ABSTRACT
The formation of granulomas is associated with the resolution of Q fever, a zoonosis due to Coxiella burnetii; however the molecular mechanisms of granuloma formation remain poorly understood. We generated human granulomas with peripheral blood mononuclear cells (PBMCs) and beads coated with C. burnetii, using BCG extracts as controls. A microarray analysis showed dramatic changes in gene expression in granuloma cells of which more than 50% were commonly modulated genes in response to C. burnetii and BCG. They included M1-related genes and genes related to chemotaxis. The inhibition of the chemokines, CCL2 and CCL5, directly interfered with granuloma formation. C. burnetii granulomas also expressed a specific transcriptional profile that was essentially enriched in genes associated with type I interferon response. Our results showed that granuloma formation is associated with a core of transcriptional response based on inflammatory genes. The specific granulomatous response to C. burnetii is characterized by the activation of type 1 interferon pathway.
Subject(s)
Coxiella burnetii/physiology , Granuloma/genetics , Granuloma/microbiology , Q Fever/genetics , Q Fever/microbiology , Adult , Aged , Coxiella burnetii/genetics , Gene Expression Profiling , Granuloma/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/microbiology , Male , Middle Aged , Q Fever/metabolismABSTRACT
BACKGROUND: The outcome of Q fever, an infectious disease caused by Coxiella burnetii, is associated with granuloma formation. Granulomas are present in patients with resolutive Q fever but are lacking in patients with chronic Q fever. METHODS: Study of granuloma formation requires invasive approaches. Here, we took advantage of a recently described method that enables in vitro generation of human granulomas specific for C. burnetii. RESULTS: Circulating mononuclear cells progressively accumulated around beads coated with C. burnetii extracts, and complete granulomas were generated in 8 days. Granuloma cells consisted of macrophages, lymphocytes, and, to a lesser extent, epithelioid cells and multinucleated giant cells. Early events that govern granuloma formation were studied using live-imaging microscopy. Monocytes migrated toward C. burnetii-coated beads independently of the presence of T lymphocytes and then recruited T lymphocytes. About 90% of patients with chronic Q fever failed to form granulomas. This deficiency was associated with defective migration of monocytes toward coated beads. CONCLUSIONS: Monocytes were involved in the early stages of granuloma formation and recruited T lymphocytes to complete granuloma formation. This article describes a direct relationship between defective granuloma formation and defective migration of monocytes.
Subject(s)
Coxiella burnetii/immunology , Coxiella burnetii/pathogenicity , Granuloma/immunology , Monocytes/immunology , Q Fever/immunology , Aged , Aged, 80 and over , Cells, Cultured , Female , Humans , Male , Middle Aged , T-Lymphocytes/immunologyABSTRACT
Image analysis tools are essential to describe and quantify dynamic biological phenomena, such as early stages of granuloma formation. Granulomas are constituted of a collection of immune cells that contain pathogens, leading to their elimination. We presented here a new method to obtain granuloma 3D reconstruction from transmitted light images. Granulomas were generated by incubating peripheral blood mononuclear cells with beads coated with sonicated Coxiella burnetii, a bacterial pathogen. Biological samples were observed under a confocal microscope, and recorded during several hours, providing a large set of data of several gigabytes. Our image processing, called Focus Detection Plugin (FDP), allowed to extract relevant images from large datasets and to perform a deblurring of image stacks. This FDP method, that was implemented as an ImageJ plugin, did not require powerful computer resources and was simple to use. To validate our FDP method, we compared our results with 3D reconstruction of fluorescent images. Both methods yielded comparable results. We concluded that our FDP method was able to generate processed images yielding robust 3D reconstruction of whole cell bodies, and presented major advantages for long-time recordings since no cell labeling was needed. This method was convenient to study the early stages of granuloma formation and may be applied to other complex biological systems.
