RESUMO
Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics.
Assuntos
Compostos de Anilina/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Neutrófilos/efeitos dos fármacos , Ácidos Nipecóticos/farmacologia , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Complemento C3/farmacologia , Convertases de Complemento C3-C5/antagonistas & inibidores , Convertases de Complemento C3-C5/metabolismo , Complemento C5a/farmacologia , Humanos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/metabolismoRESUMO
We describe a novel method of characterizing protein-RNA interactions using a fluorescence-based multiwell capillary electrophoresis platform based on microfluidic technology. As a proof of concept, we studied the binding of human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) to the transactivation-responsive RNA (TAR). We established conditions to quantify the binding of recombinant HIV-1 Tat to TAR RNA and validated the assay by demonstrating the dependence of this interaction on the presence of the UCU bulge in TAR. In addition, we showed that neomycin inhibited Tat-TAR binding in a dose-dependent manner (IC(50)=1.6-3.0µM). This microfluidic-based method is high-throughput screening compatible and may be applicable to targeting other nucleic acid-protein interactions.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética/métodos , Repetição Terminal Longa de HIV/genética , Microfluídica/métodos , RNA Viral/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Eletroforese Capilar , Humanos , Ligação ProteicaRESUMO
Activated mature B cells in which the DNA-binding activity of E-proteins has been disrupted fail to undergo class switch recombination. Here we show that activated B cells overexpressing the antagonist helix-loop-helix protein Id3 do not induce expression of the murine Aicda gene encoding activation-induced deaminase (AID). A highly conserved intronic regulatory element in Aicda binds E-proteins both in vitro and in vivo. The transcriptional activity of this element is regulated by E-proteins. We show that the enforced expression of AID in cells overexpressing Id3 partially restores class switch recombination. Taken together, our observations link helix-loop-helix activity and Aicda gene expression in a common pathway, in which E-protein activity is required for the efficient induction of Aicda transcription.
