Dexamethasone potentiates the responses of both Treg and B-1 cells to antigen immunization in the ApoE−/− mouse model of atherosclerosis

Abstract

The immunosuppressant dexamethasone was previously shown to preferentially deplete CD4⁺ Teff cells while sparing Treg cells in vivo. In the present study, we show that it also preferentially depletes B-2 cells while sparing B-1 cells. In the ApoE−/− mouse model of atherosclerosis, where both Treg and B-1 cells are thought to play an atheroprotective role, we show that HSP60-targeted immunization in the presence of dexamethasone raises Ag-reactive Treg and B-1 cells concomitantly and reduces the severity of atherosclerosis. These results indicate that dexamethasone is an adjuvant that potentiates both the Treg and the B-1 responses to immunogens. This study shows for the first time that B-1 cells with the specificity for a disease-relevant Ag can be raised in vivo by immunization.

Introduction

Autoimmune diseases and allergies are mediated by Ag-specific responses from pathogenic effector cells. Although Ag immunization represents an effective means to harness Ag-specific responses, finding the conditions that would drive Ag immunization toward tolerance remains a challenge. Our approach is to search for “tolerogenic adjuvants” that preferentially potentiate tolerogenic responses to immunogens.

Previously, Chen et al. showed that the immunosuppressant dexamethasone (Dex) affects effector T (Teff) cells and regulatory T (Treg) cells differently in that it depletes Teff cells while sparing Treg cells (1). Prompted by this finding, we evaluated the potential of Dex as a tolerogenic adjuvant and showed that Ag immunization in the presence of Dex (a strategy we named “suppressed immunization” or “SI”) expanded Ag-specific Treg cells while suppressing Ag-specific Teff cells and attenuated established allo- and auto-immunity (2). Subsequently, we showed that both the expansion of Ag-specific Treg cells and the efficacy of SI depended on tolerogenic, CD11c^loCD40^lo macrophages, which were also Dex-resistant (3). These studies thereby revealed a hitherto unappreciated selectivity of Dex in cell depletion that allows relatively intact Treg responses to immunogens.

To fully understand how Dex works as an adjuvant, we have further examined the selectivity of Dex in other cell subsets that can respond to immunization. In this report, we show that, mirroring the differential effects of Dex on the subsets of T cells, Dex affects the two major B cell subsets, B-1 and B-2, differently in that it preferentially depletes B-2 cells while sparing B-1 cells. In the ApoE−/− mouse model of atherosclerosis, where subsets of T and B cells have been reported to play different or, even, opposing roles in atherogenesis (4), we show that SI targeting the atherogenic Ag HSP60 results in the concomitant expansion of Ag-specific Treg and B-1 cells and the reduction in the severity of atherosclerosis in treated mice. Our study thus reveals that Dex’s adjuvant mechanism also involves the in vivo selection for B-1 cells.

Materials and Methods

Mice and reagents

All mouse strains were from the Jackson Laboratory and used in accordance with the institutional guidelines for animal care. The western type diet (WTD) was from Research Diets; reagents for immunostaining and immunohistochemistry, from Biolegend; and reagents for T and B cell isolation, from Miltenyi Biotec. The HP1 peptide (HSP60_292–308, KVGLQVVAVKAPGFGDN) and biotinylated HP1 were synthesized by Biomatik.

Identifying HSP60-derived peptides recognizable by both T and B cells (“dual epitoptes”)

HLA-DR and I-Ab double-restricted T-cell epitopes in human heat shock protein 60 (HSP60) were searched for using bioinformatics (5, 6). The resulting candidate epitopes were screened in ApoE−/− mice for reactive Ig by epitope-specific ELISA, which identified HP1 as a "dual epitope." HP1 overlaps partially with HSP60_291–305, which is the epitope recognized by the T cells clone 6.41 isolated from human atherosclerotic plaques (7). The HP1 sequence is identical between the human and the mouse.

