Parental smoking behavior – cellular and molecular consequences for murine offspring
Despite epidemiological data exist on adverse effects of maternal smoking during pregnancy, it remains a risk factor for increased susceptibility of pulmonary diseases like childhood asthma and chronic obstructive pulmonary disease (COPD). Infants prenatally exposed to cigarette smoke have decreased birth weights and postnatal lung function deficits. Smoking during pregnancy is frequently underreported in human studies, wherefore mild maternal smoking might be inadequately recorded. Children from mildly smoking mothers are likely to escape epidemiological assessments without showing lower birth weights and lung function declines. This study hypothesizes that mild maternal smoking during pregnancy could affect the immune system of the progeny in absence of growth retardation and lung function deficits. Furthermore, until recently, paternal influences on offspring’s disease risks have been deemed to be restricted via genetic transmission, but recent multicenter epidemiological studies have postulated that environmental factors like smoking during adolescence of fathers increase the risk of obesity and asthma in future children. At present, the underlying mechanism is unexplored. This study aims to establish a murine model of preconceptional smoking during puberty to investigate paternal and offspring’s phenotype as putative mechanisms of ancestral transmission. After intrauterine mild cigarette smoke exposure, altered T cell populations were observed in lungs and spleen throughout the life course of murine offspring. At postnatal day (PND) 21, pulmonary and splenic CD4+ T cells were increased, whereas CD8+ T cells were decreased in both organs. In addition, thymic CD4 single-positive (SP) T cells were elevated at PND21. Next-generation sequencing (NGS) data of thymic CD8SP T cells from 21-day-old offspring revealed 92 up- and 36 downregulated genes. Genes of most interest – involved in immune-regulatory pathways – were interleukin 4 receptor alpha (Il4ra), runt-related transcription factor 3 (Runx3), forkhead box protein P1 (Foxp1), interleukin 10 receptor beta (Il10rb) and phasphoinositide-3-kinase (PI3K). Eomesodermin (Eomes) expression was supported by quantitative real-time polymerase chain reaction. Adolescent smoking of fathers increased body weights in male offspring, whereas female offspring remained normal. Offspring of pubertal smoking mothers were lighter in both sexes. Frequency of spermatozoa from the epididymis and spermatogonia in testes of smoking fathers were comparable to the non-smoker’s control. NGS of spermatozoal microRNAs (miRNAs) revealed 13 upregulated and 32 downregulated miRNAs of smoking fathers. After in silico analysis, miR-340-5p, miR-204-5p and miR-96-5p were of most interest. In conclusion, this murine study of mild maternal smoking during pregnancy identified altered pulmonary and thymic T cell populations in offspring at PND21. The development of ‘innate memory’ CD8 T (TIM) cells in the thymus is IL-4-dependent and together with the expression of Runx3 and Eomes in CD8SP thymocytes, this study postulates that TIM cells could be affected by mild prenatal cigarette smoke exposure in offspring. These cells differentiate into a memory-like phenotype in the thymus and are suggested to provide support of the immune system against pathogens in young children, as their memory of peripheral adaptive immunity is insufficiently established. Parental smoking during adolescence affected the body weight of offspring in a sex-specific manner and was parent-of-origin-dependent. In this study, the hypothesis was supported that sperm-borne miRNAs can carry ancestral memory and influence early gene translation in the early zygote. Putative targets and pathways of miRNA-340-5p, miR-204-5p and miR-96-5p make these miRNAs highly interesting candidates to further investigate their possible role on the altered body weight in murine offspring, and their influence on early embryogenesis and development in zygotes. This study highlights that maternal as well as paternal smoking behavior can influence the offspring.