Study of anti- apoptotic effect of the Mycobacterium leprae in human Schwann cells
The lesions of the peripheral nervous are considered the most relevant symptoms of Leprosy, a chronic infectious disease caused by Mycobacterium leprae. The strategies employed by M. leprae to infect and multiply inside Schwann cells (SC) are, however, poorly understood. In this study we investigated whether, similarly to other obligate intracellular pathogens, M. leprae promotes an anti-apoptotic effect on the host cell. Human SC lineage ST88-14 was treated or not with the bacteria and incubated in serum-free RPMI medium to induces apoptosis. The cell viability was monitored by three methods: i) cell capacity to reduce the methyltetrazolium salt (MTT); ii) Tripan blue exclusion and iii) cell staining with fluorescein diacetate and ethidium bromide. After 24-48h incubation, cells treated with M. leprae showed 70-100% of survival, contrasting with 30-60% in the untreated cultures. M. leprae anti-apoptotic effect was shown to be dose dependent. Reduction of cells undergoing apoptosis in cultures treated with M. leprae was also observed through DiOC6 staining by flow cytometry, a method that monitors the mithocondrial membrane potential. In a next step the conditioned medium of cells treated with M. leprae was shown to mimic the anti-apoptotic effect of the bacteria, suggesting that soluble factors secreted by SC in response to M. leprae were involved in cell survival. One possible candidate for these soluble factors would be Insulin-Like Growth Factors, IGF-I and IGF-II. In fact, the capacity of M. leprae to induce the expression of IGF-I and IGF-II on SC was confirmed by RT-PCR. Also, when added to ST88-14 cells, recombinant IGF-I was able to protect the cells from apoptosis induced by serum withdrawn. IGF-I action is possibly mediated by the IGF-I receptor (IGF-IR), as ST88-14 cells were shown to express this receptor by Western blotting and FACS analysis. Finally, we showed that the pre-treatment of SC with one of the NF-kB inhibitors, thalidomide, glyotoxin and SN50 completely abolished the anti-apoptotic effect of M. leprae. Altogether, our results suggest a important strategy for the successful colonization of M. leprae in the nerve, based on the survival maintenance of the host cell through induction of IGF production and NF-kB activation.
Advisor:Maria Cristina Vidal Pessolani
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:mycobacterium leprae NF-kappa B schwann cells cell survival Insulin-Like Growth Factor I
Date of Publication:10/03/2005