Frontline Science: A reduction in DHA-derived mediators in male obesity contributes toward defects in select B cell subsets and circulating antibody

Abstract

Obesity dysregulates B cell populations, which contributes toward poor immunological outcomes. We previously reported that differing B cell subsets are lowered in the bone marrow of obese male mice. Here, we focused on how lipid metabolites synthesized from docosahexaenoic acid (DHA) known as specialized pro-resolving lipid mediators (SPMs) influence specific B cell populations in obese male mice. Metabololipidomics revealed that splenic SPM precursors 14-hydroxydocosahexaenoic acid (14-HDHA), 17-hydroxydocosahexaenoic acid (17-HDHA), and downstream protectin DX (PDX) were decreased in obese male C57BL/6J mice. Simultaneous administration of these mediators to obese mice rescued major decrements in bone marrow B cells, modest impairments in the spleen, and circulating IgG2c, which is pro-inflammatory in obesity. In vitro studies with B cells, flow cytometry experiments with ALOX5-/- mice, and lipidomic analyses revealed the lowering of 14-HDHA/17-HDHA/PDX and dysregulation of B cell populations in obesity was driven indirectly via B cell extrinsic mechanisms. Notably, the lowering of lipid mediators was associated with an increase in the abundance of n-6 polyunsaturated fatty acids, which have a high affinity for SPM-generating enzymes. Subsequent experiments revealed female obese mice generally maintained the levels of SPM precursors, B cell subsets, and antibody levels. Finally, obese human females had increased circulating plasma cells accompanied by ex vivo B cell TNFα and IL-10 secretion. Collectively, the data demonstrate that DHA-derived mediators of the SPM pathway control the number of B cell subsets and pro-inflammatory antibody levels in obese male but not female mice through a defect that is extrinsic to B cells.

Keywords: 14-HDHA; 17-HDHA; PDX; n-3 polyunsaturated fatty acids.

Conflict of interest statement

DISCLOSURES

The authors declare that they have no conflicts of interest with the contents of this article.

©2018 Society for Leukocyte Biology.

Figures

FIGURE 1. Metabolic profile of male C57BL/6J mice consuming a high fat diet.

(A) Body weights of male mice at the completion of the study. (B) Body composition assayed by Echo-MRI after 15 weeks of consuming experimental diets. (C) qRT-PCR analysis of inflammatory cytokines in the adipose tissue of control and obese mice. (D) Glucose tolerance test (GTT), performed by intraperitoneal injection of glucose after a 6 h fast. Inset shows the area under the curve (AUC), calculated by integration of the glucose curves shown in subpart (D), normalized to baseline values. (E) Fasting blood glucose and (F) insulin levels determined after a 6 h fast. (G) HOMA-IR scores. (H) Fasting leptin levels. N = 9–10 mice per diet (A, B, and D), N = 8 mice per diet (C), N = 6–8 mice per diet (E–H). Data are average ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by a Student’s unpaired t-test (A–C, E–H) or a two-way ANOVA analysis followed by a post hoc t-test (D)

FIGURE 2. DHA-derived SPM precursors and SPMs are lowered in obese male mice.

Metabololipidomic analyses of (A) DHA-derived SPM precursors and SPMs, (B) AA-derived HETES, (C) AA-derived LXA4 and leukotrienes, and (D) AA-derived thromboxanes in the spleens of mice consuming a control or high fat diet. Metabololipidomic analyses of (E) DHA-derived SPM precursors and SPMs, (F) AA-derived HETEs, (G) AA-derived LXA4 and leukotrienes, and (H) AA-derived thromboxanes in the bone marrow of control and obese mice. N.D.: not detectable. Mice consumed experimental diets for 15 weeks. N = 6 mice per diet (A–H). Data are average ± S.E.M. *P < 0.05 by an unpaired Student’s t-test

FIGURE 3. DHA-derived SPM precursors modestly increase germinal center B cells of male obese mice accompanied by increased IgG2b and a rescue of IgG2c.

(A) Sample flow cytometry plots of splenic B cell subsets for mice consuming a control, high fat, or high fat + 14-HDHA/17-HDHA/PDX including: B220+IgM+CD21lowCD23− (T1), B220+IgM+CD21−CD23+ (T2), B220+IgM+CD21highCD23+ (T2-MZ), B220+IgMlowCD21intCD23+ (Follicular), B220+IgMhighCD21+CD1dhigh (Marginal), B220+CD19+IgM+GL7+ (Germinal), B220+IgM+CD35+CD80+ (IgM+ Memory), B220lowIgM+CD138+ (Plasmablasts). (B) Percentage and (C) number of splenic B cell subsets. (D) Serum concentrations of IgG2a, IgG2b, IgG2c, and IgG3. Mice consumed experimental diets for 15 weeks. Mice received either an injection of vehicle control or a cocktail (900 ng/mouse) consisting of the SPM precursors 14-HDHA, 17-HDHA, and the SPM PDX once every day for 4 consecutive days. N = 6–10 mice per condition. Data are average ± S.E.M. *P < 0.05 by a one-way ANOVA followed by a Bonferroni post test (B and C)

FIGURE 4. DHA-derived SPM precursors and SPM rescue the decrease in the number of bone marrow B cell subsets of obese male mice.

