mast cell-derived PGD2 is a major effector of type 2 immune responses driven by TSLP and suggests that dysregulation of this innate system contributes significantly to the pathophysiology of AERD.
Assuntos
Asma Induzida por Aspirina/imunologia , Citocinas/imunologia , Mastócitos/imunologia , Prostaglandina D2/imunologia , Adulto , Idoso , Asma Induzida por Aspirina/sangue , Asma Induzida por Aspirina/urina , Células Cultivadas , Eosinofilia/sangue , Eosinofilia/imunologia , Eosinofilia/urina , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/sangue , Pólipos Nasais/imunologia , Pólipos Nasais/urina , Prostaglandinas D/urina , Rinite/sangue , Rinite/imunologia , Rinite/urina , Sinusite/sangue , Sinusite/imunologia , Sinusite/urina , Adulto Jovem , Linfopoietina do Estroma do TimoRESUMO
Aspirin-exacerbated respiratory disease (AERD), a severe eosinophilic inflammatory disorder of the airways, involves overproduction of cysteinyl leukotrienes (cysLTs), activation of airway mast cells (MCs), and bronchoconstriction in response to nonselective cyclooxygenase inhibitors that deplete homeostatic PGE2. The mechanistic basis for MC activation in this disorder is unknown. We now demonstrate that patients with AERD have markedly increased epithelial expression of the alarmin-like cytokine IL-33 in nasal polyps, as compared with polyps from aspirin-tolerant control subjects. The murine model of AERD, generated by dust mite priming of mice lacking microsomal PGE2 synthase (ptges(-/-) mice), shows a similar upregulation of IL-33 protein in the airway epithelium, along with marked eosinophilic bronchovascular inflammation. Deletion of leukotriene C4 synthase, the terminal enzyme needed to generate cysLTs, eliminates the increased IL-33 content of the ptges(-/-) lungs and sharply reduces pulmonary eosinophilia and basal secretion of MC products. Challenges of dust mite-primed ptges(-/-) mice with lysine aspirin induce IL-33-dependent MC activation and bronchoconstriction. Thus, IL-33 is a component of a cysLT-driven innate type 2 immune response that drives pathogenic MC activation and contributes substantially to AERD pathogenesis.
Assuntos
Asma Induzida por Aspirina/imunologia , Imunidade Inata , Interleucina-33/imunologia , Leucotrienos/imunologia , Mastócitos/imunologia , Adolescente , Adulto , Idoso , Animais , Asma Induzida por Aspirina/genética , Asma Induzida por Aspirina/patologia , Dinoprostona/genética , Dinoprostona/imunologia , Feminino , Humanos , Interleucina-33/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Leucotrienos/genética , Masculino , Mastócitos/patologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Prostaglandina-E Sintases , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/patologiaRESUMO
The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae through the generation of cysteinyl leukotrienes and proinflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. In this study, we demonstrate that administration of the Dectin-2 mAb solely at the time of D. farinae challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n(-/-)) sensitized with the adoptive transfer of D. farinae-pulsed wild-type (WT) bone marrow-derived dendritic cells (DCs) also had less D. farinae-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n(-/-) mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production and with no reduction in serum IgE. WT and Clec4n(-/-) mice recipients, sensitized with D. farinae-pulsed WT bone marrow-derived DCs, had comparable levels of D. farinae-elicited IL-6, IL-23, TNF-α, and cysteinyl leukotrienes in the lung. By contrast, D. farinae-elicited CCL4 and CCL8 production from pulmonary CD11c(+)CD11b(+)Ly6C(+) and CD11c(+)CD11b(+)Ly6C(-)CD64(+) monocyte-derived DCs was reduced in Clec4n(-/-) recipients. Addition of CCL8 at the time of D. farinae challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n(-/-) recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at D. farinae challenge to promote the local inflammatory response.
