Increased Mitogen-Activated Protein Kinase Activity and TNF-α Production Associated with Mycobacterium smegmatis- but Not Mycobacterium avium-Infected Macrophages Requires Prolonged Stimulation of the Calmodulin/Calmodulin Kinase and Cyclic AMP/Protein Kinase A Pathways

Abstract

Previous studies have shown the mitogen-activated protein kinases (MAPKs) to be activated in macrophages upon infection with Mycobacterium, and that expression of TNF-α and inducible NO synthase by infected macrophages was dependent on MAPK activation. Additional analysis demonstrated a diminished activation of p38 and extracellular signal-regulated kinase (ERK)1/2 in macrophages infected with pathogenic strains of Mycobacterium avium compared with infections with the fast-growing, nonpathogenic Mycobacterium smegmatis and Mycobacterium phlei. However, the upstream signals required for MAPK activation and the mechanisms behind the differential activation of the MAPKs have not been defined. In this study, using bone marrow-derived macrophages from BALB/c mice, we determined that ERK1/2 activation was dependent on the calcium/calmodulin/calmodulin kinase II pathway in both M. smegmatis- and M. avium-infected macrophages. However, in macrophages infected with M. smegmatis but not M. avium, we observed a marked increase in cAMP production that remained elevated for 8 h postinfection. This M. smegmatis-induced cAMP production was also dependent on the calmodulin/calmodulin kinase pathway. Furthermore, stimulation of the cAMP/protein kinase A pathway in M. smegmatis-infected cells was required for the prolonged ERK1/2 activation and the increased TNF-α production observed in these infected macrophages. Our studies are the first to demonstrate an important role for the calmodulin/calmodulin kinase and cAMP/protein kinase A pathways in macrophage signaling upon mycobacterial infection and to show how cAMP production can facilitate macrophage activation and subsequent cytokine production.

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages*

“…Emerging evidence suggests that IL-10 and TNF-␣ cytokines in macrophages are regulated upstream mainly by the p38 MAPK and ERK1/2 (11,62,63,92) signaling cascades. Although ERK1/2 activation is mainly required for TNF-␣ production (63,66), IL-10 induction is predominantly dependent on p38 MAPK signaling (60,65).…”

“…Although ERK1/2 activation is mainly required for TNF-␣ production (63,66), IL-10 induction is predominantly dependent on p38 MAPK signaling (60,65). Accordingly, we have observed that internalization of Mtbhsp60 through TLR2 is associated with an increased phosphorylation of p38 MAPK, whereas engagement of this protein with TLR4 induced predominantly ERK1/2 phosphorylation.…”

Mycobacterium avium Glycopeptidolipids Require Specific Acetylation and Methylation Patterns for Signaling through Toll-like Receptor 2

“…Isolation and Cultivation of Bone Marrow-derived Macrophages-Bone marrow-derived macrophages (BMM⌽s) were harvested from the bone marrow of 6-to 8-week-old C57BL/6 mice as described previously (26). Mature macrophages were harvested and stored at Ϫ140°C.…”

“…For Western blots, 10 g of native and deacetylated total lipid and purified GPLs were exposed to 3 ϫ 10 5 BMM⌽s for 4 h. BMM⌽s were then washed with icecold phosphate-buffered saline and treated for 10 min with icecold lysis buffer as previously described (26). Equal amounts of protein as determined by the MicroBCA protein assay (Pierce) were analyzed by Western blot for phosphorylated and total p38 and phosphorylated and total p65 NFB (21).…”

Mycobacterium tuberculosislipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b

“…The relative production of TNF varies among pathogenic and nonpathogenic mycobacterial species (6). Regulation of TNF biosynthesis is complex.…”