Nicotinic receptor could be target for treatment of lung inflammation

An experimental drug that stimulates a specific type of nicotinic receptor in immune cells reversed chronic and acute lung inflammation in mice.

By Karina Toledo, in Campos do Jordão (Brazil)  |  Agência FAPESP – One puff of a cigarette is enough for the nicotine inhaled with the smoke to be absorbed by the lungs, enter the bloodstream and almost instantly activate nicotinic acetylcholine receptors at the nerve cell surface, triggering the euphoric effect followed by relaxation that makes the drug addictive.

It has been amply proven that smoking can cause serious diseases such as emphysema and cancer. A new study, however, shows that pharmacological stimulation of a specific type of nicotinic receptor in cells of the immune system could be a strategy to treat inflammatory lung disease.

In this case, the therapeutic effect is associated with activation of nicotinic receptors of the alpha-7 subtype in macrophages, the immune system’s first-line cells that are central to the inflammatory response to a potential threat. 

Findings from the study, which was performed at the Federal University of São Paulo (UNIFESP) with FAPESP’s support, were presented by researcher Carla Máximo Prado, from that university’s Health & Society Institute (ISS-UNIFESP), in a presentation delivered on September 4, 2017, to the 32nd Annual Meeting of the Federation of Brazilian Societies for Experimental Biology (FeSBE) in Campos do Jordão, São Paulo State, Brazil.

“In tests with animals, specific stimulation of alpha-7 nicotinic receptors with an experimental drug called PNU-282987 reduced inflammation in a chronic allergic condition similar to asthma and in a lung inflammation model similar to acute respiratory distress syndrome (ARDS), a severe form of respiratory failure that occurs when fluid fills the air sacs in the lungs and that is usually associated with an infectious process,” Prado said in an interview given to Agência FAPESP.

Both nicotinic and muscarinic receptors are part of the cholinergic system, a branch of the nervous system in which acetylcholine is the main neurotransmitter, Prado explained.

In the lungs, acetylcholine initially became known for its role in bronchoconstriction (acute narrowing of the airways). The active ingredients in several medical drugs used to treat asthma and chronic obstructive pulmonary disease (COPD) are substances that prevent acetylcholine from binding to muscarinic receptors.

More recent research, however, suggests that acetylcholine has a protective effect on the lungs that is linked to activation of nicotinic receptors.

In a previous study involving mice that had been genetically modified so as not to express vesicular acetylcholine transporter (VAChT), a protein that mediates the release of acetylcholine at synapses, the research group from UNIFESP, working in collaboration with scientists at the University of Western Ontario in Canada, observed an exacerbated inflammatory response in the lungs, even without any kind of disease or allergy. 

“The release of acetylcholine fell 75% in these mice, and as a result, they suffered a process of inflammation and airway remodeling similar to that seen in people with asthma,” Prado said. “In addition, the cellular signaling pathways involved in the pulmonary inflammatory response were altered.”

These results were described in an article published in 2015 in the journal PLOS ONE.

Based on these findings, the group at UNIFESP decided to test the hypothesis that stimulating the cholinergic system with a drug that binds to nicotinic receptors might attenuate inflammation in the lungs of mice that had not been genetically modified so as not to express VAChT. 

Promising results

The first tests were performed using a classic mouse model of acute lung injury. The researchers injected bacterial lipopolysaccharide (LPS), a toxin extracted from the outer membrane of Gram-negative bacteria, into each mouse’s trachea.

“About 30 minutes before LPS injection, some of the mice were treated with PNU-282987, a compound that stimulates the alpha-7 nicotinic receptors. Another group was treated with the same compound six hours after LPS injection, when the inflammation reached its peak. In both cases, we observed a significant reduction in the inflammation compared with untreated mice,” Prado said.

Besides reducing pulmonary edema (lung swelling), the therapy decreased immune cell release of pro-inflammatory molecules such as interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Analysis of bronchoalveolar lavage fluid (saline instilled into the lung and then aspirated) showed a reduced presence of immune cells, especially neutrophils and macrophages.

“We also evaluated the effect of this drug on isolated lung macrophages and found a reduced percentage of M1 macrophages, which have a pro-inflammatory profile. At the same time, there was a higher proportion of M2 macrophages, which are associated with the repair of damaged tissue. This may explain the observed improvement in the lung function of the treated mice,” Prado said. 

Finally, their analysis of lung tissue showed that the treatment reduced activation of the protein NF-κB, a transcription factor that stimulates the production of inflammatory molecules by the cells of the immune system.

Some of the experiments were performed during the master’s research and PhD research of Nathalia Montouro Pinheiro, in both cases with support from FAPESP. The group also included Milton de Arruda and Iolanda Tibério, researchers at the University of São Paulo’s Medical School (FM-USP), and Niels Câmara at the same university’s Biomedical Science Institute (ICB-USP).

The findings are described in an article published in 2017 in The FASEB Journal.

According to Prado, the treatment also had positive effects in the chronic inflammation model, which used a classical asthma induction method. The procedure consists of sensitizing the immune system with two injections of ovalbumin, the main protein in egg white, associated with an adjuvant substance that boosts the immune response to this antigen. After 21 days, the animals inhale the same protein, against which their immune system has now developed antibodies, at four different times.

“In this model, the animal develops a chronic inflammatory response that eventually leads to a remodeling of the airways,” Prado explained. “Collagen is deposited in the airways, and mucus-producing and smooth muscle cells become hypertrophied. All these factors associated with the chronic inflammatory response result in loss of lung function.”

Some of the mice were treated with PNU-282987 from the 21st day after the first injection of ovalbumin, at the same time as the inhalatory challenges with the antigen began. The substance was administered by intraperitoneal injection for seven days. 

“We observed a reduction in the lung remodeling process and found that the bronchoalveolar lavage fluid contained a reduced amount of eosinophils, the main type of immune cells associated with asthma,” Prado said.

These experiments were part of Cláudia Pontes’s PhD research.

Plant cure

During the FeSBE conference, Prado also presented results from a second line of research that focuses on testing treatment with sakuranetin in asthma and ARDS models. Sakuranetin is a flavonoid isolated from Baccharis retusa, a plant in the same family as the sunflower (Asteraceae) and found in Serra da Mantiqueira, a mountain range in the states of São Paulo, Minas Gerais and Rio de Janeiro.

“We’ve shown that sakuranetin has both preventive and therapeutic effects,” she said. “We’re trying to understand the mechanisms of its action: the cellular signaling pathways it inhibits or amplifies to produce this anti-inflammatory effect” (read more at: agencia.fapesp.br/20697).

Another phytochemical Prado is studying at her laboratory is dihydrodieugenol isolated from Nectandra leucantha, a plant species belonging to the same family as the avocado (Lauraceae). “The main advantage of this compound is that it’s easy to synthesize in the lab using simple and low-cost reagents,” Prado said. “The results are promising, especially in the asthma model.” These studies are part of Rafael Cossi’s scientific initiation research with a scholarship from FAPESP, as well as Fernanda Roncon’s PhD research.

The studies involving natural compounds are being conducted in partnership with João Henrique Ghilardi Lago, a professor at the Federal University of the ABC (UFABC).