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P045. Mycobacterium avium subsp. paratuberculosis survival in macrophages from inflammatory bowel disease patients

N. Nazareth1, F. Magro2, R. Appelberg3, F. Vilas-Boas2, G. Macedo2, A. Sarmento1, 1University Fernando Pessoa, Faculty of Health Sciences, Porto, Portugal, 2Hospital de São João, Gastroenterology Department, Porto, Portugal, 3Institute for Molecular and Cell Biology (IBMC), Microbiology and Immunology of Infection, Porto, Portugal

Background

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated in Crohn's disease (CD). Increased MAP prevalence was detected in CD patients, suggesting defective handling of MAP. We studied MAP survival in macrophage cultures from healthy controls (HC) and inflammatory bowel disease (IBD) patients. Co-localization of MAP with LAMP-1 was used to evaluate the nature of intracellular MAP-containing vacuoles.

Methods

21 HC, 29 CD patients, 11 UC patients and also 22 CD patients and 22 UC patients undergoing infliximab treatment (anti-TNF-α; CD-IFX and UC-IFX), were enrolled in this study. Peripheral blood mononuclear cells were isolated using Histopaque density gradient (SIGMA). The monocyte fraction was enriched by adherence and cells were cultured for 7 days to allow differentiation into macrophages. Cultures were infected with MAP and bacterial growth was evaluated by CFU counts at infection day 7 (T7d) and day 0 (3 hours post-infection; T3h). Pre-16Sr RNA/DNA ratio was also determined at T3h and T7d, in order to confirm CFU results. For LAMP-1 co-localization studies, macrophages were infected for 24h with Syto BC-labelled MAP, fixed with 4% paraformaldehyde, permeabilized and stained with anti-human LAMP-1 antibody. Results were obtained by confocal microscopy followed by image analysis using Image J co-localization plug-in.

Results

In macrophages MAP showed constant CFU counts over 7 days. However, evaluation of the pre-16Sr RNA/DNA ratio indicated that macrophages from HC and UC patients, but not macrophages from CD patients, were able to eliminate MAP organisms shortly after infection (T3h). IFX treatment resulted in further decreased MAP killing by CD or UC macrophages. Macrophages from CD patients exhibited higher co-localization of MAP with LAMP-1, as compared to macrophages from HC. Treatment of CD or UC patients with IFX resulted in decreased MAP co-localization with LAMP-1.

Conclusion

Although MAP showed nearly constant CFU counts in macrophage cultures, results from pre-16Sr RNA/DNA ratio suggested that CD macrophages had decreased ability to eliminate MAP. The discrepancy observed between CFU and RNA/DNA ratio results might be due to the effect of clumps on MAP culturability. IFX treatment decreased MAP elimination by macrophages, suggesting a deleterious effect of IFX in MAP-colonized patients. Co-localization of MAP with LAMP-1-bearing vesicles did not correlate with MAP growth or elimination by macrophages.