Chaperonin 60 (Cpn60) is a prototypic molecular chaperone essential for cellular function due to its protein folding actions. However, over the past decade it has been established that Cpn60 can be released by human cells and by certain bacteria to act as an extracellular signalling protein. Mycobacterium tuberculosis produces two Cpn60 proteins: Cpn60.1 and Cpn60.2. We recently generated a M. tuberculosis mutant with an inactivated cpn60.1 gene and demonstrated that granuloma formation was impaired after murine/guinea pig infection. This finding suggested that Cpn60.1 may interact with the cellular organisation of the host response to M. tuberculosis bacilli. In this study, we report that recombinant M. tuberculosis Cpn60.1 has both pro- and anti-inflammatory effects on human circulating monocytes. At high concentrations, recombinant Cpn60.1 induces the synthesis of TNF-α, IL6, and IL8, and promotes the phosphorylation of NF-κBp65, p44/42MAPK and p38 MAPK. At lower concentrations M. tuberculosis Cpn60.1 inhibits lipopolysaccharide-induced release of TNF-α, and monocyte transcriptional activation program. Both effects are abrogated by proteolysis of Cpn60.1 and therefore cannot be attributed to contamination with lipopolysaccharide. Competition with LPS for binding to a common receptor, the release of IL-10 or down-regulation of TLR4 on the cell surface were excluded as explanations for the inhibitory activity of Cpn60.1. We therefore conclude that M. tuberculosis Cpn60.1 is an unusual protein with the ability to induce bipolar effects on human monocytes, which may help explain the pathology of granuloma formation in tuberculosis. We used microarrays to analyse the bipolar effectsof Cpn60.1 on human monocytes.
Overall design
To study the pro-inflammatory activities of Cpn60.1, human monocytes were purified from three healthy donors and incubated with 10 μg/ml M. tuberculosis Cpn60.1 (or 100 ng/ml LPS as a positive control or medium only as a negative control). To study the inhibitory effectds of Cpn60.1 on human monocytes, the cells were pre-exposed to 10μg/ml or 0.1ng/ml of Cpn60.1, washed and incubated with 1ng/ml LPS for further 2 hours. For positive control, the cells were pre-exposed to medium before washing and LPS stimulation while for the negative control, the cells were pre-exposed to medium, washed and again exposed to medium alone.
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