Discussion

In this study, we demonstrated that maternal undernutrition during pregnancy and lactation is associated with significant alterations in offspring development. Specifically, FR/FR offspring were markedly smaller at 3 weeks of age compared to AdLib/AdLib and FR/AdLib offspring, consistent with previous reports of growth restriction due to maternal undernutrition.^{Reference Desai, Gayle, Casillas, Boles, Ross and Ross13} Consistent with growth restriction, FR/FR offspring demonstrated reduced thymus and spleen weights. The thymus is a critical organ for T-cell development, and its reduced size in FR/FR offspring may reflect impaired thymopoiesis, a process essential for the establishment of a functional adaptive immune system. This is consistent with human studies linking prenatal undernutrition to reduced thymic function and increased susceptibility to infections.^{Reference McDade, Beck, Kuzawa and Adair16} Similarly, the smaller spleen size in FR/FR offspring may indicate impaired development of secondary lymphoid organs, further compromising immune competence. These findings highlight the importance of maternal nutrition in shaping not only the innate but also the adaptive immune system. These findings align with a growing body of evidence suggesting that early-life nutritional deficits can have profound and lasting effects on offspring health, including immune function.

Maternal food restriction significantly compromises the maternal immune system during gestation and lactation, characterized by reduced cytokine production, decreased immunoglobulin synthesis, and impaired antioxidant capacity.^{Reference Marques, O’Connor, Roth, Susser and Bjørke-Monsen17} This maternal immune dysfunction directly impacts offspring through reduced passive antibody transfer, altered maternal-fetal cytokine signaling, and epigenetic programming of immune-related genes. The compromised maternal immune environment during critical developmental windows likely contributes to the immune deficits we observed in FR/FR offspring, including reduced TNF-α responses and abnormal immune cell populations, highlighting the maternal immune system as a key mediator linking nutritional restriction to offspring immune programming.

One of the most striking findings of our study was the 50% reduction in serum TNF-α response to LPS in 3 week old FR/FR offspring compared to AdLib/AdLib and FR/AdLib neonates. This attenuated inflammatory response occurred despite higher monocyte counts in FR/FR offspring following LPS stimulation, suggesting that the defect lies not in the availability of immune cells but in their functional capacity. This is consistent with studies showing that maternal undernutrition can impair immune cell signaling pathways, such as the LPS-TLR4-NF-κB cascade, which is critical for mounting an effective innate immune response.^{Reference Levy18} For example, maternal protein restriction has been shown to alter the expression of genes involved in inflammation and immune regulation, potentially explaining the blunted TNF-α response observed in our study. These findings indicate that while monocyte numbers increase in FR/FR offspring following LPS stimulation, their ability to mount an effective inflammatory response is compromised. This reflects a state in which immune cells are numerically adequate but functionally deficient, a phenomenon linked to early-life nutritional restriction in prior studies.^{Reference Wu, Cheng, Bai and Ma14,Reference Levy18}

Another line of evidence suggests that oxidative stress and inflammation act as critical mediators linking maternal undernutrition to adverse outcomes in offspring, further underscoring the need for targeted interventions^{Reference Fioretto, Colombelli and Da Silva19}

Maternal food restriction is also known to activate the maternal–fetal stress axis, with increased glucocorticoid exposure to the fetus (partly via reduced placental 11β-HSD2), programming hypothalamic–pituitary–adrenal (HPA) reactivity in offspring. This glucocorticoid milieu can impair thymic growth and T-cell output and skew innate/adaptive responses, culminating in blunted cytokine production and altered leukocyte distributions. These HPA-mediated effects provide a biologically plausible link between maternal undernutrition and our findings of reduced LPS-stimulated TNF-α and perturbed monocyte/neutrophil–lymphocyte profiles in FR/FR pups, consistent with prior evidence that early-life undernutrition programs long-term immune dysfunction via HPA activation^{Reference Lesage, Blondeau, Grino, Bréant and Dupouy20} and with human data relating prenatal undernutrition to reduced thymic function^{Reference McDade, Beck, Kuzawa and Adair16} and the known immaturity of neonatal immune responses.^{Reference Tsafaras, Ntontsi and Xanthou10}In addition to the reduced TNF-α response, 3 week old FR/FR offspring exhibited abnormal lymphocyte and neutrophil counts following LPS stimulation. Specifically, we observed a shift from lymphocyte to neutrophil dominance in FR/FR offspring, a pattern not seen in the other groups. These differences were only apparent after LPS stimulation and not at baseline, indicating that the defect lies in the dynamic immune response rather than in steady-state immune cell populations. The exaggerated neutrophil increase combined with the lymphocyte decrease suggests a maladaptive inflammatory activation, potentially driven by altered cytokine production and stress-related hormonal pathways. The integrated visualization in Fig. 5 further highlights that only FR/FR offspring display substantial basal-to-post-LPS shifts across leukocyte subsets, reinforcing that the programmed immune deficit becomes evident only under inflammatory challenge. The minimal or absent changes in AdLib/AdLib and FR/AdLib offspring emphasize that the impaired response is specific to those exposed to nutritional restriction both prenatally and postnatally.

