Abstract
Tuberculosis (TB) is responsible for death of nearly two million people in the world annually. Upon infection, Mycobacterium tuberculosis (Mtb) causes formation of granuloma where the pathogen goes into dormant state and can live for decades before resuscitation to develop active disease when the immune system of the host is weakened and/or suppressed. In an attempt to better understand host-pathogen interactions, several groups have been developing in vitro models of human tuberculosis granuloma. However, to date, an in vitro granuloma model in which Mtb goes into dormancy and can subsequently resuscitate under conditions that mimic weakening of the immune system has not been reported. We describe the development of a biomimetic in vitro model of human tuberculosis granuloma using human primary leukocytes, in which the Mtb exhibited characteristics of dormant mycobacteria as demonstrated by (1) loss of acid-fastness, (2) accumulation of lipid bodies (3) development of rifampicin-tolerance and (4) gene expression changes. Further, when these micro granulomas were treated with immunosuppressant anti-tumor necrosis factor-alpha monoclonal antibodies (anti-TNFα mAbs), resuscitation of Mtb was observed as has been found in humans. In this human in vitro granuloma model triacylglycerol synthase 1deletion mutant (Δtgs1) with impaired ability to accumulate triacylglycerides (TG), but not the complemented mutant, could not go into dormancy. Deletion mutant of lipY, with compromised ability to mobilize the stored TG, but not the complemented mutant, was unable to come out of dormancy upon treatment with anti-TNFα mAbs. In conclusion, we have developed an in vitro human tuberculosis granuloma model that largely exhibits functional features of dormancy and resuscitation observed in human tuberculosis.
Highlights
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), remains a major threat to the world population as one-third of the world population is latently infected
We demonstrate that deletion of tgs1 (Rv3130), that has been strongly implicated in the development of dormancy, prevented Mtb from going into dormancy and deletion of lipY (Rv3097c), thought to be involved in mobilization of stored TG, prevented resuscitation caused by anti-tumor necrosis factor (TNFa) mAb treatment
The micro granulomas were positive for both CD68 and CD3 antibodies, providing further evidence that the granulomas consisted of both T cells and macrophages (Fig. 1E)
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), remains a major threat to the world population as one-third of the world population is latently infected. Mtb enters the host via aerosolization, where it infects and activates macrophages and dendritic cells in the lungs. The activated dendritic cells, present the processed antigens to CD4 T cells [1]. These activated lymphocytes and infected macrophages, in response to inflammatory cytokines and chemokines, migrate to the site of infection where they can form organized structures called granulomas in which Mtb goes into a drug-resistant dormant state. In latent TB infection, humans can harbor a small number of dormant Mtb bacilli that are likely contained in microgranuloma. These organisms are viable but in a dormant state
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