Flow cytometry

CD4⁺ T cells were isolated from the spleen by MACS (negative selection), stained with CFSE, and restimulated in culture with Ag, as we previously described (2). Ag-reactive Treg (CD4⁺Foxp3⁺CFSE^lo) and Teff (CD4⁺Foxp3⁻CFSE^lo) cells were counted as percentages of the total CD4⁺ T cells plated (2). The gating strategies for identifying non-Ag-specific and Ag-specific B cell subsets are depicted in Supplemental Figs.1A and 2, respectively.

Tetramer staining

HP1-biotin and streptavidin-phycoerythrin (SA-PE) were combined at a 2:1 molar ratio to form the HP1-tetramer (HP1-SA-PE). B cells were stained with the tetramer (0.04 µg/10⁶ cells) in PBS/0.1% BSA/1 mM EDTA, in the presence of 4 µg of SA and 0.4 µg of the SA-PE/Cy5 tandem conjugate (Biolegend) as blockers.

ELISA of sera from ApoE−/− mice

Non-coated or HP1-coated (1 µg/well) 96-well plates were blocked with 1% Ficoll-Paque (GE Healthcare) in 0.05 M NaHCO₃ (pH 9.6). The rest of the steps were performed in PBS/0.05% Tween-20. Sera from ApoE−/− C57BL/6 mice were diluted (1:500) and added to the wells (50 µl/well). Serum Ab bound to the wells was typed with a C57BL/6 Ig clonotyping kit and a panel of standards (all from SouthernBiotech). HP1-specific O.D. was calculated by subtracting the O.D. of the non-coated wells from that of the HP1-coated wells.

Suppressed immunization

Dex was injected (i.m.) on days 1 (4.5 mg/kg), 2 (2.25 mg/kg), and 4 (2.25 mg/kg). HP1 was injected (s.c., 100 µg/mouse) on days 2 and 4.

Lesion analysis and immunohistochemistry

Lesions in the aortic root were analyzed by morphometric method as described (8). Lesions in the aortic arch were analyzed by Oil-Red-O solubilization method as described (9). Macrophage infiltrated areas in the aortic root sections were quantified by MAC-2 staining and computer-assisted image analysis.

Statistic analysis

Statistic significance was assessed by the unpaired two-sided Student’s t test.

Results and discussion

It was previously reported that Dex could induce apoptosis in B cells (10). However, the prior studies did not differentiate B cell subsets; thus, the susceptibility to Dex might differ among different subsets. We sought to determine whether this is the case for the two major B cell subsets, B-1 and B-2. Initially, we isolated total B cells from the peritoneal cavity (a site harboring abundant B-1 cells) of C57BL/6 and Balb/c mice, cultured the cells in the presence of Dex, and then quantified the apoptotic cells in individual B cell subsets by flow cytometry (the gating strategy for identifying the B cell subsets is shown in Supplemental Fig. 1A). More apoptotic cells were found in the B-2 subset than in the B-1 subset, regardless whether the cells were from C57BL/6 or Balb/c mice (Fig. 1A). This result indicates that the two B cell subsets do differ in their sensitivity to Dex. This finding is consistent with a separate study by Diehl et al. showing differential responses in B-1 and B-2 cells ex vivo to glucocorticoid receptor ligation by Dex (11).

FIGURE 1.

Dex preferentially retains B-1 cells and augments their production of IL-10. (A) Total peritoneal B cells from C57BL/6 and Balb/c mice were cultured for 18 h with Dex at 1 × 10⁻⁷ M and 5 × 10⁻⁷ M, respectively, or without Dex (NT). Apoptotic (annexin V⁺) B-1 and B-2 cells were counted by flow cytometry as a percentage of their respective subsets. Shown are the combined results from at least 3 independent experiments. *p = 0.002; **p = 0.009. (B) Mice (n = 3 per group per experiment) were injected (i.m.) with Dex (4.5 mg/kg); non-treated mice were used as the control. At 20 h, B-1a, B-1b, and B-2 cells in the treated mice were counted as a percentage of their corresponding subsets in the control. Shown are the combined results from 2 – 3 independent experiments. *p ≤ 0.05 between B-2 and B-1a or B-1b. (C) Balb/c mice (n = 3 per group per experiment) were injected with Dex as in B. At 20 h, total peritoneal B cells were isolated, stimulated for 4 h with the PMA/ionomycin/brefeldin A cocktail, intracellularly immunostained for IL-10, and analyzed by flow cytometry. Shown is 1 of 2 experiments with similar results. Thick line, IL-10 staining; broken line, isotype control staining; thin line, IL-10 staining of B cells from non-Dex-injected mice.

