To our knowledge, this is the first study to demonstrate an association between cesarean section and increased neonatal secretion of IL-13 and IFN-γ. This finding provides a potential immunologic basis for previous reports of an association between cesarean section and atopy or asthma [2–8], as elevation of IL-13 [9–11] and IFN-γ [11, 12] at birth has been associated with asthma and atopy in childhood. In addition, there was a non-statistically significant trend for an inverse association between cesarean section and neonatal levels of IL-10 (a cytokine with inhibitory effects on the secretion of Th1 and Th2 cytokines in vivo) [19–21]. Although this finding should be further assessed in larger studies, it suggests that abnormal stimulation of mechanisms that downregulate both arms of the immune response (e.g., T regulatory cells [Tregs]) may influence the pathogenesis of atopy in children born by cesarean section [22–24].
The observed association between mode of delivery and neonatal immune responses may be explained by absent or reduced labor in children delivered by cesarean section. The process of labor may directly influence neonatal immune responses, thereby influencing cytokine secretion at birth. Although a relationship between labor and neonatal secretion of IL-13, IFN-γ, and IL-10 has not been shown, the stress of labor has been associated with decreased T lymphocytes and CD4+ helper T cells [25, 26], and increased neutrophils [27, 28], natural killer (NK) cells [26, 28], TNF-α , and IL-6 [13, 29] in cord blood. In contrast, cesarean section without labor has been associated with increased T lymphocytes and CD4+ helper T cells [25, 26], decreased neutrophils [27, 28], natural killer (NK) cells [26, 28], TNF-α , and IL-6 [13, 29] in neonates at birth.
Although labor itself may have important immunoregulatory effects on neonates [25–28] and thus partly explain our findings, it is also plausible that the observed neonatal cytokine profile in children born by cesarean section is due to their reduced contact with the maternal vaginal flora at birth. We measured the composition of the maternal gut flora, which is strongly correlated with that of the maternal vaginal flora.
Our findings with regard to bacterial species in the maternal intestinal flora and neonatal immune responses should be interpreted with caution because of small sample size and inability to control for confounders such as maternal diet. However, our preliminary results in children born by vaginal delivery are interesting and suggest the possibility that exposure to specific microbes in the maternal vaginal flora during passage through the birth canal influences neonatal immune responses. In particular, we found that gram-positive anaerobes and total anaerobes in maternal stool were associated with increased secretion of IL-10 by CBMCs, and that gram-negative anaerobes and gram-negative aerobes in maternal stool were associated with reduced secretion of IL-13 and IFN-γ by CBMCs.
The observed association between anaerobes in maternal stool and increased neonatal secretion of IL-10 by CBMCs (at 24 hours after stimulation with allergens) is consistent with results of experiments in murine models and in vitro studies in humans. Stimulation of cord blood lymphocytes with gram-negative bacteria for 24 hours (including anaerobes such as Bacteroides species) results in strong secretion of IL-10 . In rodents, peritoneal cells produce IL-10 after stimulation with the CPC of Bacteroides fragilis. More specifically, PSA from B. fragilis elicits IL-10 production from a population of murine Tregs beginning at 24 hours after stimulation . In murine models, the PSA molecule of B. fragilis is presented to T cells by intestinal dendritic cells (DCs) residing at mucosal surfaces, which then activate CD4+T cells and elicit appropriate cytokine secretion resulting in a balanced Th1/Th2 immune response . Moreover, DCs have been shown to mediate the secretion and activity of Tregs [34, 35].
Administration of lactobacilli to atopic children has been associated with increased production of cytokines produced by Tregs (e.g., IL-10) [36, 37]. It is thus plausible that early modulation of immune responses by specific bacteria (e.g., anaerobes) during passage of the neonate through the birth canal  results in upregulation of neonatal Tregs and/or direct downregulation of Th1 and Th2 immune responses. The effects of labor may further interact with those of the maternal gut flora on neonatal immune system development.
