Biophysical and Biochemical Outcomes of Chlamydia pneumoniae Infection Promotes Pro-atherogenic Matrix Microenvironment

Abstract

Multiple studies support the hypothesis that infectious agents may be involved in the pathogenesis of atherosclerosis. Chlamydia pneumoniae is strongly implicated in atherosclerosis, but the precise role has been underestimated and poorly understood due to the complexity of the disease process. In this work, we test the hypothesis that C. pneumoniae-infected macrophages lodged in the subendothelial matrix contribute to atherogenesis through pro-inflammatory factors and by cell-matrix interactions. To test this hypothesis, we used a 3D infection model with freshly isolated PBMC infected with live C. pneumoniae and chlamydial antigens encapsulated in a collagen matrix, and analyzed the inflammatory responses over 7 days. We observed that infection significantly upregulates the secretion of cytokines TNF-α, IL-1β, IL-8, MCP-1, MMP, oxidative stress, transendothelial permeability, and LDL uptake. We also observed that infected macrophages form clusters, and substantially modify the microstructure and mechanical properties of the extracellular matrix to an atherogenic phenotype. Together, our data demonstrates that C. pneumoniae-infection drives a low-grade, sustained inflammation that may predispose in the transformation to atherosclerotic foci.

Keywords: 3D; Chlamydia pneumoniae; atherosclerosis; collagen; endothelial dysfunction; intima; stiffness.

FIGURE 1

Chlamydia pneumoniae infection of macrophages in 3D collagen matrices. Monocytes were infected with PBS (mock) or heat-killed Cpn (HK Cpn) or Chlamydial EB of MOI 1 (Cpn) and embedded in 2 mg/ml collagen. Post gelation, cells were supplemented with media and incubated for up to 1 week. (A) Gels were stained with Calcein AM (green) and viability was estimated. A representative 2D projection confocal image of viable cells in the gel from 3D stack is shown (Scale bar: 20 μm). The results are mean ± SEM of three experiments performed in triplicate. The ^∗ and Φ symbols denote statistically significant change in viability respective groups when compared to Mock from same day and within the group relative to previous time point, respectively (P< 0.05). (B) Macrophages were stained with anti-Cpn antibody (Green), AF 633 phalloidin (Red), and DAPI (Blue). Representative confocal images of infected cells at 3 and 7 days are shown (Scale bar: 10 μm).

FIGURE 2

Pro-inflammatory environment in 3D collagen matrices with infected macrophages. (A) The supernatants were collected from the 3D gels on days 1, 3, and 7, and estimated for the levels of TNF-α, IL-1β, and IL-8 by ELISA. The results are plotted as quantity of secreted protein as obtained from standard curve. (B) MCP-1 secretion as measured from supernatants at day 3 and 7 post infection by ELISA. (C) MMP activity assayed from supernatants at day 3 and 7, using Pan-MMP activity assay kit. The results are mean ± SD of one representative experiment performed in triplicate, and the experiments were repeated three times. The ^∗ and Φ symbols denote statistically significant change in viability respective groups when compared to Mock from the same day and within the group relative to previous time point, respectively (P< 0.05).

FIGURE 3

Endothelial junction dysfunction during C. pneumoniae infection in matrices. Monocytes were infected with PBS (mock) or heat-killed Cpn (HK Cpn) or chlamydial EB of MOI 1 (Cpn) for 4 h and then embedded in 2 mg/ml collagen at a concentration of 3 million cells/ml of gel and then incubated for 24 h in a 24 well plate with trans-well containing confluent endothelial cells. (A) After incubation, the endothelial cells were stained with antibodies against VE-cadherin (green), phalloidin (red) and DAPI (blue), and visualized using Zeiss-510 confocal microscope. Representative images (Scale bar: 20 μm) with gaps (white arrow) between the cells were analyzed for gap area using Image-Pro analyzer, and results were plotted as average of three images. (B,C) In another experiment, post incubation, media from upper side of trans-well was replaced with fluorescent dextran (B) or BODIPY-LDL (C), and the plate was incubated for 1 h and supernatants were collected from bottom well. Fluorescent intensity from supernatant was measured using plate reader as an estimate of permeability. The ^∗ denote statistically significant change in the parameters between different groups, as calculated using Graph-Pad Prism (P < 0.05, ANOVA).

