Limits...
Conjugated linoleic acid induces an atheroprotective macrophage MΦ2 phenotype and limits foam cell formation.

de Gaetano M, Alghamdi K, Marcone S, Belton O - J Inflamm (Lond) (2015)

Bottom Line: However, the exact mechanisms through which CLA mediates this effect remain to be elucidated.Furthermore, this altered macrophage phenotype impacts on foam cell formation, inhibiting ox-LDL accumulation and promoting cholesterol efflux via both PPARγ and LXRα dependent pathways.The data increases the understanding of the pathways regulated by CLA in atheroprotection, namely, inhibiting the progressive acquisition of a pro-inflammatory macrophage phenotype.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical and Biomolecular Science, UCD Conway Institute, University College Dublin, Dublin, Ireland.

ABSTRACT

Background: Atherosclerosis, the underlying cause of heart attack and strokes, is a progresive dyslipidemic and inflammatory disease where monocyte-derived macrophage cells play a pivotal role. Although most of the mechanisms that contribute to the progression of atherosclerosis have been identified, there is limited information on those governing regression. Conjugated linoleic acid (CLA) is a group of isomers of linoleic acid that differ in the position and/or geometry of their double bonds. We have previously shown that a specific CLA blend (80:20 cis-9,trans-11:trans-10,cis-12-CLA) induces regression of pre-established atherosclerosis in vivo, via modulation of monocyte/macrophage function. However, the exact mechanisms through which CLA mediates this effect remain to be elucidated.

Methods: Here, we address if CLA primes monocytes towards an anti-inflammatory MΦ2 macrophage and examine the effect of individual CLA isomers and the atheroprotective blend on monocyte-macrophage differentiation, cytokine generation, foam cell formation and cholesterol metabolism in human peripheral blood monocyte (HPBMC)-derived macrophages.

Results: cis-9,trans-11-CLA and the atheroprotective 80:20 CLA blend regulates expression of pro-inflammatory mediators and modulates the inflammatory cytokine profile of macrophages and foam cells. In addition, cis-9,trans-11-CLA and CLA blend primes HPBMCs towards an anti-inflammatory MΦ2 phenotype, characterised by increased scavenger receptor (CD36) and efflux protein (ABCA-1) expression. Furthermore, this altered macrophage phenotype impacts on foam cell formation, inhibiting ox-LDL accumulation and promoting cholesterol efflux via both PPARγ and LXRα dependent pathways.

Conclusion: The data increases the understanding of the pathways regulated by CLA in atheroprotection, namely, inhibiting the progressive acquisition of a pro-inflammatory macrophage phenotype.

No MeSH data available.


Related in: MedlinePlus

CLA inhibits CD68 and increases MR expression in HPBMC-derived macrophages. (a) Freshly isolated HBPMCs were cultured for four days in the presence or absence of M-CSF and treated with c-9,t-11-CLA, t-10,c-12-CLA, CLA blend (80:20 c-9,t-11:t-10,c-12), OA, LA or TROG for a further 48 hrs. At day 6, macrophage were fixed and stained. Immunofluorescence analysis of unstimulated macrophage shows that both CLA isomers and their blend increase MR expression but do not affect CD68 expression. (b) Differentiation of HBPMCs in the presence of 100 ng/ml M-CSF showed that c-9,t-11-CLA and CLA blend inhibits CD68 expression and increases MR expression. Epifluorescent microscope images (63× magnification) are representative of three independent experiments. Blue indicates DAPI (nuclei), green indicates CD68 (macrophage marker), cyan indicates MR and red indicates phalloidin (cytoplasmic) staining. (c) Spectrophotometry quantification of CD68 and MR fluorescent signal confirms the regulation of CD68 and MR by c-9,t-11-CLA and the atheroprotective blend. Statistical analysis of three independent experiments is expressed mean % as fluorescence ± SEM vs vehicle where *p < 0.05; **p < 0.01 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4340802&req=5

