RESUMO
We demonstrate the first microfluidic-based on-chip liquefaction device for human sputum samples. Our device is based on an acoustofluidic micromixer using oscillating sharp edges. This acoustofluidic sputum liquefier can effectively and uniformly liquefy sputum samples at a throughput of 30 µL min(-1). Cell viability and integrity are maintained during the sputum liquefaction process. Our acoustofluidic sputum liquefier can be conveniently integrated with other microfluidic units to enable automated on-chip sputum processing and analysis.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Manejo de Espécimes/instrumentação , Escarro/citologia , Escarro/fisiologia , Sobrevivência Celular , Eosinófilos , Desenho de Equipamento , Citometria de Fluxo , Humanos , Técnicas Analíticas Microfluídicas/métodos , Neutrófilos , Sonicação , Manejo de Espécimes/métodosRESUMO
Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.
Assuntos
Células Epiteliais/metabolismo , Interleucina-13/fisiologia , Proteína Quinase 13 Ativada por Mitógeno/antagonistas & inibidores , Muco/metabolismo , Sistema Respiratório/metabolismo , Sítios de Ligação , Células Cultivadas , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Canais de Cloreto/fisiologia , Cristalografia por Raios X , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ligação de Hidrogênio , Cinética , Sistema de Sinalização das MAP Quinases , Proteína Quinase 13 Ativada por Mitógeno/química , Proteína Quinase 13 Ativada por Mitógeno/genética , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mucinas/genética , Mucinas/metabolismo , Naftalenos/química , Naftalenos/farmacologia , Ligação Proteica , Doença Pulmonar Obstrutiva Crônica/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Interferência de RNA , Sistema Respiratório/patologia , Via Secretória/efeitos dos fármacosRESUMO
The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.
