Maternal smoking during pregnancy is well-documented to cause long-term adverse effects on the offspring’s health outcomes in multiple organs, including respiratory, neurological, and renal systems (32). However, the sex difference in such susceptibility in respiratory disorders has not been broadly studied, perhaps due to the preference of using one gender to model asthma or COPD. Here, we showed that while maternal SE induces a persistent increase in the inflammatory response in the male’s lung, females seem to be protected from such adverse maternal effects. Against our hypothesis and our observation of maternal L-carnitine supplementation in other organs, L-carnitine only provided transient protection.
In this study, male offspring from the SE dams had smaller body weight from birth to adulthood, consistent with previous studies and human birth weight suggesting the reproducibility and human relevance of our model (33, 34). Maternal SE activated inflammatory NF-kB 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 (35), reflected by increased phosphorylation of ERK and P38 (36, 37). MAPK pathway activation can also lead to increased phosphorylation of certain transcription factors, such as NF-kB (38). In the current study, these effects in P1 male SE offspring are likely due to the chemicals including free radicals in cigarette smoke accessing the foetal circulation via the placenta. NLRP3 inflammasome activation in the male offspring at P1 is in accordance with other inflammatory pathways especially NF-kB. However, only NF-kB hyperactivation was maintained at adulthood. This may be due to a lack of a second insult after birth. As NF-kB regulates acute responses to external stimuli, its innate hyperactivation may enhance the response to postnatal environmental factors, such as an increased risk of asthma or COPD (39). This requires further investigation with additional modelling in the offspring.
It is not surprising to observe that female offspring are mostly protected from 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, 40, 41). However, sex differences are often not analysed incomparable human studies.
A recent study found that inflammasomes can be regarded as the bridge between inflammation and mitochondrial function(42). There is increasing recognition that mitochondrial dysfunction plays a key role in the development of various diseases including COPD and arthma(24, 43, 44). Maternal smoking can induce high oxidative stress levels in the developing foetus (45) persistent until adulthood which can directly damage mitochondria(14, 46). Injured mitochondria can also induce more oxidative stress and inflammation. As such, mitophagy and autophagy are key to recycle intact mitochondrial fragments and eliminate damaged ones to maintain cellular homeostasis (47).
Increased fission maker Drp-1 and autophagosome marker LC3A/B-II in the male SE offspring at birth, suggest increased damaged mitochondria due to maternal SE. The fusion marker Opa-1 was not increased accordingly suggesting less healthy fragment can be recycled. At adulthood, only LC3A/B-II remained elevated, suggesting a higher demand to eliminate other injured cellular elements by maternal SE. This may drive the development of lung disorders in the SE offspring (48). Interestingly, mitophagy markers in the lung were not changed in the female offspring at any age, again suggesting gender-specific protection from 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 (49–51). In this study, maternal L-Carnitine supplementation increased the 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 supressing NF-kB 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 on the lung did not persist until adulthood, especially in the male offspring.
The effect in the lung is against our hypothesis and the protection observed in the other organs(13, 31, 52, 53). This is surprising, but may be explained by the limitation of how we assessed the lung in this study. firstly, we did not collect BAL fluid, and as such typical markers of inflammation such as cytokine levels in bronchoalveolar lavage fluid to perform inflammatory cell counts which is a more direct way to access lung inflammation. The hyperactivation of signalling cascades may represent an increased ability to respond to external stimuli such as allergens or cigarette smoke, but in and of itself may not cause lung disorders. We did not measure ROS levels, and as such whilst it is likely that L-Carnitine supplementation acts via scavenging ROS, we can not definitively say this was the case.