Cholesterol-Ester Transfer Protein Alters M1 and M2 Macrophage Polarization and Worsens Experimental Elastase-Induced Pulmonary Emphysema

Abstract

Cholesterol-ester transfer protein (CETP) plays a role in atherosclerosis, the inflammatory response to endotoxemia and in experimental and human sepsis. Functional alterations in lipoprotein (LP) metabolism and immune cell populations, including macrophages, occur during sepsis and may be related to comorbidities such as chronic obstructive pulmonary disease (COPD). Macrophages are significantly associated with pulmonary emphysema, and depending on the microenvironment, might exhibit an M1 or M2 phenotype. Macrophages derived from the peritoneum and bone marrow reveal CETP that contributes to its plasma concentration. Here, we evaluated the role of CETP in macrophage polarization and elastase-induced pulmonary emphysema (ELA) in human CETP-expressing transgenic (huCETP) (line 5203, C57BL6/J background) male mice and compared it to their wild type littermates. We showed that bone marrow-derived macrophages from huCETP mice reduce polarization toward the M1 phenotype, but with increased IL-10. Compared to WT, huCETP mice exposed to elastase showed worsened lung function with an increased mean linear intercept (Lm), reflecting airspace enlargement resulting from parenchymal destruction with increased expression of arginase-1 and IL-10, which are M2 markers. The cytokine profile revealed increased IL-6 in plasma and TNF, and IL-10 in bronchoalveolar lavage (BAL), corroborating with the lung immunohistochemistry in the huCETP-ELA group compared to WT-ELA. Elastase treatment in the huCETP group increased VLDL-C and reduced HDL-C. Elastase-induced pulmonary emphysema in huCETP mice promotes lung M2-like phenotype with a deleterious effect in experimental COPD, corroborating the in vitro result in which CETP promoted M2 macrophage polarization. Our results suggest that CETP is associated with inflammatory response and influences the role of macrophages in COPD.

Keywords: arginase 1; cholesterol ester transfer protein; chronic obstructive pulmonary disease; inflammation; interleukin-10; macrophage—cell; pulmonary emphysema.

Figure 1

Comparison of CETP mRNA expression in bone marrow-derived macrophages from WT and huCETP Tg mice. mRNA expression was determined using RT-PCR. Results are expressed as the mean ± standard deviation. U, undetermined.

Figure 2

Gene expression of typical M1 and M2 markers from bone marrow-derived macrophages of WT and huCETP Tg mice. In vitro stimulated M1 (5 ng/ml IFNγ + 50 ng/ml LPS) and M2 (10 ng/ml IL-13 + IL-4) for 24 h. mRNA expression was determined using RT-PCR. Results are expressed as the mean ± standard deviation and were compared by unpaired Student’s t-test (n = 3–5). *P < 0.05.

Figure 3

Gene expression of factors associated with lipid metabolism from bone marrow-derived macrophages of WT and huCETP Tg mice. In vitro stimulated M1 (5 ng/ml IFNγ + 50 ng/ml LPS) and M2 (10 ng/ml IL-13 + IL-4) for 24 h. mRNA expression was determined using by RT-PCR. Results are expressed as mean ± standard deviation and were compared by unpaired Student’s t-test. (n = 3–5). *P < 0.05 (M1 vs. M2); **P < 0.05 (CETP M2 vs. WT M2).

Figure 4

Expression of cell surface markers associated with M1 (CD80) and M2 (CD206) from bone marrow-derived macrophages of WT and huCETP Tg mice. Expression of CD80 (A), CD206 (B), mean fluorescence intensity (MFI) of CD80 (C), MFI of CD206 (D), and representation of the gate strategy (E). Non-stimulated control cells (M0), stimulated M1 (5 ng/ml of IFNγ + 50 ng/ml of LPS) or M2 (10 ng/ml of IL-13 + IL-4) for 24 h. Basal fluorescence was determined using unlabeled cells, and compensation was performed with cells labeled with the respective fluorochromes on the FACSCanto II cytometer. In total, 100,000 events were analyzed. Values were expressed as mean and standard deviation. Values expressed as median and percentiles 25 and 75 were analyzed by Kruskal–Wallis test with original FDR method of Benjamini and Hochberg post-test. The values expressed as mean and standard deviation were analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test; (n = 3), P < 0.05.

