TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth

Interleukin-4 involvement in the epithelioid transformation o resident peritoneal macrophages

A diferenciacao de macrofagos em celulas epitelioides parece estar associada a secrecao local de citocinas inflamatorias tais como fator estimulador de colonias de macrofagos e granulocitos (GM-CSF), interleucina-4 (IL-4) e fator de necrose tumoral (TNF-a). O mecanismo que dirige este tipo de diferenciacao nao esta completamente entendido embora a formacao de celulas multinucleadas tenha sido obtida in vitro com o emprego de GM-CSF e IL-4. Assim, estas citocinas foram utilizadas em experimentos visando reproduzir in vitro o mecanismo de formacao de celulas epitelioides a partir de macrofagos residentes. Nossos resultados mostraram que apenas macrofagos tratados com IL-4 apresentaram morfologia caracteristica de celulas epitelioides. Estudos ultra-estruturais revelaram que estas celulas possuem reticulo endoplasmatico rugoso e complexo de Golgi proeminentes, alem de numerosas vesiculas e mitocondrias. Interdigitacoes entre macrofagos adjacentes tambem foram observadas. O tratamento com IL-4 aumentou a expressao da proteina MRP-14, de moleculas MHC de classe II e de receptores de manose. Por outro lado, houve uma acentuada reducao na producao de oxido nitrico e na fagocitose mediada por receptores Fcg. O conjunto de caracteristicas morfologicas e funcionais de macrofagos tratados durante 7 dias com IL-4 e semelhante ao observado em celulas epitelioides de lesoes granulomatosas. Isto sugere um provavel envolvimento desta citocina no mecanismo de transformacao epitelioide de macrofagosBV UNIFESP: Teses e dissertaçõe