Background
The beneficial effects of breastfeeding on infant health have been recognized for thousands of years across diverse civilizations [
1]. As breast milk is the main source of passive immunity during the early months after birth, breastfeeding is considered to be the most effective means of preventing death in young children from infectious causes [
2]. In addition, breastfeeding provides nutritional, developmental, psychological, social, economic, and environmental benefits [
3]. While there is overwhelming evidence supporting the role of breastfeeding in protecting children from most immune-mediated diseases [
4], the components in breast milk responsible for mediating this protection are not well defined.
Maternal transfer of IgG endows offspring with short-term protective immunity [
5‐
7]. The human fetus acquires a substantial amount of maternal IgG
in utero, transported across the placenta by the neonatal Fc receptor (FcRn) [
8]. In both humans and rodents, maternal IgG is acquired from breast milk [
9,
10], absorbed from the gut lumen via FcRn-dependent transcytosis in intestinal epithelial cells [
11‐
14]. It is known that mice deficient in either chain of FcRn (α-chain or β2 microglobulin) have impaired capacity to absorb maternal IgG from breast milk and accelerated decay of all IgGs, but not other Ig isotypes [
13,
15‐
19]. The structure of FcRn is well characterized [
12,
20] and several studies demonstrate a dynamic role of this receptor beyond the neonatal period [
21,
22].
It remains uncertain how maternal IgG acquired from breast milk impacts the susceptibility or severity of allergic diseases in children. It is known from animal models that offspring that receive serum fractions containing high titers of maternal antigen-specific IgG have suppressed IgE responses and enhanced IgG responses following immunization [
23]. Similarly, the presence of maternal allergen-specific IgG
1 at the time of immunization can inhibit IgE responses directed against the same allergen [
24,
25]. In contrast, passive transfer of allergen-specific IgG
1 followed by local allergen challenge within the respiratory tract can induce airway eosinophilia accompanied by hyperresponsiveness to irritants (analogous to induced bronchoconstriction in asthmatics) [
26]. The effect of passive immunization on exacerbation of allergic airway disease (AAD) appears mediated by enhanced allergen uptake in airway antigen presenting cells capable of activating proinflammatory CD4
+ T cells [
27].
We demonstrated that the breast milk from allergic mothers can protect offspring from ovalbumin (OVA)-induced AAD; with the protective effect dependent on intact maternal B cell immunity [
28]. Offspring nursed by wildtype allergic foster mothers have less severe OVA-induced AAD than offspring nursed by B cell deficient allergic foster mothers. The aim of the current study was to investigate the role of offspring FcRn in acquiring this maternal B cell-derived protective factor. We demonstrated that levels of OVA-specific IgG
1 absorbed from the gut into the circulation of breastfed offspring was determined by offspring FcRn expression. Furthermore, the allergen-specific IgG
1 absorbed from breast milk played a major role in preventing allergic sensitization in this model.
Discussion
There is overwhelming evidence supporting the role of breastfeeding in protecting children from most immune-mediated diseases [
4]. Despite this, it is not clear whether this applies to prevention of allergic disease in situations when mothers are allergic. Possible explanations for the inconsistent effects of breastfeeding on allergy and asthma prevention may be the immunologic complexities of breast milk itself and potential changes in composition in the context of maternal allergy or allergen exposure. Breast milk contains a multitude of biologically active components and some elements are thought to protect the infant from developing allergies, whereas others might promote allergic sensitization [
37].
We recently demonstrated that transmission of resistance to AAD from allergic mothers to nursing offspring is dependent on B cell-derived factors in breast milk [
28]. By comparing offspring of mothers with OVA- versus BSA-induced lung disease, we also established that the maternally transferred protection from AAD is antigen-specific [
29]. Based on these findings, we hypothesized that antigen-specific Igs in breast milk were major contributors to this protective effect. We previously demonstrated that allergen-specific IgG
1, IgA and IgE are absorbed from the neonatal gastrointestinal tract into the systemic circulation of naïve mice nursed by allergic mothers [
28,
29]. No allergen-specific IgG
2a is elicited following immunization with OVA adsorbed to Al(OH)
3 or after the aerosol challenge in our model of AAD, thus is not absorbed by offspring nursed by allergic mothers [
29]. In the present study, although maternal allergen-specific IgG
1, IgA and IgE were present at weaning in naïve FcRn-sufficient mice foster nursed by allergic mothers, it appeared that allergen-specific IgG
1 was the only isotype whose levels were sustained until allergic sensitization. This was not the case in FcRn-deficient mice where the low levels of maternal allergen-specific IgG
1 present at weaning were undetectable at the time of immunization. In mice, as in humans, the transfer of breast milk IgG across the intestinal epithelium is mediated by FcRn [
9]. Based on these and other data presented in this report, we were able to show that expression of FcRn was important for offspring to acquire sufficient levels of allergen-specific IgG
1 from the breast milk of allergic mothers to prevent allergen-specific IgE responses. A distinct experimental strategy to address this research question was recently reported by Nakata et al. [
36]. Importantly, the data derived from their studies led them to the same overall conclusion, that maternal IgG affects development of allergy in offspring. Thus, the two studies synergize to advance the understanding of the biology of FcRn as it applies to uptake of maternal IgG from the lumen of the gastrointestinal tract, and of how absorbed maternal allergen-specific IgG and offspring FcRn contribute to enhancing protection from allergic sensitization and disease.
