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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-? followed by IFN-?. Control was lost with fetal bovine serum, 20% oxygen, M-CSF, higher concentrations of cytokines, or premature exposure to IFN-?. We believe that the new culture method will enable inquiries into the antimicrobial mechanisms of human macrophages.

SUBMITTER: Vogt G

PROVIDER: S-EPMC3195467 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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In vitro differentiation of human macrophages with enhanced antimycobacterial activity.

Vogt Guillaume G Nathan Carl C

The Journal of clinical investigation 20110912 10

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. ...[more]

PMID: 21911939

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Project description:BackgroundThe milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. In the present study, we investigated the effect of CSN1S1 on the differentiation of monocytes.MethodsPrimary human monocytes were stimulated with recombinant CSN1S1 and compared to cells stimulated with GM-CSF/IL-4 or M-CSF/IFNγ. Morphological changes were assessed by microscopy and quantification of surface markers of differentiation by FACS analysis. Phagocytic activity of CSN1S1 stimulated cells was measured by quantification of zymosan labeled particle uptake. The role of mitogen activated protein kinases for CSN1S1-induced differentiation of monocytes and proinflammatory cytokine expression was assessed by supplementation of specific inhibitors.ResultsCSN1S1 at a concentration of 10 μg/ml resulted in morphological changes (irregular shape, pseudopodia) and aggregation of cells, comparable to changes observed in M-CSF/IFNγ differentiated macrophages. Surface marker expression was altered after 24 h with an upregulation of CD14 (mean 2.5 fold) and CD64 (1.9 fold) in CSN1S1 stimulated cells. CSN1S1 treated cells showed a characteristic surface marker pattern for macrophages after 120 h of incubation (CD14high, CD64high, CD83low, CD1alow) comparable to changes observed in M-CSF/IFNγ treated monocytes. Furthermore, phagocytic activity was increased 1.4 and 1.9 fold following stimulation with 10 μg/ml CSN1S1 after 24 and 48 h, respectively. Early GM-CSF, but not GM-CSF/IL-4 induced differentiation of monocytes towards dendritic cells (DC) was inhibited by addition of CSN1S1. Finally, CSN1S1 induced upregulation of CD14 was impeded by inhibition of ERK1/2, while inhibition of the mitogen activated protein kinases JNK and p38 did not influence cellular differentiation. However, JNK and p38 inhibitors impeded CSN1S1 induced secretion of the proinflammatory cytokines IL-1b or IL-6.ConclusionsCSN1S1 skews in vitro differentiation of monocytes towards a macrophage-like phenotype. Data is accumulating that functions of CSN1S1 are beyond nutritional properties and include immunomodulatory effects.

Project description:Tuberculosis (TB), a disease caused by microorganisms of the Mycobacterium tuberculosis complex, infects almost one-third of the world's population. The TB epidemic has been further exacerbated by the emergence of multi, extensively, and totally-drug-resistant (MDR, XDR, and TDRTB) strains. An effective immune response plays a crucial role in determining the establishment of TB infection. Therefore, the modulation of the immune system has been considered as a vital approach for the treatment or control of various immune-related diseases such as TB. In this study, the antimycobacterial, immunomodulatory, and apoptosis-inducing effects of six Rubiaceae species were evaluated. A twofold serial dilution method was used to determine the minimum inhibitory concentration values of the plant extracts. The effect of the extracts on the activity of 15-lipoxygenase was investigated. The levels of six different cytokines, IL-2, IL-4, IL-5, IL-10, IFN-γ, and TNF-α, were measured in LPS-activated U937 cell line while the apoptosis-inducing effect of the extracts was evaluated using an annexin V/PI assay using a flow cytometer. The results obtained revealed that all the six extracts tested had antimycobacterial activity against M. tuberculosis H37Rv, M. tuberculosis ATCC 25177, and Mycobacterium bovis ATCC 27299 strains, with MIC values ranging from 39 to 312 μg/mL. The extracts of Cremaspora triflora and Cephalanthus natalensis were the most active against M. tuberculosis (MIC = 39 μg/mL), followed by Pavetta lanceolata and Psychotria zombamontana against M. bovis (MIC = 78 μg/mL). The extracts of P. zombamontana and Psychotria capensis had remarkable IC50 values of 4.32 and 5.8 μg/mL, respectively, better than that of quercetin. The selected extracts promoted Th1/Th2 balances in an in vitro model at the tested concentration which may suggest the therapeutic value of the plant in diseases where inflammation is a significant factor such as TB. The addition of the crude extracts of C. triflora, P. capensis, and P. zombamontana at the tested concentrations to the cell culture medium induced apoptosis in a time- and dose-dependent manner. This interesting preliminary result generated from this study encourages further investigations of these extracts owing to the LOX-inhibitory effect, immunomodulatory, and apoptotic-inducing properties in addition to their antimycobacterial properties.

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In vitro differentiation of human macrophages with enhanced antimycobacterial activity.

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In vitro differentiation of human macrophages with enhanced antimycobacterial activity.

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