Delivery mode regulates microglia morphology and function via immune system priming in peri-adolescent offspring

Introduction: Among the early-life factors that impact brain development, delivery mode and mode of feeding hold an important position. C-section has been widely linked with immune disturbances and neurodevelopmental disorders in offspring [1]. Microbiota acquisition and development is altered by mode of delivery [2], [3]. The differential microbiota seeding in early life prime multiple bodily systems including immune system and brain. The gradual microbiota maturation around weaning drives the priming of the immune system, starting from the gut, during solid food introduction [4]. T-cell numbers are increased in the gut during weaning, driving the increased levels of pro-inflammatory cytokines locally [4]. In the brain, T-cells modify microglia maturation and pruning capacity in early life, with potential implications for synaptic elimination [5]. Considering the importance of immune system as a mediator of microbiota-gut-brain axis communication, we hypothesized that C-section will alter the offspring immune priming in peri-adolescence with adverse outcomes for brain development.

Methods: NIH Swiss pregnant female mice underwent either a C-section or vaginal delivery and their offspring were assessed during early adolescence (gut, mesenteric lymph nodes, plasma, and brain were collected at P18, P21, P23 and P28). Cytokines were analysed by ELISA in gut and plasma, T-cells were quantified by flow cytometry. At P21, Iba1+ cell numbers and morphology were assessed by IHC and gene expression of sorted microglia was assessed by qPCR. The data were analysed with two-way ANOVA with Bonferroni correction for multiple comparisons. Microglia morphology and gene expression (P21) were analysed with independent samples t-test.

Results: C-section males show a delay in the weaning reaction in the gut with Tnfa peaking at P23 (p=0.011) compared with controls peaking at P21 (p=0.012), while C-section females display a blunted response of immune gut reactivity (p=0.469) compared with controls at P21 (P=0.02). The increase in T-helper cells in the c-section mesenteric lymph nodes is significantly higher in c-section animals compared with controls post-weaning in both sexes (P23: p=0.005, P28: p=0.001). In the circulation, Tnfa is significantly higher pre-weaning in the c-section animals compared with controls (P18: p=0.02, P21: p=0.042), but this is transiently reversed post-weaning (P28: p=0.018) in both males and females. The number of T cells in the PFC is increased in c-section females compared with controls at P21 (p=0.021). At the same timepoint, the number of microglia is significantly decreased in c-section females compared with controls (p=0.011), while in males there is a strong trend for decrease (p=0.072). Microglia morphology is altered, with a trend for soma elongation (p=0.074), cell complexity (p=0.091) and lacunarity (p=0.055) in c-section animals compared with controls at P21. Microglia proinflammatory phenotype is increased by C-section, along with the expression of maturation and pruning-related genes (Fos, Lamp1, Klf4) in both males and females at P21.

Conclusion: The weaning reaction is altered in the gut and the circulation in response to C-section, in a sex-dependent manner. Differential immune priming might explain the altered morphological and functional changes seen in microglia in C-section offspring at weaning, with potential implications for neuronal function later in life.