CAIP-Induced ROS Production Contributes to Sustaining Atherosclerotic Process Associated with Helicobacter cinaedi Infection through Macrophages and Endothelial Cells Activation

Abstract

Several lines of evidence have linked the intestinal bacterium Helicobacter cinaedi with the pathogenesis of atherosclerosis, identifying the Cinaedi Antigen Inflammatory Protein (CAIP) as a key virulence factor. Oxidative stress and inflammation are crucial in sustaining the atherosclerotic process and oxidized LDL (oxLDL) uptake. Primary human macrophages and endothelial cells were pre-incubated with 10 µM diphenyl iodonium salt (DPI) and stimulated with 20 µg/mL CAIP. Lectin-like oxLDL receptor (LOX-1) expression was evaluated by FACS analysis, reactive oxygen species (ROS) production was measured using the fluorescent probe H2DCF-DA, and cytokine release was quantified by ELISA assay. Foam cells formation was assessed by Oil Red-O staining, and phosphorylation of p38 and ERK1/2 MAP kinases and NF-κB pathway activation were determined by Western blot. This study demonstrated that CAIP triggered LOX-1 over-expression and increased ROS production in both macrophages and endothelial cells. Blocking ROS abrogated LOX-1 expression and reduced LDL uptake and foam cells formation. Additionally, CAIP-mediated pro-inflammatory cytokine release was significantly affected by ROS inhibition. The signaling pathway induced by CAIP-induced oxidative stress led to p38 MAP kinase phosphorylation and NF-κB activation. These findings elucidate the mechanism of action of CAIP, which heightens oxidative stress and contributes to the atherosclerotic process in H. cinaedi-infected patients.

Keywords: CAIP; Helicobacter cinaedi; LOX-1; ROS; atherosclerosis; inflammation; oxidative stress.

Figure 1

LOX-1 expression in macrophages and endothelial cells. Monocytes-derived macrophages and endothelial cells (HUVECs) were treated with CAIP 20 μg/mL or saline (vehicle). After 24 and 48 h, cells were harvested and analyzed by flow cytometry for the expression of LOX-1; cells were gated on Aqua negative cells (live cells). Data are expressed as mean % of positive cells ± SEM of three independent experiments from three different donors. Significance was determined by Student’s t-test: *** p < 0.001.

Figure 2

ROS production by macrophages and endothelial cells. Monocytes-derived macrophages and endothelial cells (HUVECs) were treated with CAIP 20 μg/mL or saline (vehicle); where indicated, cells were preincubated 1 h with 20 μM DPI. After 3 and 24 h, cells were incubated 30 min with H₂DCFDA probe 10 μM and fluorescence analyzed with a fluorometer (Ex 485 nm, Em 535 nm). Data are expressed as n-fold vs vehicle ± SEM of three independent experiments from three different donors. Significance was determined by Student’s t-test: ** and °° p < 0.01 (** vs. vehicle, °° vs. CAIP + DPI).

Figure 3

ROS inhibition affects CAIP-induced LOX-1 expression in macrophages and endothelial cells. Monocytes-derived macrophages and endothelial cells (HUVECs) were treated with CAIP 20 μg/mL or saline (vehicle); where indicated, cells were preincubated 1 h with 20 μM DPI. After 24 h, cells were harvested and analyzed by flow cytometry for the expression of LOX-1; cells were gated on Aqua negative cells (live cells). Data are expressed as mean % of positive cells ± SEM of three independent experiments from three different donors. Significance was determined by Student’s t-test: *** CAIP vs. Vehicle p < 0.001; °°° CAIP vs. CAIP + DPI p < 0.001.

Figure 4

ROS are involved in CAIP-mediated foam cells formation. Monocytes-derived macrophages were exposed 24 h to CAIP or saline alone (vehicle) and preincubated, where indicated, 1 h with DPI. Cells were then fixed and stained with Oil Red O. Shown are representative images. Foam cells number was determined, and it is expressed as the percentage of total cells counted in 10 random fields. Foam cells quantification is expressed as mean value  ±  SD of three independent experiments, performed with three different cell preparations. Significance was determined by Student’s t-test between CAIP- versus vehicle-exposed cells (indicated by *) or vs CAIP + DPI-exposed cells (indicated by °). °°° p < 0.01, *** p  <  0.001.

Figure 5

The signaling pathways activated by CAIP are mediated by ROS production. Monocytes-derived macrophages and endothelial cells (HUVECs) were treated with CAIP 20 μg/mL or saline (vehicle); where indicated, cells were preincubated 1 h with 20 μM DPI. After 1 h, cells were harvested and analyzed by Western blot. MAP kinases p38 and ERK1/2, their phosphorylated forms, and IKBα were revealed by specific antibodies. β-actin was used as the endogenous reference. Blot refers to a representative of two independent experiments, performed with cells from two different donors.

Figure 6

CAIP-mediated cytokines and adhesion molecules production by macrophages and endothelial cells is affected by ROS inhibition. Monocytes-derived macrophages and endothelial cells (HUVECs) were treated with CAIP 20 μg/mL or saline (vehicle); where indicated, cells were preincubated 1 h with 20 μM DPI. After 24 h, culture supernatants were collected and analyzed by ELISA assays, and cells were harvested and analyzed by flow cytometry. For TNF-a, IL-1b, IL-6, IL-8, and CCL-2 analysis, the cytokines content was determined by ELISA following the manufacturer’s instructions. Data are expressed as mean concentration values  ±  SEM of three independent experiments with cells derived from three different donors (for both macrophages and HUVECs). The surface expression of VCAM-1 and E-selectin was evaluated on cells gated on live cells. Data are expressed as Mean Fluorescence Intensiti (MFI) ± SEM of three independent experiments, performed with cells from three different donors. Significance was determined by Student’s t-test: *** CAIP vs. Vehicle p < 0.001; CAIP vs. CAIP + DPI °° p < 0.01, °°° p < 0.001.