In vitro differentiation of human macrophages with enhanced antimycobacterial activity
Guillaume Vogt, Carl Nathan
Guillaume Vogt, Carl Nathan
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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 2

Interactive impact of O2 tension on MDM control of BCG.

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Interactive impact of O2 tension on MDM control of BCG. (A and B) Ba...
(A and B) Bacteriostatic effect of MDMs cultured in 20% O2. MDMs differentiated with RPMI–40% human plasma (no added cytokines) for 14 days were treated or not on day 14 with (A) IFN-γ or (B) GM-CSF, TNF-α, IL-13, IL-15, IL-4, IL-12, or IL-1β; infected with BCG on day 16; and lysed 3 weeks later for determination of CFU. Results are representative of 5 experiments from 2 donors. In A, “Donors” refers to pooled results for both donors. In B, percentage of control of BCG growth is relative to MDMs receiving PBS instead of cytokines. (C) Morphology of MDMs after incubation in various concentrations of O2. Monocytes were incubated without exogenous cytokines for 14 days in the indicated concentrations of O2, then treated with or without IFN-γ (5 ng/ml) and infected with BCG (MOI of 0.1) on day 16. Pictures were taken 8, 12, and 24 days after infection. Scale bars: 100 μm. (D) Control of BCG as a function of O2 concentration. Experiments as in A comparing O2 concentrations of 20%, 10%, 5%, or 1%. CFU were determined at intervals over 30 days following infection. Results were essentially the same without (shown) or with added IFN-γ (5 ng/ml; data not shown). Results in D are representative of 7 independent experiments with cells from 2 donors.