To determine the role of FcRn in the postnatal acquisition of allergic protection, it was necessary to establish that FcRn-deficient mice were competent to develop OVA-induced AAD. After OVA-immunization and aerosol challenge, FcRn
-/- AAD mice demonstrated equivalent parameters of acute disease as wildtype B6AAD mice (this report and [
36]). Of particular interest were similar titers of OVA-specific IgG
1 antibodies in FcRn
-/- AAD and B6AAD mice in serum collected 24 hours after the last aerosol exposure. Thus, despite the accelerated decay of IgG in FcRn-deficient mice [
13,
15,
16], the initial antibody titers following aerosol challenge were unaffected. These results suggest that lymphocyte responses to allergic sensitization and challenge, including the generation of OVA-specific memory CD4
+ T cells and B cells, were intact in FcRn-deficient mice. Furthermore, differentiation of memory B cells to antibody-producing plasma cells appeared unaffected by the absence of FcRn.
FcRn
-/- offspring had impaired capacity to absorb OVA-specific IgG
1 from the breast milk of allergic mothers. At 24 days of life, 10
3 - 10
4 lower levels of antigen-specific IgG
1 were detected in the serum of FcRn
-/- offspring as compared to FcRn
+/+ or FcRn
+/- offspring nursed under the same conditions. This is consistent with what is known regarding the significant role of FcRn in mediating absorption of breast milk IgG [
13,
17]. However, in the previous study, while the TNP-specific IgG
1 injected into pregnant mice was present in the serum of breastfed FcRn
+/- neonates (10-20 μg/ml), it was not detected (<80 ng/ml) in their littermate FcRn
-/- mice [
13]. Thus, the existence or impact of an FcRn-independent component of maternal IgG uptake has not been appreciated. It is possible the low levels of antigen-specific IgG
1 detected in the serum of FcRn
-/- offspring are acquired via passive diffusion across the intestinal epithelium, although it remains to be determined whether this is the case. It is known that this mucosal barrier is more permeable in neonates with gut "closure" (cessation of Ig absorption) occurring at weaning [
10].
Although we demonstrated that FcRn-independent uptake of maternal IgG can occur in neonatal mice, we found that >99.9% of IgG absorbed in wildtype mice was via an FcRn-dependent mechanism (see Figure
3). In addition to mediating transcytosis of IgG across the intestinal epithelium [
11‐
13], FcRn modulates IgG homeostasis [
13,
15,
16]. Thus, we were able to perform the first study that quantified the rate of decay of absorbed maternal IgG
1 acquired exclusively from breast milk (~8.5 days). As compared to IgG acquired via intravenous injection, it is possible that ingested IgG selected by FcRn for absorption from the gut lumen has a higher binding affinity for FcRn systemically, and thereby has increased protection from catabolism. It is not clear whether or not this is the case since a direct comparison of half-lives of the same population of IgG
1 molecules following ingestion or injection of mice at the same age has not been made. Furthermore, there appears to be a lack of consensus in the field regarding the t
1/2 of injected IgG [
13,
15,
16,
18,
19]. The explanation for the diversity in results from different laboratories is not obvious, but could be due to different routes of injection - intraperitoneal versus intravenous, or structural features of the injected monoclonal antibodies tested that contribute to their inherent strengths of interaction with FcRn.
