Background
Smoking during pregnancy is a major cause of maternal and newborn morbidity and mortality [
1], with pulmonary diseases being a major adverse outcome [
2,
3]. In-utero smoke exposure (SE) reduces lung function in human newborns [
4,
5]. Animal models have shown a decreased number of saccules, septal crests, and decreased elastin fibres in foetuses [
6] and suckling pups [
7], as well as increased airway thickness, collagen deposition, inflammation, and airway hyper-responsiveness due to intrauterine SE [
8‐
10].
In humans, certain respiratory diseases, including chronic obstructive pulmonary disease (COPD), occur disproportionately in males and females [
11]. The common pathophysiological process includes increased inflammation, oxidative stress, impaired mitochondrial renewal (mitophagy), and cellular self-cleaning mechanism (autophagy) [
12]. In keeping with this, our previous murine studies found that the changes in inflammation, oxidative stress, mitophagy, and autophagy have a marked sex difference in the offspring’s brain and kidney following in-utero SE, wherein female offspring are less affected from such adverse effects [
13,
14].
Previously, it has only been demonstrated that maternal SE causes lung inflammation in male offspring [
15]. Other studies show that prenatal SE can differentially affect DNA methylation in cord blood or lungs in males and females in both human and animal studies. For example, the gene encoding insulin-like growth factor (IGF)-1, a growth factor involved in lung development, was found to be methylated in the babies from smokers, and methylation was most pronounced in male children [
16,
17]. In an animal study, CpG site-specific hypo- and hypermethylation of
Igf1 exons was observed in the lungs from male and female offspring, respectively. However, protein transcription of IGF-1 is reduced in male offspring, even without in utero SE, which may partially explain sex differences in the susceptibility of developing certain respiratory disorders [
18]. The mechanism by which in-utero causes differential DNA methylation between males and females is not known, but a recent study in T cells has demonstrated that the parental source of the X chromosome (male versus female) affects the methylation of the X chromosome in offspring [
19], suggesting that the sex chromosomes may be involved.
Given these known differences, we hypothesised that sex differences also exist regarding the effects of in-utero SE on other pulmonary changes.
The regulation of inflammation involves several signalling pathways, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways [
20]. Three well-characterised subfamilies of MAPK include the extracellular signal-regulated kinase (ERK)1/2, Jun N-terminal kinase (JNK) stress-activated protein kinase, and p38 [
21]. NF-κB is often regarded as the master controller of inflammation [
22]. Inflammatory response requires a considerable amount of energy derived from the mitochondria [
23], whereas mitochondrial function is often compromised during this process. There is a close relationship between the activation of the nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome (increasing IL-1β activity) and mitochondrial dysfunction [
24]. Thus, the inflammasome is regarded as the bridge between inflammatory response and subsequent mitochondrial damage [
24], including oxidative stress [
24] and mitochondrial DNA impairment [
25]. This has been observed in conditions like COPD but has not been investigated in the setting of maternal SE [
26].
Mitophagy is the autophagic elimination of injured mitochondria, which is regulated by fusion and fission [
27]. The balance between fusion and fission is essential to mitochondrial integrity. Fission is to separate damaged mitochondrial fragments from the healthy part, while fusion is to generate a new mitochondrion from two healthy mitochondrial fragments [
28,
29]. We have observed dysregulated mitophagy in the brain and kidney caused by maternal SE which was associated with organ pathology [
13,
30]; however, whether this also occurs in the lung is unknown.
In-utero SE results in considerable foetal oxidative stress and inhibits the endogenous antioxidant activity [
31]. Therefore, improving antioxidant ability may alleviate the adverse effects of maternal SE. L-Carnitine has been shown to attenuate age-related disorders by reducing oxidative stress and increasing antioxidant capacity in rats [
32,
33]. A clinical study also showed that L-Carnitine supplementation can suppress serum levels of inflammatory cytokines in humans [
34]. We have shown that maternal L-Carnitine supplementation during pregnancy and lactation can alleviate brain [
13] and renal dysfunction [
35] in offspring from the SE mothers. As such, this approach may also ameliorate the adverse impact of maternal SE on lung health in the offspring.
Given the known differences in the susceptibility of developing lung diseases between males and females [
36], we hypothesised that in-utero smoke exposure would result in chronic hyperactivation of inflammatory markers and dysregulated autophagy and mitophagy in male offspring, but not in female offspring. Maternal L-carnitine may ameliorate the adverse impact of maternal SE on the offspring’s lung.
Discussion
Maternal smoking during pregnancy is well-documented to cause long-term adverse effects on the health outcomes in multiple organs in the offspring, including respiratory, neurological, and renal systems [
37]. However, the sex difference in respiratory disorders has not been broadly studied, perhaps due to the preference of using one gender to model asthma or COPD.
