Short-term consumption of n-3 PUFAs increases murine IL-5 levels, but IL-5 is not the mechanistic link between n-3 fatty acids and changes in B-cell populations

Abstract

N-3 polyunsaturated fatty acids (PUFAs) exert immunomodulatory effects on B cells. We previously demonstrated that n-3 PUFAs enhanced the relative percentage and/or frequency of select B2 cell subsets. The objectives here were to determine if n-3 PUFAs (a) could boost cytokines that target B-cell frequency, (b) enhance the frequency of the B1 population and (c) to identify the mechanism by which n-3 PUFAs modify the proportion of B cells. Administration of n-3 PUFAs as fish oil to C57BL/6 mice enhanced secretion of the Th2 cytokine IL-5 but not IL-9 or IL-13. N-3 PUFAs had no influence on the percentage or frequency of peritoneal B1 or B2 cells. Subsequent experiments with IL-5(-/-) knockout mice showed n-3 PUFAs decreased the percentage of bone marrow B220(lo)IgM(hi) cells and increased the proportion and number of splenic IgM(+)IgD(lo)CD21(lo) cells compared to the control. These results, when compared with our previous findings with wild-type mice, suggested IL-5 had no role in mediating the effect of n-3 PUFAs on B-cell populations. To confirm this conclusion, we assayed IL-5 secretion in a diet-induced obesity model in which n-3 PUFAs enhanced the frequency of select B-cell subsets. N-3 PUFA supplementation as ethyl esters to obesogenic diets did not alter circulating IL-5 levels. Altogether, the data establish that n-3 PUFAs as fish oil can increase circulating IL-5 in lean mice, which has implications for several disease end points, but this increase in IL-5 is not the mechanistic link between n-3 PUFAs and changes in B-cell populations.

Keywords: B cells; DHA; EPA; IL-5; n-3 PUFAs.

Figure 1. N-3 PUFAs enhance serum IL-5 levels in C57BL/6 mice

Serum levels of IL-5, IL-9, and IL-13 from mice fed n-3 PUFAs for 4 weeks. Mice were injected with 1μ of TNP-LPS on week 3 and serum was isolated 7 days post-injection. Values are average ± S.E. from 5-6 independent experiments. Asterisk indicates statistical significance relative to the control diet: *p<0.05.

Figure 2. N-3 PUFAs have no influence on the percentage of B1 and B2 cells in the peritoneal cavity of wild type mice

(A) Flow cytometry gating strategy for analyzing B1 and B2 cells in the peritoneal cavity. (B) Percentage and (C) frequency of B1 and B2 cell subsets. Mice were injected with 1μg of TNP-LPS on week 3 and analyses were conducted one week later. Values are average ± S.E. from 4 independent experiments.

Figure 3. N-3 PUFAs modify the percentage and frequency of splenic IgM⁺IgD^loCD21^lo and IgM⁺IgD^hiCD21^hi B cells in IL-5^−/− mice

(A) Sample gating strategy for analysis for differing B cell subsets. (B) Percentage and (C) frequency of differing IgM⁺ B cell subsets from control and n-3 PUFA fed mice. Values are average ± S.E. from 5-6 independent experiments. Asterisks indicate statistical significance relative to the control diet: *p<0.05, **p<0.01, ***p<0.001.

Figure 4. N-3 PUFAs lower splenic B cell surface IgD expression of IL-5^−/− mice

(A) Flow cytometry gating strategy for analysis of surface IgM and IgD expression. (B) Normalized mean intensity of surface IgM and IgD of B cells. Fluorescence intensity values required normalization since experimental settings were adjusted between experiments. Values are average ± S.E. from 5-6 independent experiments. Asterisks indicate statistical significance relative to the control diet: **p<0.01.