24 February 2021
 

Development of a 3D Intestinal Model for Assessing the Immunomodulatory Properties of Anti-inflammatory Compounds

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Hi, this is Diego Marescotti from Philip Morris International, and in the next few slides, I'm going to show you our work done on the Development of a 3D Intestinal Model for Assessing the Immunomodulatory Properties of Anti-inflammatory Compounds

The best way to avoid harm from smoking is to never start and for those who smoke to quit, smoking is causally linked to serious disease, including cancer, cardiovascular, and pulmonary diseases. The link between smoking and ulcerative colitis (UC) is, however, less well understood, with epidemiological and clinical evidence pointing to an inverse association between smoking and the development of UC.

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Several studies have proposed the activation of nicotine receptor mediated cholinergic anti-inflammatory pathways by nicotine. Moreover, recent observations have highlighted the potential anti-inflammatory role of other tobacco alkaloids, such as anatabine, in a mouse model of colitis. 

During smoking, nicotine and other alkaloids are inhaled together with a large number of harmful constituents generated when tobacco burns. It is therefore important to decouple the harmful constituents to understand the immunomodulating properties of inhaled nicotine and other alkaloids in the context of intestinal inflammation. 

As nicotine and other alkaloids can now be delivered by alternative products that heat rather than burn tobacco, this work focuses on the development of a suitable model for screening anti-inflammatory compounds. 

We developed a 3D in vitro intestinal model which mimics a healthy intestine and a stable barrier integrity. The model consists of a differentiated epithelial layer, composed by Caco-2 and HT-29, and immune competent cells represented by differentiated THP-1 cells. The culture conditions of the epithelial layer were developed to ensure optimal response to pro-inflammatory stimuli such as TNF-α. In particular, we monitored transepithelial electrical resistance, tissue permeability, and cytokine release to assess the response to the inflammatory stimuli. Various conditions of the macrophage differentiation protocol were also evaluated. In particular, different PMA concentration and resting time after differentiation were assessed to identify optimal conditions to obtain a specific, LPS (Lipopolysaccharides) induced inflammatory response. Finally, pre-culture assembly conditions were further defined to obtain a healthy model with stable barrier integrity, with the ability to respond to LPS induced inflammatory stimulus. 

Overall, a controlled and reversible inflammatory state was triggered by LPS administration, resulting in impairment of barrier integrity and release of pro-inflammatory cytokines. Several endpoints, such as cytokine release, tissue permeability, and cell viability can be used to fully characterize the model response to pro-inflammatory stimuli. With the use of known immunomodulatory drugs, of which only Budesonide is shown here, conditions for compound screening were defined. Using the approach developed and verified with the reference compound, we assessed the anti-inflammatory properties of known tobacco alkaloids, such as nicotine and anatabine. Of note, anatabine was tested as a citrate salt in in both racemic and S forms. 

Although the anti-inflammatory effect of nicotine has been previously described, we observed no such effect within the concentration range tested in this study. Unlike nicotine, racemic anatabine  

showed concentration dependent effect on both epithelial membrane integrity and TEER. The observed effect is most likely due to an anti-inflammatory effect exerted on THP-1 cells, as evident from the decrease in cytokine release. The S form exhibited comparable anti-inflammatory effect to those of the racemate.

Nonetheless, S form mediated effects appear to be unspecific, because the S form also induced greater cytotoxicity in the immune cells. Comparison of the result of racemic and S form of anatabines suggests the R form is most likely less toxic, and more specific with regard to anti-inflammatory effect.  

In conclusion, intestinal triculture with 3D geometry, generated by using Transwell system allowed us to produce an in vitro model with several advantages. First of all, less laborious cell culture handling compared to previously published models. Then a direct immune-to-epithelial cell pro-inflammatory activation. And finally, a set of functional readouts that recapitulate in vivo phenotype. We also prove the suitability of the model for use as a screening tool for anti-inflammatory compounds by assessing known anti-inflammatory drugs. And we finally use the model to further characterize known tobacco alkaloids such as nicotine and anatabine, which has previously been shown to have anti-inflammatory abilities in animal models of intestinal diseases.  

I finally thank you for your time and I thank the organizing committee for the opportunity to share our work.

Important: This presentation is for the purpose of publishing and disseminating scientific information about Philip Morris International’s efforts to develop and assess products that have the potential to reduce individual risk and population harm associated with tobacco use. This presentation is for audiences of scientists, public health and regulatory communities, and other stakeholders with an interest in tobacco policy. The purpose is not advertising or marketing. It is not intended for use by consumers. 

Nothing in this presentation should be construed as making any representation, express or implied, that the FDA has approved or has otherwise endorsed IQOS.

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