Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice - PubMed (original) (raw)
. 2003 Oct;112(8):1176-85.
doi: 10.1172/JCI18580.
Manolis Pasparakis, Marion J J Gijbels, Monique N Vergouwe, Iris Partouns-Hendriks, Remond J A Fijneman, Björn E Clausen, Irmgard Förster, Mark M Kockx, Klaus Rajewsky, Georg Kraal, Marten H Hofker, Menno P J de Winther
Affiliations
- PMID: 14561702
- PMCID: PMC213488
- DOI: 10.1172/JCI18580
Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice
Edwin Kanters et al. J Clin Invest. 2003 Oct.
Abstract
Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kappaB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-kappaB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.
Figures
Figure 1
Plasma lipoprotein profiles of IKK2fl- and IKK2del-transplanted _Ldlr_–/– mice after 8 weeks of high-fat feeding. Indicated are cholesterol levels (Chol) and triglyceride levels (TG) after size fractionation of pooled plasma samples.
Figure 2
Atherosclerosis in IKK2fl- and IKK2del-transplanted _Ldlr_–/– mice. (a) Representative lesions from Ldlr–/– mice transplanted with IKK2fl and (b) IKK2del BMM using MOMA-2. (c) Atherosclerotic lesion area in IKK2fl-transplanted (open circles, n = 20) and IKK2del-transplanted (closed circles, n = 18) _Ldlr_–/– mice. Circles indicate individual mice. *P < 0.01.
Figure 3
Necrosis, apoptosis, T cells, and collagen in atherosclerotic lesions from IKK2fl- and IKK2del-transplanted _Ldlr_–/– mice. (a) Representative lesions from IKK2fl- or (b) IKK2del-transplanted mice, showing signs of necrosis (arrows) in the IKK2del-transplanted mice. (c) Number of mice for each group that showed signs of necrosis (pyknosis, karyorrhexis, or complete absence of nuclei). (d) Apoptosis was detected by TUNEL staining. Bars represent number of TUNEL-positive cells per lesion area. Shown are positive cells in the lesions from IKK2fl- (n = 15) and IKK2del- (n = 11) transplanted _Ldlr_–/– mice. (e) T cells were detected by staining for CD4 and CD8. Shown are cumulative numbers of positive cells in the lesions from IKK2fl- (n = 20) and IKK2del- (n = 18) transplanted _Ldlr_–/– mice. (f) Collagen in the lesions was detected by Sirius red staining and quantified using Scion Image (Scion Corp.). Shown is the Sirius red-positive area as a percentage of the total lesion area in the advanced plaques in lesions from IKK2fl- (n = 16) and IKK2del- (n = 14) transplanted _Ldlr_–/– mice. Error bars indicate SEM.
Figure 4
Lesion classification and analysis of early lesions. (a) Lesions were typed according to their severity. Shown is the lesion distribution as a percentage of the total number of lesions. (b) Size of early lesions in IKK2fl- (n = 16) and IKK2del- (n = 15) transplanted _Ldlr_–/– mice. (c) Number of cells in early lesions in IKK2fl- (n = 16) and IKK2del- (n = 15) transplanted _Ldlr_–/– mice. (d) Number of cells per lesion area in early lesions. Error bars indicate SEM. *P < 0.05.
Figure 5
In vitro characterization of IKK2fl and IKK2del macrophages. (a) Southern blot of IKK2fl and IKK2del macrophages. Indicated are the presence or absence (+ or –) of Cre-recombinase in the macrophages, the floxed and deleted allele, and the percentage of deletion calculated by quantification of the ratio between the floxed and deleted band. (b) Cells were stimulated with LPS for the indicated times, and p65 activation was quantified in nuclear extracts using Trans-am assay. Shown are absorbances after background subtraction. (c) Expression of macrophage differentiation markers was quantified by staining for the indicated markers. (d) Uptake of different multiplicity of infection of green fluorescent protein expressing E. coli by IKK2fl and IKK2del macrophages. (e) Uptake of DiI-labeled oxidized LDL (oxLDL) and acetylated LDL (acLDL) by IKK2fl and IKK2del macrophages. (f) Cell death of IKK2fl and IKK2del macrophages after indicated incubations with oxLDL (25 μg/ml) and/or LPS (10 ng/ml), determined by propidium iodide (PI) staining. au, arbitrary units; gm, geomean; cytD, cytochalasin D. Error bars indicate SEM. Figures are representative for two experiments. *P < 0.01.
Figure 6
Cytokine production by LPS-stimulated BMM from IKK2fl and IKK2del mice. (a) IKK2fl and (b) IKK2del macrophages were left untreated (white) or stimulated with LPS (gray). TNF production was detected by intracellular cytokine staining and analyzed by FACS. (c) IKK2fl and IKK2del macrophages were stimulated with LPS. TNF production was detected by intracellular cytokine staining and analyzed by FACS. Results are representative for at least two experiments. (d) IKK2fl and IKK2del macrophages were stimulated overnight with LPS. Cytokines were measured in the supernatants. Shown are the changes in cytokine production by IKK2del macrophages as compared with IKK2fl macrophages. Results are the average of five to eight experiments. Error bars indicate SEM. *P < 0.01; #P < 0.01 by paired t test.
Figure 7
IL-6 production and effects of IL-10. (a) IKK2fl and IKK2del macrophages were stimulated with LPS for indicated times. IL-6 was measured in the supernatants. (b) IKK2fl and IKK2del macrophages were stimulated with LPS for 24 hours in combination with indicated doses of recombinant mouse IL-10. IL-6 was measured in the supernatants. (c) IKK2fl and IKK2del macrophages were stimulated with LPS for 24 hours, in the absence or presence of blocking anti-IL-10 antibody or IgG control. IL-6 was measured in the supernatants. Figures are representative for two experiments. Error bars indicate SEM. *P < 0.05, **P < 0.01.
Figure 8
Characterization of resident peritoneal cells and thioglycollate-elicited peritoneal macrophages. (a) Southern blot analysis of deletion in FACS-purified resident peritoneal CD11bhigh cells from mice with the indicated genotypes and (b) in thioglycollate-elicited IKK2fl and IKK2del peritoneal macrophages. Indicated are the presence or absence (+ or –) of Cre-recombinase in the macrophages, the WT, floxed (fl), and deleted (d) allele, and the percentage of deletion calculated by quantification of the ratio between the floxed and deleted band. IKK2fl (c) and IKK2del (d) thioglycollate-elicited macrophages were untreated (white) or stimulated with LPS (gray). TNF production was detected by intracellular cytokine staining and analyzed by FACS. (e) IKK2fl and IKK2del macrophages were stimulated with LPS, and TNF production was detected by intracellular cytokine staining and analyzed by FACS. Macrophages were identified as F4/80+. Results are representative for at least two experiments. Error bars indicate SEM. *P < 0.01.
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