Innate response activator B cells aggravate atherosclerosis by stimulating T helper-1 adaptive immunity

Abstract

Background: Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose, and complement each other's function and shape the course of disease can illuminate our understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of granulocyte macrophage colony-stimulating factor-secreting cells of hitherto unknown function in atherosclerosis.

Methods and results: Here, we show that IRA B cells arise during atherosclerosis in mice and humans. In response to a high-cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell-derived granulocyte macrophage colony-stimulating factor develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classic dendritic cells, which then generate interferon γ-producing T helper-1 cells. This IRA B cell-dependent T helper-1 skewing manifests in an IgG1-to-IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins.

Conclusions: Granulocyte macrophage colony-stimulating factor-producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a T helper-1-associated milieu that aggravates atherosclerosis.

Keywords: B-lymphocytes; T-lymphocytes; atherosclerosis; dendritic cells; granulocyte-macrophage colony-stimulating factor; immunology.

Figure 1

GM-CSF producing IRA B cells expand in atherosclerosis. (A) Identification of GM-CSF⁺ B220⁺ IgM^high IRA B cells in spleens of Ldlr^−/− and Apoe^−/− mice after 3 months of HCD by flow cytometry and (B) immunofluorescence histochemistry. (C) Flow cytometry based enumeration of IRA B cells in peripheral blood (per ml), total bone marrow, spleen, peritoneal lavage, four para-aortic lymph nodes and aorta in aged-matched Ldlr^−/−, C57Bl/6 WT and Apoe^−/− mice after 3 months of normal chow diet (gray) and HCD (white), respectively (n ≥ 3 mice per group). Cell counts are presented as mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, comparing chow vs. HCD per organ. (D) Kinetics of IRA B cell development in spleens of Apoe^−/− mice. 8 week old Apoe^−/− mice were placed on HCD and sacrificed after 4, 8, 12 and 24 weeks on HCD to quantify IRA B cell numbers (n ≥ 3 mice per time point). Cell counts are presented as mean ± SEM. (E) On the left, identification of IgM⁺ GM-CSF⁺ IRA B cells in the spleen of a patient with atherosclerosis. On the right, quantification of IRA B cells in spleen sections of patients with (white) or without (gray) symptomatic cardiovascular disease (CVD). Cells were counted in 12 randomly selected visual fields of 0.1mm² per sample. The combined number of IRA B cells in all 12 visual fields per patient was divided by the total area analyzed (12 × 0.1mm²). Results are presented as means ± SEM, ** p ≤ 0.01, n = 4 per group. For all flow cytometric plots, the ticks represent 0, 10², 10³, 10⁴, 10⁵ fluorescence units, except axes labeled “SSC,” for which the ticks represent 0, 50 000, 100 000, 150 000, 200 000, and 250 000 fluorescence units.

Figure 2

IRA B cells promote atherosclerosis. (A) For generation of mixed bone marrow chimeras with B cell restricted GM-CSF deficiency (IRA B KO) lethally irradiated 8 week old Ldlr^−/− mice were reconstituted with a 50:50 mixture of GM-CSF deficient (Csf2^−/−) and B cell deficient (µMT) bone marrow (white). Control mice were reconstituted with a 50:50 mixture of GM-CSF deficient (Csf2^−/−) and WT bone marrow (gray). After 6 weeks of reconstitution mice were placed on HCD for another 10 weeks. (B) Validation of B cell restricted GM-CSF deficiency in IRA B KO mice after reconstitution and 10 weeks of HCD. Identification of GM-CSF (Csf2) mRNA expression by semi-quantitative reverse transcription PCR in sorted CD3⁺ (T cells), CD19⁺ (B cells) and CD11b⁺ (myeloid cells) splenocytes from control and IRA B KO mice. Rpl19 serves as the housekeeping gene. (C) En face Oil-Red-O (ORO) staining of excised aortas from control and IRA B KO mice after 10 weeks of HCD on the left and quantification of lesion area on the right (n = 7 per group). Results are presented as means ± SEM, ** p ≤ 0.01, gray color for control, white color for IRA B KO mice. (D) Representative H&E staining of aortic root sections from control (gray) and IRA B KO (white) mice after 10 weeks of HCD with quantification of lesion size in two independent experiments (n ≥ 20 per group). Results are presented as means ± SEM. In addition immunohistology depicting ORO-, Mac3-, smooth muscle actin (SMA)-, Masson’s trichrome (Masson) and CD4-positive staining of aortic root lesions representative of both groups with quantification of n ≥ 10 samples per group. Results are presented as mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

