Cytoadherence-dependent induction of inflammatory responses by Mycoplasma pneumoniae

Abstract

Pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Mycoplasma pneumoniae shows strong cytoadherence to host cells and this cytoadherence is thought to be involved in the progression of pneumonia. However, the interaction between the cytoadherence and the immune responses is not known in detail. In this study, we demonstrated that the induction of pro-inflammatory cytokines in the human monocyte cell line THP-1 is dependent on the property of cytoadherence of M. pneumoniae. A wild-type strain of M. pneumoniae with cytoadherence ability induced pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1β (IL-1β). Whereas, heat-killed M. pneumoniae and cytoadherence-deficient mutants of M. pneumoniae caused significantly less production of pro-inflammatory cytokines than the wild-type strain. The wild-type strain induced pro-inflammatory cytokines in an endocytosis-independent manners, but the induction by heat-killed M. pneumoniae and cytoadherence-deficient mutants was dependent on endocytosis. Moreover, the wild-type strain induced caspase-1 production and ATP efflux, promoting the maturation of IL-1β and release of the pro-IL-1β precursor, whereas heat-killed M. pneumoniae and the cytoadherence-deficient mutants failed to induce them. These data suggest that the cytoadherence ability of M. pneumoniae activates immune responses and is involved in the pathogenesis of M. pneumoniae infection.

© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

Figures

Figure 1

Induction of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by heat-killed Mycoplasma pneumoniae. The M. pneumoniae was killed by heating at 60° for 30 min. THP-1 cells (2 × 105 cells/500 μl) were treated with 50 μl living or heat-killed M. pneumoniae (OD595 = 0.1). After 6 hr incubation, amounts of TNF-α (a) and IL-1β (b) in culture medium were measured by ELISA. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with living M. pneumoniae by multiple comparison.

Figure 2

Induction of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by cytoadherence-deficient mutants of Mycoplasma pneumoniae. THP-1 cells (2 × 105 cells/500 μl) were infected with 50 μl of the indicated amounts of wild-type or mutants of M. pneumoniae. After 6 hr incubation, amounts of TNF-α (a) and IL-1β (b) in culture medium were measured by ELISA. THP-1 cells (2 × 105 cells/500 ml) were infected with 50 ml of M. pneumoniae (OD595 = 0.1). Amounts of TNF-α (c) and IL-1β (d) in culture medium were measured by ELISA at the indicated time post-infection. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with wild-type of M. pneumoniae by multiple comparison.

Figure 3

Induction of Toll-like receptor 2 (TLR2) signalling. (a) 293T cells (2 × 105 cells/500 μl) were transfected with the 0.1 μg/ml pFLAG-TLR2, 0.01 μg/ml pNF-kB-luc and 0.01 μg/ml pRL-TK. After 24 hr incubation, the cells were infected with 50 μl wild-type or mutants of Mycoplasma pneumoniae (OD595 = 0.1). After 6 hr incubation, the activity of luciferase was measured as described in the Materials and methods. All values represent the means and SD of three assays. (b, c) THP-1 cells (2 × 105 cells/500 μl) were treated with 10 μg/ml anti-TLR2 monoclonal antibody and infected with 50 μl wild-type or mutants of M. pneumoniae (OD595 = 0.1). After 6 hr incubation, amounts of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in culture medium were measured by ELISA. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with control IgG by multiple comparison (d) THP-1 cells (2 × 105 cells/500 μl) were treated with 50 μl living or heat-killed M. pneumoniae (OD595 = 0.1). After 6 hr incubation, THP-1 cells were harvested and the induction of pro-IL-1β precursor was investigated by Western blotting.

Figure 4

Endocytosis-independent induction of pro-inflammatory cytokines. THP-1 cells (2 × 105 cells/500 μl) were cultured in the presence of 1.0 μm cytochalasin D or 100 nm bafilomycin A1 for 1 hr, and treated with 50 μl of living or heat-killed Mycoplasma pneumoniae (OD595 = 0.1). After 6 hr incubation, amounts of tumour necrosis factor-α (TNF-α) (a) and interleukin-1β (IL-1β) (b) in culture medium were measured by ELISA. Cells were harvested and the induction levels of pro-IL-1β precursor were determined by Western blotting (c). All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with PBS treatment by multiple comparison.

Figure 5

Involvement of the inflammasome. (a, b) 1000 ng/ml of FAM20 was incubated with 1% of lipofectamin. THP-1 cells (2 × 105 cells/500 μl) were transfected with 100 ng/ml FAM20. After 6 hr incubation, the amount of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the culture medium was measured by ELISA. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with control by multiple comparison. (c) THP-1 cells (2 × 105 cells/500 μl) were infected with 50 μl of indicated Mycoplasma pneumoniae (OD595 = 0.1). After 6 hr incubation, levels of caspase-1 in culture medium were measured by ELISA. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with M129 by multiple comparison. (d) THP-1 cells (2 × 105 cells/500 μl) were cultured in the presence of 100 μm z-Val-Ala-Asp(oMe)-CH2F (z-VAD-FMK) or Ac-Trp-Glu-His-Asp-H (Ac-WEHD-CHO) for 1 hr, and infected with 50 μl M129 (OD595 = 0.1). After 6 hr incubation, amounts of IL-1β in culture medium were measured using ELISA. All values represent the means and SD of three assays. (e) THP-1 cells (2 × 105 cells/500 μl) were infected with 50 μl of the indicated M. pneumoniae (OD595 = 0.1). After 6 hr incubation, the IL-1β levels in the supernatants or infected cells were determined by Western blotting.

Figure 6

Involvement of ATP in the interleukin-1β (IL-1β) induction. (a) THP-1 cells (2 × 105 cells/500 μl) were cultured in the presence of 200 μm ecto-ATPase inhibitor, ARL for 1 hr, and infected with 50 μl of the indicated Mycoplasma pneumoniae sOD595 = 0.1). After 6 hr incubation, concentrations of ATP were measured as described in the Materials and methods. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.05 for a comparison with M129 by multiple comparison. (b) THP-1 cells (2 × 105 cells/500 μl) were cultured in the presence of 300 μm oxidized ATP for 1 hr and infected with 50 μl M129 (OD595 = 0.1). After 6 hr incubation, THP-1 cells were harvested and the induction levels of pro-IL-1β precursor were determined by Western blotting. (c) THP-1 cells were treated with oxidized ATP and infected with M129 as described above. Release levels of IL-1β were measured by ELISA. All values represent the means and SD of three assays. An asterisk indicates that the P-value is < 0.01 for a comparison with PBS treatment by multiple comparison.