CCL20 is overexpressed in Mycobacterium tuberculosis-infected monocytes and inhibits the production of reactive oxygen species (ROS)

Abstract

CCL20 is a chemokine that attracts immature dendritic cells. We show that monocytes, cells characteristic of the innate immune response, infected with Mycobacterium tuberculosis express the CCL20 gene at a much higher level than the same cells infected with non-tuberculous mycobacteria. Interferon (IFN)-γ, a fundamental cytokine in the immune response to tuberculosis, strongly inhibits both the transcription and the translation of CCL20. We have also confirmed that dendritic cells are a suitable host for mycobacteria proliferation, although CCL20 does not seem to influence their intracellular multiplication rate. The chemokine, however, down-regulates the characteristic production of reactive oxygen species (ROS) induced by M. tuberculosis in monocytes, which may affect the activity of the cells. Apoptosis mediated by the mycobacteria, possibly ROS-dependent, was also inhibited by CCL20.

Fig. 1

Measurement of CCL20 expression. (a) Relative gene expression measured by quantitative polymerase chain reaction (qPCR). Data represent the mean ± standard deviation (s.d.) of the relative gene expression log in infected groups in comparison with control groups (non-infected cells) in monocytes (n = 4). Relative gene expression level in the control groups is always 1·0, its log 0·0, and has no s.d. Tukey's honestly significant difference (HSD) test was performed for pairwise comparisons. (b) Protein expression measured by enzyme-linked immunosorbent assay (ELISA). Bars represent the median. Dunn's test was performed for pairwise comparisons. Comparisons versus‘no infection’ group with *P < 0·05 is considered significant.

Fig. 2

Chemotactic response of dendritic cells. Data represent the migration index mean ± standard deviation (s.d.) from supernatants of infected cells (a, n = 3) or from purified CCL20 (b, n = 4). Controls are the supernatant from non-infected cells (a) and medium without chemokine (b), respectively. Migration index in the control group is always 1·00 and has no s.d. Dunnett's T3 test for pairwise comparison in (b) versus control group and versus CCL20 + anti-CCL20 group with *P < 0·05 is considered significant.

Fig. 3

Influence of CCL20 in the apoptosis mediated by Mycobacterium tuberculosis in infected monocytes. Monocytes were infected with a multiplicity of infection of 5, and a negative control (no infection) was included. Data are the proportions (expressed as percentage) of fluorescein-labelled cells (apoptotic) with respect to 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI)-labelled cells (total) and represent the mean ± standard deviation (s.d.) of five independent experiments. Tukey's honestly significant difference (HSD) test was used for comparisons between the negative control and the other groups. *P < 0·05 is considered significant.