RESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Interleucina-17/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Terapia de Imunossupressão , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.
Assuntos
Pulmão/imunologia , Mucina-5B/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Animais , Asma/imunologia , Asma/metabolismo , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Cílios/fisiologia , Orelha Média/imunologia , Orelha Média/microbiologia , Feminino , Inflamação/patologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mucina-5AC/deficiência , Mucina-5AC/metabolismo , Mucina-5B/deficiência , Mucina-5B/genética , Fagocitose , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Staphylococcus aureus/imunologia , Análise de SobrevidaRESUMO
RATIONALE: The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity. OBJECTIVES: To test the inducibility of lung defenses against bacterial challenge. METHODS: Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol. MEASUREMENTS AND MAIN RESULTS: Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48-72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid. CONCLUSIONS: We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.
Assuntos
Adjuvantes Imunológicos/farmacologia , Extratos Celulares/farmacologia , Imunidade Inata , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/prevenção & controle , Aerossóis , Animais , Bactérias , Líquido da Lavagem Broncoalveolar/imunologia , Extratos Celulares/administração & dosagem , Contagem de Colônia Microbiana , Relação Dose-Resposta Imunológica , Feminino , Hospedeiro Imunocomprometido , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos , Mucosa Respiratória/imunologia , Análise de SobrevidaRESUMO
Mucus hypersecretion contributes to morbidity and mortality in many obstructive lung diseases. Gel-forming mucins are the chief glycoprotein components of airway mucus, and elevated expression of these during mucous metaplasia precedes the hypersecretory phenotype. Five orthologous genes (MUC2, MUC5AC, MUC5B, MUC6, and MUC19) encode the mammalian gel-forming mucin family, and several have been implicated in asthma, cystic fibrosis, and chronic obstructive pulmonary disease pathologies. However, in the absence of a comprehensive analysis, their relative contributions remain unclear. Here, we assess the expression of the entire gel-forming mucin gene family in allergic mouse airways and show that Muc5ac is the predominant gel-forming mucin induced. We previously showed that the induction of mucous metaplasia in ovalbumin-sensitized and -challenged mouse lungs occurs within bronchial Clara cells. The temporal induction and localization of Muc5ac transcripts correlate with the induced expression and localization of mucin glycoproteins in bronchial airways. To better understand the tight regulation of Muc5ac expression, we analyzed all available 5'-flanking sequences of mammalian MUC5AC orthologs and identified evolutionarily conserved regions within domains proximal to the mRNA coding region. Analysis of luciferase reporter gene activity in a mouse transformed Clara cell line demonstrates that this region possesses strong promoter activity and harbors multiple conserved transcription factor-binding motifs. In particular, SMAD4 and HIF-1alpha bind to the promoter, and mutation of their recognition motifs abolishes promoter function. In conclusion, Muc5ac expression is the central event in antigen-induced mucous metaplasia, and phylogenetically conserved 5' noncoding domains control its regulation.