Abstract
The accurate assessment of immune competence through ex vivo analysis is paramount to our understanding of those immune mechanisms that lead to protection or susceptibility against a broad range of human pathogens. We have developed a flow cytometry-based, whole blood phagocyte functional assay that utilizes the inflammatory inducer zymosan, coupled to OxyBURST-SE, a fluorescent reporter of phagosomal oxidase activity. The assay measures both phagocytic uptake and the superoxide burst in the phagocyte populations in whole blood. We utilized this assay to demonstrate impaired superoxide burst activity in the phagocytes of hospitalized HIV-positive patients with laboratory-confirmed tuberculosis. These data validate the use of the assay to assess the immune competence of patients in a clinical setting. The method is highly reproducible with minimal intraindividual variation and opens opportunities for the rapid assessment of cellular immune competence in peripheral blood in a disease setting.
Highlights
Bacterial killing assays in whole blood are well established and allow ex vivo assessment of immune function in patients, in the context of assessing response to vaccines or evaluating new bactericidal therapies [1,2,3,4]
We report the application of this novel reporter platform to quantify the phagocytic and superoxide burst functions of phagocytes in whole blood obtained from individuals in a clinical setting
It is interesting to note that the reduced superoxide burst in the phagocytes from HIV/ TB-coinfected individuals observed in this study is consistent with a recent report of impaired innate immune function of monocytes from HIV/TB-coinfected patient cohort in South Africa [31]
Summary
Bacterial killing assays in whole blood are well established and allow ex vivo assessment of immune function in patients, in the context of assessing response to vaccines or evaluating new bactericidal therapies [1,2,3,4]. The main read out of these assays is microbial killing measured via culture and colony counting, or fluorescence if reporter strain organisms are used. An extensive range of dynamic assays of phagosome function have been developed that are capable of providing a broad range of physiological readouts from the phagosome [6, 7] These assays have mostly utilized inert beads derivatized with different fluorescent reporters and focused on human alveolar macrophages or murine bone marrow-derived macrophages in culture [8,9,10]. We utilized zymosan derivatized with the oxidation-sensitive fluorescent reporter, OxyBURST-SE, to quantify phagosomal oxidase activity in peripheral blood phagocytes in situ.
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