RESUMO
Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.
Assuntos
Antígeno CD11b/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Receptores Toll-Like/imunologia , Animais , Antígeno CD11b/genética , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores Toll-Like/genéticaRESUMO
We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC(50) of 10.5 microM and in vitro selectivity for binding to the recombinant alphaA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding alphaA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.
Assuntos
Anti-Inflamatórios não Esteroides/química , Antígeno CD11b/efeitos dos fármacos , Antígenos CD18/efeitos dos fármacos , Furanos/química , Tiazolidinedionas/química , Anti-Inflamatórios não Esteroides/farmacologia , Antígeno CD11b/química , Antígeno CD11b/metabolismo , Antígenos CD18/química , Antígenos CD18/metabolismo , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Furanos/farmacologia , Humanos , Conformação Proteica , Relação Estrutura-Atividade , Tiazolidinedionas/farmacologiaRESUMO
During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin alphaIIbbeta3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin alphaIIbbeta3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin alphaIIbbeta3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future.
