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. 2018 Nov 20;49(5):943-957.e9.
doi: 10.1016/j.immuni.2018.09.011. Epub 2018 Oct 30.

An Interleukin-23-Interleukin-22 Axis Regulates Intestinal Microbial Homeostasis to Protect from Diet-Induced Atherosclerosis

Aliia R Fatkhullina  1 , Iuliia O Peshkova  1 , Amiran Dzutsev  2 , Turan Aghayev  1 , John A McCulloch  2 , Vishal Thovarai  3 , Jonathan H Badger  2 , Ravi Vats  4 , Prithu Sundd  4 , Hsin-Yao Tang  5 , Andrew V Kossenkov  6 , Stanley L Hazen  7 , Giorgio Trinchieri  2 , Sergei I Grivennikov  8 , Ekaterina K Koltsova  9
Affiliations

An Interleukin-23-Interleukin-22 Axis Regulates Intestinal Microbial Homeostasis to Protect from Diet-Induced Atherosclerosis

Aliia R Fatkhullina et al. Immunity. .

Abstract

Although commensal flora is involved in the regulation of immunity, the interplay between cytokine signaling and microbiota in atherosclerosis remains unknown. We found that interleukin (IL)-23 and its downstream target IL-22 restricted atherosclerosis by repressing pro-atherogenic microbiota. Inactivation of IL-23-IL-22 signaling led to deterioration of the intestinal barrier, dysbiosis, and expansion of pathogenic bacteria with distinct biosynthetic and metabolic properties, causing systemic increase in pro-atherogenic metabolites such as lipopolysaccharide (LPS) and trimethylamine N-oxide (TMAO). Augmented disease in the absence of the IL-23-IL-22 pathway was mediated in part by pro-atherogenic osteopontin, controlled by microbial metabolites. Microbiota transfer from IL-23-deficient mice accelerated atherosclerosis, whereas microbial depletion or IL-22 supplementation reduced inflammation and ameliorated disease. Our work uncovers the IL-23-IL-22 signaling as a regulator of atherosclerosis that restrains expansion of pro-atherogenic microbiota and argues for informed use of cytokine blockers to avoid cardiovascular side effects driven by microbiota and inflammation.

Keywords: IL-22; IL-23; atherosclerosis; cytokines; host-microbe interaction; inflammation; microbiome; myeloid cells.

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Conflict of interest statement

