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Coeliac disease (CD) is a chronic inflammatory and autoimmune disorder, primarily affecting the small intestine, developed in genetically susceptible individuals upon gluten ingestion. The only effective treatment so far is a lifelong, strict gluten-free diet. However, difficulties to follow dietary compliance can lead to complications, highlighting the unmet need for adjuvant therapies.
Recently in Gut, we described a novel m6A-XPO1-NFκB pathway that is activated in patients with CD. Specifically, YTHDF1 m6A reader was found to selectively bind the 5’ UTR of XPO1 mRNA and induce its translation, increasing XPO1-mediated inflammation in intestinal cells both in vitro and in vivo.1 These findings opened the door to new therapeutic approaches directed to m6A machinery proteins, already in use for the treatment of other disorders.2
Interestingly, novel studies have described salvianolic acid (SAC) as a selective inhibitor of YTHDF1, which can rescue Fragile X syndrome linked defects in neural progenitor cells.3 In this study, we used our previously developed in vitro and in vivo gluten exposure models1 in order to test whether two forms of SAC (termed Y20 and Y22) could be used to ameliorate intestinal inflammation. Our in vitro data show a reduction of the pepsin-trypsin digested gliadin (PTG)-induced inflammation, represented by the enhanced XPO1, NFκB and IL8, at both RNA and protein levels, in cells treated with YTHDF1 inhibitors (figure 1A,B). Additionally, mice treated with PTG and SAC presented lower levels of Xpo1, NFκB and Mip2a, Cxcl5 and Cxcl1 cytokines (homologues for human IL8) than those exposed only to PTG, which showed induced intestinal inflammation (figure 1C–E, online supplemental figure 1A). Small intestinal epithelium morphometric and histologic quantification of intestinal response to PTG was also addressed (figure 1F). While PTG treatment showed a significant decrease of the villus height to crypt depth ratio (figure 1 F and G), a slight recovery can be observed in Y20 treated mice group when compared with PTG treated mice (figure 1G), suggesting that this inhibitor could help protecting intestinal disruption during inflammation. Th1 response related cytokines, Ifng and Il21, and the CD45+ intraepithelial lymphocyte-specific gene expression was also augmented in PTG treated mice but was reduced in SAC treated mice (figure 1H, online supplemental figure 1B); suggesting a decrease in the coeliac characteristic immune cell infiltration after SAC treatment. Moreover, in the intestinal biopsy ex vivo model from newly diagnosed CD patients, a reduction of XPO1, NFκB and IL8 was observed when incubated with the inhibitors (figure 1I,J). All these in vitro, in vivo and ex vivo results show that SAC based selective YTHDF1 inhibitors can help ameliorate gluten-induced intestinal inflammation.
In addition, we were able to show that both SAC forms do not show toxicity in our in vivo model. No significant changes were observed between control and treated mice regarding their size and weight. Moreover, no gastrointestinal effects could be detected in terms of diet consumption or faeces weight in treated mice groups (online supplemental figure 1C-E). In addition to the lymphocyte markers (figure 1H, online supplemental figure 1B), we could also confirm that our in vivo PTG stimulation activates an inflammatory response by the increased number of goblet cells present in the epithelial cells as well as eosinophil counts in the lamina propia4 5 (online supplemental figure 1F). In mice treated with PTG and either YTHDF1 inhibitor, these counts were reverted to control values, pointing again that these SAC molecules can, at least partially, protect from intestinal inflammation (online supplemental figure 1F). Interestingly, in another intestinal inflammatory scenario (on IFNG stimulation), both inhibitors also showed the ability to reduce inflammatory IL8 chemokine levels (online supplemental figure 1G), showing that these small molecules could also be useful in other gluten-independent intestinal inflammatory conditions.
To sum up, here, we present two different selective YTHDF1 inhibitors that have the ability to reduce gluten-induced inflammation in intestinal cells without apparent side effects in vivo. Although further exploration of other conventional CD pathways is still needed, this study paves the way for the development of promising therapeutic strategies for intestinal inflammatory disorders as CD.
Patient consent for publication
This study involves human participants and this study was approved by the Basque Country Ethics Committee CEIm-E with reference number PI2019133. Participants gave informed consent to participate in the study before taking part.
AO-G and HR-M are joint first authors.
Twitter @HRMrqz, @AinaCastellanos
Contributors Conceptualisation: AC-R. Methodology: AOG, HRM, MMR, PR, AAL, YC, MJP, LH, DS, CH and ACR. Investigation: AOG, HRM, MMR, PR, AAL, YC and ACR. Resources: PR, MJP, LH, DS, CL, LB, CH and ACR. Patient recruitment and sample collection: LB. Writing–original draft: AO-G and AC-R. Writing–review and editing: AOG, HRM, MMR, PR, AAL, YC, MJP, LH, DS, CL, LB, CH and ACR. Supervision: AC-R. Project administration: AC-R. Funding acquisition: LH, DS, CL, LB, CH and AC-R.
Funding This study was supported by the Spanish Ministry of Science, Universities and Innovation (Grants PGC2018-097573-A-I00 to AC-R, and PID2020-114953RB-C21 to LH and DS cofunded by the European Regional Development Fund (ERDF)), the Biomedical Research Centre in Pathophysiology of Obesity and Nutrition (CIBEROBN) (Grant CB06/03/0001 to LH), the Merck Health Foundation (to LH), and the Government of Catalonia (2021SGR00367 to LH). CL was funded by National Administration of Traditional Chinese Medicine (ZYYCXTD-202004). CH is a Howard Hughes Medical Institute Investigator and has been funded by the National Institute of Health HG008935. AO-G was funded by postdoctoral fellowships from the University of the Basque Country (ESPDOC21/56). HR-M was funded by predoctoral grant from the Spanish Ministry of Science, Universities and Innovation (PRE2019-089350). Ciberehd is funded by the Instituto de Salud Carlos III.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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