In this US birth cohort study, we found that both higher levels of cord blood n-3 EPA and n-6 AA were associated with attenuation of cord blood lymphocyte proliferation and decreased IFN-γ secretion in response to allergen stimulation. The evidence for independent effects of both EPA and AA on attenuated immune responses was stronger for IFN-γ than for SI (lymphoproliferative response). Although n-3 EPA and n-6 AA had similar inverse associations with SI and IFN-γ, the ratio of AA/EPA also influenced IFN-γ production, with lower amounts of AA relative to EPA predicting lower levels of IFN-γ. Within class, individual n-6 FAs differed in their influence on allergen-stimulated secretion of IFN-γ and IL-13. Increased levels of the n-6 LA (but not AA) were associated with increases of allergen-stimulated secretion of the TH2 cytokine IL-13.
Our findings of reduced cord blood lymphocyte proliferation responses with increased n-3 FA levels are qualitatively similar to the findings in the Australian randomized, double-blind, placebo-controlled trial of 98 atopic pregnancy women by Dunstan et al,7
although their associations of lymphocyte proliferation with n-3 FAs were not statistically significant. In contrast to the Dunstan group, we found specific n-3 (EPA) FA-associated suppression of IFN-γ rather than overall suppression of allergen-stimulated cytokine secretion (although as with lymphocyte proliferation, in the Dunstan study associations with cytokines were generally not statistically significant).
Although Dunstan et al7
had initially hypothesized an influence of n-3 PUFAs on the balance between TH
1 and TH
2 cytokine expression, they interpreted their findings to suggest a more global influence of n-3 PUFAs on T-cell regulation. A number of investigators have demonstrated that increased intake of EPA ± DHA can lead to diminished lymphoproliferative responses, IFN-γ responses, or both to mitogens (eg, concanavalin A) and antigens (eg, influenza virus and Listeria monocytogenes
) in rats, mice, or adult human subjects.8,11–13
Our data and data from other studies suggest that the effects of FAs on suppression of lymphocyte proliferation and some cytokine production might not be isolated-specific to the n-3 class of PUFAs. N-6 FAs, like AA and its prostaglandin (PG) byproducts, might have effects similar to those of EPA. Concanavalin A− or LPS-stimulated lymphocyte proliferation and IFN-γ production were inhibited by PG subtypes metabolized either from n-3 EPA (PGE3
) or n-6 AA (PGE2
) in a study by Dooper et al.14
The investigators concluded that the immunomodulatory effects of PUFAs might not be caused by a shift in the subtype of PGE and that certain n-3 and n-6 PUFAs might have similar immunomodulatory effects. This conclusion would be consistent with our findings of similar effects for n-3 EPA and n-6 AA. Other investigators have demonstrated, as we have, different immunomodulatory effects for AA versus LA, demonstrating that it is also an over-simplification to generalize about the effects of the overall class of n-6 (or n-3) FAs.11
The implications of our findings for the development of respiratory infections or allergic inflammatory diseases have yet to be defined. Newson et al5
have questioned the importance of n-3 FAs in the development of wheeze and have challenged the Black and Sharpe hypothesis3
that n-6/n-3 imbalance promotes atopic disease through increases in PGE2
and IgE levels. Newson et al5
argue that although PGE2
might inhibit TH
1 responses and enhance TH
2 responses of some sorts, it can also inhibit IgE production by B cells and protect against airway inflammation and bronchoconstriction.