Declaration of interest: None

Figures

Figure 1.
Figure 1.. Aggravated atherosclerosis and increased immune cells infiltration in aortas of IL23 deficient mice.
A. Images of aortic arch from Ldlr−/− mice transplanted with Il23−/− or WT BM and fed with WD for 16 weeks. Representative images of aortic root sections (B) and quantitative comparison of atherosclerotic lesion size (C) of Il23−/− ➔Ldlr−/− (n=13) or WT ➔Ldlr−/− (n=13) mice. D. Immune cell composition of aortas isolated from Il23−/− ➔Ldlr−/− (n=13) and WT ➔Ldlr−/− (n=11) was analyzed by flow cytometry. Percentage (left panel) and absolute cell number (right panel) of CD45+ hematopoietic cells, and among them CD11b+, CD11b+CD11c+ and CD11c+ myeloid cells and CD4+ TCRβ+ cells. Relative gene expression in the aortas (E) and intestines (F) of Il23−/− ➔Ldlr−/− (n=10) and WT➔Ldlr−/− (n=10) mice. Gene expression was normalized to RpL32 and then to gene expression in WT➔Ldlr−/− mice. Data are mean ± SEM from at least 3 independent experiments. *p<0.05, **p<0.001, ***p<0.0001. Student’s t-test. See also Figures S1,2,3.
Figure 2.
Figure 2.. Ablation of IL-22 exacerbates atherosclerosis and IL-22 administration suppresses the disease development in IL-23 deficient mice.
A. Images of aortic arch from Ldlr−/− mice transplanted with Il22−/− or WT BM and fed with WD for 16 weeks. Representative images of aortic root sections (B) and quantitative comparison of atherosclerotic lesion size of Il22−/− ➔Ldlr−/− (n=16) or WT➔Ldlr−/− (n=12) mice (C). D. Immune cell composition of aortas isolated from Il22−/− ➔Ldlr−/− (n=7) and WT➔Ldlr−/− (n=5) was analyzed by flow cytometry. Percentage (left panel) and absolute cell number (right panel) of CD45+ hematopoietic cells, and among them CD11b+, CD11b+CD11c+ and CD11c+ myeloid cells and CD4+ TCRβ+ cells. Relative gene expression in the aortas (E) and intestines (F) of Il22−/− ➔Ldlr−/− (n=10) or WT➔Ldlr−/− (n=10) mice was normalized to RpL32 and then to gene expression in WT➔Ldlr−/− mice. G. Scheme of experiment. Il23−/− ➔Ldlr−/− and WT➔Ldlr−/− were administered with mIL-22-Ig for 4 weeks starting at 8 weeks of WD feeding. H. Representative images of aortic root sections and quantitative comparison of atherosclerotic lesion size from Il23−/− ➔Ldlr−/− (n=13 (control), n=12 (mIL-22-Ig)) and WT➔Ldlr−/− (n=9 (control), n=8 (mIL-22-Ig)) mice treated with mIL-22-Ig or control. ***p<0.0001; *p<0.05. Data are mean ± SEM from 3 independent experiments. *p<0.05, **p<0.001, ***p<0.0001. Student’s t-test. I. IF representative image from 3 independent experiments of aortic root sections from Il23−/−➔Ldlr−/− or WT➔Ldlr−/− mice after mIL-22-Ig treatment demonstrates the reduction of CD11b+, CD11c+, and CD11b+CD11c+ myeloid cells. A-adventitia, M-media, P-atherosclerotic plaque. See also Figure S2,3.
Figure 3.
Figure 3.. IL-23 or IL-22 deficiency reduces antimicrobial peptide expression and contributes to microbiome alterations in the gut of atherosclerotic mice.
A. Relative antimicrobial peptides gene expression in the intestines of WT➔Ldlr−/− (n=12), Il23−/− ➔Ldlr−/− (n=12) and Il22−/− ➔Ldlr−/− (n=11) mice was normalized to RpL32 and then to gene expression in WT➔Ldlr−/− mice. Data are mean ± SEM from 3 independent experiments. *p<0.05, **p<0.001, ***p<0.0001. Student’s t-test. B. Representative IHC staining of RegIIIγ in colon tissue of WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− mice (top panel), scale bar is 100μm. Representative mucin staining in colon tissue sections of WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− mice stained for Mucin2 (green) and DAPI (blue) (middle panel), scale bar is 100μm. Mucin layer staining after Carnoy’s Fixation (bottom panel), scale bar is 50μm. Images are representative from 