(A) Sample flow cytometry plots for total CD19+ B cells in the bone marrow of mice consuming a control, high fat, and high fat diet +14-HDHA/17-HDHA/PDX. (B) Percentage and (C) number of CD19+ cells in the bone marrow. (D) Sample flow cytometry plots for B cell subsets in the bone marrow including CD19+CD43+CD24+IgM− (pro), CD19+CD43−CD24+IgM− (pre), CD19+CD24+IgM+IgD− (immature), CD19+IgM+IgD+ (mature), and CD19+CD138+ (long-lived plasma cells, LLPCs). (E) Percentage and (F) number of B cell subsets in the bone marrow. Mice received either an injection of vehicle control or an SPM cocktail (900 ng/mouse) consisting of 14-HDHA, 17-HDHA, and PDX once every day for 4 consecutive days. N = 7–10 mice per condition. Data are average ± S.E.M. *P < 0.05, **P < 0.01 by a one-way ANOVA followed by a Bonferroni post test

FIGURE 5. The reduction in SPM precursor production of male mice is not driven by a defect in dietary DHA or B cell production of 14-HDHA and 17-HDHA.

(A) qRT-PCR analysis of SPM receptors in splenocytes and splenic B cells. (B) qRT-PCR analysis of 5-LOX and 12/15-LOX in splenocytes and splenic B cells. (C) Concentration of B cell SPM precursor production with or without substrate (DHA) relative to the whole spleen. (D) GC/MS analyses of splenic ALA (18:3 n-3), EPA (20:5 n-3), and DHA (22:6 n-3). (E) Fatty acid analysis of the major splenic n-6 polyunsaturated fatty acids including linoleic acid (LA) (18:2 n-6), eicosadienoic acid (EDA) (20:2 n-6), dihomo-gamma-linolenic acid (DGLA) (20:3 n-6), and arachidonic acid (AA) (20:4 n-6). Fatty acid analyses show milligrams of fatty acid (FA) per gram of spleen. Mice consumed experimental diets for 15 weeks. N = 7 mice per diet (A and B), N = 3 mice per condition (C) and N = 10 mice per diet (D and E). Data are average ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by an unpaired Student’s t-test

FIGURE 6. Metabolic profile of female mice consuming a high fat diet.

(A) Body weights of female mice at the completion of the 15-week study. (B) Body composition assayed by Echo-MRI. (C) qRT-PCR analysis of inflammatory markers from white adipose tissue of control and obese mice. (D) Glucose tolerance test (GTT), performed by intraperitoneal injection of glucose after a 6 h fast. (Inset) Area under the curve (AUC), calculated by integration of the curve normalized to baseline values. (E) Fasting blood glucose levels determined after a 6 h fast. (F) Fasting insulin levels determined after a 6 h fast. (G) HOMA-IR scores. (H) Fasting leptin levels. N = 8–10 mice per diet (A–D), N = 6–8 mice per diet (E–H). Data are average ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by a Student’s unpaired t-test (A–C, E–H) or a two-way ANOVA analysis followed by a post hoc t-test (D)

FIGURE 7. Obese female mice display small changes in splenic and bone marrow B cell subsets.

(A) Percentages and (B) numbers of B cell subsets in the spleen of control and obese female C57BL/6J mice. (C) Percentage and (D) number of total CD19+ B cells in the bone marrow. (E) Percentages and (F) number of B cell subsets in the bone marrow. Mice were fed lean control or high fat diets for 15 weeks. N = 7–8 mice per diet (A and B) and N = 9–10 for (C–F). Data are average ± S.E.M. *P < 0.05 by a Student’s unpaired t-test

FIGURE 8. Obese females have increased levels of plasma B cells relative to nonobese females and elevated levels of select cytokines.

(A) Sample flow cytometry data showing the percentage of CD45+CD14−CD19+ (B cells), CD45+CD3+CD8+ (cytotoxic T cells), CD45+CD3+CD4+ (helper T cells), and CD45+CD3−CD14+ (monocytes) in peripheral blood mononuclear cells. (B) Quantification of the percentages of B cells, monocytes, helper T cells, and cytotoxic T cells. (C) Sample flow cytometry plots of memory (CD19+IgD+CD27+) and plasma B cells (CD19+CD38+IgD−CD27+). (D) Quantification of the percentages of naïve, memory, and plasma B cells in peripheral blood mononuclear cells. (E) Ex vivo B-cell cytokine secretion upon CpG-ODN + anti-IgM or PAM3CSK4 stimulation. (F) Ex vivo IgM and IgG concentrations upon stimulation of B cells with CpG-ODN + anti-IgM for nonobese and obese females. N = 10 female human subjects per group for (A–D). N = 9 female human subjects per group for (E and F). Data are average ± S.E.M. *P < 0.05 by unpaired Student’s t test