The observed association between gram-negative anaerobes and increased secretion of IL-13 (at baseline and after stimulation with Fel d 1 and LPS) and IFN-γ (after stimulation with LPS) by CBMCs in children born by cesarean section is intriguing and unexplained. Although it may be due to chance or confounding by unmeasured variables, this association may also be due to poorly understood effects of the maternal gut flora on the immune system of mothers not undergoing labor, which may ultimately influence neonatal immunity.
Similar to previous studies, we found an independent association between maternal history of atopy and secretion of IL-13 [9–11] and IFN-γ [11, 12] by CBMCs. This association was not related to maternal gut colonization or cesarean section delivery, as we did not find evidence that the maternal stool flora or mode of delivery differed between atopic and nonatopic mothers. Although the allergic status of the mother was not confirmed by measurement of serum IgE specific to allergens or by allergy skin testing, self-reported atopic diseases have been shown to be correlated with objective markers of atopy in mothers of children participating in similar studies . The reason for frequently finding the strongest and/or most statistically significant associations between the exposures of interest (e.g., cesarean section) and particular patterns of cytokine secretion by CBMCs after stimulation with dust mite (Der f 1) and cat (Fel d 1) allergens but not after stimulation with mitogen or LPS is not clear. It is plausible that these two inhalant allergens are more widely distributed in the environment and therefore maternal exposure to these allergens during pregnancy result in transplacental transfer of these allergens to the fetus leading to allergen-specific T cell priming in utero [41, 42].
We recognize several additional limitations to our findings. First (as previously mentioned), the data has to be interpreted as preliminary because of small sample size resulting in limited statistical power and inability to adequately control for potential confounders. However, we found consistent associations between cesarean section and neonatal secretion of cytokines. Furthermore, we found consistent correlations between various bacterial species in maternal stool and specific cytokine patterns in neonatal cord blood that differed by mode of delivery. Second, we did not have adequate statistical power to test whether the pattern of cytokine secretion at birth is different in children born by elective versus non-elective cesarean section. Because a majority of the study participants who had a cesarean section had it scheduled, elective cesarean section is likely responsible for our findings. Third, although we did not select our cohort based on a family history of atopy, approximately 60% of the mother reported having a history of atopy which is higher than reported from the general population; therefore the results may not be generalizable to the general population. Fourth, we recognize that IL-10 is secreted by cells other than Tregs (e.g., Th2 cells). However, we found an inverse association between cesarean section and IL-13 (a Th2 cytokine) and differences in the association between specific microbes in maternal stool and secretion of IL-13 vs. IL-10 by CBMCs, suggesting that neonatal IL-10 was likely secreted by Tregs (a major source of this cytokine) [19–21, 43]. Other studies have found increased secretion of IL-10 and IFN-γ by CBMCs in response to stimulation with LPS , peptidoglycan (a cell wall component of gram-positive bacteria) , and mycobacterial extract (PPD) . Together with our findings, these results suggest that different microbial stimuli can impact specific subsets of T cells in the fetal immune system such as Th1 cells and T regulatory cells. Fifth, potential contamination with LPS may influence the interpretation of cytokine responses in cord blood. However, the endotoxin contents present in the allergens (Der f 1 and Fel d 1 ) and mitogen (PHA) samples (≤ 1.23 EU/mg) were 1 × 106 times less than the concentration of LPS stimulus used in this study. Finally, maternal infection or antibiotic use around the time of birth was not an exclusion criterion in our study. However, the white blood cell (WBC) count of participating mothers at the time of delivery (median = 10 × 109 cells/L) was not significantly associated with secretion of any cytokine by CBMCs (P > 0.20 in all cases). In addition, there was no significant difference in WBC count between mothers who had a vaginal delivery and those who had a cesarean section (P = 0.82).
In conclusion, our findings suggest that cesarean section leads to abnormal neonatal immune responses (increased secretion of IL-13 and IFN-γ) that may precede the development of asthma and atopy. In addition, results of our exploratory analyses suggest that exposure to specific microbes (e.g., lactobacilli and bifidobacteria) in the maternal vaginal flora may influence neonatal immune responses in children born by vaginal delivery. These hypotheses will need to be examined in future longitudinal studies with large sample size.