DHA-enriched fish oil targets B cell lipid microdomains and enhances ex vivo and in vivo B cell function

Abstract

DHA is a n-3 LCPUFA in fish oil that generally suppresses T lymphocyte function. However, the effect of fish oil on B cell function remains relatively understudied. Given the important role of B cells in gut immunity and increasing human fish oil supplementation, we sought to determine whether DFO leads to enhanced B cell activation in the SMAD-/- colitis-prone mouse model, similar to that observed with C57BL/6 mice. This study tested the hypothesis that DHA from fish oil is incorporated into the B cell membrane to alter lipid microdomain clustering and enhance B cell function. Purified, splenic B cells from DFO-fed mice displayed increased DHA levels and diminished GM1 microdomain clustering. DFO enhanced LPS-induced B cell secretion of IL-6 and TNF-α and increased CD40 expression ex vivo compared with CON. Despite increased MHCII expression in the unstimulated ex vivo B cells from DFO-fed mice, we observed no difference in ex vivo OVA-FITC uptake in B cells from DFO or CON mice. In vivo, DFO increased lymphoid tissue B cell populations and surface markers of activation compared with CON. Finally, we investigated whether these ex vivo and in vivo observations were consistent with systemic changes. Indeed, DFO-fed mice had significantly higher plasma IL-5, IL-13, and IL-9 (Th2-biasing cytokines) and cecal IgA compared with CON. These results support the hypothesis and an emerging concept that fish oil enhances B cell function in vivo.

Figure 1.. Lipid microdomain organization in B cells from SMAD−/− mice.

(A) Representative fluorescent images of lipid microdomains on purified B cells from SMAD−/− mice fed the CON (left) or DFO (right) diet. Cells were stained with CTxB conjugated to FITC for visualization of clustered GM1 molecules. (B) Lipid microdomain size on a micron scale, measured with the feret's diameter, of purified B cells from SMAD−/− fed the CON (open bar) or DFO (solid bar) diet. Data are represented as mean ± sem; n = 10. Data indicate significant differences between the DFO diet and the CON diet: ***P < 0.001.

Figure 2.. Expression of surface markers associated with cell signaling and cytokine production in LPS-stimulated, purified B cells from SMAD−/− mice.

Aliquots of splenic B cells, purified using negative selection and identified above as B220⁺, were cultured for 24 h in media containing 1 μg/mL LPS. (A) Cell surface marker expression of LPS-stimulated B cells in SMAD−/− mice fed the CON (open bars) or DFO (solid bars) diet. (B) Cytokine production in the supernatants of LPS-stimulated B cells from SMAD−/− mice fed the CON or DFO diet was assayed. Data are represented as mean ± sem; n = 8–10. Data indicate significant differences between the DFO diet and the CON diet: *P < 0.05; **P < 0.01.

Figure 3.. Uptake of OVA as a model antigen in purified B cells from SMAD−/− mice.

Aliquots of B cells were resuspended with 50 μg/mL OVA conjugated to FITC (OVA-FITC) at 37°C for 0, 60, and 90 min before being placed on ice. Purified B cells were negatively selected from the spleens of SMAD−/− fed the CON (open bars) diet or the DFO (solid bars) diet. (A) Representative histograms displaying the change in fluorescence over time after incubation with OVA-FITC in CON (left)- and DFO (right)-fed SMAD−/− mice B cells. (B) Change in MFI of OVA-FITC on purified B cells over time. Data are represented as mean ± sem; n = 10.

Figure 4.. In vivo distribution of B220⁺ MHCII⁺ lymphocytes in varying tissues of SMAD−/− mice.

Flow cytometry was performed on the spleen (Spln), MsLN, and PP of SMAD−/− mice fed the CON (open bars) diet or the DFO (solid bars) diet. Represented as a percentage of total lymphocytes, B220⁺ MHCII⁺ lymphocytes of dounce-homogenized tissues were identified. Data are represented as mean ± sem; n = 3–5. Data indicate significant differences: *P < 0.05.

Figure 5.. Surface marker expression on B cells from SMAD−/− mice.

An in vivo characterization was performed on the spleen, MsLNs, and PPs of SMAD−/− mice fed the CON (open bars) diet or the DFO (solid bars) diet. The MFI of MHCII and CD11c was determined on B cells, identified as CD8a⁻ Gr1⁻ B220⁺ MHCII⁺ lymphocytes. (A) Staining for MHCII on B220^hi B cells. (B) Staining for MHCII on B220^lo B cells. (C) Staining for CD11c on B220^hi B cells. (D) Staining for CD11c on B220^lo B cells. Data are represented as mean ± sem; n = 3–5. Data indicate significant differences: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6.. Systemic immunological outcomes of SMAD−/− mice fed CON or DFO diets.

(A) Th2-associated cytokines from the plasma of SMAD3−/− mice fed the CON (open bars) or DFO (solid bars) diet. Plasma samples were assayed using a MILLIPLEX MAP mouse cytokine/chemokine assay. (B) Secretory IgA in the cecal contents of SMAD−/− mice fed the CON (open bar) diet or the DFO (solid bar) diet. Cecal contents were homogenized with protease inhibitors and spun twice at 16,000 g to remove debris. Supernatants were analyzed using ELISA. Data are represented as mean ± sem, (A) n = 7–8; (B) n = 4–5. Data indicate significant differences between the DFO diet and the CON diet: *P < 0.05.