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CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival

Limor Landsman, Liat Bar-On, Alma Zernecke, Ki-Wook Kim, Rita Krauthgamer, Erdenechimeg Shagdarsuren, Sergio A. Lira, Irving L. Weissman, Christian Weber and Steffen Jung

Data supplements

  • Supplemental materials for: Landsman et al

    Files in this Data Supplement:

    • Figure S1. Differential effect of enforced Bcl2 expression on the two monocyte subsets (JPG, 56.2 KB) -
      (A) Expression of hBcl2 transgene by monocyte subsets. MACS purified CD115+ blood cells from cx3cr1gfp∕+;hMRP8bcl2 and cx3cr1gfp∕+ littermates were stained with anti-Gr1 and anti-human Bcl2 antibodies. Dot plot shows Gr1hi (R7 gated cell) and Gr1low (R8 gated cell) monocyte populations. Histograms show hBcl2 expression by the subsets of hMRP8bcl2 transgenic (empty histogram) and non-transgenic (gray filled histogram) mice. (B) Comparison of blood monocyte populations of wt (black filled circles) and hMRP8bcl2 (empty circles) mice. Diagram shows the numbers of total Gr1hi and Gr1low monocytes (identified as indicated in Figure 1A) in 1 ml of blood. Note that bcl2 expression rescues more Gr1low than Gr1hi monocytes.





    • Figure S2. Reduced atherogenesis in the absence of CX3CR1 (JPG, 37.4 KB) -
      Analysis of atherosclerotic lesion area in apoe−∕− recipients of wt, cx3cr1gfp∕+ sand cx3cr1gfp∕gfp BM cells, Atherosclerotic plaques were analyzed in the aorta after Oil-red-O staining. (A) Aorta of representative mouse for each group. Titles indicate BM donor genotype. (B) Quantitative analysis of apoe−∕− recipients of wt (empty circles), cx3cr1gfp∕+ (gray circles) and cx3cr1gfp∕gfp (black circles) BM cells. n=5. *pValue<0.01, ns=non significant (Student�s t-Test).

Article Figures & Data

  • Figure 1

    Reduced numbers of Gr1low monocytes in CX3CR1-deficient mice under steady-state conditions. (A) Flow cytometric analysis of blood monocytes. Left dot plot shows total Ficoll-fractionated wt blood cells. Cells gated in region R1 are living, nongranular white blood cells (ngWBCs). Middle and right dot plots show CD115 and Gr1 staining of blood cells gated in R1. Region R2 gates monocytes, as indicated by their CD115 expression. Cells gated in R3 and R4 regions are Gr1hi and Gr1low monocytes, respectively. (B) Comparison of blood monocyte population size of wt (●) and cx3cr1gfp/gfp (○) mice. Diagram shows number of monocytes, identified as CD115+ cells (R2 gated cells), in 1 mL blood. Each circle represents a single mouse. (C,C′) Comparison between monocyte population size of wt (■), cx3cr1gfp/+ (Embedded Image) and cx3cr1gfp/gfp (Embedded Image) mice. Bar diagram (C) shows for each group of mice the percentage of total blood monocytes (R2 gated cells), Gr1hi monocytes (R3 gated cells) and Gr1low monocytes (R4 gated cells) out of ngWBC gated in R1. (C′) Alternative presentation of the data showed in panel C. Bar diagram shows the percentage of Gr1low monocytes (R4 gated cells) among total monocytes (R2 gated cells) for each of the mouse strains; n = 5. (D) Comparison between blood monocytes of wt (■), cx3cr1gfp/gfp (Embedded Image) and cx3cl1−/− mice (□). Bar histogram shows for each group the percentage of Gr1low monocytes (R4 gated cells) of total monocytes (R2 gated cells) for each mouse strain; n = 5. *P < .05, **P < .005, **P < 5 × 10−5, all as compared with wt mice (Student t test).