Other studies demonstrate that under the appropriate experimental conditions, breast milk may be protective against [
28,
36,
38‐
41] or increase susceptibility to [
42] the development of allergic disease in offspring. It is important to understand the mechanistic basis for differences in the effect of breast milk during this early period of immune maturation. In our studies, it is possible that maternal antigen-specific IgG
1 absorbed into the systemic circulation of offspring, neutralized the antigen - clearing it from the circulation prior to its recognition by cells of the adaptive immune system. This is supported by data from offspring nursed by allergic mothers where few, if any, FcRn-sufficient offspring produced allergen-specific IgE at 7 days after the second intraperitoneal immunization (data not shown). Neutralization of pathogens is known to occur when infants receive certain live vaccines (e.g. measles virus) in the presence of preexisting maternal antibodies. This is a major factor for delaying infant immunization until the majority of maternal antibodies have disappeared [
43,
44]. The functions of maternal antibodies in determining immune parameters in offspring can be influenced by the presence or absence of antigen and the ratio between them [
45]. Interestingly, in some experiments performed in this and our other related studies evaluating how allergic mothers influence parameters of allergic disease in offspring, allergen-specific IgG
1 and IgE responses were virtually absent without profound effects on airway eosinophilia. Since airway eosinophilia can occur in the complete absence of B cells [
28], this implies that protection from AAD acquired from allergic mothers in our model has more robust downstream effects on B cell than T cell parameters of disease. Thus, in addition to allergen neutralization, it is likely that maternal antigen-specific IgG
1/allergen immune complexes contribute to determining the outcome of offspring responses to allergic sensitization [
46].
It should be noted that not all maternal Igs have beneficial effects in progeny. Recently, a murine model of peanut allergy demonstrated that maternal derived anti-peanut IgG
1 is associated with anaphylactic reactions in offspring [
47]. In addition, several autoimmune diseases such as systemic lupus erythematosus are known to result in transmission of maternal IgG's that have deleterious effects in progeny [
48]. The ability of maternal IgG to mediate differential effects in offspring may be related to affinities for individual FcγRs resulting from Fc glycosylation. IgG is known to contain a single N-linked gycan at Asn
297 of the Fc domain, and variations of this covalently linked complex carbohydrate determines binding interactions with individual FcγRs [
49,
50]. Fc sialyation of IgG
1 results in a reduced binding affinity for the activating receptor FcγRIII and promotes anti-inflammatory effects through the inhibitory receptor FcγRIIB [
49]. It is known that several autoimmune diseases are associated with individual glycoforms of IgG [
51,
52]. Perhaps the structure of glycans on antigen-specific IgGs varies during the pathogenesis of allergic disease, or is influenced by the environment at the site of the allergen challenge (such as the lung or gut mucosa). Control of post-translational modification of carbohydrate residues on IgGs could be determined during plasma cell differentiation from memory B cells, and/or modified by glycosylation or de-glycosylation enzymes unique to specific tissue environments or disease states. It is possible that physiological changes during lactation itself play a role in the characteristics of maternal IgG acquired by offspring to influence whether maternal IgG transfers increased risk or protection from allergic disease. Recent results from Victor et al. [
41] could be supportive of this idea. In their study, neonates nursed by immunized mothers exhibit marked inhibition of B and T cell responses following immunization. In contrast, postnatal injected anti-allergen IgG (purified from serum of immunized mice) failed to modulate expression of FcγRIIB or regulate B or T cell cytokine production.
Our findings suggest a serum concentration limit of absorbed maternal antigen-specific IgG sufficient to protect offspring from AAD. This concept is supported by data from FcRn
+/+ or FcRn
+/- offspring, where serum levels of antigen-specific IgG
1 of 10
5 - 10
6 ng/ml at 52 days of life appeared sufficient to protect offspring from AAD initiated one week later. Significantly reduced levels of antigen-specific IgG
1 in the serum of weanling FcRn
-/- mice, that decayed to negligible levels prior to immunization, resulted in the absence of protection from AAD. Interestingly, the concentration limit of maternal IgG needed to protect offspring from AAD appears to be dependent on the severity of disease elicited in murine models, with lower levels of absorbed allergen-specific IgG
1 (60-90 ng/ml) being sufficient to protect from mild disease [
36]. Additional experimentation aimed at defining the contributions of serum concentrations of maternal IgG, immune complexes and structural glycoforms sufficient to protect offspring from allergic sensitization will be important.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
APM supervised the animal experiments, participated in the immunoassays and study design, performed the statistical analysis, and drafted the manuscript. RST reviewed the histopathology and helped to draft the manuscript. ER performed the majority of immunoassays, PCR, and sample collections. EGL helped to direct the animal experiments and immunoassays. LP conceived of the study, participated in its design, coordination, and data analysis; and helped to draft the manuscript. All authors have read and approved the final manuscript.