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Atherogenic dyslipidemia promotes autoimmune follicular helper T cell responses via IL-27

Nature Immunology volume 19, pages 583–593 (2018)Cite this article

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A Publisher Correction to this article was published on 19 June 2018

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Abstract

The incidence of atherosclerosis is higher among patients with systemic lupus erythematosus (SLE); however, the mechanism by which an atherogenic environment affects autoimmunity remains unclear. We found that reconstitution of atherosclerosis-prone Apoe–/– and Ldlr–/– mice with bone marrow from lupus-prone BXD2 mice resulted in increased autoantibody production and glomerulonephritis. This enhanced disease was associated with an increase in CXCR3+ follicular helper T cells (TFH cells). TFH cells isolated from Apoe–/– mice had higher expression of genes associated with inflammatory responses and SLE and were more potent in inducing production of the immunoglobulin IgG2c. Mechanistically, the atherogenic environment induced the cytokine IL-27 from dendritic cells in a Toll-like receptor 4 (TLR4)-dependent manner, which in turn triggered the differentiation of CXCR3+ TFH cells while inhibiting the differentiation of follicular regulatory T cells. Blockade of IL-27 signals diminished the increased TFH cell responses in atherogenic mice. Thus, atherogenic dyslipidemia augments autoimmune TFH cell responses and subsequent IgG2c production in a TLR4- and IL-27-dependent manner.

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Fig. 1: Autoantibodies and autoimmune glomerulonephritis in Apoe–/– and Ldlr–/– recipients of BXD2 bone marrow.
Fig. 2: Analysis of GC reactions and TFH cell responses in Apoe–/– recipients of BXD2 bone marrow.
Fig. 3: Transcriptomic and functional analyses of TFH cells isolated from atherogenic mice.
Fig. 4: Atherogenic dyslipidemia induces IL-27 production by DCs.
Fig. 5: TLR4 and LXR regulate IL-27 production by DCs from atherogenic mice.
Fig. 6: IL-27 is necessary for the increased TFH cell responses and GC reactions in atherogenic mice.
Fig. 7: Patients with hypercholesterolemia exhibit increased autoantibodies and IL-27.

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  • 19 June 2018

    In the version of this article initially published, the third label along the horizontal axis of Fig. 4b (Il13a) and the middle label above each plot in Fig. 6k (Stat–/–) were incorrect, and the hash marks along the horizontal axis for Fig. 6i were spaced incorrectly. Also, the statistical results in the citation for Supplementary Fig. 5a (*P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired Student’s t-test)) in the fifth subsection of Results were incorrect. The correct label for Fig. 4b is Il23a and for Fig. 6k is Stat1–/–, and the right hash mark along the horizontal axis for Fig. 6i should be beneath the data points at right. The correct citation of the statistical results is as follows: “(P < 0.05 and P < 0.01 (unpaired Student’s t-test); Supplementary Fig. 5a).” The errors have been corrected in the HTML and PDF version of the article.

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Acknowledgements

We thank K.-W. Kim and S.-J. Bae for support in flow cytometry; S. Akira (Osaka University) and S-Y. Sung (Seoul National University) for Stat3fl/fl and Tlr4–/– mice; H. S. Kim (Ulsan University) for Stat1–/– mice; J.-H. Choi (Hanyang University) for Apoe–/– mice; S-A. Yoo (The Catholic University of Korea) for animal models; the entire Chung lab for suggestion and discussion; and J. Reynolds, G. Martinez and D.-S. Kuen for proofreading the manuscript. Supported by the National Research Foundation of Korea (research grants 2014R1A2A1A11054364, 2017R1A2B3007392 and 2017K1A1A2004511 to Y.C., and 2015R1D1A1A01059719 to M.C.).

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Authors and Affiliations

  1. Laboratory of Immune Regulation, Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea

    Heeju Ryu, Hoyong Lim, Garam Choi, Young-Jun Park, Minkyoung Cho, Hyeongjin Na & Yeonseok Chung

  2. BK21 Plus program, College of Pharmacy, Seoul National University, Seoul, Republic of Korea

    Heeju Ryu, Garam Choi, Young-Jun Park, Hyeongjin Na, Chul Won Ahn, Young Chul Kim & Yeonseok Chung

  3. Laboratory of Toxicology, College of Pharmacy, Seoul National University, Seoul, Republic of Korea

    Chul Won Ahn & Young Chul Kim

  4. Center for Integrative Rheumatoid Transcriptomics and Dynamics, The Catholic University of Korea, Seoul, Republic of Korea

    Wan-Uk Kim

  5. Division of Cardiology, Department of Internal Medicine, Severance Hospital, College of Medicine, Yonsei University, Seoul, Republic of Korea

    Sang-Hak Lee

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Contributions

H.R., H.L., G.C. and H.N. performed the in vitro and in vivo experiments; Y.-J.P. generated Ebi3–/–Apoe–/– mice; M.C. generated the Tlr4–/– bone marrow chimeras; C.W.A. and Y.C.K. provided Ldlr–/– mice; H.R., W.-U.K. and Y.C. analyzed the data; S.-H.L. provided the human plasma samples and patient information; H.R. and Y.C. wrote the manuscript; and all authors complied in submission of the manuscript for publication.

