RESUMO
Inhibition of cytokine gene expression by the hormone-activated glucocorticoid receptor (GR) is the key component of the anti-inflammatory actions of glucocorticoids, yet the underlying molecular mechanisms remain obscure. Here we report that glucocorticoid repression of cytokine genes in primary macrophages is mediated by GR-interacting protein (GRIP)1, a transcriptional coregulator of the p160 family, which is recruited to the p65-occupied genomic NFκB-binding sites in conjunction with liganded GR. We created a mouse strain enabling a conditional hematopoietic cell-restricted deletion of GRIP1 in adult animals. In this model, GRIP1 depletion in macrophages attenuated in a dose-dependent manner repression of NFκB target genes by GR irrespective of the upstream Toll-like receptor pathway responsible for their activation. Furthermore, genome-wide transcriptome analysis revealed a broad derepression of lipopolysaccharide (LPS)-induced glucocorticoid-sensitive targets in GRIP1-depleted macrophages without affecting their activation by LPS. Consistently, conditional GRIP1-deficient mice were sensitized, relative to the wild type, to a systemic inflammatory challenge developing characteristic signs of LPS-induced shock. Thus, by serving as a GR corepressor, GRIP1 facilitates the anti-inflammatory effects of glucocorticoids in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Anti-Inflamatórios/imunologia , Citocinas/antagonistas & inibidores , Regulação da Expressão Gênica/imunologia , Glucocorticoides/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores de Glucocorticoides/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Immunoblotting , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Análise de Sobrevida , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: One of the most devastating complications following implant-based breast reconstruction is periprosthetic infection. Making a prompt and accurate diagnosis has been a challenge as plastic surgeons are limited by nonspecific systemic markers of infection, clinical examination findings, or imaging modalities. OBJECTIVES: The aim of this study is to evaluate the use of periprosthetic fluid using cell count and differential as an aid in the diagnosis of infection. METHODS: This is a retrospective chart review. The authors selected patients who underwent breast reconstruction and had periprosthetic fluid analysis during the previous 10 years based on CPT 89051 (cell count and differential, body fluid). Only patients with clinical concerns for infection were included (cellulitis, fever, etc.); all others were excluded. RESULTS: A total of 54 samples were included in the study. Twenty-seven samples were associated with periprosthetic breast infections based on positive cultures or intraoperative findings consistent with infection. On fluid analysis, those with infection had a significantly higher neutrophil percentage (84.2% vs 19.3%, P < 0.0001). A cutoff value of 77% neutrophils had a sensitivity of 89% and a specificity of 93% in diagnosing infection. Delayed treatment in patients with high neutrophil percentage was associated with poorer outcomes. Lastly, there was a strong correlation between higher neutrophil percentage and increased rate of capsular contracture. CONCLUSIONS: Early and accurate diagnosis of periprosthetic breast infections can lead to earlier treatment and potentially improved the outcomes. Aspiration and analysis of periprosthetic fluid for neutrophil percentage can be a reliable method to guide clinical decision making.
RESUMO
Much of the regulatory diversity in eukaryotic transcription is provided by coregulators, which are recruited by DNA-binding factors to propagate signaling to basal machinery or chromatin. p160 family members, including the glucocorticoid receptor (GR)-interacting protein 1 (GRIP1), function as coactivators for GR, a ligand-dependent transcription factor of the nuclear receptor superfamily. Unlike other p160s, GRIP1 also potentiates GR-mediated repression of AP1 and NF-κB targets and, surprisingly, transcriptional activation by interferon regulatory factors. What enables GRIP1 activating or repressing properties or discrimination between physiologically antagonistic pathways is unknown. We found that endogenous GRIP1 in mammalian cells undergoes glucocorticoid-induced, GR interaction-dependent phosphorylation and identified one constitutive and six inducible phosphorylation sites and two putative GRIP1 kinases, casein kinase 2 and cyclin-dependent kinase 9. We raised phosphospecific antibodies to the four closely spaced sites in a previously uncharacterized part of GRIP1 which, combined with mutagenesis, revealed the conservation of GRIP1 phosphorylation across several cell types and species and its functional relevance to GR-activated transcription and to response element-specific recruitment of phospho-GRIP1 to native GR targets. We propose that cofactor engagement by GR is neither passive nor stochastic; rather, GR actively imparts modifications that dictate GRIP1 function in a subset of complexes, adding a layer of specificity to GR transcriptional control.