Differential regulation of DAP12 and molecules associated with DAP12 during host responses to mycobacterial infection

Abstract

DAP12 and its associating molecules MDL-1, TREM-1, and TREM-2 are the recently identified immune regulatory molecules, expressed primarily on myeloid cells including monocytes/macrophages, dendritic cells, NK cells, and neutrophils. However, little is known about the regulation of their expression during host antimicrobial responses. We have investigated the effect of pulmonary mycobacterial infection and type 1 cytokines on the expression of these molecules both in vivo and in vitro. While DAP12 was constitutively expressed at high levels in the lungs, the MDL-1, TREM-1, and TREM-2 molecules were inducible during mycobacterial infection. Their kinetic expression was correlated with that of the type 1 cytokines tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma). In primary lung macrophage cultures, high constitutive levels of DAP12 and TREM-2 were not modulated by mycobacterial or type 1 cytokine exposure. In contrast, expression of both MDL-1 and TREM-1 was markedly induced by mycobacterial infection and such induction was inhibited by concurrent exposure to IFN-gamma. On mycobacterial infection of TNF-alpha(-/-) and IFN-gamma(-/-) mice in vivo or their lung macrophages in vitro, TNF-alpha was found to be critical for mycobacterially induced MDL-1, but not TREM-1, expression whereas IFN-gamma negatively regulated mycobacterially induced MDL-1 and TREM-1 expression. Our findings thus suggest that DAP12 and its associating molecules are differentially regulated by mycobacterial infection and type 1 cytokines and that MDL-1- and TREM-1-triggered DAP12 signaling may play an important role in antimicrobial type 1 immunity.

FIG. 1.

(A) In vivo mRNA expression of DAP12 and associating molecules in lung tissue during mycobacterial infection. mRNA expression was examined by RT-PCR at the indicated time points after BCG infection (two C57BL/6 mice per time point). (B) Relative quantity of mRNA of DAP12 and associating molecules in the lung tissue during mycobacterial infection. Quantitation was carried out, relative to the expression of the GAPDH housekeeping gene by real-time quantitative PCR with the same total RNA samples as for the experiment in panel A. (C) Detection of DAP12 protein in the lung tissue during mycobacterial infection. Tissue lysates from each mouse lung (2 mg) were immunoprecipitated (IP) with anti-DAP12 antibodies and analyzed by Western blotting (IB) using anti-DAP12 antibody. Representative results from two separate experiments are shown.

FIG. 2.

Levels of type 1 cytokines in the lungs of C57BL/6 mice on day 27 postinfection. The levels of type 1 cytokines in the lungs were determined by ELISA of samples of BAL fluids. Results are expressed as means and standard errors of the mean for three to four mice per time point.

FIG. 3.

Regulation of DAP12 and associating-molecule mRNA expression in lung macrophages by various stimuli. Isolated lung macrophages from naive C57BL/6 mice were incubated with live BCG bacilli, LPS, TNF-α, IFN-γ or a combination of TNF-α and IFN-γ or BCG and IFN-γ for 40 h and then examined by real-time quantitative PCR (TNF-α or IFN-γ, 5 ng/ml; LPS, 1 μg/ml; BCG, 2 CFU/cell). Gene expression was quantitated relative to the expression of GAPDH. Similar results were also obtained by RT-PCR (data not shown). Representative results from three separate experiments are shown.

FIG. 4.

Role of TNF-α and IFN-γ in the regulation of DAP12 and its associating molecule expression in the lungs during mycobacterial infection. (A) Expression of DAP12 and associating molecules in the lungs of C57BL/6, TNF-α^−/−, and IFN-γ^−/− mice. Mice were infected with 0.5 × 10⁶ CFU of BCG intratracheally and sacrificed on day 25 postinfection. The lungs were examined by real-time quantitative PCR (two mice per strain per time point). Gene expression was quantitated relative to the expression of GAPDH. Similar results were also obtained by RT-PCR (data not shown). (B) Detection of DAP12 protein in the lungs of C57BL/6, TNF-α^−/−, and IFN-γ^−/− mice during mycobacterial infection. Tissue lysates (1.5 mg) from each mouse lung were immunoprecipitated with anti-DAP12 antibodies and analyzed by Western blotting. Representative results of two separate experiments are shown.

FIG. 5.

Role of TNF-α and IFN-γ in the regulation of DAP12 and associating molecules in lung macrophages during in vitro mycobacterial infection. Isolated lung macrophages from naive C57BL/6, TNF-α^−/−, and IFN-γ^−/− mice were incubated with BCG and/or the indicated cytokines (1 or 5 ng/ml) for 40 h and then examined by RT-PCR. The experiments were repeated twice, with similar results, and representative results are shown.