Fig2: CLA inhibits CD68 and increases MR expression in HPBMC-derived macrophages. (a) Freshly isolated HBPMCs were cultured for four days in the presence or absence of M-CSF and treated with c-9,t-11-CLA, t-10,c-12-CLA, CLA blend (80:20 c-9,t-11:t-10,c-12), OA, LA or TROG for a further 48 hrs. At day 6, macrophage were fixed and stained. Immunofluorescence analysis of unstimulated macrophage shows that both CLA isomers and their blend increase MR expression but do not affect CD68 expression. (b) Differentiation of HBPMCs in the presence of 100 ng/ml M-CSF showed that c-9,t-11-CLA and CLA blend inhibits CD68 expression and increases MR expression. Epifluorescent microscope images (63× magnification) are representative of three independent experiments. Blue indicates DAPI (nuclei), green indicates CD68 (macrophage marker), cyan indicates MR and red indicates phalloidin (cytoplasmic) staining. (c) Spectrophotometry quantification of CD68 and MR fluorescent signal confirms the regulation of CD68 and MR by c-9,t-11-CLA and the atheroprotective blend. Statistical analysis of three independent experiments is expressed mean % as fluorescence ± SEM vs vehicle where *p < 0.05; **p < 0.01 and ***p < 0.001.

Mentions: To validate the hypothesis that transcriptional changes may influence protein expression, we also performed immunocytochemistry of the pan-macrophage marker CD68 and of the MΦ2-type receptor MR, in HPBMCs either unstimulated (Figure 2a) or under M-CSF-stimulated conditions (Figure 2b). Immunofluorescence microscopy and subsequent spectrophotometric quantification of the fluorescent signals (Figure 2c) showed that, in unstimulated conditions, CLA had no effect on CD68 expression but upon M-CSF stimulation, both the c-9,t-11 isomer and CLA blend decreased CD68 (by 10 ± 1%, p < 0.01 and 18 ± 1%, p < 0.001, respectively). Importantly, in mature macrophages following M-CSF stimulation, c-9,t-11-CLA and CLA blend increased MR expression (by 57 ± 7% p < 0.01 and 55 ± 9%, p < 0.01, respectively). Finally, t-10,c-12-CLA, OA and linoleic acid had no effect on CD68 or MR expression under either condition. This confirms that the atheroprotective CLA isomer and blend primes monocytes to an MΦ2 phenotype during differentiation, thus altering the macrophage phenotype. Negative controls for immunofluorescence experiments are shown in “Additional Information” (Additional file 3).Figure 2


Conjugated linoleic acid induces an atheroprotective macrophage MΦ2 phenotype and limits foam cell formation.

de Gaetano M, Alghamdi K, Marcone S, Belton O - J Inflamm (Lond) (2015)

CLA inhibits CD68 and increases MR expression in HPBMC-derived macrophages. (a) Freshly isolated HBPMCs were cultured for four days in the presence or absence of M-CSF and treated with c-9,t-11-CLA, t-10,c-12-CLA, CLA blend (80:20 c-9,t-11:t-10,c-12), OA, LA or TROG for a further 48 hrs. At day 6, macrophage were fixed and stained. Immunofluorescence analysis of unstimulated macrophage shows that both CLA isomers and their blend increase MR expression but do not affect CD68 expression. (b) Differentiation of HBPMCs in the presence of 100 ng/ml M-CSF showed that c-9,t-11-CLA and CLA blend inhibits CD68 expression and increases MR expression. Epifluorescent microscope images (63× magnification) are representative of three independent experiments. Blue indicates DAPI (nuclei), green indicates CD68 (macrophage marker), cyan indicates MR and red indicates phalloidin (cytoplasmic) staining. (c) Spectrophotometry quantification of CD68 and MR fluorescent signal confirms the regulation of CD68 and MR by c-9,t-11-CLA and the atheroprotective blend. Statistical analysis of three independent experiments is expressed mean % as fluorescence ± SEM vs vehicle where *p < 0.05; **p < 0.01 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340802&req=5

