Materials and methods
Differentiated THP-1 macrophages have been widely used as an in vitro model of human macrophages. There are some reports about optimizing the PMA concentrations required for differentiating monocytes while minimizing gene upregulation. According to these reports, treatment of THP-1 Puromycin with PMA at 2.5–100 ng/mL concentration for 48 hours can differentiate monocytes from macrophages; however, the minimal concentration of PMA required for stable differentiation without being overwhelmed by undesirable gene upregulation is 5 ng/mL. Therefore, the same concentration of PMA was used in all experiments prior to treating the cells with recombinant proteins.
The data presented here show that the recombinant mycobacterial secretory proteins, ESAT-6, CFP-10, and their fusion, improve the adhesion of macrophages to ECM components, with no effect on the viability of human THP-1 cells (Fig. 1), leading to granuloma formation. We also found that α4β1 integrin plays a key role in the adhesion of macrophages to FN. Recruitment of immune cells such as macrophages within pulmonary granulomas depends on their adhesion to the ECM. Macrophages use integrins to adhere to FN as a major ECM component. The most common receptors for FN are the α4β1 and α5β1 integrins. The interaction between FN and these integrins is effective in adhesion, spreading, and focal contact formation in monocytes/macrophages. Despite the good understanding of the structural organization of granulomas structure, the molecular and cellular mechanism of formation of granulomas and the role of ECM proteins in this process are not well defined. A predominant role of mycobacterial glycolipids envelop in the formation of granulomas has been recently uncovered. It has been shown that adhesion and fusion of macrophages are mediated by Toll-like receptor-2 (TLR2) and is dependent on the β1 integrin. In the same study, using microarray analysis, Puissegur et al showed that the expression of α4β1 integrin has been induced in granuloma cells. Rojas et al also indicated that the mycobacterial glycolipids envelop such as phosphatidylinositol mannoside binds α5β1 on T cells and induces adhesion to FN. Using transparent zebrafish embryo for monitoring the infection process in real time, it has been shown that RD1-deficient bacteria fail to elicit efficient granuloma formation, but macrophages infected with virulent mycobacteria produce an RD1-dependent signal, which directs macrophages to aggregate into granulomas. These observations indicated that the mycobacterium RD1 locus induces infected macrophages to send chemotactic signals for aggregation of macrophages, which in turn affect adhesion and other downstream events, resulting in granuloma formation. Despite the obvious inducing role of RD1 locus in macrophages aggregation, little is known about the effects of RD1 antigens, individually. In addition, the role of integrins is not clear in this process. Therefore, we aimed to investigate the role of recombinant M. tuberculosis antigens in the interactions and adhesion between macrophages and FN mediated by FN receptor integrins (α4β1 and α5β1) using a series of biochemical methods. The results obtained from cell attachment assay showed that differentiated THP-1 cell attachment to FN by 52 ± 3.08% is likely due to expression of α5β1 and α4β1 integrins. As shown in Fig. 2, the treatment of these cells with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins significantly increased the attachment on FN by 80 ± 1.2%, 74 ± 2.6%, and 90 ± 1.7%, respectively (p < 0.001). These results indicate that THP-1 cells adhere to FN and the recombinant antigens are able to increase attachment of this cell type on FN. According to the attachment assay results in the cells treated with different antigens, adhesion on FN was significantly increased when compared with untreated cells (p < 0.001). To examine whether treating cells with recombinant M. tuberculosis antigens leads to changes in F-actin organization and focal contact formation, immunofluorescent assay was performed. Results showed that THP-1 cells treated with all recombinant antigens used in this study form well-organized fibers and focal contacts containing vinculin in comparison with untreated cells. Accordingly, ESAT-6 and ESAT-6/CFP-10 showed better results in increasing focal contacts than CFP-10 (Fig. 3). Because adhesion of macrophages to ECM components, especially FN, depends on the two integrins, α4β1 and α5β1, it is suggested that the induction of stress fibers and formation of focal contacts after treatment with these recombinant antigens could be through inducing the expression of these two integrins.
Materials and methods