3 independent experiments. C. Microbiome compositions in Il23−/− ➔Ldlr−/− and Il22−/− ➔Ldlr−/− mice compared to WT➔Ldlr−/− cage mate controls as determined by whole metagenome shotgun sequencing followed by principal component analysis (PCA). D. Heat map of cecum microbiome composition in Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− and WT➔Ldlr−/− mice as determined by shotgun sequencing. Each column represents a single mouse. Red – upregulated bacterial genes, green – downregulated bacterial genes. E, F. Functional characterization of luminal bacteria from Il23−/− ➔Ldlr−/−, Il22−/− ➔Ldlr−/− and WT➔Ldlr−/− mice as determined by metagenome shotgun sequencing. E. Principal component analysis (PCA) and (F) heat map of enzymatic activities (Enzyme Commission (EC) numbers) of sequenced microbiota G. Relative bacterial 16S rRNA expression in the intestines of Il23−/− ➔Ldlr−/− (n=7), Il22−/− ➔Ldlr−/− (n=7) or WT➔Ldlr−/− (n=6) mice. H. Heat map of tissue adhesive bacteria gene expression in Il23−/− ➔Ldlr−/− and WT➔Ldlr−/− mice as determined by 16S rRNA sequencing. Red – upregulated bacteria, blue – downregulated bacteria. D, F, H. Only statistically significantly different results are shown. *p<0.05. See also Figure S4.
Figure 4.
Figure 4.. Cytokine-mediated alterations in microbiota drive atherosclerosis progression in Il23−/− ➔Ldlr−/− and Il22−/− ➔Ldlr−/− mice.
A. Scheme of experiment. WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− mice fed with WD for 16 weeks were maintained for the last 4 weeks of WD feeding on regular (Reg) or broad-spectrum antibiotic (Abx) containing water. B. Representative images of aortic root sections (left) and (C) quantitative comparison of atherosclerotic lesion size in WT➔Ldlr−/− (n=8 (Reg), n=5 (Abx)), Il23−/− ➔Ldlr−/− (n=9 (Reg), n=6 (Abx)) or Il22−/− ➔Ldlr−/− (n=12 (Reg), n=6 (Abx)) mice maintained on regular or antibiotic water. Data are average from 3 independent experiments. D. Relative gene expression in aortas from Il23−/− ➔Ldlr−/− (n=5) or Il22−/− ➔Ldlr−/− (n=5) mice maintained on antibiotic-containing or regular water was normalized to RpL32 and then to average gene expression in untreated mice of the same genotype. E. Scheme of microbiota transfer experiment. F. Representative images of aortic root sections and quantitative comparison (G) of atherosclerotic lesion size in WT➔Ldlr−/− (n=7–8) or Il23−/− ➔Ldlr−/− (n=7–9) mice that received microbiome from either WT➔Ldlr−/− or Il23−/− ➔Ldlr−/− mice with advanced atherosclerosis. *p<0.05, **p<0.001. Data are average from 3 independent experiments. H. Relative gene expression in the aortas from WT ➔Ldlr−/− mice that received microbiota from WT ➔Ldlr−/− (n=7) or Il23−/− ➔Ldlr−/− (n=8) atherosclerotic mice was normalized to RpL32 and then to average gene expression in WT ➔Ldlr−/− mice reconstituted with WT microbiota. Data are mean ± SEM from 3 independent experiments Student’s t-test. *p<0.05, **p<0.001. See also Figure S 4,5.
Figure 5.
Figure 5.. Bacterial signaling pathways and metabolites in Il23−/− ➔Ldlr−/− and Il22−/− ➔Ldlr−/− mice.
A-C. Protein domain structure prediction for luminal bacteria from Il23−/− ➔Ldlr−/−, Il22−/− ➔Ldlr−/− and WT➔Ldlr−/− mice as determined by metagenome shotgun sequencing. A. Principal component analysis (PCA). B. Heat map. Red – upregulated, green – downregulated domains. Only statistically significantly changed genes are shown. *p<0.05. C. Presence of Lipopolysaccharide assembly protein A gene domain in microbiota of Il23−/− ➔Ldlr−/−, Il22−/− ➔Ldlr−/− and WT➔Ldlr−/− mice. B, C Only statistically significantly different results are shown. D. Serum Endotoxin (LPS) in WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− (n=22–25). E. Serum Endotoxin (LPS) in