Mycobacterium tuberculosis (Mtb) infects many people yet only causes disease in a subset of these individuals. The development of active disease in immunocompetent people appears due to the progression of individual granulomas in the face of a robust, systemic immune response. Progression of a granuloma is marked by destructive tissue remodeling, and culminates in the release of infectious bacilli into the airways when the granuloma cavitates. The factors that determine which granulomas progress to active disease are unknown, although macrophages appear to be actively involved in the process. We propose exploiting microarray and immunohistological examination of human TB granulomas to identify the candidates implicated in the process. These data will be used to "inform" our experimental murine granuloma model to enable us to identify both the inducers and the effectors of this late stage tissue damage, which will then be tested in a rabbit infection model. The central hypothesis is that pathogen-derived molecules, like trehalose dimycolate, induce host factors that stimulate destructive invasion in macrophages, and this destructive invasion contributes to the late-stage damage that leads to active tuberculosis. 1. Functional analysis of hydrolase activities expression in "progressing" human TB granulomas and active disease in the rabbit model. a. Histological analysis of human TB granulomas. Analysis of human TB granulomas from the extensive tissue collection archived at the Groote Schuur Hospital, University of Cape Town. b. In situ zymography of matrix metalloproteinase (MMP) and cathepsin activities. c. Development of PET probes for in vivo visualization of granuloma progression. A collaboration with Dr. Clif Barry, NIAID, in developing MMP-specific PET probes for non-invasive analysis of granuloma progression in rabbits treated with anti-inflammatory drugs 2. Establishment of an experimentally-accessible murine model for destructive invasion. We have developed a murine model for probing the granulomatous response to Mtb-derived molecules. The conditioned medium from this granuloma induces destructive monocyte migration through a Matrigel plug, forming the basis of an experimental model for identification of the factors active in this process. 3. Phenotypic characterization of the destructive macrophages in vitro and in vivo. We will determine the functional phenotype of these destructive macrophages, define the signaling pathways involved in their activation and compare these data to known states of macrophage polarization.
Progression of tuberculosis to an active disease in immuno-competent people is determined locally, at the level of the individual granuloma, yet we know little about the factors that drive this process. We propose studies on human tissue and animal models to enable identification and experimental validation of the host factors that mediate progression to active disease. Finally, we will investigate the use of anti-inflammatory and anti-tissue remodeling compounds, as a means of modulating progression of tuberculosis in the rabbit model.
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