Demystifying the impact of prenatal tobacco exposure on the placental immune microenvironment: Avoiding the tragedy of mending the fold after death
Authors: Zhao Xiaoxuan , Jiang Yuepeng , Ma Xiao , Yang Qujia , Ding Xinyi , Wang Hanzhi , Yao Xintong , Jin Linxi , Zhang Qin 
Published: September 2023

Prenatal tobacco exposure (PTE) correlates significantly with a surge in adverse pregnancy outcomes, yet its pathological mechanisms remain partially unexplored. This study aims to meticulously examine the repercussions of PTE on placental immune landscapes, employing a coordinated research methodology encompassing bioinformatics, machine learning and animal studies. Concurrently, it aims to screen biomarkers and potential compounds that could sensitively indicate and mitigate placental immune disorders. In the course of this research, two gene expression omnibus (GEO) microarrays, namely GSE27272 and GSE7434, were included. Gene set enrichment analysis (GSEA) and immune enrichment investigations on differentially expressed genes (DEGs) indicated that PTE might perturb numerous innate or adaptive immune‐related biological processes. A cohort of 52 immune‐associated DEGs was acquired by cross‐referencing the DEGs with gene sets derived from the ImmPort database. A protein–protein interaction (PPI) network was subsequently established, from which 10 hub genes were extracted using the maximal clique centrality (MCC) algorithm (JUN, NPY, SST, FLT4, FGF13, HBEGF, NR0B2, AREG, NR1I2, SEMA5B). Moreover, we substantiated the elevated affinity of tobacco reproductive toxicants, specifically nicotine and nitrosamine, with hub genes through molecular docking (JUN, FGF13 and NR1I2). This suggested that these genes could potentially serve as crucial loci for tobacco's influence on the placental immune microenvironment. To further elucidate the immune microenvironment landscape, consistent clustering analysis was conducted, yielding three subtypes, where the abundance of follicular helper T cells (p < 0.05) in subtype A, M2 macrophages (p < 0.01), neutrophils (p < 0.05) in subtype B and CD8+ T cells (p < 0.05), resting NK cells (p < 0.05), M2 macrophages (p < 0.05) in subtype C were significantly different from the control group. Additionally, three pivotal modules, designated as red, blue and green, were identified, each bearing a close association with differentially infiltrated immunocytes, as discerned by the weighted gene co‐expression network analysis (WGCNA). Functional enrichment analysis was subsequently conducted on these modules. To further probe into the mechanisms by which immune‐associated DEGs are implicated in intercellular communication, 20 genes serving as ligands or receptors and connected to differentially infiltrating immunocytes were isolated. Employing a variety of machine learning techniques, including one‐way logistic regression, LASSO regression, random forest and artificial neural networks, we screened 11 signature genes from the intersection of immune‐associated DEGs and secretory protein‐encoding genes derived from the Human Protein Atlas. Notably, CCL18 and IFNA4 emerged as prospective peripheral blood markers capable of identifying PTE‐induced immune disorders. These markers demonstrated impressive predictive power, as indicated by the area under the curve (AUC) of 0.713 (0.548–0.857) and 0.780 (0.618–0.914), respectively. Furthermore, we predicted 34 potential compounds, including cyclosporine, oestrogen and so on, which may engage with hub genes and attenuate immune disorders instigated by PTE. The diagnostic performance of these biomarkers, alongside the interventional effect of cyclosporine, was further corroborated in animal studies via ELISA, Western blot and immunofluorescence assays. In summary, this study identifies a disturbance in the placental immune landscape, a secondary effect of PTE, which may underlie multiple pregnancy complications. Importantly, our research contributes to the noninvasive and timely detection of PTE‐induced placental immune disorders, while also offering innovative therapeutic strategies for their treatment.