WT➔Ldlr−/− and Il23−/−➔Ldlr−/− administered with mIL-22-Ig (n=6 and 10 respectively) or control (n=20). F. Serum Endotoxin (LPS) in WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− mice on regular (n=20) and antibiotics (n=7, 10 and 9 respectively) containing water. G. Serum Endotoxin (LPS) in mice after fecal transplant from WT➔Ldlr−/− or Il23−/− ➔Ldlr−/− into WT➔Ldlr−/− (n=6 and 9 respectively) or Il23−/− ➔Ldlr−/− (n=12 and 9 respectively) mice. H. Presence of TMA lyase bacterial gene in microbiota of Il23−/− ➔Ldlr−/−, Il22−/− ➔Ldlr−/− and WT➔Ldlr−/− mice as determined by shotgun sequencing. I. Cumulative scheme demonstrating upregulation and statistical analysis for multiple metabolites of TMAO pathway in serum of WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− mice (circle) and mice after fecal transplant (diamond). J. Serum TMAO Ldlr−/− x Il23+/+ (n=5) and Ldlr−/− x Il23−/− (n=8) mice fed WD with 1% choline. Data are mean ± SEM from 2–3 independent experiments. Student’s t-test. ANOVA for multiple comparisons. *p<0.05, **p<0.001, ***p<0.0001. See also Figure S6.
Figure 6.
Figure 6.. Osteopontin is required for enhanced atherosclerosis in Il23−/− ➔Ldlr−/− and Il22−/−</P/> ➔Ldlr−/− mice.
A. Relative gene expression of Spp1 in the aortas of WT➔Ldlr−/− (n=12), Il23−/− ➔Ldlr−/− (n=13) or Il22−/− ➔Ldlr−/− (n=13) was normalized to RpL32 and then to gene expression in WT➔Ldlr−/− mice. B. Intracellular OPN staining in myeloid cells isolated from aortas of Il23−/− ➔Ldlr−/− mice. C. Q-PCR gene expression analysis of Spp1 in aortas of Il23−/− ➔Ldlr−/− (n=6) or Il22−/− ➔Ldlr−/− (n=5) mice, treated with regular (Reg) or antibiotic (Abx) water for the last 4 weeks of WD feeding. Normalized to RpL32 and then to average gene expression in untreated mice (n=6 per group) of the same genotype. D. OPN gene expression in WT ➔Ldlr−/− recipients of fecal transplant from WT ➔Ldlr−/− or Il23−/− ➔Ldlr−/− atherosclerotic mice. E. Scheme- WT➔Ldlr−/−, Il23−/− ➔Ldlr−/− or Il22−/− ➔Ldlr−/− mice were administered with anti-OPN-antibody or control every 3 days for the last 2.5 weeks of WD feeding. Representative images of aortic root sections (F) and quantification of atherosclerotic lesion size (G) from WT➔Ldlr−/− (n=7–10), Il23−/− ➔Ldlr−/− (n=8–13) or Il22−/− ➔Ldlr−/− (n=7–11) mice treated with antiOPN-antibody or control (Black – PBS, Blue – IgG1k isotype control). H. Confocal microscopy of myeloid cells in aortic roots of indicated anti-OPN-treated or control mice. I. Relative gene expression in aortas from Il23−/− ➔Ldlr−/− (n=6) and Il22−/− ➔Ldlr−/− (n=4) mice, injected with anti-OPN-antibody or control. Gene expression was normalized to RpL32 gene expression and then normalized to average of gene expression of untreated mice of the same genotype. Data are mean ± SEM and images are representative from 3 independent experiments. Student’s t-test. *p<0.05, **p<0.001, ***p<0.0001.See also Figure S7.
Figure 7.
Figure 7.. In vivo administration of LPS and TMAO accelerates atherosclerosis development.
Representative images of aortic root sections (A) and quantitative comparison of atherosclerotic lesion size (B) from Ldlr−/− mice fed with WD for 6 weeks followed by administration with LPS and TMAO or PBS control for 3 weeks every 3 days. C. Spp1, Cd36 and Il1b genes expression in the aortas of Ldlr−/− mice treated with LPS+TMAO or PBS control. D. Spp1 expression in circulating Ly6Chi monocytes in response to LPS and TMAO administration (n=4–6). Gene expression was normalized to RpL32 and then to average gene expression in controls. Images are representative from 2 independent experiments. Data are mean ± SEM. Student’s t-test. *p<0.05, **p<0.001. See also Figure S7.
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