Assuntos
Células Dendríticas/imunologia , Dermatophagoides farinae/imunologia , Lectinas Tipo C/imunologia , Pneumonia/imunologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Dermatophagoides/imunologia , Antígenos Ly/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Quimiocina CCL4/biossíntese , Quimiocina CCL4/metabolismo , Quimiocina CCL8/biossíntese , Quimiocina CCL8/metabolismo , Cisteína/imunologia , Células Dendríticas/transplante , Eosinófilos/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Leucotrienos/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Receptores de IgG/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, tissue eosinophilia, overproduction of cysteinyl leukotrienes (cysLTs), and respiratory reactions to nonselective cyclooxygenase (COX) inhibitors. Ex vivo studies suggest that functional abnormalities of the COX-2/microsomal prostaglandin (PG)E2 synthase-1 system may underlie AERD. We demonstrate that microsomal PGE2 synthase-1 null mice develop a remarkably AERD-like phenotype in a model of eosinophilic pulmonary inflammation. Lysine aspirin (Lys-ASA)-challenged PGE2 synthase-1 null mice exhibit sustained increases in airway resistance, along with lung mast cell (MC) activation and cysLT overproduction. A stable PGE2 analog and a selective E prostanoid (EP)2 receptor agonist blocked the responses to Lys-ASA by â¼90%; EP3 and EP4 agonists were also active. The increases in airway resistance and MC products were blocked by antagonists of the type 1 cysLT receptor or 5-lipoxygenase, implying that bronchoconstriction and MC activation were both cysLT dependent. Lys-ASA-induced cysLT generation and MC activation depended on platelet-adherent granulocytes and T-prostanoid (TP) receptors. Thus, lesions that impair the inducible generation of PGE2 remove control of platelet/granulocyte interactions and TP-receptor-dependent cysLT production, permitting MC activation in response to COX-1 inhibition. The findings suggest applications of antiplatelet drugs or TP receptor antagonists for the treatment of AERD.
Assuntos
Asma Induzida por Aspirina/metabolismo , Plaquetas/metabolismo , Dinoprostona , Oxirredutases Intramoleculares/genética , Receptores de Leucotrienos/metabolismo , Resistência das Vias Respiratórias/genética , Animais , Asma Induzida por Aspirina/tratamento farmacológico , Asma Induzida por Aspirina/genética , Asma Induzida por Aspirina/patologia , Asma Induzida por Aspirina/fisiopatologia , Plaquetas/patologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Humanos , Oxirredutases Intramoleculares/metabolismo , Mastócitos/metabolismo , Mastócitos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Inibidores da Agregação Plaquetária/uso terapêutico , Prostaglandina-E Sintases , Receptores de Leucotrienos/genéticaRESUMO
Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4(+/+) animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4(-/-) mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4(-/-) mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4(+/+) mice. In addition, lung DCs of challenged Lilrb4(-/-) mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4(-/-) mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.
Assuntos
Células Dendríticas/imunologia , Regulação para Baixo , Glicoproteínas de Membrana/deficiência , Pneumonia/imunologia , Receptores Imunológicos/deficiência , Animais , Apresentação de Antígeno , Quimiocina CCL21/metabolismo , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina/imunologia , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores Imunológicos/genética , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Mast cells (MC) and basophils share expression of the high-affinity receptor for IgE (FcεRI) but can be distinguished by their divergent expression of KIT and CD49b. In BALB/c mice, MC lineage cells expressing high levels of FcεRI by flow cytometry were seen only in bone marrow whereas those expressing intermediate levels of FcεRI were present in bone marrow and spleen of naive mice and in mesenteric lymph nodes (mLN) of Trichinella spiralis-infected mice. These FcεRI(+)KIT(+)CD49b(-) cells had a membrane phenotype similar to i.p. connective tissue-type MC, but were smaller and hypogranular by flow cytometry forward and side scatter profiles, respectively. Consistent with this, they lacked the prominent secretory granules identified by histochemistry and immunodetection for the MC-specific granule proteases that are readily seen in mature jejunal mucosal MC that also are induced by the infection and present at the same time. The concentration of these MC lineage cells in mLN determined by flow cytometry was comparable to that of MC progenitors (MCp) measured by limiting dilution and clonal expansion with maturation. We observed upregulation of IL-4 transcription by MCp in mLN and spleens of helminth-infected 4get mice, and we demonstrated by intracellular cytokine staining production of IL-4 and IL-6 by the mLN MCp in helminth-infected mice. Furthermore, treatment of helminth-infected mice with anti-FcεRI mAb, a protocol known to deplete basophils, also depleted mLN MCp. Thus, this study identifies a hypogranular subset of MCp recruited to mLN by helminth infection that may be an important unrecognized source of cytokines.