IRA B cells promote the generation of T_H1 effector cells in atherosclerosis. (A) Representative dot plots showing gating for CD3⁺ CD4⁺ CD44^high CD62L^low T effector (T_eff) cells and CD3⁺ CD4⁺ Foxp3⁺ regulatory T cells (T_reg) in blood. (B) Kinetics of T_eff and T_reg cell development in blood and spleen as well as proportion of T_eff cells in para-aortic lymph nodes during 10 week HCD feeding of IRA B KO (white) and control (gray) mice. Results are presented as mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, comparing IRA B KO vs. control mice at 10 weeks, n ≥ 6 per group. (C) Representative dot plots showing gating for CD3⁺ CD4⁺ IFNγ⁺ T cells in blood. (D) Quantification of IFNγ-producing T cells in blood, spleen and para-aortic lymph nodes after 10 week HCD feeding of IRA B KO (white) and control (gray) mice. Results are presented as mean ± SEM, * p ≤ 0.05, n ≥ 10 per group. (E) Kinetics of total IgG and IgM serum levels during 10 week HCD feeding of IRA B KO (white) and control (gray) mice. Results are presented as mean ± SEM, n ≥ 6 per group and time point. (F) Quantification of IgG_2c antibody titers against MDA-LDL and copper-oxidized LDL (CuOxLDL) in 1:25 diluted individual serum samples, n ≥ 10 per group. Results are presented as mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01. (G) Quantitative ratio of IgG_2c and IgG₁ titers against MDA-LDL and copper-oxidized LDL (CuOxLDL) in 1:25 diluted individual serum samples, n ≥ 10 per group. Results are presented as mean ± SEM fold changes of the IgG_2c : IgG₁ ratio to illustrate shifts in isotype switching between control and IRA B KO mice, * p ≤ 0.05, ** p ≤ 0.01.

Figure 4

IRA B cells promote the generation of classical dendritic cells (cDC) in atherosclerosis. (A) Representative dot plots showing gating for CD19⁻ MHCII^high CD11c^high classical (c)DC, CD8⁻ CD11b⁺, CD8⁺ CD11b⁻ and CD8⁺ CD103⁺ subsets in the spleen. (B) Kinetics of splenic cDC subset development during 10 week HCD feeding of IRA B KO (white) and control (gray) mice. Results are presented as mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, comparing IRA B KO vs. control mice at 10 weeks, n ≥ 6 per group. (C) Identification and quantification of the proportion of cDC in para-aortic lymph nodes. Results are presented as mean ± SEM, * p ≤ 0.05, n ≥ 10 per group. (D) Quantification of IL-12p40 expression in splenic cDC sorted from IRA B KO (white) and control (gray) mice by real-time PCR. Results are presented as mean ± SEM fold change of 2^ΔCt, * p ≤ 0.05, n ≥ 10 per group. (E) Ldlr^−/− mice were lethally irradiated, reconstituted with a 50:50 mixture of CD45.1⁺ WT (black) and CD45.2⁺ Csf2rb^−/− (white) bone marrow and placed on HCD for 3 months. (F) Assessment of chimerism for CD45.1 (WT in black) and CD45.2 (Csf2rb^−/− in white) in CD11b⁺ and CD8⁺ splenic cDC. Results are presented as mean ± SEM, * p ≤ 0.05, n = 5 per group. (G) Quantification of IL-12p40 expression in sorted CD45.1 (WT in black) and CD45.2 (Csf2rb^−/− in white) splenic cDC by real-time PCR. Results are presented as mean ± SEM fold change of 2^ΔCt, * p ≤ 0.05, n = 5 per group. (H) Flow assisted cell sorting of CD23^low IgM^high CD43^high CD138^high cells from WT and Csf2^−/− mice after 4 × 25mg/day LPS i.p. Representative dot plot showing enrichment for GM-CSF⁺ IRA B cells in WT mice. Dashed lines represent isotype controls. (I) Representative dot plot showing MHCII and CD11c expression in lineage depleted (Lin = CD3, CD90.2, CD19, B220, NK1.1, Ly6G) CD45.1⁺ bone marrow cells before in vitro culture. Dashed lines represent isotype controls. (J) Representative dot plot showing high MHCII, CD11c, CD86 and CD40 expression on bone marrow derived DC (BMDC) generated through co-culture with IRA B cells and IL-4 over 8 days. Dashed lines represent isotype controls. (K) Enumeration of MHCII⁺ CD11c⁺ BMDC after co-culture with medium alone (dark gray), medium plus IL-4 (black), IRA B cells and IL-4 (gray), or corresponding B cells from LPS challenged GM-CSF^−/− mice with IL-4 (white). Results are presented as mean ± SEM, * p ≤ 0.05, comparing WT vs. all other groups by ANOVA, n ≥ 3 per group. (L) Evaluation of dendritic cell morphology of BMDC generated with IRA B cells or Csf2^−/− B cells. Representative phase contrast microscopy images are shown on the left. Quantification of cells with typical dendritiform protrusions per visual field is shown on the right. Results are presented as mean ± SEM analyzed in 6 visual fields per well and group, * p ≤ 0.05, ** p ≤ 0.01. (M) CD4⁺ CD25⁻ OT-II cells were co-cultured with IRA B cell-generated BMDC loaded with ovalbumin (OVA; 100µg/ml) or BSA (100µg/ml) for 4 days. Representative histograms show cell divisions of OT-II T cells labeled with a cell tracer dye.