  • Figure 2

    Reduction of Gr1low monocytes in absence of CX3CR1 is due to impaired cell survival. (A) Comparison of wt and cx3cr1gfp/gfp monocytes in mixed BM chimera blood. BM cells were isolated from either wt or cx3cr1gfp/gfp donors, mixed to 1:1 ratio from each genotype and transferred into irradiated wt recipients. Eight weeks after transfer recipient mice were bled, and their blood content was analyzed. Dot plot shows discrimination between GFP-positive cx3cr1gfp/gfp monocytes (R5 gated cells) and GFP-negative wt monocytes (R6 gated cells). Bar diagram shows percentage of Gr1low monocytes (as gated in R4, Figure 1A) of either total wt (R6 gated cells, ■) or cx3cr1gfp/gfp (R5 gated cells, Embedded Image) monocytes; n = 8. (B) Comparison of monocyte levels in presence and absence of CX3CL1. Wt and cx3cl1−/− irradiated mice received 1:1 mix of BM cells from wt and cx3cr1gfp/gfp mice, and were bled 8 weeks later. Bar histogram shows percentage of Gr1low monocytes (R4 gated cells, Figure 1A) of total monocytes (R2 gated cells, Figure 1A) of wt (■) and cx3cl1−/− (□) recipients of either wt or cx3cr1gfp/gfp BM cells; n = 5. (C) Comparison of blood and BM monocytes of cx3cr1gfp/+ (Embedded Image) and cx3cr1gfp/gfp (Embedded Image) littermates. Bar diagram shows the percentage of Gr1low cells (gated in R4, Figure 1A) out of total blood or BM monocytes (cells gated in R2, Figure 1A); n = 4. (D) Comparison of monocytes of hMRP8bcl2-transgenic cx3cr1gfp/gfp and cx3cr1gfp/+ mice. Bar histogram shows percentage of Gr1low monocytes (cells gated in R4, Figure 1A) of total monocytes (cells gated in R2, Figure 1A) of cx3cr1gfp/+ (Embedded Image) and cx3cr1gfp/gfp (Embedded Image) mice, either hMRP8bcl2-transgenic (right bars) or nontransgenic (left bars); n = 3. *P < .0013 **P < .001, ***P < < .005 (Student t test).

  • Figure 3

    CX3CL1 rescues human blood monocytes from experimentally induced death. (A) Flow cytometric analysis of cultured human blood monocytes stained with propidium iodide (PI). Cells gated in region R7 (left dot plot) represent total analyzed population. Region R8 (right dot plot) gates PI+ cells. All bar diagrams shown in this figure present the portion of R8 gated PI+ dying cells out of R7 gated total cells. (B) Human blood monocytes (CD14++CD16) were incubated in RPMI supplemented with 10% FCS, 10 nM soluble recombinant CX3CL1 (rCX3CL1), or nonsupplemented medium for 4 hours; n = 3. (C) Human blood monocytes (CD14++CD16) were incubated in serum-supplemented RPMI either further supplemented or nonsupplemented with 10 nM soluble recombinant CX3CL1 (rCX3CL1). Six hours later, 30 μM 7-β-hydroxycholesterol (7β-OH-CH) was added to the cultures, and cells were analyzed after additional 16 hours; n = 3. (D) Human blood monocytes (CD14++CD16) were incubated with RPMI supplemented with 1, 5, 10, or 100 nM CX3CL1. Diagram shows percentage of PI+ cells; n = 3. (E) Human blood monocytes (CD14+CD16+) were incubated in RPMI supplemented with 1 μg/mL pertussis toxin (PTx) for 1 hour followed by incubation with 10% FCS, 10 nM soluble recombinant CX3CL1 (rCX3CL1) or unsupplemented medium for additional 4 hours; n = 3. (F) Flow cytometric analysis of isolated CD14++CD16 (top dot plot) and CD14+CD16+ (bottom dot plot) human blood monocytes, before culture. Numbers indicate percentage of gated cells out of total population. (G) Isolated CD14++CD16 and CD14+CD16+ human blood monocytes incubated in RPMI (Medium; □) or RPMI supplemented with either 10% FCS (+FCS; ■) or 10 nM recombinant CX3CL1 (+CX3CL1; Embedded Image); n = 3. *P < .005, **P < .01, ***P < .05, NS = not significant (Student t test).

