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A critical view of monocyte subpopulations in human hypercholesterolemia

  • Sam C. Latet
    Correspondence
    Corresponding author. Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital Wilrijkstraat 10, 2650 Edegem, Belgium.
    Affiliations
    Cardiovascular Diseases, Department of Translational Pathophysiological Research, University of Antwerp, Belgium

    Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital, Belgium
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  • Amaryllis H. Van Craenenbroeck
    Affiliations
    Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital, Belgium

    Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Belgium
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  • Paul L. Van Herck
    Affiliations
    Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital, Belgium
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  • Emeline M. Van Craenenbroeck
    Affiliations
    Cardiovascular Diseases, Department of Translational Pathophysiological Research, University of Antwerp, Belgium

    Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital, Belgium
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  • Christiaan J. Vrints
    Affiliations
    Cardiovascular Diseases, Department of Translational Pathophysiological Research, University of Antwerp, Belgium

    Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital, Belgium
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  • Vicky Y. Hoymans
    Affiliations
    Cardiovascular Diseases, Department of Translational Pathophysiological Research, University of Antwerp, Belgium

    Laboratory of Cellular and Molecular Cardiology, Department of Cardiology, Antwerp University Hospital, Belgium
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      We have read with great interest the article by Fadini et al. reporting that circulating monocyte–macrophages (MoMas) show an imbalance toward pro-inflammatory M1 MoMas in patients with hypercholesterolemia and atherosclerosis compared to healthy subjects [
      • Fadini G.P.
      • Simoni F.
      • Cappellari R.
      • Vitturi N.
      • Galasso S.
      • Vigili de Kreutzenberg S.
      • et al.
      Pro-inflammatory monocyte-macrophage polarization imbalance in human hypercholesterolemia and atherosclerosis.
      ]. Also, non-familiar hypercholesterolemia (NFH) was associated with a more severe M1/M2 MoMas imbalance than familiar hypercholesterolemia (FH). The authors speculated that adverse diet and lifestyle, factors contributing to hypercholesterolemia in NFH, were even more detrimental than genetics for the development of inflammation. No significant differences were observed among groups in the distribution of three monocyte subsets, defined as classical (CD14++CD16; or Mon1), intermediate (CD14++CD16+; or Mon2) and non-classical monocytes (CD14+CD16+; or Mon3) [
      • Fadini G.P.
      • Simoni F.
      • Cappellari R.
      • Vitturi N.
      • Galasso S.
      • Vigili de Kreutzenberg S.
      • et al.
      Pro-inflammatory monocyte-macrophage polarization imbalance in human hypercholesterolemia and atherosclerosis.
      ].
      Even though the work highlights an important field in atherosclerosis research, we would like to formulate some remarks.
      Fadini et al. identified the monocyte population solely on physical features, i.e. on a FSC vs. SSC density plot on flow cytometric analysis. Albeit CD68 and CX3CR1 were chosen to selectively gate subsets, no additional pan-monocytic marker was used to distinguish monocytes from other cells such as neutrophils and NK cells. Yet, precise gating of the real total monocyte population is of value when trying to obtain consistent quantitative information of any monocyte subset. For this purpose, approaches that include an extra pan-monocyte marker, such as CD86 or HLA-DR, have been designed [
      • Zawada A.M.
      • Rogacev K.S.
      • Schirmer S.H.
      • Sester M.
      • Bohm M.
      • Fliser D.
      • et al.
      Monocyte heterogeneity in human cardiovascular disease.
      ,
      • Heimbeck I.
      • Hofer T.P.
      • Eder C.
      • Wright A.K.
      • Frankenberger M.
      • Marei A.
      • et al.
      Standardized single-platform assay for human monocyte subpopulations: lower CD14+CD16++ monocytes in females.
      ]. Next, optimal discrimination between the intermediate and non-classical monocyte subsets by flow cytometry has been a matter of debate. In his article, Fadini et al. preferred to adhere to a CD14 vs. CD16 plot with rectangular gating. However, in order to optimize the distinction between CD16+ monocyte subsets, Shantsila et al. recommended in 2011 to include the CCR2 marker [
      • Shantsila E.
      • Wrigley B.
      • Tapp L.
      • Apostolakis S.
      • Montoro-Garcia S.
      • Drayson M.T.
      • et al.
