In vitro differentiation of human macrophages with enhanced antimycobacterial activity
Guillaume Vogt, Carl Nathan
Guillaume Vogt, Carl Nathan
Published September 12, 2011
Citation Information: J Clin Invest. 2011;121(10):3889-3901. https://doi.org/10.1172/JCI57235.
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Technical Advance Infectious disease

In vitro differentiation of human macrophages with enhanced antimycobacterial activity

Abstract

Mycobacterium tuberculosis causes widespread, persistent infection, often residing in macrophages that neither sterilize the bacilli nor allow them to cause disease. How macrophages restrict growth of pathogens is one of many aspects of human phagocyte biology whose study relies largely on macrophages differentiated from monocytes in vitro. However, such cells fail to recapitulate the phenotype of tissue macrophages in key respects, including that they support early, extensive replication of M. tuberculosis and die in several days. Here we found that human macrophages could survive infection, kill Mycobacterium bovis BCG, and severely limit the replication of M. tuberculosis for several weeks if differentiated in 40% human plasma under 5%–10% (physiologic) oxygen in the presence of GM-CSF and/or TNF-α followed by IFN-γ. Control was lost with fetal bovine serum, 20% oxygen, M-CSF, higher concentrations of cytokines, or premature exposure to IFN-γ. We believe that the new culture method will enable inquiries into the antimicrobial mechanisms of human macrophages.

Authors

Guillaume Vogt, Carl Nathan

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Figure 6

Comparison of 10% and 5% O2, and use of GM-CSF and TNF-α in combination during differentiation for control and killing of M. tuberculosis by MDMs activated with various cytokines.

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Comparison of 10% and 5% O2, and use of GM-CSF and TNF-α in combination ...
(A) CFU for cultures incubated in 10% O2. MDMs were differentiated for 14 days with no exogenous cytokines (left panel) or with GM-CSF plus TNF-α (0.5 ng/ml each) (right panel) under 10% O2. Cells were activated with the indicated cytokines (50 ng/ml each) on day 14, infected with M. tuberculosis (MOI of 0.1) on day 16, and lysed 2 weeks later for CFU. (B) CFU for cultures incubated in 5% O2. Experiments were performed as in A, except that the O2 concentration was further reduced. (C) Morphology of MDMs. Cells were prepared as in A and B, except that MDMs were differentiated with GM-CSF plus TNF-α (0.5 ng/ml each) for the first 14 days and then activated with TNF-α (50 ng/ml). Micrographs were taken 14 days after infection. Scale bars: 100 μm; 50 μm (insets). (D) MDM markers. MDMs were differentiated for 14 days with GM-CSF plus TNF-α (0.5 ng/ml each) (left panel) or M-CSF (50 ng/ml) (right panel) under 5% O2, then analyzed by flow cytometry. Percentages indicate the proportion of positive cells relative to the isotype control. Asterisk denotes a distinct subpopulation that was positive for CD40, although the median fluorescence of the whole population was close to that of the isotype control. Results are means for MDMs from 2 different donors in 1 of 3 independent experiments, each with 2 donors.