  • Figure 4

    CX3CR1-deficient atheromas show changes in cellular composition and increased apoptosis. Irradiated apoe−/− recipients of indicated BM cells were subjected to high-fat diet for 12 weeks, after which their plaque content was analyzed. (A) Histology of atherosclerotic plaque of representative recipient of cx3cr1gfp/+, cx3cr1gfp/gfp, and cx3cr1gfp/gfp;hMRP8bcl2 (cx3cr1gfp/gfp;hBcl2) BM cells. CX3CR1/GFP expression is shown in green; DAPI stain in blue. (B) Immunohistochemistry of atherosclerotic plaque of representative recipient of cx3cr1gfp/+ BM cells. CX3CR1/GFP expression is shown in green, anti–MOMA-2 staining in red, and DAPI in blue. Brightfield shows orange oil (Oil Red O) deposit. Arrows indicate large CX3CR1/GFP+ cells also positive for MOMA-2 expression and orange oil deposit. (C) Histology-based quantification of CX3CR1/GFP+ cells per total plaque areas. Bar diagram shows recipients of cx3cr1gfp/+ (Embedded Image) and cx3cr1gfp/gfp (■), both on either wt (non-tg) or hMRP8bcl2-transgenic background (hBcl2 tg); n = 12. (D) Immunohistochemistry-based quantification of MOMA-2 positive area out of total plaque area. Bar diagram shows recipient of wt (□) and cx3cr1gfp/gfp (■), both either on wt (non tg) or hMRP8bcl2 (hBcl2 tg)–transgenic background; n = 12. (E) Immunohistochemistry-based quantification of TUNEL+ cells out of total plaque cells (identified by DAPI staining). Bar diagram shows recipient of wt (□) and cx3cr1gfp/gfp (■), both on either wt (non-tg) or hMRP8bcl2-transgenic background (hBcl2 tg); n = 12. (F) Immunohistochemistry of atherosclerotic plaque of cx3cr1gfp/gfp BM cell recipient. CX3CR1/GFP expression is shown in green, TUNEL staining is shown in red. Right panel shows higher magnification of area marked by white rectangular. Arrows indicate cells double positive for CX3CR1/GFP and TUNEL. (G) Immunohistochemistry of atherosclerotic plaque of wt (non-tg) or hMRP8bcl2-transgenic (hBcl2 tg) BM were stained with an antibody specific for human Bcl2 (red). DAPI is shown in blue. Arrow indicates human Bcl2-positive cells. (H) Histology-based quantification of acellular plaque area. Bar diagram shows recipient of wt (□) and cx3cr1gfp/gfp (■), both on either wt (non-tg) or hMRP8bcl2-transgenic background (hBcl2 tg); n = 12. *P < .05, **P < .01, ***P < .005 (Student t test).

  • Figure 5

    Reduced numbers of Gr1low monocytes in CX3CR1-deficient mice under atherosclerotic condition and rescue by Bcl2 expression. Blood monocyte levels of irradiated apoe−/− recipients of wt (□) and cx3cr1gfp/gfp (■) BM cells, either transgenic (hBcl2 tg) or nontransgenic (non-tg) for hMRP8bcl2. (A) Summary of total monocytes, identify as CD115+ cells (R2 gated cells, Figure 1A), as a percentage of ngWBC (R1 gated cells, Figure 1A); n = 12. (B) Summary of Gr1low monocytes levels (R4 gated cells, Figure 1A) as a percentage of total CD115+ monocyte levels (R2 gated cells, Figure 1A); n = 12. *P < .01, **P < .05 (Student t test).

  • Figure 6

    Enforced cell survival promotes atherogenesis in the absence of CX3CR1. Quantitative analysis of atherosclerotic lesion area in apoe−/− recipients of wt (○) and cx3cr1gfp/gfp (●) BM cells, either from hMRP8bcl2-transgenic (hBcl2 tg) or nontransgenic (non tg) donors. Mice were subjected to high-fat diet for 12 weeks, after which atherosclerotic plaques were analyzed in aortic roots (A,A') and en face prepared aortas (B,B′) by Oil Red O staining. Panels A′ and B′ show a summary of Oil Red O+ plaque area for each group, a representative of which is shown in panels A and B, respectively; n = 12, representative of 2 independent experiments. *P < .05, **P < .01 (Student t test).

Supplementary Materials

  • Figure S1

    Supplementary PDF file available online.

  • Figure S2

    Supplementary PDF file available online.