Figure 5

Transfer of GM-CSF competent B cells aggravates atherosclerosis. (A) Experimental strategy for B cell adoptive transfer. Naive IRA B KO mice were divided into three groups receiving either 2.5 × 10⁷ CD19⁺ B cells from WT (gray) or Csf2^−/− (white) mice (n = 7 per group) or vehicle (DPBS) alone (black) (n = 5) at week 0 and 4 of a 8 week period of HCD feeding. (B) Representative dot plots showing identification of IRA B cells in the spleen of a CD45.2⁺ IRA B KO recipient on HCD 8 weeks after transfer of 25 × 10⁶ CD45.1⁺ WT B cells twice, 4 weeks apart. (C) Quantification of GM-CSF (Csf2) expression in whole spleen tissue of IRA B KO mice 8 weeks after transfer of WT (gray), Csf2^−/− (white) (n = 7 per group) or no B cells (black; n = 5) by real-time PCR. Results are presented as mean ± SEM fold change of 2^ΔCt, * p ≤ 0.05, comparing WT vs. the other groups by ANOVA. (D) Enumeration of spleen cDC, blood T effector cells, and blood IFNγ-producing T cells in recipients of WT (gray) cells compared to those receiving Csf2^−/− (white) B cells (n = 7 per group) or vehicle (black; n = 5) after 8 weeks HCD feeding. Results are presented as mean ± SEM, * p ≤ 0.05, comparing WT vs. the other groups by ANOVA. (E–G) Quantification of T_H1-associated Tbet and IFNγ, T_reg-associated Foxp3, TGFβ₁ and IL-10, and T_H2- and T_H17-associated GATA3, IL-4, RORγT and IL-17 expression in aortic tissue of WT (gray) versus Csf2^−/− (white) B cell recipients (n = 7 per group) and vehicle group (black; n = 5) by real-time PCR. Results are presented as mean ± SEM fold change of 2^ΔCt, * p ≤ 0.05, comparing WT vs. the other groups by ANOVA. (H) Quantification of ORO-rich areas in aortic root sections of recipients of WT (gray) versus Csf2^−/− (white) B cells (n = 7 per group) and vehicle group (black; n = 5) on the right and representative images on the left. Results are presented as mean ± SEM, * p ≤ 0.05, comparing WT vs. the other groups by ANOVA. (I–L) Representative images and quantification of Mac3-, CD4-, SMA-positive and Masson’s trichrome staining in aortic root sections of the three groups. Results are presented as mean ± SEM, * p ≤ 0.05, comparing WT vs. the other groups by ANOVA.

Figure 6

Model of IRA B cell-dependent T_H1 skewing during atherosclerosis. During atherosclerosis IRA B cells arise in secondary lymphoid organs via Myd88-dependent signaling and promote the generation of classical IL-12 producing classical dendritic cells (cDC). CD4⁺ T-helper cells that recognize disease related antigens (i.e. oxidation specific epiptopes) presented by these cDC differentiate into IFNγ-producing T_H1 cells. T_H1 cells infiltrate atherosclerotic lesions and stimulate macrophages. Antigen-specific interaction between T_H1 cells and B cells leads to IFNγ-dependent isotype switching from IgG1 to IgG2a/c which carry the highest Fcγ-receptor mediated activation capacity. By instructing T_H1-priming cDC IRA B cells aid in bridging innate and adaptive immunity. Solid arrows depict functional relationship and dashed arrows depict spatial relationship.