Protect Your Unborn or Newborn Baby: Secondhand Smoking’s Association to Asthma

Do you know someone with asthma? Chances are that you do. In 2009, over 8% of the US population reported that they currently had asthma. Interestingly, asthma rates are the highest among children and teenagers (Akinbami et al., 2011). Allergic asthma in particular is the type of asthma that is most commonly found in children. Allergic asthma, also known as atopic asthma, affects the lower respiratory tracts. Inhalation of an allergen leads to the release of granules from sensitized cells called mast cells that are located in the mucus of a person’s nose or bronchi (which serve as passageways into his or her lungs). The release of these granules and other molecules favour inflammation. This causes abundant amounts of mucus to be secreted and the tightening of a person’s airways. Asthmatics routinely report feelings of constriction in their chest as well as wheezing. Given this information, you might ask the following question: what factors contribute to the development of allergic asthma? Exposure to cigarette smoke during fetal development and during a child’s infancy has been shown to be a risk factor for allergic asthma (DiFranza et al., 2004). Mice exposed to secondhand smoke (SS) early-on after birth also seem to develop respiratory infections (Phaybouth et al., 1006), and can be used as a model for this disease. The specific contributions of exposure to smoke that is either prenatal (before birth) or early postnatal (after birth) in the development of allergic asthma, however, is not well understood. Also, the specific way in which exposure to cigarette smoke leads to asthma is not understood in great detail. To shed some light on these topics, researchers decided to use a mouse model to study the development of allergic asthma (Singh et al. 2011).

Pregnant mice were exposed to smoke released from the burning end of a cigarette (SS) or filtered air (FA) for six hours per day and seven days a week. This level was the same level of secondhand smoke exposure that a pregnant mother would be exposed to if she spent three hours per day in a smoking bar. The pregnant mice were exposed to one of the conditions for the entire duration of their pregnancy (as well as for two weeks before mating) to cause prenatal fetal exposure. After birth the mice pups were exposed to either FA or SS for eight to ten weeks. (Thus, there were four combinations of postnatal/prenatal exposures in total: FA/FA, FA/SS, SS/FA, and SS/SS). Researchers discovered that prenatal, but not postnatal, exposure to secondhand smoke increased airways’ hyperreactivity (measured with a system that calculates airway resistance or tightness) in the mice after they were exposed to an allergen (which contains an antigen that can lead to allergic responses) called A. fumigatus. Therefore, secondhand smoke before birth seems to have made the lungs of the mice pups more constricted and hyperreactive.

The researchers also desired to observe the presence of Th2 cytokines (which are small proteins) in the lung. After all, allergic asthma is characterized by the presence of Th2 cells, which can be seen both in human (Holt et al., 2010) and mouse (Cates et al., 2007) models of the disease. What exactly are Th2 cells though? Th2 cells originate from Th cells. Th cells are found in your body and can be involved in mounting an immune response against a pathogen. Naïve Th cells become “activated” through a series of events including the recognition of specific complexes called pMHC complexes, which contain an antigen (that was originally part of a pathogen) that can be identified by a receptor on the Th cell. Other events such as costimulation between both the cell that is presenting the pMHC complexes and the naïve Th cell, and the release of cytokines, fully activate the Th cell; this cell is then referred to as a Th0 cell. Extracellular pathogens can cause an unknown cell type to secrete one specific kind of cytokine, called IL-4. IL-4 causes the activated Th0 cells to turn into Th2 cells. These Th2 cells, in turn, release specific cytokines which help to fight off said extracellular pathogens. IL-13 and IL-4 are two of these cytokines that seem to be critical to allergic asthma (Wills-Karp et al., 1998). A previous study by the authors, however, showed that Th2 responses and airway hyperreactivity seem to be regulated by different mechanisms (Singh et al., 2009). That is why the authors decided to investigate whether prenatal and early postnatal SS exposure could differentially affect the process by which Th0 cells become Th2 cells, which is known as Th2 polarization.

