Human macrophage gamma interferon decreases gene expression but not replication of Mycobacterium tuberculosis: analysis of the host-pathogen reciprocal influence on transcription in a comparison of strains H37Rv and CMT97

Abstract

Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages (MPhi). Host cells have developed different mycobactericidal mechanisms, including the production of inflammatory cytokines. The aim of this study was to compare the MPhi response, in terms of cytokine gene expression, to infection with the M. tuberculosis laboratory strain H37Rv and the clinical M. tuberculosis isolate CMT97. Both strains induce the production of interleukin-12 (IL-12) and IL-16 at comparable levels. However, the clinical isolate induces a significantly higher and more prolonged MPhi activation, as shown by reverse transcription-PCR analysis of IL-1beta, IL-6, IL-10, transforming growth factor beta, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) transcripts. Interestingly, when IFN-gamma transcription is high, the number of M. tuberculosis genes expressed decreases and vice versa, whereas no mycobactericidal effect was observed in terms of bacterial growth. Expression of 11 genes was also studied in the two M. tuberculosis strains by infecting resting or activated MPhi and compared to bacterial intracellular survival. In both cases, a peculiar inverse correlation between expression of these genes and multiplication was observed. The number and type of genes expressed by the two strains differed significantly.

FIG. 1

Southern blotting of RT-PCR products relative to the cytokines produced by resting uninfected human macrophages and by resting macrophages infected with two strains of M. tuberculosis (MTB), H37Rv and the CMT97 clinical isolate. Three time points were analyzed for each sample: 1, 3, and 7 days of culture for the controls, and 1, 3 and 7 days after infection for the infected cells. In each of the three groups, the first lane represents uninfected macrophages, the second represents H37Rv-infected macrophages, and the third represents CMT97-infected macrophages. As it comes out from the kinetics of RT-PCR products, the majority of cytokines decrease as a function of time, while the clinical strain induces a stronger and more prolonged production of TNF-α and IFN-γ compared with the laboratory strain H37Rv.

FIG. 2

(A) Immunofluorescence of H37Rv-infected macrophages. The top panel shows anti-CD14 staining in nonpermeabilized macrophages; the bottom panel shows anti-IFN-γ staining in permeabilized macrophages. The arrowheads indicate macrophages that are positive for both antibodies. (B) Immunofluorescence of H37Rv- and CMT97-infected macrophages. Top and bottom panels, macrophages infected with the CMT97 strain. Middle panel, macrophages infected with the H37Rv strain. The left column shows anti-CD14 staining, and the right column shows anti-IFN-γ staining. (C) ELISA titration of IFN-γ released in culture supernatants of human macrophages infected with H37Rv and CMT97. The macrophages were prepared as described in the text and infected for 3 h at different MOIs (50:1,10:1, and 1:1); after 1 and 3 days of infection, the culture supernatants were collected, and IFN-γ protein was measured. Compared with the laboratory strain H37Rv, clinical strain CMT97 infection induces higher and more prolonged IFN-γ production.

FIG. 3

(A) RT-PCR Southern blotting for cytokine production in IFN-γ- and LPS-activated human macrophages. The cDNAs of human macrophages infected for 3 days with M. tuberculosis H37Rv and the clinical strain CMT97 were assayed with cytokine-specific primers. The housekeeping gene was β2 microblobulin (bottom line). The cytokines induced by CMT97 in the activated cells are only IL-1β and TNF-α, while laboratory strain H37Rv infection induces the production of all the cytokines analyzed. (B) Graphic representation of the modulation of CFU, bacterial gene expression, and IFN-γ production relative to H37Rv- and CMT97-infected macrophages, On the y axis are reported arbitrary units, ranging from 1 (assigned to the minimum value obtained) to 10 (assigned to the maximum value), independent of the absolute values of each group of observations. In particular, for CFU the values represent the bacterial colonies obtained from infected-cell lysates; for gene expression they reflect the number of M. tuberculosis-expressed genes out of the total number analyzed; for IFN-γ expression we considered the minimum and maximum optical density of the RT-PCR bands obtained.