Assuntos
Grânulos Citoplasmáticos/imunologia , Interleucina-4/biossíntese , Interleucina-6/biossíntese , Linfonodos/imunologia , Mastócitos/imunologia , Triquinelose/imunologia , Animais , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Grânulos Citoplasmáticos/parasitologia , Grânulos Citoplasmáticos/patologia , Regulação para Baixo/imunologia , Genes Reporter , Interleucina-4/genética , Linfonodos/parasitologia , Linfonodos/patologia , Mastócitos/parasitologia , Mastócitos/patologia , Mesentério/imunologia , Mesentério/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Células-Tronco/imunologia , Células-Tronco/parasitologia , Células-Tronco/patologia , Trichinella spiralis , Triquinelose/parasitologia , Triquinelose/patologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Defining mast cell (MC) function has been clouded by use of mice with Kit mutations. In this and a previous issue of Immunity, two studies (Dudeck et al., 2011; Feyerabend et al., 2011) clarify MC function by using two unique mouse models.
RESUMO
We have previously shown that group V secretory phospholipase A(2) (sPLA(2)) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. In this study, we report that group V sPLA(2) (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to D. farinae compared with WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by D. farinae had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of D. farinae-challenged mice. Adoptively transferred D. farinae-loaded Pla2g5-null BMDCs were less able than D. farinae-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null D. farinae-loaded BMDCs exhibited significantly reduced local inflammatory responses to D. farinae, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA(2) in APCs regulates Ag processing and maturation of DCs and contributes to pulmonary inflammation and immune response against D. farinae. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA(2) is upregulated by D. farinae, and whose function is also regulated by group V sPLA(2).
Assuntos
Células Dendríticas/metabolismo , Hipersensibilidade/imunologia , Fosfolipases A2 Secretórias/imunologia , Pneumonia/imunologia , Pyroglyphidae/imunologia , Animais , Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Fosfolipases A2 do Grupo V/imunologia , Hipersensibilidade/complicações , Hipersensibilidade/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/enzimologia , Pneumonia/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We previously established that the inhibitory receptor LILRB4 mitigates LPS-induced, neutrophil-dependent pathologic effector mechanisms in inflammation. We now report that LILRB4 on dendritic cells (DCs) counterregulates development of an adaptive Th2 immune response and ensuing inflammation in a model of allergic pulmonary inflammation, initiated by inhalation sensitization with OVA and LPS followed by airway challenge with OVA. We found that Lilrb4(-/-) mice had significantly exacerbated eosinophilic pulmonary inflammation, as assessed in bronchoalveolar lavage and lung tissue, as well as elevated levels of OVA-specific IgE and Th2 cytokines produced by OVA-restimulated lymph node cells. LILRB4 was preferentially expressed on MHC class II(high)CD86(high) OVA-bearing DCs in lung-draining lymph nodes after sensitization or challenge. Moreover, the lymph nodes of Lilrb4(-/-) mice had significantly more of these mature DCs after challenge with OVA, which was accompanied by significantly more IL-4-producing lymphocytes, compared with Lilrb4(+/+) mice. Sensitization of naive Lilrb4(+/+) mice by transfer of OVA-LPS-pulsed Lilrb4(-/-) bone marrow-derived DCs was sufficient to confer exacerbated allergic lung pathology upon challenge with OVA, compared with mice that received Lilrb4(+/+) bone marrow-derived DCs. Our findings establish that maturation and migration of pulmonary DCs to lymph nodes in response to Ag and an innate immune stimulus is associated with upregulated expression of LILRB4. In addition, this receptor attenuates the number of these mature DCs and attendant IL-4-producing lymphocytes in the lymph nodes, and accordingly, the ability of DCs to elicit pathologic Th2 pulmonary inflammation.