To study the effect of prenatal and early postnatal exposure on Th2 polarization, the authors looked at the production of IL-13 and IL-4 in the four aforementioned groups of mice after they were sensitized with the allergen A. fumigatus. The mice exposed to smoke postnatally had a relatively small but significant increase in the levels of the Th2 cytokines. The levels of IL-13 and IL-4 in the lungs, however, were much lower in the mice exposed to SS postnatally when compared to the levels seen in mice exposed to SS prenatally. (These levels were determined using a biochemical technique called an ELISA assay, which relies on antibodies and enzymes to produce a color change which can be quantified and indicate the levels of the cytokines in the samples derived from the mouse lungs.) Therefore, prenatal SS exposure led to a strong Th2 cytokine response in the lungs.

The researchers then wanted to know the specific mechanisms that contribute to the differentiation of Th0 cells into Th2 cells. GATA3 is a transcription factor (which is a protein that affects the expression of specific genes) involved in Th2 differentiation. GATA3 can also cause the expression of genes that are needed to make Th2 cytokines (Zhang et al, 1997). Proteins with the names of Lck, ERK, and STAT6 control the activity of GATA3 (Zhu et al., 2004) (Yamashita et al., 2004), (Kemp et al., 2010). Given these previous findings, the researchers wanted to observe the expression of GATA3 and its influencing proteins in the lungs. To do so, they used techniques called Western Blotting and/or quantitative PCR. Western blotting involves using a gel and electricity to separate different kinds of proteins, which can be transferred onto a membrane and detected using antibodies and reactions that produce color or luminescence. Quantitative PCR is a technique used to tell researchers what the expression level of something (such as DNA or RNA) is. Increased levels of activated (phosphorylated) GATA3 were seen in animals exposed to SS prenatally but not after birth. The same findings were seen with regards to phosporylated ERK 1 / 2, phosphorylated Lck, and STAT6. For these reasons, we now believe that prenatal, but not postnatal exposure to secondhand smoke activates Th2 differentiation through the GATA3 pathway.

The researchers were also interested in the way that SS exposure, either prenatally or postnatally, affected the formation of mucus in the lungs of mice. The mucus in your lungs can serve as an anatomical barrier to physically prevent inhaled pathogens from causing disease and is cleared out regularly. Mucus formation, however, can be impaired in diseases such as asthma. In control animals, exposure to the A. fumigatus allergen caused noticeable increases in the mucus content in the lungs. In all conditions involving secondhand smoke exposure, however, marked decreases of mucus levels in the airways were seen. These levels were quantified by staining tissue with a substance that colored mucus-producing cells pink and then counting these cells to calculate their density. Exposure to prenatal and/or postnatal smoke was shown to limit mucus production, which could hinder an animal’s defence mechanisms against inhaled pathogens.

Mucus in the lungs is made by goblet cells. Goblet cells can also manufacture and release into the mucus certain antibacterial molecules which can break down the cell walls of bacterial pathogens or take away nutrients needed for bacterial growth. SPDEF is a transcription factor which is involved in the development of goblet cells (Chen et al., 2009). Singh and his colleagues looked at the expression of SPDEF in airway epithelial cells in mice using immunohistochemistry. Immunohistochemistry uses antibodies to detect specific proteins in tissues and an antibody-antigen interaction can then be visualized through color-producing reactions. Sensitization with A. fumigatus in control mice strongly upregulated the expression of SPDEF. This would make sense because they would need more goblet cells to make more mucus in an attempt to fight off pathogens. However, the prenatally SS-exposed mice could not increase the expression of SPDEF in their airways. The SS/FA animals had levels of SPDEF that were actually lower than the levels seen in the non-sensitized control mice! Sensitized mice exposed to SS postnatally also had much lower levels of SPDEF than sensitized controls. These results seem to tell us that different expression levels of SPDEF may affect goblet cells and impair mucus production in mice exposed to secondhand smoke.

These researchers certainly shed some light on the mechanisms that are related to both prenatal and postnatal secondhand smoke and the development of allergic asthma. Prenatal SS causes a strong Th2 response in the lung (probably through a GATA3 pathway) and increases SS airway hyperreactivity (which was not seen with postnatal SS). Additionally, prenatal and/or early postnatal SS exposure leads to reduced mucus formation, probably through an impairment of the SPDEF transcription factor and its effect on goblet cells. This study, however, could be extended further. Given that hundreds of different chemicals are found in cigarette smoke, a relevant future direction for these researchers might be to assess which specific chemicals appear to primarily account for the deleterious effects of cigarette smoking as it relates to allergic asthma. It would also be interesting to see if any therapeutic measures could be taken to reverse some of the harmful effects caused by prenatal and/or postnatal secondhand smoking.