FIGURE 4

Low density lipoprotein uptake of C. pneumoniae infected macrophages in collagen matrices. (A) The gels after 7 days of infection were stained for ROS (green) and SOD (red) as per manufacturer’s instructions. A representative image of Cpn infected macrophages in gels and the graph showing quantification of fluorescence intensity from the gels. Scale bar: 10 μm. (B) Monocytes were infected with mock PBS or HK/Live Chlamydial EB (MOI 1) for 4 h and embedded in 2 mg/ml of collagen with 10 mg/dl of BODIPY-LDL (green) and incubated for up to 1 week. Macrophages were fixed and stained for actin (Red), and nucleus (Blue). A representative confocal image of macrophages 7 days after infection (Scale bar: 10 μm), and quantification of LDL uptake by macrophages. The results are mean ± SD of one representative experiment performed in triplicate, and the experiments were repeated three times. The ^∗ denote statistically significant change in the parameters between different groups, as calculated using Graph-Pad Prism (P < 0.05, ANOVA).

FIGURE 5

Chlamydia pneumoniae infection alters macrophage phenotype in collagen matrices. After infection for 4 h, monocytes were embedded in collagen and incubated for up to 1 week. (A,B) Macrophages in 3D gels were fixed and stained for actin (Red), and DAPI (Blue). (A) Representative confocal image of infected cells at 7 days post infection (Scale bar: 10 μm), with graph showing the area of spread cells. (B) Visualization of quantification of cellular actin. (C) Macrophages were stained with Calceine AM and Z-stack confocal images of gels at 7 days after infection are shown (Scale bar: 20 μm) with graph showing area occupied by cell clusters. (D) Gels were stained for nicks in collagen (green), actin (pink), and DAPI (blue), and visualized by confocal microscopy. A representative 3D image (Scale bar: 20 μm), corresponding 3D surface plot showing nicked–collagen (green), and its localization with cells (pink/blue), and quantification of intensity of collagen breaks as obtained from image analysis using Image Pro Analyzer. The results are mean ± SD of three experiments performed in triplicates and ^∗ denote statistically significant change in the parameters between different groups, as calculated using Graph-Pad Prism (P < 0.05, ANOVA).

FIGURE 6

Macrophages with C. pneumoniae infection alter collagen micro environment. After infection for 4 h, monocytes were encapsulated in collagen matrix doped with 10% FL–collagen and incubated for up to 1 week. (A) Representative 3D images of fluorescent collagen fibers from confocal microscopy (Scale bar: 20 μm), with quantification of: fluorescent collagen fiber gray scale area (B); filament thickness (C); filament length (D); and fiber angle (E) from image analysis using Image J analyzer. The results are mean ± SD of one experiment performed in triplicate, and the experiment was repeated at least three times.

FIGURE 7

Matrix mechanical changes due to live C. pneumoniae infection in intima. Monocytes were infected with mock PBS or HK/Live Chlamydial EB (MOI 1) for 4 h. Post infection, cells were embedded in 200 μl of 2 mg/ml collagen, and supplemented with media and incubated for up to 1 week. (A) Visual inspection of gel integrity after 1 week of incubation before rheological analysis; (B–F) stiffness and strength of gels was estimated by dynamic shear rheometer. (B) Stiffness; (C) Representative amplitude sweep with linear viscoelastic region (black arrow), yield point onset (red arrow), cross over point (green arrow), (D) Dross-over point, and (E) Yield point onset. The results are mean ± SD of five experiments performed in triplicates. The ^∗ and Φ denote statistically significant change in viability respective groups when compared to Mock from same day and within the group relative to previous time point, respectively, as calculated using Graph-Pad Prism (P < 0.05, ANOVA) (F) In another experiment, the gels were prone to a shear stress of 50 dyn/cm² in a micro-fluidic chamber using syringe pump. The gels were fixed and analyzed by scanning electron microscopy with representative images (Scale bar: 5 μm) from different conditions.