Fishmeal supplementation during ovine pregnancy and lactation protects against maternal stress-induced programming of the offspring immune system

Adverse uterine environments caused by maternal stress and infection can alter the planned programming of various tissues, organs and systems of the fetus. When the re-programming of these systems do not match the predicted environment, the offspring may be more susceptible to inflammatory diseases such as atopy, cardiovascular disease and type II diabetes in later life [1–10]. This hypothesis has recently become one of the main focuses in atopic disease research.

With the increase in atopic disorders, such as asthma, food allergies and atopic dermatitis in the Westernized population, it is speculated that genetic predisposition itself cannot be solely responsible, and focus is being placed on in utero events and environmental factors that may be playing a contributing role [11–13]. Prenatal stress and the associated rise in glucocorticoids (GCs), as well as the high concentration of pro-inflammatory mediator omega-6 polyunsaturated fatty acid (n-6 PUFA) has been found to be a factor contributing to the susceptibility to atopic diseases by altering the programming of both the immune system and hypothalamic-pituitary-adrenal axis (HPAA) [14, 15]. For example, alterations in the HPAA through fetal programming have been shown to increase the occurrence of respiratory, and skin diseases [16–18]. These alterations in HPAA programming may be responsible for the typical increase in T helper type 2 (Th2) lymphocytes as well as the associated cytokines and chemokines observed in individuals who were prenatally stressed and those with atopic disease [19, 20]. During normal pregnancy the dominant immune response is of Th2 origin and this helps to facilitate maternal tolerance for the fetus. Shortly after parturition the balance between Th2:Th1 is restored. However, in prenatally stressed individuals, it has been suggested that this shift may be delayed, which may increase the susceptibility to atopic diseases [11].

Recent studies suggest that supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) may help to alleviate atopic disorders during both childhood and adulthood [21–23]. Unlike n-6 PUFAs, n-3 PUFAs promote anti-inflammatory mediators and may help protect against inflammatory challenges. For example, n-3 PUFAs have been shown to alter T lymphocyte gene expression profiles by suppressing their differentiation. Their function is also inhibited due to decreased concentrations of cytokines, chemokines and immunoglobulins associated with these responses [24–26]. However, it appears that the timing, type and dosage of n-3 PUFA supplementation may be crucial in the treatment of atopic disease, as various studies have also shown no beneficial affects with supplementation [27, 28]. Previous studies have focused their efforts on postnatal impacts, however the role of n-3 during pregnancy and an activation of protection is ill defined.

Therefore, the purpose of this study was to investigate whether maternal fishmeal (FM) supplementation rich in n-3 PUFA can protect the offspring’s immune system from simulated maternal infection. It was hypothesized that maternal supplementation with n-3 PUFAs would protect the offspring from maternal endotoxin challenge and will decrease the dermal immune response and antibody-specific response to novel antigens. In order to test this objective a sheep model will be used. Sheep are an excellent model for humans as their offspring are a similar size at birth, and their brain development occurs during fetal development.

All lambs survived this study with no significant treatment differences in body weight. The plasma n-3 PUFA concentrations at weaning (50 days of age), where the least square means of EPA concentrations were 1.33 for FM and 1.109 for SM offspring and DHA concentrations were 1.72 for FM and 1.53 for SM offspring. At the time of hypersensitivity testing (135 days of age) there was no difference in plasma n-3 concentrations where EPA concentrations were 0.528 for FM and 0.520 for SM offspring and DHA concentrations were 1.16 for FM and 1.26 for SM offspring [32].

Dermal hypersensitivity response to OVA and CAA Antigens

All lambs responded to both OVA and CAA dermal hypersensitivity challenge with a significant increase in skinfold thickness represented by both linear and quadratic over time (P < 0.05, Fig. 1). Offspring born to SM + LPS mothers demonstrated the greatest increase in skinfold thickness trend to both antigens compared to their control counterparts (SM + CON) as well as both FM treatment groups (FM + LPS, FM + CON, P < 0.05; Fig. 1). There was no difference in contrasts for skinfold thickness trends to either OVA or CAA between FM treatment groups (FM + LPS, FM + CON; P > 0.05; Fig. 1). There was also a three-way interaction of diet by treatment by quadratic time between the FM + LPS lambs and the SM + LPS lambs to the CAA antigen (P < 0.05; Fig. 1 b, d). Therefore, quadratic time trends differed between dietary groups but also between treatment groups.

