Study of intestinal inflammation

Intestinal inflammation, caused by genetic or environmental factors, is the basis of pathologies such as irritable bowel syndrome (IBS), Crohn’s disease and ulcerative colitis, as well as being an important risk factor for the development of tumors. The inflammatory bowel state is characterized by an uncontrolled immune response to the normal resident microflora, leading to abdominal pain and chronic diarrhea that often persist for an extremely long period of time. The inflammatory process in the intestine is regulated by complex interactions between the immune system and other cells located in the epithelial barrier.

Cells of the immune system exchange cellular signals through the production of cytokines, growth factors and adhesion molecules, which facilitate and amplify the process. In epithelial cells stimulated by the typical cytokines of innate immunity (TNF-α, IL-1β) involved in the response to bacterial infections, different pro-inflammatory pathways are activated, through the over-expression of cyclo-oxygenase-2 (COX-2 ), inducible NO synthase (iNOS), and MMP-9. Several pro-inflammatory enzyme products (nitric oxide, prostaglandin E2) and the cytokines themselves lead to the loss of integrity of the epithelial barrier, which thus becomes susceptible to infection by various pathogens.

The inflammatory process of IBD is characterized by an important infiltration of neutrophils, lymphocytes and macrophages. These leukocytes are attracted by chemokines produced in the intestinal mucosa of patients with IBD, among which, IL-8 attracts neutrophils, CCL2 attracts macrophages, CXCL10 attracts lymphocytes. Resident macrophages are thought to be extremely relevant in the first communication between intestinal epithelium and lymphocytes, which are mainly responsible for chronic inflammation. Recent studies have shown that the production of IL-12 and IL-23 by macrophages leads to the activation of lymphocytes, which produce high levels of cytokines involved in autoimmune processes such as TNF-α, IFN-γ and IL-17A . These mediators, together with those described above, have important effector functions on the intestinal epithelium: the main goal of the process is the resolution of the infection, but uncontrolled inflammation leads to chronic pain, mucosal erosion and intestinal ulcers, typical of IBD. The inhibition of TNF-α and IL-23, but not of IL-12 and IL-17A, by monoclonal antibodies (infliximab, ustekinumab) leads to very relevant clinical improvements in patients. Even at the intestinal level, as at the gastric level , the activation of nuclear factor κB (NF-κB) in epithelial cells and its translocation from the cytoplasm to the nucleus represent the primary process regulating the transcription of inflammatory mediators.

Furthermore, many cytokines involved in chronic inflammatory and autoimmune processes activate the transcription factor STAT (IL-12, IL-23, IL-6, IFN-γ): its inhibition also represents a therapeutic target for several new drugs.

In vitro assays

The assays involve the use of a cell culture of human intestinal epithelium (Caco-2) both undifferentiated and differentiated into enterocytes, the main intestinal cell population. These cells represent the main in vitro model described in the literature to study the anti-inflammatory activity in the intestinal tract.

1. Measurement of NF-κB or AP-1-driven transcription following pro-inflammatory stimulation with TNF-α, IL-1β, IFN-γ, Escherichia coli or Salmonella typhimurium bacterial LPS.
2. Measurement of the translocation of NF-κB from the cytoplasm to the nucleus, an event capable of triggering the pro-inflammatory cascade at the gastric level, following a pro-inflammatory stimulus with TNF-α, IL-1β, IFN-γ and Escherichia coli or Salmonella typhimurium bacterial LPS.
3. Release of chemokines (CCL2, IL-8, CXCL10) by intestinal epithelial cells.
4. Secretion, gene expression and catalytic activity of metalloprotease-9 following pro-inflammatory stimuli typical of intestinal pathologies.
5. Measurement of nuclear translocation of NRF2.
6. Effect on the release of reactive oxygen species (ROS).
7. Study of intestinal permeability in Caco-2 cells using enterocyte cultures on Transwell.
8. Evaluation of the expression of proteins involved in intestinal intercellular junctions (TJ) in Caco-2 cells on Transwell.
9. In vitro digestion with intestinal enzymes, in order to establish how the intestine modifies products of vegetable origin.