TGF-b-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates Carolina L’Abbate 1 , Ivone Cipriano 2 , Elizabeth Cristina Pe ´ rez-Hurtado 1 , Sylvia Cardoso Lea ˜o 3 , Ce ´ lia Regina Whitaker Carneiro 1 , Joel Machado Jr. 4 * 1 Disciplina de Imunologia, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brasil, 2 Disciplina de Biologia do Desenvolvimento, Departamento de Morfologia e Gene ´ tica, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brasil, 3 Disciplina de Microbiologia, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brasil, 4 Departamento de Cie ˆ ncias Biolo ´ gicas, Campus de Diadema, Universidade Federal de Sa ˜o Paulo, Sa ˜ o Paulo, Brasil Abstract Transforming growth factor beta (TGF-b) 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-b 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-b and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-b produced by ECs are sufficient to induce a self-sustaining autocrine TGF-b signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-b to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-b receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-b 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-b 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-b production. In summary, our findings indicate that the amplitude of TGF-b signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth. Citation: L’Abbate C, Cipriano I, Pe ´ rez-Hurtado EC, Lea ˜o SC, Carneiro CRW, et al. (2011) TGF-b-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates. PLoS ONE 6(6): e21465. doi:10.1371/journal.pone.0021465 Editor: Guillaume Dalmasso, Emory Unviersity, United States of America Received December 22, 2010; Accepted June 1, 2011; Published June 22, 2011 Copyright: ß 2011 L’Abbate et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜ o Paulo (FAPESP), grant numbers 06/01533-9 and 02/6935-7. CL was awarded a fellowship by the Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´ gico (CNPq). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joel.jr@unifesp.br Introduction Bacteria of the Mycobacterium avium complex are facultative intracellular microorganisms that mainly infect mononuclear phagocytes and cause disseminated infection in immunocompro- mised patients [1,2]. The mycobacteria gain access to host tissues by a variety of portals [3,4], and invade the mucosa via epithelial cells [5,6]. Once the bacteria are transported into the deeper tissues by macrophages and perhaps other phagocytic cells, additional macrophages gather at individual infected foci to form granulomas. Granulomas likely begin as aggregates of mononu- clear phagocytes that surround individual infected macrophages [7,8]. These macrophages become activated, a transformation reflected by an increase in their size and subcellular organelles, ruffled cell membranes, and enhanced phagocytic and microbici- dal capabilities [9,10]. A common feature of all Mycobacterium granulomas is the differentiation of macrophages into epithelioid cells that have tightly interdigitated cell membranes in zipper-like arrays linking adjacent cells [7,10]. It is speculated that mycobacteria enter into a latent phase within mature granulomas and remain contained within these structures for prolonged periods of time [11]. Several studies have shown that the transforming growth factor b (TGF-b) is produced by macrophages upon infection with Mycobacterium and also with other pathogens and that the presence of TGF-b favors virulence, probably through its immunosuppres- sive action by impairing the response of macrophages to cytokines such as TNF-a or by suppression of nitric oxide (NO), and reactive oxygen intermediates [12,13,14]. TGF-b is a pleiotropic cytokine produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells [15,16]. The action of TGF-b on these cells is dependent not only on the cell type and its state of differentiation, but also on the milieu of cytokines present [17], suggesting that perturbations of the balance PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21465