Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment
Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn’s disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.
KeywordsInflammatory bowel disease Mycobacterium avium subsp. paratuberculosis Macrophages Phagosomal maturation
This work was funded by the Portuguese Science Foundation (FCT) and COMPETE Program subsidized by FEDER, through the PIC/IC/82802/2007 Grant. The monoclonal antibody H4A3-c (antihuman LAMP-1), developed by J. Thomas August and James E. K. Hildreth, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242. The authors wish to thank Paula Sampaio, for helping with confocal image acquisition and processing. The authors also wish to thank all patients and controls for collaborating in this study.
Conflict of interest
The authors declare that they have no competing interests.
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