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Correspondence to Yeonseok Chung.

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Supplementary Figure 1 Apoe–/– recipients of BXD2 bone marrow did not exhibit augmented autoantibody production without HFD.

(a-b) Bone marrow chimeric mice were generated as described in Fig. 1 and were subjected to normal chow (NC). (a) ELISA of autoantibodies against dsDNA, and histone in the sera. (b) ELISA of autoreactive IgG subclass autoantibodies against dsDNA. (c) Gating strategy used for flow cytometry analysis of each cell types. (d-g) Representative flow cytometry data of germinal center B (d), plasma (e), TFH cells (f), and TFR (g) cells in the spleens of the bone marrow chimeric mice depicted in Fig. 2b,c. Data are one representative experiment of four independent experiments with n=5 per group.

Supplementary Figure 2 Germinal center reactions and TFH cell responses against exogenous antigen are augmented in atherogenic mice.

(a,b) Wild-type or Apoe–/– mice were subjected to HFD for four weeks before being immunized with chicken type II collagen in CFA. (a) Levels of collagen-specific antibodies in the sera of the indicated mice. (b) Flow cytometric analysis of germinal center B cells, plasma cells, and TFH cells in the lymph nodes of the indicated mice. Data are one representative experiment of two independent experiments with n=5 per group. (c-e) Wild-type or Apoe–/– mice were subjected to HFD for four weeks, immunized with OVA-NP and analyzed after seven days. (c) ELISA of NP-specific global affinity antibodies (NP29) in the sera of the indicated mice. (d) ELISA of NP-specific high affinity antibodies (NP7) in the sera of the indicated mice. (e) Flow cytometric analysis of germinal center B cells, plasma cells, and TFH cells in the lymph nodes of the indicated mice. Data are one representative experiment of four independent experiments with n=5 per group. *, p<0.05; **, p<0.01; ***, p<0.001 in comparison with WT (unpaired Student’s t-test, mean+SEM).

Supplementary Figure 3 CXCR3+ TFH cells are more potent in inducing IgG2c production in an IFNγ-dependent manner.

(a-c) CXCR3– or CXCR3+ TFH cells from HFD-fed Apoe–/– mice were flow-sorted and were co-cultured with naive B cells for seven days in the presence of anti-CD3 and anti-IgM. Amounts of IgG1 (a), IgG2c (b) or IgG2c/IgG1 (c) in the supernatant were determined. (d-f) CXCR3+ TFH cells were co-cultured with naive B cells as described above. Cells were treated with anti-IFNγ antibody or corresponding isotype control every other day. Amounts of IgG1 (d), IgG2c (e) or IgG2c/IgG1 (f) in the supernatant were determined. Data are representative of two independent experiments with n=4 per group. (g) Absolute numbers of TH1, TH17, and TFH cells of the indicated mice. (h) TFH cells or non-TFH (CD4+CD44+PD-1-CXCR5–) cells from HFD-fed Apoe–/– mice were flow-sorted and were co-cultured with naive B cells for seven days in the presence of anti-CD3 and anti-IgM. Amount of IgG2c in the supernatant were determined. (i) Heat map of the indicated genes that were differentially expressed subjected to RNA-seq analysis. Data are representative of two independent experiments with n=4 per group. *, p<0.05; **, p<0.01; ***, p<0.001 between the two groups (unpaired Student’s t-test, mean+SEM).

Supplementary Figure 4 Atherogenic condition promotes IL-27 production by CD11b+ dendritic cells through expression of pattern recognition receptors.

(a) Bone marrow-ablated wild-type or Apoe–/– mice were i.v. transferred with bone marrow cells from BXD2 mice, were subjected to normal chow (NC) or high fat diet (HFD). Levels of IL-27 in the sera of the indicated mice. Data are representative of two pooled independent experiments with n=5 per group. (b,c) Linear-regression analysis between serum IL-6 (b) or IL-27 (c) and TFH. Data are one representative experiment of four independent experiments with n=5 per group. (d-f) Bone marrow-ablated wild-type, Apoe–/– or Ebi3–/–Apoe–/– mice were i.v. transferred with bone marrow cells from congenic mice (WTSJL, ApoESJL, or Ebi3xApoESJL, respectively), and were subjected to high fat diet (HFD). (d) Levels of IL-27 in the sera of the indicated mice. (e,f) Flow cytometric analysis of lymphocytes (e) and dendritic cells (f). Data are one representative experiment of two independent experiments with n=4 per group. (g) Bone marrow chimeric mice were generated and treated as described in Fig. 1 (n=5). CD11c+ dendritic cells were isolated and quantitative RT-PCR analysis of the indicated genes were determined. Data are representative of two independent experiments with n=4 per group. *, p<0.05; **, p<0.01; ***, p<0.001 in comparison with WT (unpaired Student’s t-test, mean+SEM).