In this study, male offspring from the SE dams had smaller body weight from birth to adulthood, consistent with previous animal studies and humans suggesting the reproducibility and human relevance of our model [
38,
39]. Maternal SE activated inflammatory NF-κB and MAPK pathways, which were more prominent in the male offspring at P1. It is well known that cigarette smoking can induce inflammation via the MAPK signalling cascade [
40], reflected by increased phosphorylation of ERK and P38 [
41,
42]. MAPK pathway activation can also lead to increased activation of certain transcription factors, such as NF-κB [
43]. In the current study, these effects in P1 male SE offspring are likely due to the chemicals in cigarette smoke including free radicals reaching the foetus via the placenta. NLRP3 inflammasome activation in the male offspring at P1 is in accordance with other inflammatory pathways, especially NF-κB. However, only NF-κB hyperactivation was maintained at adulthood. This may be due to a lack of a second insult after birth. As NF-κB regulates acute responses to external stimuli, its innate hyperactivation may enhance the response to postnatal environmental factors, such as allergens to increase the risk of asthma or tobacco smoking to increase the risk of COPD [
44]. This requires further investigation with additional modelling in the offspring.
It is not surprising to observe that female offspring are less affected by the adverse effect of maternal SE compared with the male littermates. Such a lack of response in the females is consistent with our previous observations in the brain and kidney [
14,
45,
46]. In adults
, there are known differences in innate and adaptive immune responses between males and females which might be related to immune related genes encoded upon the X chromosome being differentially expressed in males and females. Furthermore the sex hormone oestrogen can protect females from developing several diseases [
46,
47].
A recent study found that inflammasomes can be the bridge between inflammation and mitochondrial function [
47]. There is increasing recognition that mitochondrial dysfunction plays a key role in the development of various diseases including COPD and asthma [
27,
48,
49]. Maternal smoking can induce a high level of oxidative stress in the developing foetus [
50] which is persistent until adulthood to directly damage the mitochondria [
14,
51]. Injured mitochondria can also induce more oxidative stress and inflammation. As such, mitophagy and autophagy are key to recycle mitochondrial fragments and eliminate defective mitochondria to maintain cellular homeostasis [
52]. Increased fission maker Drp-1 and autophagosome marker LC3A/B-II in the male SE offspring at birth suggests an increased number of damaged mitochondria due to maternal SE. The fusion marker Opa-1 was not increased suggesting less mitochondrial biogenesis. At adulthood, only LC3A/B-II remained elevated, suggesting higher demand to eliminate injured cellular elements resulting from maternal SE. This may drive the development of lung disorders in the SE offspring [
53]. Interestingly, mitophagy markers in the lung were not changed in the female offspring at any age, suggesting a gender-specific response to maternal SE. These results are consistent with our previous research in other organs [
13].
In vivo and in vitro studies have demonstrated that L-Carnitine can prevent oxidative stress-induced injury [
54‐
56]. In this study, maternal L-Carnitine supplementation increased birth weight in both male and female SE offspring. This suggests that L-Carnitine can ameliorate in-utero underdevelopment caused by maternal SE. Additionally, maternal L-Carnitine supplementation exhibited some anti-inflammatory effects in newborns from the SE dams, by partially suppressing NF-κB activation and NLRP3 inflammasome formation in the males, as well as MAPK pathway and IL-1β in the females. This may be due to its ability to inhibit oxidative stress induced by maternal SE in utero. However, the protection of maternal L-Carnitine supplementation during gestation at birth did not persist until adulthood, especially in the male offspring. The result of increased IL-1β in the male offspring lung, especially in the group with maternal L-Carnitine supplementation is intriguing as there was no increase in inflammasome activation. IL-1β is encoded by its own gene and produced as an in-active protein which is converted into an active protein by the inflammasome. Without a concomitant increase in inflammasome levels our results suggest at 13 weeks that either there is an increase in inactive IL-1β, not active IL-1β or the IL-1β measured by western blotting is produced external to the lungs.
The protective effects of maternal L-Carnitine supplementation have been observed in the other organs, including the brain, kidney, and liver [
13,
30,
35,
57], however not in the lung as shown in this study. This is surprising, but may be explained by the limitation of how we assessed the lung in this study. Firstly, we did not collect bronchoalveolar lavage (BAL) fluid, which can provide information on inflammatory cytokine changes and inflammatory cell counts which are more direct ways to assess lung inflammation. We only used one dose of L-carnitine during pregnancy and lactation, which cannot provide the correlation between L-carnitine dose and inflammation. The multiple-dose regime needs to be tested in our future study. The hyperactivation of inflammatory signalling cascades may represent an increased ability to respond to external stimuli such as allergens or cigarette smoke, but itself may not necessarily cause lung diseases. We did not measure reactive oxygen species levels, and as such whilst it is likely that L-Carnitine supplementation acts via scavenging reactive oxygen species, we cannot definitively say this was the case.
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