Marine fish oils are not equivalent with respect to B-cell membrane organization and activation

Abstract

We previously reported that docosahexaenoic-acid (DHA)-enriched fish oil (DFO) feeding altered B-cell membrane organization and enhanced B-cell function. The purpose of this study was to evaluate whether menhaden oil (MO) and eicosapentaenoic-acid (EPA)-enriched fish oil (EFO) alters B-cell function/phenotype similarly. Mice were fed control (CON), MO, EFO or DFO diets for 5weeks. We evaluated the fatty acid composition of B-cell phospholipids, membrane microdomain organization, ex vivo B-cell functionality and in vivo B-cell subsets. Red blood cells and B cells were found to be strongly (r>0.85) and significantly (P<.001) correlated for major n-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFAs). Compared to CON, MO and DFO resulted in decreased clustering of membrane microdomains, whereas EFO increased clustering. All fish oil treatments had 1.12-1.60 times higher CD40 expression following stimulation; however, we observed 0.86 times lower major histocompatibility complex class II expression and 0.7 times lower interleukin (IL)-6 production from EFO, but 3.25 times higher interferon-γ from MO and 1.5 times higher IL-6 from DFO. By 90min of incubation, MO had 1.11 times higher antigen uptake compared to CON, whereas EFO was 0.86 times lower. All fish oil treatments resulted in decreasingly mature splenic and bone marrow B-cell subsets. We conclude that diets high in n-3 LCPUFAs may elicit similar B-cell phenotypes but different organizational and functional outcomes. More specifically, these data suggest that the EPA and DHA content of a diet influences immunological outcomes, highlighting the importance of understanding how specific n-3 LCPUFAs modulate B-cell development and function.

Keywords: B cell; Fish oil; Immunomodulation; Inflammation; Membrane.

Figure 1. n-3/6 LCPUFA composition of experimental diets and phospholipids from murine red blood cells and B cells

(A) The EPA, DHA, and AA content of the experimental diets (CON, MO, EFO, and DFO) were analyzed by gas chromatography. Triplicates were run on a single batch of each diet. Correlations of the (B) EPA, (C) DHA, (D) AA, and (E) EPA+DHA content between red blood cell phospholipids and B cell phospholipids from mice fed either CON, MO, EFO, or DFO diets was performed. A Pearson's r was used to assess the linear correlation between the two samples from each animal; n = 4 mice/group.

Figure 2. Clustering of lipid microdomains on murine B cells

(A-D) Representative fluorescent images of lipid microdomains on purified, splenic B cells from SMAD3-/- mice fed CON, MO, EFO, or DFO diets. Cholera toxin subunit B conjugated to FITC was used to visualize GM1, an extensively used reporter of lipid rafts. Blinded scoring analysis of fluorescent lipid microdomains on cells as (E) clustered or (F) non-clustered. Data are represented as mean ± SEM, whereby 10 cells were scored per animal and n = 10-15 mice/group. Different letters denote statistically significant differences at the P < 0.05 level.

Figure 3. Expression of B cell surface markers and secreted cytokines in LPS-stimulated murine B cells

Purified, splenic B cells from SMAD3-/- mice fed CON, MO, EFO, or DFO diets were cultured for 24 h in the presence of 1μg/mL LPS. Flow cytometry on LPS-stimulated B cells was used to assess expression of B cell surface markers: (A) CD40 and (B) MHCII expression as a fold-change over CON. Flow-based multiplex assay was used to quantify secreted cytokines in the supernatants of LPS-stimulated B cells, including: (C) IL-6, (D) TNF-α, and (E) IFN-γ as a fold-change of cytokine production over CON. Data are represented as mean ± SEM; n = 5-15 mice/group. Each of the fish oil treatments were performed separately and therefore have their own controls. Different letters denote significant difference between treatment fold-changes in response to stimulation. Asterisks indicate significant differences compared to the CON diet: * P < 0.05

Figure 4. Antigen uptake in murine B cells

Uptake of ovalbumin conjugated to FITC (OVA-FITC) was used as a model antigen to assess B cell antigen uptake. Purified, splenic B cells from SMAD3-/- mice fed CON, MO, EFO, and DFO diets were incubated at 37°C for 0, 60, and 90 min. (A+C+E) Representative histograms demonstrate an increase in the MFI of OVA-FITC from 0 min (filled, light grey) to 60 min (dashed, dark grey) to 90 min (solid black). (B+D+F) Change over time of OVA-FITC MFI on purified B cells. Data are represented as mean ± SEM; n = 10-15 mice/group. Each of the fish oil treatments were performed separately and therefore have their own controls. Asterisks indicate significant differences compared to the CON diet: * P < 0.05 *** P < 0.001

Figure 5. in vivo B cell subset phenotyping of spleen and bone marrow

Flow cytometric phenotyping of B cell subsets was performed on spleen and bone marrow tissues in SMAD3-/- mice fed CON, MO, EFO, or DFO diets. (A) Splenic B cells (B220+) subsets were phenotyped as T1 B cells (CD23-CD24^Bright CD21-), T2 B cells (CD23- CD24^Bright CD21^Bright/Dim and CD23+ CD24^Bright/Dim CD21^Bright), follicular B cells (CD23+/- CD24^Dim CD21^Dim), and marginal zone B cells (CD23- CD24^Bright/Dim CD21^Bright). (B) Bone marrow precursor and developmental B cells (B220+) subsets were phenotyped as pre-pro-B cells (IgD- IgM- CD24+ CD43-), pro-B cells (IgD- IgM- CD24+ CD43+), pre-B cells (IgD-IgM- CD24- CD43), immature B cells (IgM+ IgD-), and mature B cells (IgM+ IgD+). Data are represented as mean ± SEM; n = 10 mice/group. Asterisks indicate significant differences compared to the CON diet: * P < 0.05 ** P < 0.01