Because of this difference, we reasoned that Dex, if injected into animals, would preferentially deplete B-2 cells in vivo. To test this possibility, we injected a single pharmacological dose of Dex (4.5 mg/kg) into mice; at 20 h, we analyzed via flow cytometry the B cells in blood, the spleen, and the peritoneal cavity (Supplemental Fig. 1A). In this experiment, we divided B-1 cells further into the B-1a and B-1b subsets. In both of the mouse stains, Dex depleted significantly more B-2 cells than either B-1a or B-1b cells, regardless whether we counted the cells relatively as a percentage of the same subset in non-treated controls (Fig. 1B), or absolutely as count per site (Supplemental Fig. 1B). Given that B-1a and B-1b cells showed a similar degree of resistance to the depletion (Fig. 1B), we treated them as a single B-1 subset for the rest of this study. Additional in vivo experiments further showed that the preferential depletion of B-2 cells was Dex dose-dependent; as a result, B-1 cells were progressively enriched at the expense of B-2 cells within the Dex dose range of 1.5 – 13.5 mg/kg (Supplemental Fig. 1C). Lastly, the effect of Dex was abolished when the mice were injected with mifepristone, a specific inhibitor of the glucocorticoid receptor (Supplemental Fig. 1D).

An important property of both murine and human B-1 cells is that they constitutively produce the anti-inflammatory cytokine IL-10 as an autocrine growth factor (12, 13). We assessed the IL-10 production in the B-1 cells retained after the Dex treatment. Compared with those from untreated mice, the B-1 cells from Dex-treated mice showed increased IL-10 production (Fig. 1C) and IL-10 secretion (Supplemental Fig. 1E). This finding is similar to our previously reported upregulation of IL-10 production in the Dex-resistant macrophages (3). However, it remains unknown whether Dex increases IL-10 production by inducing de novo synthesis or by preferentially retaining a cell subpopulation expressing higher levels of IL-10.

Echoing our previous findings from T cells (2), we reasoned that Dex, when used as a tolerogenic adjuvant in Ag immunization, should preferentially potentiate B-1 (over B-2) responses to immunizing Ags. If this is true, SI, which was previously thought to induce Ag-specific Treg responses, should actually induce Ag-specific Treg and B-1 responses concomitantly.

We examined this possibility in the ApoE−/− C57BL/6 mouse model of atherosclerosis. Previous studies in this model had suggested that both Treg and B-1 cells play an atheroprotective role (4, 14–16), although this may not be the case in other disease settings; for example, a subpopulation of B-1 cell cells have been shown to play a pathogenic role in lupus (17). To that end, we decided to target HSP60 in SI, because it is a known atherogenic auto-Ag in both humans and the mouse models (18). To target both T and B cell axes simultaneously, we used a human HSP60-derived “dual epitope” (that can be recognized by both T and B cells), namely HP1 (HSP60_292–308). Initial experiments with HP1 showed that CD4⁺ T cells from HSP60-immunized ApoE−/− mice responded to HP1 restimulation in an Ag-specific manner (Fig. 2A), which indicates that HP1 matches an endogenously produced HSP60 epitope. Moreover, by staining with an HP1-tetramer (HP1-biotin:streptavidin-PE) and flow cytometry (the gating strategy is illustrated in Supplemental Fig. 2), we detected HP1-reactive B cells in the blood of non-immunized mice in an HP1-specific, B cell receptor (BCR)-dependent manner (Fig. 2B). This indicates a spontaneous B cell reactivity to HP1 in this model. Furthermore, compared with mice fed a normal chow diet (Chow), mice fed an atherogenic, western type diet (WTD) for 8 weeks showed increased percentages of HP1-tetramer⁺ B cells (Fig. 2C). Interestingly, in both Chow- and WTD-fed mice, B-2 cells (CD19⁺CD23⁺) were enriched in the HP1-tetramer⁺ B cells. While constituting the majority (~ 96%) of total blood B cells (data not shown), they constituted nearly all (~ 99%) of the tetramer⁺ B cells (Fig. 2C). Consistent with this finding, while the Chow- and WTD-fed groups showed similar levels of HP1-reactive serum IgM, IgG1, IgG2b, and IgG3 (data not shown), the WTD group showed a significantly higher level of HP1-reactive serum IgG2c (Fig. 2D), an isotype produced by activated B-2 cells. Collectively, these results suggest that WTD, while promoting atherogenesis, also promotes B-2 responses to HP1.