      Immunophenotypic characterization of human monocyte subsets: possible implications for cardiovascular disease pathophysiology.
      ]. Other efforts have been based on CCR5 [
      • Ancuta P.
      • Rao R.
      • Moses A.
      • Mehle A.
      • Shaw S.K.
      • Luscinskas F.W.
      • et al.
      Fractalkine preferentially mediates arrest and migration of CD16+ monocytes.
      ,
      • Rogacev K.S.
      • Seiler S.
      • Zawada A.M.
      • Reichart B.
      • Herath E.
      • Roth D.
      • et al.
      CD14++CD16+ monocytes and cardiovascular outcome in patients with chronic kidney disease.
      ], ACE [
      • Ulrich C.
      • Heine G.H.
      • Garcia P.
      • Reichart B.
      • Georg T.
      • Krause M.
      • et al.
      Increased expression of monocytic angiotensin-converting enzyme in dialysis patients with cardiovascular disease. Nephrology, dialysis, transplantation.
      ], and TLR2 [
      • Urra X.
      • Villamor N.
      • Amaro S.
      • Gomez-Choco M.
      • Obach V.
      • Oleaga L.
      • et al.
      Monocyte subtypes predict clinical course and prognosis in human stroke. Journal of cerebral blood flow and metabolism.
      ]. By doing so, it has become apparent across studies and different disease conditions that the intermediate and non-classical monocytes account for ≤20% of blood monocytes. As such, these ‘CD16+ monocyte subsets’ are much less abundant in the periphery than classical monocytes, which generally constitute up to 80–90% of blood monocytes [
      • Shantsila E.
      • Wrigley B.
      • Tapp L.
      • Apostolakis S.
      • Montoro-Garcia S.
      • Drayson M.T.
      • et al.
      Immunophenotypic characterization of human monocyte subsets: possible implications for cardiovascular disease pathophysiology.
      ,
      • Rogacev K.S.
      • Seiler S.
      • Zawada A.M.
      • Reichart B.
      • Herath E.
      • Roth D.
      • et al.
      CD14++CD16+ monocytes and cardiovascular outcome in patients with chronic kidney disease.
      ,
      • Ziegler-Heitbrock L.
      • Ancuta P.
      • Crowe S.
      • Dalod M.
      • Grau V.
      • Hart D.N.
      • et al.
      Nomenclature of monocytes and dendritic cells in blood.
      ,
      • Heine G.H.
      • Ulrich C.
      • Seibert E.
      • Seiler S.
      • Marell J.
      • Reichart B.
      • et al.
      CD14(++)CD16+ monocytes but not total monocyte numbers predict cardiovascular events in dialysis patients.
      ,
      • Rogacev K.S.
      • Cremers B.
      • Zawada A.M.
      • Seiler S.
      • Binder N.
      • Ege P.
      • et al.
      CD14++CD16+ monocytes independently predict cardiovascular events: a cohort study of 951 patients referred for elective coronary angiography.
      ]. In the paper by Fadini et al., classical monocytes reached 20–30% of total monocytes, the intermediate monocyte subset amounted to 60%, whereas the non-classical monocyte subset represented about 5–10%. Still, we cannot rule out the possibility that the choice of CD16 antibody clone is critical for monocyte research, and therefore that the divergence in results might be explained by differences in antibody clone. A final concern relates to the terminology and phenotyping of MoMas. Although resident in the bone marrow and in lymphoid tissue [
      • Swirski F.K.
      • Nahrendorf M.
      • Etzrodt M.
      • Wildgruber M.
      • Cortez-Retamozo V.
      • Panizzi P.
      • et al.
      Identification of splenic reservoir monocytes and their deployment to inflammatory sites.
      ], the Nomenclature Committee of the International Union of Immunological Societies approved in 2010 that for the monocyte/macrophage system, the term ‘monocyte’ is reserved principally for cells in blood [
      • Ziegler-Heitbrock L.
      • Ancuta P.
      • Crowe S.
      • Dalod M.
      • Grau V.
      • Hart D.N.
      • et al.
      Nomenclature of monocytes and dendritic cells in blood.
      ]. The panel also agreed that monocytes circulate in blood before they migrate into (non-lymphoid) tissue where they develop into different types of macrophages or dendritic cells. For this reason, it was also felt that it is not always critical to use the label ‘blood’ when referring to monocytes [
      • Ziegler-Heitbrock L.
      • Ancuta P.
      • Crowe S.
      • Dalod M.
      • Grau V.
      • Hart D.N.
      • et al.
      Nomenclature of monocytes and dendritic cells in blood.
      ]. Still, the aim of the article by Fadini et al. to quantify circulating monocyte–macrophages (MoMas) subsets remains confusing since it seems to imply that macrophages are present in peripheral blood. In particular, Fadini et al. defined circulating M1 MoMas as CD68+CCR2+ cells and M2 MoMas as CX3CR1+CD163+/CD206+ cells. To our knowledge, there are no studies available showing direct proof for macrophage emigration from the vasculature into the blood stream.