FAs can influence immune responses and airway inflammation through mechanisms other than PGE2
. Our finding that higher levels of n-6 LA were correlated with increased allergen-stimulated IL-13 levels provides weak evidence for increased TH
2 cytokine production with this specific n-6 FA. However, we do not yet know whether increased neonatal IL-13 levels will be predictive of increased risk for later allergic or asthmatic disease. Some studies suggest that increased TH
2 cytokine levels at birth might in fact reflect a normal pregnancy.15
Similarly, it is as yet uncertain whether FA-associated suppression of lymphocyte proliferation and IFN-γ in fetal life represents “normal” protective T-cell regulation, as suggested by Dunstan et al.7
A number of investigators have found reduced IFN-γ levels in early childhood to be predictive of increased risk of allergic disease or asthma.16
Although the implication of our findings for the risk of allergy or asthma is uncertain, our study and the Dunstan study7
suggest that if fetal n-3 or n-6 FAs influence the risk of allergy or asthma through immune modulation, the immunomodulatory pathways are unlikely to be through simple alteration of the TH
2 balance of cytokine secretion. If both n-3 EPA and n-6 AA serve similar immunomodulatory functions, it might be wise to take the advice of Herrerra1
in his review on FAs, placental metabolism, and fetal and postnatal development. He suggested being cautious before recommending long-chain n-3 FA supplementation in pregnancy. Both EPA and AA are needed for normal development, and fish oil EPA supplementation might reduce levels of AA, which is needed for normal fetal development and antioxidant capacity in the fetus.
Our study has several limitations. The moderate correlation between EPA and AA made it possible to distinguish some, but not all, of the independent effects of EPA from AA on lymphoproliferative responses and cytokine levels. In the case of Bla g 2, low power related to the low level of cytokine production in response to allergen stimulation might have limited our potential to separate EPA from AA effects. Potential contamination with LPS was also considered as a limitation in interpretation of proliferative and cytokine responses. The allergens Der f 1, Bla g 2, and OVA, as well as the mitogen PHA, were tested for endotoxin contents by means of Limulus assay. We found low concentrations of endotoxin (<0.01 EU/mL = 0.002 ng/mL). These concentrations did not significantly change lymphocyte proliferation or cytokine secretion in CBMCs. In separate assays we tested the lymphoproliferative response of CBMCs to endotoxin components (eg, LPS and lipid A, an active component of LPS) in a dose-response analysis (ie, 0.01 ng to 100 ng/mL). Increased lymphocyte proliferation could only be detected with doses of lipid A of greater than 1 ng/mL. In addition, we also assessed cytokine secretion of TNF-α and IL-6, 2 innate cytokines typically increased after stimulation with lipid A. These cytokines were not increased at doses of lipid A as low as 0.002 ng/mL, as detected in our reagents. Thus our data suggest that endotoxin content had minimal influence on the outcomes of lymphoproliferation and cytokine secretion. Even if LPS did not influence our allergen-stimulated responses, it is possible that these responses are not antigen-allergen specific because the presence of allergen-specific T-cell memory has been questioned and challenged. In in vitro
studies of small samples of newborns, others have found that naive T regulatory cells predominate, with a relative or absolute absence of measurable conventional T memory cells in cryopreserved CBMCs.17
Nevertheless, even if the lymphoproliferative and cytokine responses to in vitro
antigen-allergen stimulation do not involve conventional T memory cells, it is still possible that these in vitro
responses to antigen stimulation reflect the neonate’s in vivo
early immune development. And it is still possible that these responses are influenced by cord blood FA levels. Although our study results could have been influenced by perinatal events, only 2 of the babies in the analyses were not healthy after birth, and sensitivity analyses eliminating these children from analyses did not significantly alter results. We report results only at one time point and one dose of stimulants, but dose-response and time-kinetic experiments lead to the choice of the time point and dose. Cells other than T cells can also be affected by FAs, and it is possible that some of our responses were, in fact, dependent on interaction between the different types of mononuclear cells. We are limited by lack of knowledge about maternal sensitization to dust mite and cockroach, although we did not find that maternal asthma was a confounder of the relation of FAs with cellular responses to allergen stimulation. Although cord blood FA levels are likely to reflect the in utero
exposure to FAs, it is possible that unmeasured perinatal events influenced those levels.
In conclusion, our data strongly suggest that increased fetal-neonatal levels of EPA or AA attenuate immunologic responses, specifically allergen-stimulated lymphocyte proliferation and IFN-γ production. Follow-up is needed to evaluate whether FA-associated attenuation of immune responses in the neonate can influence the risk of subsequent allergy and asthma in later childhood.