Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression:implications for atherosclerosis research

Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE−/− mice, their M1/M2 phenotype, inflammatory status, and lipid metabolism signatures were compared. oxLDL accumulation was similar in PEMs and BMDMs. On protein expression level, BMDMs showed an M2-like CD206(high)CD11c(low) profile, while cholesterol loading led to enhanced CD11c expression and reduced MCP-1 secretion. In contrast, PEMs expressed low levels of CD206 and CD11c, and responded to cholesterol loading by increasing CD11c expression and MCP-1 secretion. mRNA expression of M1/M2 markers was higher in PEMS than BMDMs, while lipid metabolism genes were similarly expressed. Whole aorta flow cytometry showed an accumulation of M2-like CD206(high)CD11c(low) macrophages in advanced versus early atherosclerotic disease in ApoE−/− mice. In isolated lesions, mRNA levels of the M2 markers Socs2, CD206, Retnla, and IL4 were downregulated with increasing disease severity. Likewise, mRNA expression of lipid metabolism genes (SREBP2, ACSL1, SRB1, DGAT1, and cpt1a) was decreased in advanced versus early lesions. In conclusion, PEMs and BMDMs are phenotypically distinct and differ from macrophages in lesions with respect to expression of M1/M2 markers and lipid metabolism genes

Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice

<div><p>Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosclerosis in uremic settings, insight into new treatment options with effects on both parameters is warranted. The GLP-1 analogue liraglutide improves glucose homeostasis, and is approved for treatment of type 2 diabetes. Animal studies suggest that GLP-1 also dampens inflammation and atherosclerosis. Our aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 μg/kg, s.c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68⁺F4/80^-), CD4⁺, and CD8⁺ T-cells, suggesting that moderate uremia induced kidney inflammation. Furthermore, markers of fibrosis (i.e. Col1a1 and Col3a1) were upregulated, and histological examinations showed increased glomerular diameter in NX mice. Importantly, liraglutide treatment attenuated atherosclerosis (~40%, p < 0.05) and reduced kidney inflammation in NX mice. There was no effect of liraglutide on expression of fibrosis markers and/or kidney histology. This study suggests that liraglutide has beneficial effects in a mouse model of moderate uremia by reducing atherosclerosis and attenuating kidney inflammation.</p></div

Gene expression depicted as ΔCT and fold change in NX relative to control (SHAM) in column 4 and in NX LIRA relative to NX in column 5.

<p>Results are shown as mean±SEM.</p

Liraglutide attenuates atherosclerosis in uremic LDLr-/- mice.

<p>Uremic LDLr-/- mice were treated with vehicle (NX; n = 14) or liraglutide (NX LIRA; n = 15) and sham operated control LDLr-/-mice were treated with vehicle (SHAM; n = 14). After 11 weeks of treatment with full dose (1000 μg/kg), atherosclerosis was quantified as the relative plaque area in % of the total aortic arch area in all mice (<b>A</b>) and in NX mice with urea levels >20 (<b>B</b>). Depicted values are mean±SEM. *p<0.05 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 14–15 mice per group.</p

Immune cell composition is changed in kidneys from uremic LDLr-/- mice.

<p>Sixteen weeks after induction of uremia, flow cytometry was performed on kidneys from control (SHAM) and uremic (NX) LDLr-/- mice (n = 5 mice/group). The number of CD4⁺ T-cells (<b>A</b>), CD8⁺ T-cells (<b>B</b>), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (<b>C and D</b>) relative to kidney weight is depicted. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (<b>E</b>) or the M2 marker CD206 (<b>F</b>) was detected. Depicted values are mean±SEM. *p<0.05 as determined by unpaired students t-test.</p

Uremia increases glomerular size, but not cortical collagen content in LDLr-/- mice.

<p>Representative pictures of kidney sections from control (SHAM), uremic (NX) and liraglutide treated uremic (NX LIRA) mice stained with Massons trichrome (<b>A</b>). Scale bar = 200 μm for top row and 100 μm for bottom row. Glomerular diameters were measured (<b>B</b>) (25–49 glomeruli were assessed per kidney, n = 7–8 in each group) and collagen deposition in the kidney cortex was quantified (<b>C</b>) (n = 5–7 in each group) using the Visiopharm software. **p<0.01, as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test.</p

Liraglutide attenuates NX mediated kidney inflammation in LDLr-/- mice.

<p>A-F: flow cytometry analysis of kidneys from control (SHAM; n = 7), uremic (NX; n = 7) or liraglutide treated uremic (NX LIRA; n = 7) LDLr-/- mice showing the number of CD4⁺ T-cells (<b>A</b>), CD8⁺ T-cells (<b>B</b>), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (<b>C</b> and <b>D</b>) relative to kidney weight. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (<b>E</b>) or the M2 marker CD206 (<b>F</b>) was detected. Depicted values are mean±SEM. *p<0.05, **p<0.01, ***p<0.005 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 6 mice per group.</p