      To end, we report our preliminary results on monocyte subsets analysis in patients with hypercholesterolemia. Blood samples were collected from 26 subjects referred for elective coronary angiography at the department of Cardiology, UZA, Belgium. Exclusion criteria were: age >80 years, acute coronary syndromes, autoimmune diseases, infections, anti-inflammatory therapy and oncological processes. Hypercholesterolemia was defined as a documented total cholesterol value ≥ 220 mg/dl or an ongoing treatment with a statin. The procedure was approved by the local ethics committee and all participants gave written informed consent. We used the following antibodies: anti-CD14 phycoerythrin (PE; Clone: MφP9; BD Biosciences, Erembodegem, Belgium), anti-CD16 fluorescein isothiocyanate (FITC; Clone: 3G8; BD Biosciences), anti-CD86 peridinin chlorophyll cyanin 5.5 (PerCP-Cy5.5; Clone: 2331 (FUN-1); BD Biosciences), anti-CD45 allophycocyanin-Hilite 7 (APC-H7; Clone: 2D1; BD Biosciences) and anti-CCR2 allophycocyanin (APC; Clone: IgG2B 48607; R&D systems, Abingdon, UK). Flow cytometry was performed as previously described [
      • Latet S.C.
      • Van Herck P.L.
      • Van Craenenbroeck A.H.
      • Van Ackeren K.
      • Van Craenenbroeck E.M.
      • Vrints C.J.
      • et al.
      Altered expression of monocyte and dendritic cell membrane-associated antigens following coronary angiography.
      ,
      • Van Craenenbroeck A.H.
      • Van Ackeren K.
      • Hoymans V.Y.
      • Roeykens J.
      • Verpooten G.A.
      • Vrints C.J.
      • et al.
      Acute exercise-induced response of monocyte subtypes in chronic heart and renal failure.
      ]. Monocyte subsets were defined as CD86+CD14++CD16CCR2+ (classical, Mon1), CD86+CD14++CD16+CCR2+ (intermediate, Mon2) and CD86+CD14+CD16++CCR2- (non-classical, Mon3) in accordance with the latest nomenclature [
      • Shantsila E.
      • Wrigley B.
      • Tapp L.
      • Apostolakis S.
      • Montoro-Garcia S.
      • Drayson M.T.
      • et al.
      Immunophenotypic characterization of human monocyte subsets: possible implications for cardiovascular disease pathophysiology.
      ]. The gating strategy is summarized in Fig. 1. Patient characteristics are shown in Table S1 (Supplementary material). Comparison between patients with and without hypercholesterolemia revealed that cholesterol values were non-significant between groups, indicating that the ongoing treatment with statins had normalized blood cholesterol values. The results of the flow cytometry analysis of the different monocyte subsets are listed in Table S1 and Fig. 2. Mon1 turns out to be the largest subset (median: 85.8% of monocytes), whereas Mon2 and Mon3 subsets are remarkably smaller (median: 3.6% and 10.8%, respectively). Total monocytes, expressed as a % of leukocytes, nor the distribution of the three subsets did not differ significantly between patients with and without hypercholesterolemia. In addition, there was no correlation of monocytes (subsets) with on-treatment total, HDL or LDL cholesterol levels.
      Figure thumbnail gr1
      Fig. 1Representative gating strategy for monocyte subsets. Monocytes were identified by the expression of the panmonocytic marker CD86 together with the scatter profile on a forward/side scatter dot plot (a). Subsequently, monocytes were subdivided into their subsets based on the surface expression of CD14, CD16 and CCR2 (b), which resulted into three distinct subsets (c).
      Figure thumbnail gr2
      Fig. 2Monocyte subsets in patients with and without hypercholesterolemia
      In conclusion, even though we have some methodological remarks relating to the flow cytometric assessment of blood monocytes in the article by Fadini et al., the finding that there are no significant differences in the percentages of classical, intermediate, non-classical, and total monocytes between patients with and without hypercholesterolemia was confirmed by our data.

      Appendix A. Supplementary data

      The following is the supplementary data related to this article:

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      Linked Article

      • Pro-inflammatory monocyte-macrophage polarization imbalance in human hypercholesterolemia and atherosclerosis
        AtherosclerosisVol. 237Issue 2
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          Monocyte-macrophages (MoMas) play a major role in atherosclerosis. In mice, hypercholesterolemia increases pro-inflammatory monocytes that promote plaque growth, but whether this is true also in humans in unknown. We herein analyzed monocyte subsets and MoMa phenotypes in familiar (FH, n = 22) and non-familiar (NFH, n = 20) hypercholesterolemic compared with normocholesterolemic (CTRL, n = 20) patients. We found that FH and NFH had higher circulating pro-inflammatory CD68+CCR2+ M1 MoMas than CTRL, while anti-inflammatory CX3CR1+CD163+/CD206+ M2 MoMas were reduced only in NFH.
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