FIGURE 2.

HP1 matches an endogenously generated T-cell epitope that is recognized spontaneously by B cells. (A) ApoE−/− C57BL/6 mice were immunized with HSP60 (whole protein) in CFA. After 3 weeks, splenic CD4⁺ T cells (pooled from ≥ 2 mice per experiment) were restimulated in vitro with either HP1 (test) or MOG_35–55 (Ag specificity control). Splenic CD4⁺ T cells from non-immunized mice were stimulated with HP1 as the immunization control. T cells responding to restimulation were quantified as a percentage of the total CD4⁺ T cells plated. Shown are the combined results from 3 experiments. *p ≤ 0.04 between the test and either control. (B) Blood leukocytes from Chow-fed ApoE−/− mice (pooled from 3 mice) were stained with HP1-SA-PE ("Tetramer"). The resulting PE⁺ B cells were counted by flow cytometry as a percentage of the total B cells examined. As controls, leukocytes were stained with SA-PE (for HP1-independent binding) or blocked with either goat anti-mouse Ig Ab ("αmIg") or goat anti-human Ig Ab ("αhIg") prior to the tetramer staining (for BCR-independent binding). Shown are the combined results from 14 experiments. *p = 0.0001; **p = 0.004. (C) ApoE−/− mice (n = 3 per group per experiment) fed WTD or Chow for 8 weeks were analyzed for HP1-tetramer⁺ B cells in blood (filled bar) and the B-2 fraction in the tetramer⁺ B cells (open bar). Shown are the combined results from 5 experiments. *p = 0.03 between WTD- and Chow-fed mice. (D) Sera from the mice in C were immunotyped by ELISA, using HP1 as the capture Ag. Shown are the combined results from 4 experiments. *p = 0.03.

To determined whether SI could preferentially raise HP1-reactive Treg and B-1 cells despite the existing B-2 responses, we fed ApoE−/− mice WTD for 8 weeks first, and then treated them with 2 regimens of SI using Dex and HP1 ("SI"). At the start of the treatment, WTD was replaced with Chow for the rest of the experiment, because prolonged atherogenic stress from high lipids could mask the effect of T and B cells (4) and low-fat diets are more relevant clinically for any patients seeking an anti-atherosclerosis treatment. As controls, mice were non-treated (“NT”, injected with PBS), treated with Dex alone (“Dex”), or treated with HP1 alone (“HP1”). Two weeks after the completion of the treatment, the mice were examined for HP1-reactive T and B cells. Consistent with our previous observation in T cells (2, 3), only SI expanded Ag-specific Treg cells (Fig. 3A). In the B cell subsets, while appearing to reduce HP1-reactive B-2 cells slightly (p ≤ 0.08), SI expanded HP1-reactive B-1 cells (p ≤ 0.02) in both blood and the spleen; whereas HP1 failed to expand the B-1 cells at the same sites. In the peritoneal cavity, SI did not expand HP1-reactive B-1 cells, whereas HP1 increased HP1-reactive B-2 cells (p ≤ 0.02) (Fig. 3B). Collectively, these results demonstrate that SI can expand Ag-reactive Treg and B-1 cells concomitantly and that Dex is required for the selective expansion of these two cell subsets.

