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. 2013 Oct;69(1):1-6.
doi: 10.1111/2049-632X.12058. Epub 2013 Oct 1.

Chlamydia pneumoniae induces expression of pro-atherogenic factors through activation of the lectin-like oxidized LDL receptor-1

Lee A Campbell  1 , Amy W Lee  2 , Michael E Rosenfeld  3 , Cho-Chou Kuo  2
Affiliations

Chlamydia pneumoniae induces expression of pro-atherogenic factors through activation of the lectin-like oxidized LDL receptor-1

Lee A Campbell et al. Pathog Dis. 2013 Oct.

Abstract

Several lines of evidence have associated Chlamydia pneumoniae with cardiovascular disease including acceleration of atherosclerotic lesion progression in hyperlipidemic animal models by infection. Many of the pro-atherogenic effects of oxidized low-density lipoprotein (ox-LDL) occur through the activation of the lectin-like ox-LDL receptor-1 (LOX-1). Chlamydia pneumoniae upregulates the expression of the LOX-1 mRNA, promotes the uptake of ox-LDL, and utilizes the LOX-1 receptor for infectivity. The overall goal of this study was to determine whether C. pneumoniae organisms upregulated LOX-1 protein expression in vascular cells and whether upregulation of pro-atherogenic factors by C. pneumoniae occurred through LOX-1. Chlamydia pneumoniae induced LOX-1 protein expression in both endothelial cells and RAW macrophages. Upregulation was prevented by preincubation of cells with LOX-1 antibody prior to infection. Similarly, C. pneumoniae upregulated protein expression of adhesion molecules, MMP-1, and MMP-3, which was mitigated by anti-LOX-1 antibody. Prior treatment of organisms with PNGase, which removes the chlamydial glycan that is N-linked to the major outer membrane, abolished C. pneumoniae upregulation of LOX-1. These studies suggest that activation of LOX-1 expression occurs through binding of the chlamydial glycan and provides one mechanism by which C. pneumoniae infection could play a role in the pathogenesis of atherosclerosis.

Keywords: Chlamydia; LOX-1 receptor; adhesion molecules; atherosclerosis; glycan.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Time course of LOX-1 protein expression upregulation by Chlamydia pneumoniae. Endothelial cells (HUVEC, Panel A) and RAW macrophages (Panel B) were plated in 24 well plates to a confluency of 4 × 105 cells and infected with C. pneumoniae at a MOI of 10. At various times post-infection, infected cells were harvested and cell lysates prepared. LOX-1 protein expression was determined in uninfected TNF-α stimulated cells (positive control), uninfected cells (base level control, labeled as C), infected cells (Cpn), and cells pretreated with anti-LOX-1 antibody for 2 hrs at 37 °C with gentle rocking prior to infection (Cpn+ Ab). C. pneumoniae upregulated LOX-1 expression in both cell types in comparison to uninfected cells, which was negated by preincubation of cells with anti-LOX-1 antibody prior to infection.
Figure 2
Figure 2
Chlamydia trachomatis does not up regulate LOX-1 protein expression. Endothelial cells (HMEC, Panel A) and RAW macrophages (Panel B) were plated in 24 well plates to a confluency of 4 × 105 cells and infected with C. trachomatis (Ctr) or C. pneumoniae (Cpn) at a MOI of 10. LOX-1 protein expression was determined in uninfected TNF-α stimulated cells (positive control), uninfected cells (base level control, labeled as C), infected cells, and infected cells pretreated with anti-LOX-1 antibody for 2 hrs at 37 °C with gentle rocking prior to infection (Ctr+Ab or Cpn+Ab). LOX-1 protein expression was visualized by immunoblot using anti-LOX-1 specific antibodies and normalized to cellular B-actin expression.
Figure 3
Figure 3
Upregulation of protein expression of proatherogenic factors by Chlamydia pneumoniae occurs through LOX-1 activation. Endothelial cells (HMEC, Panel A and HUVEC, Panel B) and RAW macrophages (Panel C) were plated in 24 well plates to a confluency of 4 × 105 cells and infected with C. pneumoniae (Cpn) at a MOI of 10. Protein expression was determined through immunoblot analysis using specific antibodies against Intercellular Adhesion Molecule-1 (ICAM), Vascular Cellular Adhesion Molecule-1 (VCAM), E-selectin (E-Sel), matrix metalloproteinase (MMP-1) and matrix metalloproteinase 3 (MMP-3). Protein expression was determined in uninfected TNF- α or LPS stimulated cells (positive control), uninfected cells (base level control, labeled as C), infected cells (Cpn), and cells pretreated with anti-LOX-1 antibody for 2 hrs at 37°C with gentle rocking prior to infection (Cpn+Ab). C. pneumoniae upregulation of adhesion molecule protein expression was prevented by prior treatment of cells with anti-LOX-1 antibody.
Figure 4
Figure 4
Upregulation of LOX-1 by C. pneumoniae does not occur when the chlamydial glycan is removed by N-glycanase treatment. Endothelial cells (HMEC, Panel A) and RAW macrophages (Panel B) were plated in 24 well plates to a confluency of 4 × 105 cells and infected with C. pneumoniae (Cpn) at a MOI of 10. LOX-1 protein expression was determined in uninfected TNF-α stimulated cells (positive control), uninfected cells (base level control, labeled as C), infected cells, and infected cells exposed to N-glycanase (Cpn+) PNGase with anti-LOX-1 antibody for 2 hrs at 37 °C with gentle rocking prior to infection (Cpn+Ab). LOX-1 protein expression was visualized by immunoblot using anti-LOX-1 specific antibodies and normalized to cellular B-actin expression.
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