Figure 5

Cytokine secretion, CETP expression, and secretion in cell culture medium. Bone marrow-derived macrophages from WT and huCETP mice, unpolarized (M0), or stimulated to M1 (5 ng/ml IFNγ + 50 ng/ml LPS) or to M2 (10 ng/ml IL-13 + IL-4) for 24 h. TNF-α (A), IL-6 (B), IL-10 (C), CETP mRNA expression in macrophages (D) and CETP secretion (E). Concentrations were determined by ELISA, and CETP expression was determined by RT-PCR. Values expressed as median and percentiles 25 and 75 were analyzed by Kruskal–Wallis test with original FDR method of Benjamini and Hochberg post-test. Values were expressed as mean and standard deviation analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test; (n = 5–8). P < 0.05. *M0 vs. M1 and M2.

Figure 6

Gene expression of typical M1 and M2 markers in bone marrow-derived macrophages from WT mice stimulated in the presence of recombinant CETP in vitro. Macrophages were stimulated using 5 ng/ml IFNγ + 50 ng/ml LPS (M1) or 10 ng/ml IL-13 + IL-4 (M2) for 24 h. mRNA expression was determined by RT-PCR. Results are expressed as the mean ± standard deviation and compared by unpaired Student’s t-test. (n = 3–5). *P < 0.05 for 24 h.

Figure 7

Expression of cell surface markers associated with M1 (CD80) and M2 (CD206) from bone marrow-derived macrophages of WT mice in the presence of recombinant CETP. Expression of CD80 (A), CD206 (B), mean fluorescence intensity (MFI) of CD80 (C), MFI of CD206 (D), and representation of the gate strategy (E). Non-stimulated control cells (M0), stimulated M1 (5 ng/ml of IFNγ + 50 ng/ml of LPS) or M2 (10 ng/ml of IL-13 + IL-4) for 24 h. Basal fluorescence was determined using unlabeled cells, and compensation was performed with cells labeled with the respective fluorochromes on the FACSCanto II cytometer. In total, 100,000 events were analyzed. Values were expressed as mean and standard deviation. These data were analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test; (n = 3). P < 0.05.

Figure 8

Cytokine secretion in cell culture medium. Bone marrow-derived macrophages from WT mice, unpolarized (M0), or stimulated to M1 (with 5 ng/ml IFNg + 50 ng/ml LPS), or to M2 (with 10 ng/ml IL-13 + IL-4) in the absence (control) or presence of recombinant human CETP (1 µg/ml) for 24 h. TNF (A), IL-6 (B) and IL-10 (C).

Figure 9

Lung mechanics in WT and huCETP mice after elastase-induced emphysema (ELA), or saline (SAL). (A) Respiratory system resistance (Rrs); (B) Tissue elastance (Htis); (C) Respiratory system elastance (Ers); (D) Airway resistance (RAW). (E) Lung tissue resistance (Gtis); (F) Exhaled nitric oxide. Values expressed as median and percentiles 25 and 75 were analyzed by Kruskal–Wallis test with original FDR method of Benjamini and Hochberg post-test. Values were expressed as mean and standard deviation analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test; (n = 8). P < 0.05.

Figure 10

Total and differential leukocyte count in broncho-alveolar lavage (BAL), and cytokine levels in WT and huCETP mice after elastase-induced emphysema (ELA) or saline (SAL). Total cells (A), eosinophils (B), neutrophils (C), macrophages (D), lymphocytes (E), cytokines in BAL: TNF (F), IL-6 (G), IL-10 (H), cytokine plasma (I). Values were expressed as mean and standard deviation. These data were analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test. P < 0.01: *CETP Ela vs. all groups, (n = 8).

Figure 11

Photomicrograph of stained slides for immunohistochemistry analysis of inflammatory markers from WT and CETP Tg mice and from huCETP mice after elastase-induced emphysema (ELA) or saline (SAL). TNF (A), IL-10 (B), iNOS (C) and Arginase 1 (D). Representative images of micrographs at ×1,000 magnification. Values were expressed as mean and standard deviation. These data were analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test. p < 0.01: (n = 8).

Figure 12

Photomicrograph of stained slides for immunohistochemistry analysis of CETP in airways (A), lung parenchyma (B) and mean linear intercept (LM) by dot count of airway spreads (C) from WT and huCETP mice after elastase-induced emphysema (ELA) or saline (SAL). Representative images of photomicrographs: CETP magnification ×1,000 and (LM) ×400. Values expressed as median and percentiles 25 and 75 were analyzed by Kruskal–Wallis test with original FDR method of Benjamini and Hochberg post-test. The values were expressed as mean and standard deviation were analyzed by ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post-test. p < 0.01, (n = 8).

Figure 13

Plasma lipoprotein analysis by high-performance liquid chromatography (FPLC) in WT and huCETP mice after elastase-induced emphysema (ELA) or saline (SAL). Average of two to three pools (n = 4).