It is important to mention that the mechanisms which were seen at play in the aforementioned rodent model also probably play a role in human babies. Practically speaking, the researchers showed that although prenatal secondhand smoke appears to have the most harmful effects if it occurs prenatally as opposed to merely postnatally, damage by postnatal secondhand smoke is also serious. Conscientious parents must be careful not only to avoid smoking themselves, for the sake of their newborn or unborn children, but also to keep their children sheltered from secondhand smoke. If these measures are not taken, children may develop allergic asthma or a variety of other respiratory infections.

Primary Reference:

Singh, S. P., Gundavarapu, S., Pena-Philippides, J. C., Rir-Sima-Ah, J., Mishra, N. C., Wilder, J. A., . . . Sopori, M. L. 2011. Prenatal secondhand cigarette smoke promotes th2 polarization and impairs goblet cell differentiation and airway mucus formation. Journal of Immunology 187(9): 4542-4552.

Additional References:

Akinbami, L., Moorman, J., & Liu, X. 2011. Asthma prevalence, health care use, and mortality: United states, 2005-1009. National Health Statistics Reports. 32: 1-16.

Cates, E. C., R. Fattouh, J. R. Johnson, A. Llop-Guevara, and M. Jordana. 2007. Modeling responses to respiratory house dust mite exposure. Contrib. Microbiol. 14: 42–67.

Chen, G., T. R. Korfhagen, Y. Xu, J. Kitzmiller, S. E. Wert, Y. Maeda, A. Gregorieff, H. Clevers, and J. A. Whitsett. 2009. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production. J. Clin. Invest. 119: 2914–2924.

DiFranza, J. R., C. A. Aligne, and M. Weitzman. 2004. Prenatal and postnatal environmental tobacco smoke exposure and children’s health. Pediatrics 113(4, Suppl.)1007–1015.

Holt, P. G., J. Rowe, M. Kusel, F. Parsons, E. M. Hollams, A. Bosco, K. McKenna, L. Subrata, N. de Klerk, M. Serralha, et al. 2010. Toward improved prediction of risk for atopy and asthma among preschoolers: a prospective cohort study. J. Allergy Clin. Immunol. 125: 653–659, 659, e1–e659, e7.

Kemp, K. L., S. D. Levin, P. J. Bryce, and P. L. Stein. 2010. Lck mediates Th2 differentiation through effects on T-bet and GATA-3. J. Immunol. 184: 4178–4184.

Phaybouth, V., S. Z. Wang, J. A. Hutt, J. D. McDonald, K. S. Harrod, and E. G. Barrett. 2006. Cigarette smoke suppresses Th1 cytokine production and increases RSV expression in a neonatal model. Am. J. Physiol. Lung Cell. Mol. Physiol. 290: L222–L231.

Singh, S. P., N. C. Mishra, J. Rir-Sima-Ah, M. Campen, V. Kurup, S. Razani-Boroujerdi, and M. L. Sopori. 2009. Maternal exposure to secondhand cigarette smoke primes the lung for induction of phosphodiesterase-4D5 isozyme and exacerbated Th2 responses: rolipram attenuates the airway hyperreactivity and muscarinic receptor expression but not lung inflammation and atopy. J. Immunol. 183: 2115–2121.

Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, and D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Science 282: 2258–2261.

Yamashita, M., R. Shinnakasu, H. Asou, M. Kimura, A. Hasegawa, K. Hashimoto, N. Hatano, M. Ogata, and T. Nakayama. 2005. Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitinproteasome pathway. J. Biol. Chem. 280: 29409–29419.

Zhang, D. H., L. Cohn, P. Ray, K. Bottomly, and A. Ray. 1997. Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272: 21597–21603.

Zhu, J., B. Min, J. Hu-Li, C. J. Watson, A. Grinberg, Q. Wang, N. Killeen, J. F. Urban, Jr., L. Guo, andW. E. Paul. 2004. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat. Immunol. 5: 1157–1165.