Mycobacterium bovis Bacillus Calmette–Guérin (BCG) stimulates IL-10 production via the PI3K/Akt and p38 MAPK pathways in human lung epithelial cells

https://doi.org/10.1016/j.cellimm.2008.03.002Get rights and content

Abstract

Infection of human cells with mycobacteria has been shown to result in the production of anti-inflammatory cytokines. However, the signaling pathways that regulate the Mycobacterium bovis BCG-induced interleukin (IL)-10 production are currently unknown. In the present study, we investigated the involvement of phosphatidylinoditol 3-kinase (PI3K)/Akt and the p38 MAPK signaling pathways in the secretion of IL-10 in human lung epithelial cells (A549) after infection with M. bovis BCG. Treatment of A549 cells with LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and wortmannin, two PI3K inhibitors, inhibited M. bovis BCG-induced IL-10 production. Stimulation of cells with M. bovis BCG caused an increase in Akt phosphorylation in a time-dependent manner, which was inhibited by wortmannin. In addition, treatment of A549 cells with an Akt inhibitor significantly blocked M. bovis BCG-induced IL-10 production. Moreover, the p38 inhibitor SB203580 significantly decreased IL-10 production in a dose-dependent manner, whereas M. bovis BCG-induced IL-10 secretion was completely unaffected by the MEK inhibitor PD98059. Finally, the inhibition of PI3K did not significantly affect p38 MAPK activation in M. bovis BCG-infected cells, indicating that PI3K activity is not required for the M. bovis BCG-induced phosphorylation of p38 MAPK. Collectively, these data suggest that the PI3K/Akt and p38 MAPK signaling pathways play an important role in the regulation of M. bovis BCG-induced IL-10 secretion in A549 cells.

Introduction

Mycobacterium bovis bacillus Calmette–Guérin (BCG) vaccine comprises a live strain of M. bovis that was attenuated by Calmette and Guérin for use in vaccination against Mycobacterium tuberculosis[1]. It has been demonstrated higher interleukin (IL)-10 production in systemic T cells and monocytes of BCG-vaccinated children [2], suggesting activation of the anti-inflammatory immune axis. In addition, a recent study of tuberculosis patients and healthy BCG-vaccinated subjects has shown elevated pro- as well as anti-inflammatory cytokines by human cells in response to mycobacterial antigens, represented by significant levels of IFN-γ, TNF-α and IL-10 in both groups [3]. IL-10 is a Class II α-helical cytokine [4]. At the in vitro level mycobacterial antigens have been shown to induce IL-10 production in macrophages [5], [6]. The synthesis of IL-10 is increased in mycobacterial infection [7] and has been detected in tuberculous lesions [8]. IL-10 has been well characterized for its ability to suppress the synthesis of proinflammatory cytokines and aid in the development of Th2 immunity [9]. IL-10 reduces antigen presentation and inhibits T-cell activation [10], [11]. Several pathogens, including leishmania [12], vaccinia virus [13], and mycobacteria [14], exploit the immunosuppressive activity of IL-10 to establish infection. Whereas the ability of IL-10 to regulate different aspects of immunity are well defined, there is limited information on the signaling pathways that regulate its production in lung epithelial cells.

Phosphatidylinoditol 3-kinase (PI3K) cascade pathways contribute to the transmission of extracellular signals that ultimately result in many cellular processes [15]. Activation of PI3K occurs via the regulatory subunit binding to phosphotyrosine residues present within cellular receptors located on the plasma membrane [16]. Once activated, PI3K catalyzes the production of PI (3,4,5)P3, which allows for recruitment of signaling proteins, including the serine/threonine kinase Akt [17], [18]. After recruitment, Akt becomes phosphorylated at Thr308 and Ser473[19]. Previous studies have shown that the PI3K/Akt pathway plays a pivotal role in regulating production of inflammatory mediators in both human and mouse macrophages [20], [21]. A role of PI3K and its downstream serine/threonine kinase, Akt, has been reported in macrophage phagocytosis of mycobacteria [22]. Previous studies have demonstrated that PI3K/pathway modulate the activation of the mitogen-activated protein kinase (MAPK) [23]. To date, seven MAPK kinase homologs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK6, and MKK7) and four distinct groups of MAPKs (ERK1/2, JNK, p38, ERK5) have been identified. ERK1 has been found in epithelial cells [24]. The activation of these kinases has been implicated in mycobacteria-induced proinflammatory cytokine signaling in human cells [24], [25]. There is limited information, however, on the role and regulation of these signaling pathways in M. bovis BCG-induced anti-inflammatory cytokine production in lung epithelial cells. Therefore, in the present study, we attempted to elucidate the roles of PI3K/Akt and the MAPK signaling pathways in M. bovis BCG-mediated IL-10 production in human lung epithelial cells. Our findings revealed that intracellular network of PI3K/Akt, and p38 MAPK signaling pathways plays an important role for regulating M. bovis BCG-induced IL-10 secretion in human lung epithelial cells.

Section snippets

Specific reagents

LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), wortmannin, the Akt inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]), SB203580 and PD98059 were purchased from Calbiochem–Novabiochem (San Diego, CA, USA). Dimethyl sulfoxide (DMSO; Sigma–Aldrich) was added to cultures at 0.01% (vol/vol) as a solvent control. Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/Ham’s F-12), fetal calf serum, penicillin/streptomycin, and Lipofectamine Plus™ reagent

PI3K/Akt pathway is involved in M. bovis BCG-induced IL-10 secretion in A549 cells

To understand the connection between IL-10 secretion of M. bovis BCG and its PI3K/Akt signaling pathway, we sought to determine whether PI3K/Akt inhibition affects the induction of IL-10 secretion. Two different PI3K inhibitors, LY 294002 [26] and wortmannin [27], were used. As shown in Fig. 1a, IL-10 production was dose-dependently inhibited by the PI3K inhibitor Ly294002. The Ly294002 was added to the cells 30 min after infection due to its known effect on phagocytosis. In addition, we

Discussion

Association of PI3K/Akt activation with reduced inflammatory responses has been shown in a murine sepsis model [30]. Moreover, it has been demonstrated that the PI3K/Akt pathway plays an important role in human monocyte antimycobacterial activity [31]. In this study, we investigated the role of the PI3K/Akt signaling pathway in M. bovis BCG-induced IL-10 production by human lung epithelial cells. To our knowledge this is the first report showing the functional significance of the PI3K/Akt

Acknowledgments

This research work was supported by grants from the Coordinación General de Posgrado e Investigación de el IPN (CGPI). P.M.S. is a COFAA, EDI, and SNI fellow.

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