Control of mycobacterial replication in human macrophages: roles of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways

Abstract

Intracellular persistence of mycobacteria may result from an intricate balance between bacterial replication and signaling events leading to antimicrobial macrophage activities. Using human monocyte-derived macrophages, we investigated the relevance of mitogen-activated protein kinase activation for the growth control of Mycobacterium avium isolates differing in their abilities to multiply intracellularly. The highly replicative smooth transparent morphotype of M. avium strain 2151 induced significantly less p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation than the smooth opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. Inhibition of the p38 pathway by highly specific inhibitors did not significantly affect mycobacterial replication within macrophages, regardless of the in vitro virulence of the M. avium strain. However, repression of the ERK1/2 pathway further enhanced intracellular growth of highly replicative M. avium strains, although it did not increase survival of the poorly replicating M. avium isolate. Inhibition of the ERK1/2 pathway resulted in decreased tumor necrosis alpha (TNF-alpha) secretion irrespective of the virulence of the M. avium isolate used for infection, revealing that TNF-alpha could have been only partially responsible for the control of intracellular M. avium growth. In conclusion, ERK1/2- and TNF-alpha-independent pathways are sufficient to limit intramacrophage growth of less-virulent M. avium strains, but early ERK1/2 activation in infected macrophages is critically involved in controlling the growth of highly replicative M. avium strains.

FIG. 1.

Uptake and intracellular replication of M. avium 2151 SmT and SmO in human macrophages. Human monocyte-derived macrophages were infected with the smooth transparent (SmT) or the smooth opaque (SmO) morphotype of M. avium strain 2151. (A) CFU counts in lysed macrophages were determined 4 h after infection. (B) Intracellular mycobacterial replication was determined by CFU counts after 7 days and compared to initial intracellular bacterial numbers (dotted line). Macrophage (Mφ) numbers in indicator areas were determined. (C) To meaningfully compare actual intracellular mycobacterial replication rates within macrophages, CFU were correlated to macrophage numbers at day 7. Relative CFU were calculated by dividing CFU per well by the number of macrophages in indicator areas (as described in Materials and Methods). Means of duplicates ± SD of one typical experiment out of six (six different donors) are shown.

FIG. 2.

Differential phosphorylation of MAP kinases by M. avium 2151 SmT and SmO. Human monocyte-derived macrophages were incubated with either the smooth transparent (SmT) or smooth opaque (SmO) morphotype of M. avium strain 2151 or LPS for 30 min. Cells were lysed, and aliquots of cell lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted onto nitrocellulose membranes. The membranes were incubated with specific anti-phospho-ERK1/2 and anti-phospho-p38 antibodies and then incubated with a peroxidase-coupled secondary antibody. Visualization was performed by enhanced chemiluminescence. As controls, the amounts of total ERK1/2 and p38 were detected in the same lysates. One representative experiment out of three independent experiments (three different donors) is shown.

FIG. 3.

PD98059, but not SB203580, enhances intracellular mycobacterial growth. Cultures of human monocyte-derived macrophages were preincubated with 3 or 10 μM PD98059 (•) or SB203580 (○) or 0.1% (vol/vol) DMSO as solvent control for 60 min. Macrophages were infected with M. avium 2151 smooth transparent (SmT) or smooth opaque (SmO) (A) or M. avium strain SE01 (B). The number of intracellular viable bacteria correlated to macrophage numbers (relative CFU) was determined 7 days postinfection. Values represent means of duplicates ± SD of one representative experiment out of six. (C and D) Results obtained from different donors. Macrophages were preincubated with 10 μM MAP kinase inhibitor or 0.1% (vol/vol) DMSO. Cells were infected with 2151 SmT or 2151 SmO (C) or with SE01 (D). For each donor, CFU counts 7 days postinfection were correlated to macrophage numbers and are shown as relative CFU. The boxes indicate the 25th and 75th percentiles. Whiskers above and below the boxes indicate the 90th and 10th percentiles. Medians are marked. All outlying points are graphed. Data shown were obtained from six independent experiments (six different donors) performed in duplicate. *, P < 0.05; n.s., not significant.

FIG. 4.

TNF-α and IL-10 release in cultures of human macrophages infected with M. avium strain 2151 SmT or SmO. Human monocyte-derived macrophages were incubated with increasing numbers of M. avium 2151 smooth transparent (SmT) or smooth opaque (SmO) or LPS as positive control. Supernatants were harvested after 18 h (TNF-α) and additionally from the same cultures 44 h (IL-10) postinfection. Cytokine concentrations were measured by ELISA. Means of duplicates (± SD) of one representative experiment out of three (three different donors) are shown.

FIG. 5.

PD98059 reduces M. avium-induced TNF-α formation; SB203580 reduces M. avium-induced IL-10 release. Human monocyte-derived macrophages were preincubated with medium containing 3 or 10 μM PD98059 (A) or SB203580 (B), or 0.1% (vol/vol) DMSO as solvent control for 60 min. Cultures were incubated with M. avium 2151 smooth transparent (SmT) or smooth opaque (SmO) or LPS as a positive control. Supernatants were harvested after 18 h (TNF-α) or 44 h (IL-10), and cytokine concentrations were determined by ELISA. To compare results from different experiments showing substantial donor-dependent variation of cytokine concentrations, secreted amounts of TNF-α and IL-10 are shown as percent of control cultures stimulated in the presence of DMSO (normalized to 100%). Data shown are the mean ± SD of three independent experiments (three different donors) performed in duplicate. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n.s., not significant.