Linear and quadratic sex difference trends were also observed following both CAA and OVA dermal sensitivity challenge. The diet by treatment by sex contrast demonstrated an increase in skinfold thickness trends, which was greater in female and male offspring born to SM + LPS dams as compared to all other treatment groups within the same sex (P < 0.05; Fig. 1). There was also a significant difference in the trends over time with female and male FM + LPS lambs having a slightly greater change in skinfold thickness to the CAA antigen compared to the FM + CON lambs of the same sex (P < 0.05; Fig. 1 b, d). This trend was not present at the OVA test site over time in the FM offspring in which both groups responded slightly to the same degree in both sexes (Fig. 1 a, c). A sex difference trend was observed between female and male offspring born to SM + LPS dams over time during the CAA challenge. Quadratic trends demonstrated that SM + LPS female offspring had a greater skinfold thickness response over time to CAA antigen compared to male offspring (P < 0.05; Fig. 1 b, d). There were no differences over time observed between male and female offspring born to FM + LPS or FM + CON dams (P > 0.05).

Serum OVA-specific IgG response

There was an increase in OVA IgG concentrations over time across all treatment groups demonstrating that the inoculation protocol worked to induce both a primary and secondary IgG immune response (P < 0.05; Fig. 2). Offspring from SM + LPS mothers had an increased OVA- specific IgG response during both the primary (d 10) and secondary (d 21) immune response compared to all other treatment groups (P < 0.05). Unlike the SM treatment groups, a difference was not observed between offspring born to FM + LPS or FM + CON mothers.

Sex differences were only observed for the secondary IgG immune response. Female offspring from SM + LPS mothers had a greater secondary IgG response compared to their male counterparts (P < 0.05; Fig. 2). This trend was not observed in any of the other treatment groups. Additionally, SM + LPS female offspring had a greater concentration of OVA-specific IgG antibodies compared to the female offspring born to both SM + CON and FM + LPS mothers. This response was not observed between male offspring. However, SM + LPS male offspring tended to have a greater IgG response following the primary immune response compared to SM + CON male offspring (P < 0.10).

Maternal infection during pregnancy has been shown to increase the offspring’s susceptibility to atopic disease [1]. Recent studies have shown that n-3 PUFA supplementation may provide protection from atopic disease because of its immunomodulatory properties [33–35]. This study investigated whether maternal supplementation with FM (high in n-3) could protect the offspring’s immune system from maternal stress caused by a simulated bacterial infection using endotoxin. The offspring’s immune response was assessed by cutaneous hypersensitivity tests to novel antigens OVA and CAA, as well as the IgG response to OVA. All offspring responded to both antigens with an increase in skinfold thickness, and a primary and secondary response to the OVA antigen. As expected offspring born to mothers supplemented with SM and challenged with endotoxin had the greatest secondary antibody response to OVA antigen as well as the greatest increase in skinfold thickness to both OVA and CAA compared to all other treatment groups. Other studies have demonstrated alterations in both the dermal hypersensitivity response and Ab-mediated response but this was during n-3 PUFA supplementation. One study showed that fish oil supplemented cats had a significantly lower skinfold thickness response to an intradermal injection of histamine compared to the control group [36]. While Lauritzen et al. [37], demonstrated that fish oil supplementation to mouse dams decreased their offspring’s IgG1 antibody response to OVA compared to dams consuming an n-3 PUFA deficient diet or a linseed diet (rich in n-6 PUFA) [37]. The IgE antibody response to OVA has also been shown to be decreased following supplementation with fish oil in mice [38]. Additionally, many studies looking at atopic dermatitis and allergies in humans have also showed improvement in the disorder following supplementation with n-3 PUFAs [28, 39, 40]. Therefore, it appears that n-3 PUFA supplementation may improve atopic disease, however, further research is required to determine the proper dietary inclusion level as well as timing of treatment, as multiple studies that have demonstrated no differences or even increases in both the skinfold thickness and the Ab response with n-3 PUFAs [41, 42]. One should also note that FM and fish sources of n-3 PUFAs might also contain various other fatty acids and amino acids that could also influence the immune response of the offspring.

