Nonhuman primates were used to develop an animal model that closely mimics human Mycobacterium tuberculosis infection. Cynomolgus macaques were infected with low doses of virulent M. tuberculosis via bronchoscopic instillation into the lung. All monkeys were successfully infected, based on tuberculin skin test conversion and peripheral immune responses to M. tuberculosis antigens. Progression of infection in the 17 monkeys studied was variable. Active-chronic infection, observed in 50 to 60% of monkeys, was characterized by clear signs of infection or disease on serial thoracic radiographs and in other tests and was typified by eventual progression to advanced disease. Approximately 40% of monkeys did not progress to disease in the 15 to 20 months of study, although they were clearly infected initially. These monkeys had clinical characteristics of latent tuberculosis in humans. Low-dose infection of cynomolgus macaques appears to represent the full spectrum of human M. tuberculosis infection and will be an excellent model for the study of pathogenesis and immunology of this infection. In addition, this model will provide an opportunity to study the latent M. tuberculosis infection observed in ϳ90% of all infected humans.

Background Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen.Methodology/Principal FindingsWe developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells.Conclusions/SignificanceThe new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

Little is known regarding the early events of infection of humans with Mycobacterium tuberculosis. The cynomolgus macaque is a useful model of tuberculosis, with strong similarities to human tuberculosis. In this study, eight cynomolgus macaques were infected bronchoscopically with low-dose M. tuberculosis; clinical, immunologic, microbiologic, and pathologic events were assessed 3 to 6 weeks postinfection. Gross pathological abnormalities were observed as early as 3 weeks, including Ghon complex formation by 5 weeks postinfection. Caseous granulomas were observed in the lung as early as 4 weeks postinfection. Only caseous granulomas were observed in the lungs at these early time points, reflecting a rigorous initial response. T-cell activation (CD29 and CD69) and chemokine receptor (CXCR3 and CCR5) expression appeared localized to different anatomic sites. Activation markers were increased on cells from airways and only at modest levels on cells in peripheral blood. The priming of mycobacterium-specific T cells, characterized by the production of gamma interferon occurred slowly, with responses seen only after 4 weeks of infection. These responses were observed from T lymphocytes in blood, airways, and hilar lymph node, with responses predominantly localized to the site of infection. From these studies, we conclude that immune responses to M. tuberculosis are relatively slow in the local and peripheral compartments and that necrosis occurs surprisingly quickly during granuloma formation.

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.

Mycobacterium tuberculosis (Mtb) is an extraordinarily successful human pathogen, one of the major causes of death by infectious disease worldwide. A key issue for the study of tuberculosis is to understand why individuals infected with Mtb experience different clinical outcomes. To better understand the dynamics of Mtb infection and immunity, we coupled nonhuman primate experiments with a mathematical model we previously developed that qualitatively and quantitatively captures important processes of cellular priming and activation. These processes occur between the lung and the nearest draining lymph node where the key cells mediating this process are the dendritic cells (DC). The nonhuman primate experiments consist of bacteria and cell numbers from tissues of 17 adult cynomolgus macaques (Macaca fascicularis) that were infected with Mtb strain Erdman (∼25 CFU/animal via bronchoscope). The main result of this work is that delays in either DC migration to the draining lymph node or T cell trafficking to the site of infection can alter the outcome of Mtb infection, defining progression to primary disease or latent infection and reactivated tuberculosis. Our results also support the idea that the development of a new generation of treatment against Mtb should optimally elicit a fast DC turnover at the site of infection, as well as strong activation of DCs for maximal Ag presentation and production of key cytokines. This will induce the most protective T cell response.