(T0930-10-66) Impact of Aging on Relative Gene Expression of Tight Junction Proteins in Duodenal and Colonic Epithelium: Preliminary Data

Purpose: Claudin-2, occludin, ZO-1, and JAM-A are TJ proteins that are essential for TJ assembly but also regulate paracellular permeability of water, ions, and small molecules across the pore and the leak pathways [1-4]. There is also evidence that the proinflammatory cytokines IL-6 and IL-1B can affect the expression levels of claudin-2 and occludin when overexpressed, thus indirectly affecting permeability [5,6]. Few studies have focused on age-related changes in the expression levels of TJ and TJ-related proteins of the human intestinal epithelium, resulting indifferent data for the ileum and the sigmoid colon [7,8]. Since other intestinal regions have not been investigated yet, our aim is to compare expression levels of TJ and TJ-related proteins known to regulate paracellular permeability in older and young adults using biopsies from the duodenum, the ascending colon and the descending colon.

Methods: The study protocol was submitted and approved by the Scientific Committee (ΑP 664 |15-02-22), and the Executive Committee (ΑP 12846 |25-05-22) of the Hospital. Biopsies were collected from young (18-45 years) and older adults (≥65 years) undergoing gastroscopy or colonoscopy for medical reasons. Biopsies were taken from macroscopically healthy epithelia of the duodenum, the ascending colon and the descending colon. To study expression levels in duodenum, data from 4 older adults were compared with data from 4 young adults. Biopsies from the ascending and the descending colon were taken in pairs from the same individuals, and expression levels were assessed in 3 young and 3 older adults. RNA was extracted from biopsies using the MagMAX^TM mirVana Total RNA Isolation Kit (Thermo Fisher Scientific). Extracted RNA was used for reverse transcription into cDNA (QuantiTec® Reverse Transcription Kit, Qiagen). For quantitative real-time polymerase chain reaction (qPCR) set up, QuantiNova LNA PCR Custom Panels (Qiagen) containing high specific LNA enhanced-primer assays in combination with the QuantiNova® SYBR® Green PCR Kit (Qiagen) were used. qPCR reactions to measure relative expression levels of Claudin-2, Occludin, ZO-1, JAM-A, IL-6 and IL-1B were run on the Rotor Gene Q (Qiagen). Target gene expressions were normalized to the reference genes beta-actin and hypoxanthine phosphoribosyltransferase. The delta-delta CT method was used to calculate age-related fold changes in gene expression. A 50-100% increase of expression in older adults was considered as potential increase. Analogously, a 25-50% decrease in expression was considered as potential decrease.

Results: In duodenum, no change in expression level was observed for claudin-2, occludin, ZO-1, JAM-A, IL-6 and IL-1B in older adults compared to young adults (Figure 1). In the ascending colon the expression of IL-6 was potentially increased in older adults, whereas no change between the two age groups was observed for claudin-2, occludin, ZO-1, JAM-A, and IL1B (Figure 1). In the descending colon, expression of claudin-2 was potentially increased and expression of ZO-1 potentially decreased in older adults compared to young adults. No change in descending colon was observed for occludin, JAM-A, IL-6 and IL1B (Figure 1).

Conclusion: Preliminary data indicate that there are no age-related changes in the expression of TJ and TJ-related proteins in the duodenum. However, more samples need to be measured to confirm or refute these results. Apparently, the limited increase in IL-6 in the ascending colon of older adults had no effect on claudin-2 expression. However, the increase in claudin-2 in biopsies from the descending colon of older adults may contribute to increased flux via the pore pathway [1], and the decreased expression of ZO-1 in samples from the descending colon of older adults may indicate increased permeability via the leak pathway [3]. Further measurements are needed to confirm the observed changes in gene expression of TJ and TJ-related proteins in the colon.

References: [1] Weber et al., eLife 4:e09906 (2015)
[2] Al-Sadi et al., Am J Physiol Gastrointest Liver Physiol 300:G1054-64 (2011)
[3] Van Itallie et al., J. Cell Sci. 121:298–305 (2008)
[4] Mandell et al., J. Biol. Chem. 280, 11665–11674 (2005)
[5] Al-Sadi et al., PLoS One 9:e85345 (2014)
[6] Rawat et al., Gastroenterology 159:1375 (2020)
[7] Man et al., Clin. Sci. 129:515 (2015)
[8] Wilms et al., Sci Rep 10:475 (2020)

Acknowledgements:This work has received funding from Horizon 2020 Marie Sklodowska-Curie Innovative Training Networks programme under grant agreement No 956146.

Figure 1