FIGURE 3.

SI with HP1 induces HP1-reactive Treg and B-1 cells concomitantly. (A) ApoE−/− mice (4 weeks of age) were fed WTD for 8 weeks, switched to Chow, and treated as indicated (n = 3 per group per experiment). After 2 weeks, splenic CD4⁺ T cells were purified, and the HP1-reative Treg and Teff cells were analyzed in culture as percentages of the total CD4⁺ T cells plated. Shown are the combined results from 2 experiments. *p ≤ 0.02 between SI and any of the other treatments in Treg cell count. (B) ApoE−/− mice treated as described in A (n ≥ 2 per group per experiment) were restimulated in vivo with an injection (i.p.) of the HP1 peptide (100 µg/mouse). Three days later, blood, spleen, and peritoneal cavity cells were isolated and stained with the HP1-tetramer. Tetramer⁺ B cells were counted as the absolute count per each of the anatomic sites. Shown are the combined results from 4 experiments. *p ≤ 0.02 between SI and any of the other treatments in B-1 cell count; **p ≤ 0.02 between HP1 and NT or SI in B-2 cell count.

We examined the severity of atherosclerosis in all the treated groups. Only the SI group showed a moderate (~ 30%), but statistically significant, reduction in aortic lesions (Fig. 4, A–C) and a similar reduction in lesional infiltration by macrophages (Fig. 4D). One mechanism by which B-1 cells play atheroprotective roles is the secretion of IgM (14, 19). Hence, we also analyzed Ab responses with serum samples, collected at the time of the aorta examination. Compared with the NT group, only the SI group showed an increase in HP1-reactive IgM (Fig. 4E), whereas the IgG2c levels were statistically equivalent among all groups. Thus, the co-expansion of HP1-reactive Treg and B-1 cells, and/or the relative suppression of the HP1-reactive Teff and B-2 cells, is associated with atheroprotection.

FIGURE 4.

SI using Dex and HP1 reduces atherosclerosis. ApoE−/− mice (4 weeks of age) were fed WTD for 8 weeks, switched to Chow, and treated as indicated. At 24 weeks of age, the atheroprotective efficacy of the treatment was analyzed. Data shown are from 2 experiments with a total of 8 – 10 mice per group. (A) Representative cross-sections of the aortic root stained with ORO. (B) Quantitation of the lesions in the aortic root. Lesion coverage was calculated as the percentage of the areas in a cross-section that were stained red with ORO. *p ≤ 0.02. The p values in B–D are all for the difference between SI and any of the other groups. (C) Quantitation of the lesions in the aortic arch. The aortic arch was stained with ORO; the incorporated stain was extracted with a solvent and quantified colorimetrically as the ORO concentration (µM) per mg of tissue extracted. *p ≤ 0.05. (D) Macrophage infiltration at the aortic root was quantified as the percentage of the areas (in a cross-section) stained positive with the MAC-2 mAb. *p ≤ 0.005. (E) Sera from the treated mice were analyzed by ELISA for HP1-reactive IgG2c and IgM. *p ≤ 0.04 between SI and NT in IgM.

In summary, we have shown that Dex preferentially depletes B-2 cells and potentiates the response of B-1 cells to immunogens. In the ApoE−/− mice, SI combining Dex and an HSP60-derived dual epitope induces the co-expansion of epitope-reactive Treg and B-1 cells, which is correlated with atheroprotection. It remains to be determined what respective roles these cell subsets each play in the atheroprotection. To achieve this goal, methods still need to be developed for selectively depleting the Ag-specific B-1 or Treg cells and for obtaining Ag-specific B-1 or Treg cells at quantifies sufficient for adoptive transfer. Nonetheless, the present study demonstrates the feasibility to raise B-1 cells in vivo with the specificity for a disease-relevant Ag. The possibility to potentially improve therapeutic efficacy via resetting the B-1/B-2 cell balance in an Ag-specific way emerges.