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Sexual dimorphism in lung transcriptomic adaptations in fetal alcohol spectrum disorders
Vishal D Naik1, Dylan J Millikin1, Daniel Moussa1
1Department of Obstetrics and Gynecology, C.S. Mott Center for Human Growth and Development, School of Medicine, Wayne State University, 275 E Hancock St, Rm 195, Detroit, MI, 48201, USA.
Insights
Developmental alcohol exposure impacts fetal lung development, causing sexually dimorphic changes in gene expression and immune responses. Female fetal lungs show significant alterations in T cell regulation, while both sexes exhibit impaired lung morphology and reduced alveolarization.
Area of Science:
- Reproductive biology
- Developmental toxicology
- Immunology
Background:
- Fetal alcohol spectrum disorders (FASD) research predominantly focuses on neurological effects, neglecting pulmonary complications.
- Respiratory infections are a major cause of newborn morbidity and mortality, with limited understanding of FASD's impact on fetal lung immunity.
- Existing studies on FASD and lung adaptations show decreased surfactant protein A, impaired alveolar macrophage function, and increased pneumonia risk, but lack investigation into sexual dimorphism.
Purpose of the Study:
- To investigate the sexually dimorphic effects of developmental alcohol exposure on fetal lung morphology and immune adaptations.
- To identify specific gene expression patterns and immune pathways affected by prenatal alcohol exposure in male and female fetal lungs.
Main Methods:
- Pregnant rats were exposed to alcohol or pair-fed controls during critical gestational periods.
- Fetal lung tissues were analyzed for gene expression using RNA sequencing (Illumina NovaSeq S4).
- Histopathological assessments were performed to evaluate lung morphology and differentiation.
Main Results:
- Alcohol exposure led to significantly reduced fetal weight, crown-rump length, and placental efficiency in exposed pups.
- Gene expression analysis revealed a sex-linked dichotomy, with significantly more altered genes in female fetal lungs compared to males.
- Females exposed to alcohol showed downregulated genes related to T cell activation, regulation, and differentiation, including decreased CD8+ T cell numbers. Both sexes exhibited stunted lung differentiation, hypoplasia, reduced alveolar size, and decreased alveolar capillarization.
Conclusions:
- Prenatal alcohol exposure induces sexually dimorphic changes in fetal lung development and immune gene expression.
- Female fetal lungs are disproportionately affected, with significant dysregulation of T cell-related genes.
- These findings underscore the importance of considering sexual dimorphism in FASD research and highlight potential long-term risks for respiratory health in affected individuals.
Abstract:
Current fetal alcohol spectrum disorders (FASD) studies primarily focus on alcohol's actions on the fetal brain although respiratory infections are a leading cause of morbidity/mortality in newborns. The limited studies examining the pulmonary adaptations in FASD demonstrate decreased surfactant protein A and alveolar macrophage phagocytosis, impaired differentiation, and increased risk of Group B streptococcal pneumonia with no study examining sexual dimorphism in adaptations. We hypothesized that developmental alcohol exposure in pregnancy will lead to sexually dimorphic fetal lung morphological and immune adaptations. Pregnant rats were orogastrically treated once daily with alcohol (4.5 g/kg, gestational day [GD] 4 to 10, peak BAC, 216 mg/dl; 6.0 g/kg, GD 11 to 20, peak BAC, 289 mg/dl) or 50% maltose dextrin (isocalorically matched pair-fed controls) to control for calories derived from ethanol. Male and female fetal lung RNA from a total of 20 dams were assessed using the TapeStation (Agilent) and Qubit RNA broad-range assay. Samples with RNA Integrity Numbers (RINs) > 8 were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), xGen Broad-range RNA Library Prep (IDT), and xGen Normalase UDI Primer Plate 2 (IDT). Final libraries were checked for quality and quantity by Qubit hsDNA and LabChip. The samples were sequenced on the Illumina NovaSeq S4 Paired-end 150 bp. Fetal lung tissue were analyzed for histopathological assessments. Mean fetal weight, crown-rump length and placental efficiency of the alcohol-administered rats were significantly lower (P < 0.05) than the pair-fed control pups. Differentially expressed genes indicated a sex-linked gene regulation dichotomy with a significantly higher number of genes altered in the female fetal lungs compared to the male. Network analysis plot of downregulated genes in the females exposed to alcohol in utero showed a negative impact on T cell activation and regulation, T cell differentiation, decrease in CD8+ T cell number etc. The most altered genes were Cd8b, Ccl25, Cd3e, Cd27, Cd247, Cd3d, Ccr9, Cd2, Cd8a and were decreased by a log2fold change of > 2 (P < 0.05) in the female fetal lungs. KEGG analyses showed that male and female fetal lungs had downregulated genes associated with development and mitosis, whereas the females alone showed dysregulation of T cell genes. Comparison of gross appearance and histopathologic morphology showed that the developing lungs of both male and female fetal pups, displayed stunted differentiation, were relatively hypoplastic, and displayed a diminution of alveolar size and air spaces. Similarly, in both sexes, decreased alveolar capillarization was also evident in the alcohol-exposed fetal lungs. These data provide novel information in a growing area focused on alcohol effects on the offspring lung and its influence on appropriate fetal/neonatal immune responses and highlights the importance of examining sexual dimorphism in developmental adaptations.
