We found that maternal nicotine exposure during pregnancy significantly reduces SP-A gene expression and protein level in pre-term born lambs, while has no effect in naturally delivered, full-term lambs. It is likely that the additional gestational time allows more complete transcriptional activation or enhanced cellular differentiation and proliferation of SP-A producing cells in full-term lambs. Nicotine exposure did not alter SP-D gene expression and protein level in pre-term or full-term born lambs. The qPCR results show wide standard errors of mean. This may be explained by the fact that sheep used in this experiment are outbred. Individual genetic variability in these animals tends to be wide in contrast to commonly used experimental animals such are mice and rats, which are inbred and have less genetic variation within the same strains. Although the wide errors of mean may question the validity of qPCR data, the protein levels determined by Western blot analysis closely parallel the qPCR findings which confirm biological significance of changes determined at mRNA levels. The IHC analysis did not show any statistically significant difference in SP-A protein level in PTN lambs when compared to PTC group; the significant difference was not present between FTN and FTC groups. This finding does not closely match the Western blot results in which the SP-A protein level was reduced in nicotine-exposed lambs born pre-maturely compared to the controls. This may be explained by the fact that the IHC scoring system includes only proteins located within the intracellular compartment (intracytoplasmic SP-A) and does not take into account proteins located within the extracellular compartment (SP-A secreted into intraalveolar space). In contrast, Western blot analysis measures protein located in both, intra-cellular and extracellular compartments and therefore represents more sensitive and more reliable method of lung surfactant protein level determination.
Surfactant proteins are considered to be the key aspect of secretory lung innate immunity, and nicotine-induced suppressive effects may explain, in part, the increased susceptibility of pre-term born, nicotine-exposed neonates to lower respiratory infections. Although lung epithelia secrete many anti-microbial peptides, proteins, and molecules, SP-A and SP-D are among the most extensively studied pulmonary host defense/innate proteins. They are constitutively expressed and secreted by ATII cells and Clara cells and belong to a subgroup of collectin-containing mammalian C-type lectins, called collectins
SP-A and SP-D can bind, aggregate and opsonize many microorganisms including Gram-negative and Gram-positive bacteria, enveloped viruses like Influenza AVirus (IAV), RSV, non-enveloped viruses like Rotavirus and fungal organisms in order to protect lung from potential infection.13
In animal studies, the deficiency of SP-A, SP-D, or both were associated with increased susceptibility to pulmonary Pseudomonas aeruginosa
Elevated levels of SP-A and SP-D mRNA in the lungs of neonatal lambs are associated with clearance of Parainfluenza virus-3 (PIV-3).13
Also, reduced clearance of RSV in lambs is associated with pre-maturity and inadequate expression of pulmonary collectins12
while SP-A has been shown to enhance RSV clearance in mice.16
In addition, when SP-A and SP-D were administered to the lung in a murine model of invasive pulmonary aspergillosis, the mortality rate of infected animals was significantly reduced.13
In rats, administration of SP-A reduces the oxidative damage of ventilated lungs.17
Published data on the influence of smoking and nicotine on ATII cells, lung surfactant, and surfactant proteins are conflicting. There are several reports clearly indicating that cigarette smoking or nicotine alone affects alveolar ATII cells and therefore, possibly lung surfactant metabolism. In a study published by Maritz and Thomas,18
intrauterine exposure of rat pups to nicotine resulted in increased proliferation of ATII cells. Nicotine-exposed ATII cells exhibited obvious mitochondrial swelling and damage which may have disrupted adequate cellular metabolism, including metabolism of lung surfactant and surfactant proteins. In the most recent study published by Rehan et al.,19
nicotine exposure accelerated ATII cell proliferation, differentiation and metabolism in rat embryonic lung and stimulated synthesis of surfactant lipids. However, in both of these studies the influence of nicotine on SP-A and SP-D was not investigated. A study conducted in healthy adult smokers showed reduced content of SP-A and SP-D in BAL fluid when compared to non-smokers.20
A similar study conducted in rats confirmed this finding since rats chronically exposed to cigarette smoke had significantly reduced SP-A and SP-C levels in BAL fluid.21
Several studies conducted in rats showed that maternal nicotine or cigarette smoke exposure alters the expression of surfactant proteins in neonatal pups. In one study, maternal nicotine exposure increased SP-A and SP-D expression in neonatal pups on postnatal day 7 and decreased SP-A and SP-D expression on postnatal day 14.22
Yet, in another study conducted in rats, maternal side-stream cigarette smoke exposure showed reduced levels of SP-A in BAL of pups on postnatal day 1 and increased levels of SP-A in BAL on postnatal day 21.23
However, there are reports that suggest that maternal smoking does not influence lung surfactant proteins of exposed neonates. For example, neonates from mothers who smoked during pregnancy did not show reduced levels of SP-A according to amniotic fluid measurements.24
There is also a report indicating that smoking in healthy individuals increases levels of SP-A when measured in serum.25
In addition, there is scientific evidence that pre-natal nicotine exposure has permanent detrimental effect on later, postnatal lung development and function. It has been shown that in utero exposure to nicotine influences the ‘‘programmed’’ lung aging and maintenance thus accelerates aging and pre-disposes lung to parenchymal damage.18
In addition, there is controversy related to the safety of nicotine patch use during pregnancy. Although the nicotine patch is highly recommended as nicotine replacement therapy in pregnant women25
during the smoking cessation, to our knowledge there are no studies conducted to evaluate the effect of nicotine patch on developing fetus. In this study, we have shown that nicotine delivered to the mother by nicotine patch does have an undesirable effect on fetal lung epithelial development.