Supplementary Figure 5 Lipid accumulation in dendritic cells alters metabolic profile and differs in LXR expression.

(a) Bone marrow chimeric mice were generated as described in Fig. 5. The levels of NP-specific antibodies were analyzed (a). Data are one representative experiment of two independent experiments with n=4 per group. (b-f) CD11c+ dendritic cells were isolated from the indicated mice and were analyzed for ECAR profile (b), glycolysis, glycolytic capacity (c), OCR profile (d), basal respiration, ATP production, and maximal respiration (e). Energy map of dendritic cells isolated from the indicated mice (f). Data are representative of three independent experiments with two technical replicates (n=5 per group). (g) BODIPY staining of each cell types. Data are one representative experiment of three independent experiments with n=5 per group. (h-j) Splenocytes from indicated mice were enriched with CD11c (h) and flow-sorted (i). The expression of LXRβ was examined by Immunoblot analysis. Quantified values of the band intensities are presented at the bottom of each blot. Cropped blot images (h). Quantitative RT-PCR analysis of the indicated gene were determined (i). Original gel image from Supplementary Fig. 6h (j). Data are one representative experiment of three independent experiments with n=5 per group. *, p<0.05; **, p<0.01 between the two groups except in a. (a) *, p<0.05; **, p<0.01; ***, p<0.001 in comparison with ApoEWT. #, p<0.05; ##, p<0.01 in comparison with WTWT mice (unpaired Student’s t-test, mean+SEM).

Supplementary Figure 6 IL-27 plays an essential role on the antibody production.

(a) Mice were generated and treated as described in Fig. 6. Levels of total IgG, IgG1 and IgG2c in the sera of the indicated mice. (b,c) Mice were generated and treated as described in Supplementary Figure 2a,b. Levels of collagen-specific antibodies in the sera (b). Flow cytometric analysis of germinal center B cells, plasma cell and TFH cells in the lymph nodes of the indicated mice (c). Data are one representative experiment of three independent experiments with n=5 per group. (d,e) Quantitative RT-PCR analysis of selected genes from the RNA-seq (d) or TFH-related genes (e) in the sorted TFH cells. Data are one representative experiment of two independent experiments with n=4 per group. Box-and-whisker: center line, media; box limits, min to max. (f,g) Bone marrow-ablated Apoe–/– mice were i.v. transferred with bone marrow cells from mixture of congenic and Il27ra–/– mice, and were subjected to high fat diet. Flow cytometric analysis of TFH cells, each TFH subset (f) and TFR cells (g). (h-k) Apoe–/– mice were fed with HFD for four weeks, immunized with KLH and analyzed after seven days. Each group of mice were injected with control IgG or anti-IL-6 antibody intraperitoneally every other day. (h) ELISA of KLH-specific antibodies in the sera of the indicated mice. (i-k) Flow cytometric analysis of germinal center B cells, TFH cells (i), TFH subset (j), and indicated CD4+ T cell subsets (k) in the lymph nodes of the indicated mice. (l) Flow cytometric analysis of TFH cells in the lymph nodes of the mice as described in Fig. 6j. Data are one representative experiment of two independent experiments with n=5 per group. *, p<0.05; **, p<0.01; ***, p<0.001 between the two groups (unpaired Student’s t-test, mean+SEM).

Supplementary Figure 7 A graphical summary of the present study.

Our study demonstrates that atherogenic condition triggers the secretion of IL-27 and IL-6 from CD11b+ dendritic cells in a TLR4-dependent manner. IL-27 activates STAT1 and STAT3 signaling pathway in T cells and increases the numbers of CXCR3+ TFH cells while suppressing TFR cells. These CXCR3+ TFH cells, in turn, stimulate the differentiation of autoreactive B cells into IgG2c-secreting plasma cells, and pathogenic IgG2c autoantibodies exacerbate autoimmune lupus. Thus hyperlipidemia not only induces cardiovascular diseases but also promotes autoimmune lupus.

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Ryu, H., Lim, H., Choi, G. et al. Atherogenic dyslipidemia promotes autoimmune follicular helper T cell responses via IL-27. Nat Immunol 19, 583–593 (2018). https://doi.org/10.1038/s41590-018-0102-6

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