This suggests that maternal undernutrition may disrupt the balance between different immune cell populations, potentially compromising the offspring’s ability to respond appropriately to infections or other inflammatory challenges. These findings are supported by work showing that maternal undernutrition can induce long-term immune dysfunction through mechanisms involving oxidative stress and inflammation.^{Reference Fioretto, Colombelli and Da Silva19}

These findings of abnormal immune response in offspring following maternal undernutrition confirm prior reports. Gotardo et al., focusing on the humoral immune response rather than the innate immune response in Wistar rats, demonstrated that food restriction between days 6–17 of gestation resulted in alterations in delayed-type hypersensitivity response and humoral immune response against sheep red blood cells in 21-day-old offspring.^{Reference Gotardo, Dipe, Hueza and Górniak21} Interestingly, in their study, the adjusted weight of the thymus was increased in FR as compared to AdLib pups, while in our study, thymus-adjusted weights were similar between groups. This discrepancy may reflect differences in the timing and duration of maternal undernutrition, as well as species-specific responses to nutritional stress. Badr et al. demonstrated that early undernutrition, particularly during the prenatal and lactation periods, attenuated T cell survival and the immune response in 8-week adult male rats.^{Reference Badr and Mohany22} Their findings align with our observation of reduced thymus weight in FR/FR offspring, suggesting that maternal undernutrition may impair thymic function and T-cell development. This is further supported by studies showing that maternal undernutrition can induce epigenetic modifications^{Reference Zelko, Zhu and Roman23} that alter immune-related gene expression, potentially explaining the long-term immune deficits observed in offspring.

The immune deficits observed in FR/FR offspring were not seen in FR/AdLib offspring, which exhibited responses similar to AdLib/AdLib neonates. This contrasts with our previous findings in female offspring at 9 months of age, where both FR/FR and FR/AdLib groups showed attenuated immune responses.^{Reference Desai, Gayle, Casillas, Boles, Ross and Ross13} This discrepancy may reflect differences in the timing of immune assessment (neonatal period vs. adulthood) rather than dose-dependent effects. Neonatal immune responses are inherently immature and differ significantly from those of adults, which may explain the observed differences.^{Reference Tsafaras, Ntontsi and Xanthou10} Additionally, sex-specific effects cannot be ruled out, as male and female offspring may respond differently to maternal undernutrition.

The immaturity of the neonatal immune system further complicates the interpretation of these findings. Neonates rely heavily on innate immunity during the first weeks of life, as their adaptive immune system is not fully developed.^{Reference Tsafaras, Ntontsi and Xanthou10} This immaturity makes them particularly vulnerable to the effects of maternal undernutrition, which may disrupt the delicate balance of immune cell populations and signaling pathways required for effective host defense. For example, maternal undernutrition has been shown to alter the gut microbiota, which plays a critical role in shaping neonatal immune development.^{Reference Jin, Zhang and Jia24} These early-life disruptions may have long-term consequences, increasing the risk of infections and inflammatory diseases later in life.^{Reference Lesage, Blondeau, Grino, Bréant and Dupouy20} Moreover, maternal low-protein diets can induce sex-specific developmental programming in offspring, highlighting the importance of evaluating both male and female progeny in future investigations.^{Reference Cavariani, de Mello Santos and Chuffa25}

While some of our findings confirm previous reports of altered immune function following maternal undernutrition,^{Reference Desai, Gayle, Casillas, Boles, Ross and Ross13,Reference Badr and Mohany22} this study provides new insights into the early neonatal period and the role of postnatal nutrition. By assessing immune parameters at both day 1 and day 21 and using a cross-fostering design, we distinguished the effects of gestational versus combined gestational and lactational undernutrition. This novel approach revealed that prolonged maternal undernutrition throughout pregnancy and lactation leads to persistent defects in TNF-α production and immune cell distributions, changes not seen with gestational restriction alone. These findings expand our understanding of how the timing and duration of maternal undernutrition shape neonatal immune development