Assuntos
Imunidade Adaptativa , Células Dendríticas/imunologia , Glicoproteínas de Membrana/fisiologia , Pneumonia/imunologia , Receptores Imunológicos/fisiologia , Células Th2/imunologia , Animais , Movimento Celular , Interleucina-4/biossíntese , Lipopolissacarídeos , Pneumopatias/patologia , Linfonodos/patologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Ovalbumina , Pneumonia/induzido quimicamente , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Regulação para Cima/genéticaRESUMO
Phospholipase A(2) (PLA(2)) hydrolyzes the sn-2 position of cell membrane phospholipids to release fatty acids and lysophospholipids. We have previously reported that group V secretory PLA(2) (sPLA(2)) translocates from the Golgi and recycling endosomes of mouse peritoneal macrophages to newly formed phagosomes and regulates the phagocytosis of zymosan, suggesting a role in innate immunity. Here we report that in macrophages lacking group V sPLA(2), phagosome maturation was reduced 50-60% at early time points while the binding of zymosan was unimpaired. The ability of group V sPLA(2) to regulate phagocytosis extended to phagocytosis of IgG- and complement-opsonized sheep RBC. Moreover, macrophages lacking group V sPLA(2) had delays in phagocytosis, phagosome maturation, and killing of Candida albicans. Cytokine production and eicosanoid generation were not impaired by the lack of group V sPLA(2). Furthermore, in a model of systemic candidiasis, mice lacking group V sPLA(2) had an increased fungal burden in the kidney, liver, and spleen at day 7 postinfection and increased mortality. Thus, group V sPLA(2) regulates phagocytosis through major phagocytic receptors and contributes to the innate immune response against C. albicans by regulating phagocytosis and killing through a mechanism that is likely dependent on phagolysosome fusion.
Assuntos
Candida albicans/imunologia , Fosfolipases A2 do Grupo V/metabolismo , Imunidade Inata/imunologia , Fagossomos/enzimologia , Fagossomos/imunologia , Animais , Candidíase/genética , Candidíase/imunologia , Candidíase/metabolismo , Candidíase/patologia , Fosfolipases A2 do Grupo V/deficiência , Fosfolipases A2 do Grupo V/genética , Lectinas Tipo C , Macrófagos/enzimologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Fagocitose , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/biossíntese , Zimosan/metabolismoRESUMO
Little is known about the serine/threonine kinase protein kinase D (PKD)1 in mast cells. We sought to define ligands that activate PKD1 in mast cells and to begin to address the contributions of this enzyme to mast cell activation induced by diverse agonists. Mouse bone marrow-derived mast cells (BMMC) contained both PKD1 mRNA and immunoreactive PKD1 protein. Activation of BMMC through TLR2, Kit, or FcepsilonRI with Pam(3)CSK(4) (palmitoyl-3-cysteine-serine-lysine-4), stem cell factor (SCF), and cross-linked IgE, respectively, induced activation of PKD1, as determined by immunochemical detection of autophosphorylation. Activation of PKD1 was inhibited by the combined PKD1 and protein kinase C (PKC) inhibitor Gö 6976 but not by broad-spectrum PKC inhibitors, including bisindolylmaleimide (Bim) I. Pam(3)CSK(4) and SCF also induced phosphorylation of heat shock protein 27, a known substrate of PKD1, which was also inhibited by Gö 6976 but not Bim I in BMMC. This pattern also extended to activation-induced increases in mRNA encoding the chemokine CCL2 (MCP-1) and release of the protein. In contrast, both pharmacologic agents inhibited exocytosis of beta-hexosaminidase induced by SCF or cross-linked IgE. Our findings establish that stimuli representing innate, adaptive, and growth factor pathways activate PKD1 in mast cells. In contrast with certain other cell types, activation of PKD1 in BMMC is largely independent of PKC activation. Furthermore, our findings also indicate that PKD1 preferentially influences transcription-dependent production of CCL2, whereas PKC predominantly regulates the rapid exocytosis of preformed secretory granule mediators.