Fig2: CLA inhibits CD68 and increases MR expression in HPBMC-derived macrophages. (a) Freshly isolated HBPMCs were cultured for four days in the presence or absence of M-CSF and treated with c-9,t-11-CLA, t-10,c-12-CLA, CLA blend (80:20 c-9,t-11:t-10,c-12), OA, LA or TROG for a further 48 hrs. At day 6, macrophage were fixed and stained. Immunofluorescence analysis of unstimulated macrophage shows that both CLA isomers and their blend increase MR expression but do not affect CD68 expression. (b) Differentiation of HBPMCs in the presence of 100 ng/ml M-CSF showed that c-9,t-11-CLA and CLA blend inhibits CD68 expression and increases MR expression. Epifluorescent microscope images (63× magnification) are representative of three independent experiments. Blue indicates DAPI (nuclei), green indicates CD68 (macrophage marker), cyan indicates MR and red indicates phalloidin (cytoplasmic) staining. (c) Spectrophotometry quantification of CD68 and MR fluorescent signal confirms the regulation of CD68 and MR by c-9,t-11-CLA and the atheroprotective blend. Statistical analysis of three independent experiments is expressed mean % as fluorescence ± SEM vs vehicle where *p < 0.05; **p < 0.01 and ***p < 0.001.
Mentions: To validate the hypothesis that transcriptional changes may influence protein expression, we also performed immunocytochemistry of the pan-macrophage marker CD68 and of the MΦ2-type receptor MR, in HPBMCs either unstimulated (Figure 2a) or under M-CSF-stimulated conditions (Figure 2b). Immunofluorescence microscopy and subsequent spectrophotometric quantification of the fluorescent signals (Figure 2c) showed that, in unstimulated conditions, CLA had no effect on CD68 expression but upon M-CSF stimulation, both the c-9,t-11 isomer and CLA blend decreased CD68 (by 10 ± 1%, p < 0.01 and 18 ± 1%, p < 0.001, respectively). Importantly, in mature macrophages following M-CSF stimulation, c-9,t-11-CLA and CLA blend increased MR expression (by 57 ± 7% p < 0.01 and 55 ± 9%, p < 0.01, respectively). Finally, t-10,c-12-CLA, OA and linoleic acid had no effect on CD68 or MR expression under either condition. This confirms that the atheroprotective CLA isomer and blend primes monocytes to an MΦ2 phenotype during differentiation, thus altering the macrophage phenotype. Negative controls for immunofluorescence experiments are shown in “Additional Information” (Additional file 3).Figure 2

Bottom Line: However, the exact mechanisms through which CLA mediates this effect remain to be elucidated.Furthermore, this altered macrophage phenotype impacts on foam cell formation, inhibiting ox-LDL accumulation and promoting cholesterol efflux via both PPARγ and LXRα dependent pathways.The data increases the understanding of the pathways regulated by CLA in atheroprotection, namely, inhibiting the progressive acquisition of a pro-inflammatory macrophage phenotype.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical and Biomolecular Science, UCD Conway Institute, University College Dublin, Dublin, Ireland.

ABSTRACT

Background: Atherosclerosis, the underlying cause of heart attack and strokes, is a progresive dyslipidemic and inflammatory disease where monocyte-derived macrophage cells play a pivotal role. Although most of the mechanisms that contribute to the progression of atherosclerosis have been identified, there is limited information on those governing regression. Conjugated linoleic acid (CLA) is a group of isomers of linoleic acid that differ in the position and/or geometry of their double bonds. We have previously shown that a specific CLA blend (80:20 cis-9,trans-11:trans-10,cis-12-CLA) induces regression of pre-established atherosclerosis in vivo, via modulation of monocyte/macrophage function. However, the exact mechanisms through which CLA mediates this effect remain to be elucidated.

Methods: Here, we address if CLA primes monocytes towards an anti-inflammatory MΦ2 macrophage and examine the effect of individual CLA isomers and the atheroprotective blend on monocyte-macrophage differentiation, cytokine generation, foam cell formation and cholesterol metabolism in human peripheral blood monocyte (HPBMC)-derived macrophages.

Results: cis-9,trans-11-CLA and the atheroprotective 80:20 CLA blend regulates expression of pro-inflammatory mediators and modulates the inflammatory cytokine profile of macrophages and foam cells. In addition, cis-9,trans-11-CLA and CLA blend primes HPBMCs towards an anti-inflammatory MΦ2 phenotype, characterised by increased scavenger receptor (CD36) and efflux protein (ABCA-1) expression. Furthermore, this altered macrophage phenotype impacts on foam cell formation, inhibiting ox-LDL accumulation and promoting cholesterol efflux via both PPARγ and LXRα dependent pathways.

Conclusion: The data increases the understanding of the pathways regulated by CLA in atheroprotection, namely, inhibiting the progressive acquisition of a pro-inflammatory macrophage phenotype.

No MeSH data available.


Related in: MedlinePlus