When comparing to the above-mentioned studies, this is the first study to investigate whether FM supplementation during gestation and lactation can protect the offspring from programming following simulated maternal infection during late gestation. This study is different from others as supplementation with n-3 PUFAs occurred solely through the dam and the offspring n-3 PUFA concentrations were not different across treatments at the time of challenge with OVA and CAA. n-3 PUFA EPA enrichment was observed at 50 days of age with greater concentrations of EPA in the plasma of offspring from FM supplemented dams demonstrating that maternal supplementation during gestation and lactation does in fact cause enrichment in the lambs. However, these differences were small and warrant further investigation into PUFA partitioning in the body to assess whether greater enrichment occurred in other tissues or organs. Nonetheless this suggests that n-3 PUFA supplementation during pregnancy could help reduced the risk of topic disease later in life as well as reducing the risk of infection-induced programming.

Alterations in the ratio of Th1:Th2 lymphocytes and associated mediators have been reported as a factor in the development of atopic disease and it has been suggested that n-3 PUFAs may be acting in a way to restore the balance. For example, various animal models have demonstrated that maternal supplementation with n-3 PUFAs may reverse the skewed Th2 biased response as they have been shown to promote a Th1 population with the increase in IL-2 and IFNγ [25, 43]. This shift in the Th population may explain the differences observed in the skinfold thickness response and Ab-response to OVA in the current study as OVA is typically thought of as a Th2 antigen. However, it does account for the observed decrease in the skinfold thickness response to the CAA, a known Th1 antigen. Therefore, this suggests that a different mechanism may be at work.

Recently, T regulatory cells (T regs) have been shown to play a role in the reduction of atopic disease. T regs are found in the lymph nodes, spleen, and peripheral blood, help to maintain tolerance again allergens [44]. T regs act to suppress the proliferation of Th1 and Th2 lymphocytes as well as their function [44, 45]. It was previously suggested that pregnancy hormones supported a Th2 dominant immune profile, however, new evidence suggests that Tregs induce maternal tolerance to the fetus and help to maintain a successful pregnancy [46]. It has also been shown that maternal stress can reduce the number of T regs at the maternal-fetal interface and this may influence programming of the fetal immune system to one that is less tolerant to novel antigens [11, 47, 48]. Interestingly, supplementation with both DHA and EPA increases the expression of IL-10 and TGF-β as well as Foxp3, a transcription factor required for the production and function of T regs [49]. With this in mind it is speculated that n-3 PUFAs suppress the Th1 and Th2 response by up-regulating T regs and T reg mediators. This may also explain the observed hypersensitivity and Ab suppression in the FM + LPS from the present study offspring and not the SM + LPS offspring to both OVA and CAA antigens, although further research is needed to validate this hypothesis.

Sex differences were also observed in this study between male and female offspring born to SM + LPS mothers. To our knowledge this is the first study to investigate the sex effects of offspring born to mothers supplemented with PUFAs and challenged with endotoxin. It was observed that female offspring had a greater skinfold measurement to the CAA antigen compared to the male offspring. Female offspring also had a greater IgG response to OVA antigen compared to their male counterparts. There was no difference between FM groups observed. This is not surprising, as females have typically demonstrated greater antibody responses to allergens and vaccine. For example, female mice exposed to environmental tobacco smoke, which is known to increase allergic sensitization, and then sensitized with OVA demonstrated greater OVA specific IgG1 and IgE in their serum compared to males [50, 51]. Although females in this study demonstrated the greatest increase in OVA specific IgG, one cannot conclude that the males were not also susceptible to this type of programming, as the male offspring born to SM + LPS mothers also demonstrated great differences in the skinfold measurements to both antigens. This emphasizes the fact that the timing and type of stressor as well as species differences may need to be taken into account for when using animal models. Based on these results of this study, however, it appears that maternal supplementation with FM helps to counteract the in utero programming of offspring’s risk of allergic disease later in life regardless of sex.

Overall this study has provided insight into the potential benefits of supplementing maternal diets with FM to reduce the risk of atopy. It is apparent that FM provides a protective effect from maternal endotoxin challenge during late gestation as indicated by the decrease in offspring change in skinfold thickness in response to OVA and CAA as well as a decreased IgG response to OVA in female offspring. Future studies should focus on the immune and epigenetic mechanisms involved in this protective effect.