Assuntos
Adaptação Fisiológica/imunologia , Mastócitos/imunologia , Proteína Quinase C/metabolismo , Transdução de Sinais/imunologia , Fator de Células-Tronco/imunologia , Animais , Sítios de Ligação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Carbazóis/farmacologia , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Exocitose/efeitos dos fármacos , Imunidade Inata/imunologia , Imunoglobulina E/imunologia , Indóis/farmacologia , Ligantes , Lipopeptídeos , Maleimidas/farmacologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator de Células-Tronco/farmacologia , Relação Estrutura-AtividadeRESUMO
We previously reported that joint swelling, synovial thickening, and cartilage matrix depletion induced by the injection of anti-collagen monoclonal antibodies and lipopolysaccharide (LPS) in BALB/c mice are increased in the absence of inhibitory leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4; formerly gp49B1) in a neutrophil-dependent manner. Because both mast cells and neutrophils express LILRB4, we sought a mast cell requirement with mast cell-deficient mouse strains, but unexpectedly obtained full arthritis in Kit(W-sh) mice and full resistance in Kit(W/KitW-v) mice. Kit(W-sh) mice were indeed mast cell deficient as assessed by histology and the absence of IgE/mast cell-dependent passive cutaneous anaphylaxis in the ear and joint as well as passive systemic anaphylaxis. Deletion of LILRB4 in Kit(W-sh) mice exacerbated anti-collagen/LPS-induced joint swelling that was abolished by neutrophil depletion, establishing a counterregulatory role for LILRB4 in the absence of mast cells. Whereas blood neutrophil levels and LPS-elicited tissue neutrophilia were equal in Kit(W-sh) and Kit+ mice, both were impaired in Kit(W/KitW-v) mice. Although both strains are mast cell deficient and protected from IgE-mediated anaphylactic reactions, their dramatically different responses to autoantibody-mediated, neutrophil-dependent immune complex arthritis suggest that other host differences determine the extent of mast cell involvement. Thus, a conclusion for an absolute mast cell role in a pathobiologic process requires evidence from both strains.
Assuntos
Anticorpos , Artrite Experimental/genética , Artrite Experimental/imunologia , Mastócitos/imunologia , Animais , Colágeno/imunologia , Artropatias/imunologia , Lipopolissacarídeos/imunologia , Mastócitos/patologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/imunologiaRESUMO
Leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4)(previously termed gp49B1) is a member of the Ig superfamily expressed constitutively on the surface of mast cells, neutrophils, and macrophages. LILRB4 inhibits IgE-dependent activation of mast cells in vitro through its two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit the src homology domain type-2-containing tyrosine phosphatase 1 into the cell membrane. Accordingly, Lilrb4(-/-) mice exhibit greater incidence and severity of IgE- and mast cell-dependent anaphylactic inflammation compared with mice that express LILRB4. In addition, mast cell-dependent inflammation induced by the interaction of stem cell factor (SCF) with its receptor Kit is also more severe in Lilrb4(-/-) mice, indicating that the counterregulatory function of LILRB4 extends beyond inflammation induced by Fc receptors, which signal through ITIMs, to responses initiated through a receptor tyrosine kinase. Indeed, pathologic inflammatory responses induced by activation of neutrophils with lipopolysaccharide (LPS) alone or with tissue-specific autoantibodies are greatly exacerbated in Lilrb4(-/-) mice. The rapid upregulation of LILRB4 expression on neutrophils in Lilrb4(+/+) mice in response to LPS suggests it is an innate counterregulatory response designed to reduce pathologic inflammation. Nevertheless, LILRB4 also serves a similar purpose for inflammation induced by the humoral adaptive immune response that is manifested through effector cells bearing Fc receptors.
Assuntos
Inflamação/imunologia , Mastócitos/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Animais , Humanos , Imunoglobulina E/imunologia , Células Matadoras Naturais/imunologia , Ligantes , Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Ratos , Receptores Imunológicos/agonistas , Receptores Imunológicos/genéticaRESUMO
The immune system must effectively regulate the balance between beneficial and detrimental inflammation. This process is achieved in part through cell surface receptors that rapidly integrate activating and inhibitory signals. The inhibitory members of the leukocyte Ig-like receptor (LILR) family, termed LILRBs, are broadly distributed among cell populations in the immune system and potently counterregulate cell activation induced by stimuli of innate and adaptive immune responses. Studies in mice and humans indicate that LILRBs appreciably downregulate harmful inflammatory responses induced by microbial, allergic, and cytotoxic mechanisms. Hence, the LILRBs likely play significant roles in regulating the incidence and severity of many inflammatory diseases, making them potential targets for therapeutic interventions.