Differences of the above studies with the lamb model are likely related to differences in animal lung maturation at the various stages of lung development, duration of nicotine or cigarette smoke exposure, as well as in the techniques used to determine surfactant protein levels. Although cigarette smoking during pregnancy remains a significant cause of neonatal mortality and morbidity, previous studies frequently utilized rodents as animal models which do not adequately parallel fetal lung development in humans. This is especially relevant in alveolar and pulmonary surfactant development research area. At birth, the lung of rodents is in saccular stage and alveoli with lung surfactant develop during first 3 weeks of postnatal life.26
In contrast, gestating ewes and their offspring are well-suited for these studies since: (1) alveolar development in lambs occurs pre-natally as in humans26
(2) lambs can be derived pre-term and survive, unlike many other animal models,12
(3) ovine respiratory epithelia of airways, distal bronchioles and alveoli are similar to those of human lung including similar numbers of Clara cells (which can reach 50% of the epithelium of mice), and the ovine lung has submucosal glands not present in some animal models,10,27
(4) expression of SP-A and SP-D and other innate immune genes of ovine lung are well-characterized and are very similar to human orthologs,10
and (5) pre-term lambs are susceptible to RSV, a known pathogen of pre-mature infants, and maternal cigarette smoking is a risk for pre-term birth. Finally, sheep have long been used for respiratory studies related to human disease as well as for studying the effects of nicotine on pulmonary function.
Nicotine is the major cigarette constituent that crosses the placenta and accumulates in the fetal compartment to influence fetal lung growth and development.9,28
The transdermal patch as a nicotine delivery method was used for several reasons. First, when fitted to the prepared skin area, the patch slowly and constantly releases nicotine for at least a 12 hr time period, which closely resembles the smoking habit in humans. In contrast, other nicotine delivery techniques such as intravenous administration and nicotine releasing subcutaneous pumps are invasive and may result in excessive nicotine blood levels which are difficult to control. Second, according to several published studies, nicotine metabolism in pregnant women is significantly accelerated compared to non-pregnant females and males.29
Also, pregnant women tend to quit or at least significantly reduce the number of cigarettes smoked per day. According to a study published by Peacock et al.,30
pregnant women who smoked during the last trimester of pregnancy most frequently had CPL ranging from 15–75 ng/ml. Similar levels were present in maternal blood in this study. In addition, depending on cigarette brand and strength, this plasma cotinine level would be equivalent to 2–6 cigarettes smoked per day. Lastly, although administering nicotine during entire length of gestation would more closely mimic the human behavior, we have chosen to administer nicotine during the last 6 weeks of ovine gestation since this time frame corresponds to the time frame of pulmonary surfactant development in fetal lambs.31
In conclusion, nicotine exposure during the last trimester of pregnancy in sheep is associated with a significant reduction in SP-A gene expression and protein level in lambs born pre-term. This finding may have clinical significance and will allow us to further investigate RSV infection as a means to address the hypothesis that nicotine exposure during pregnancy may increase RSV susceptibility in human infants. To our knowledge, there are no publications describing similar animal model in lung development research area. In this work we have described an innovative animal model that may be used by broad research community focused on neonatal nicotine-related pathological processes such as respiratory distress syndrome, impaired intrauterine lung growth, and other pulmonary abnormalities during postnatal life.