Introduction

Celiac disease (CeD) is triggered by gluten peptides that bind to HLA-DQ2 or -DQ8 in the small intestine leading to gluten specific Th1 T cell expansion and villous atrophy. The oral transglutaminase 2 inhibitor Zed1227 blocks the deamidation of gliadin peptides, a prerequisite for effective gliadin antigen presentation. The CEC-3 clinical trial demonstrated that Zed1227 effectively prevented mucosal damage in 160 CeD patients in remission challenged with 3 g gluten daily for 6 weeks (Schuppan D et al, NEJM 2021).

Aims & Methods

We analyzed regulatory T cell (Treg) gene expression pre and post gluten challenge in the 100 mg/d Zed1227 vs placebo treated patients using RNAseq from the patients’ small intestinal biopsies (Dotsenko V et al, Nat Immunol 2024). We focused on genes of Treg subtypes and related cells. Subanalysis was performed via different gene set enrichments for Treg subsets, regulatory pathways and cell types. Genes that were enhanced in favorable vs unfavorable villous height to crypt depth (V/C) ratio outcomes, also after placebo treatment, were analyzed. We further analyzed pre-disposing genes before challenge, favoring a beneficial or negative post challenge V/C outcome.

Results

93% of the top 100 most significant regulatory genes were upregulated in Zed1227-treated patients compared to those treated with a placebo. These regulatory genes included TGF-beta signaling genes such as Smad7 and MERTK, cholesterol- and fatty acid related genes, such as ACAT1 and NPC1L1, specific autophagy/apoptosis related genes such as VPS35, SQSTM1, BMF, and inhibitory genes like SOCS2. Zed1227 treated patient samples with a maintained post challenge V/C ratio showed evidence for upregulated iTreg genes. Interestingly, Treg genes from placebo-treated subjects were upregulated in correlation with better inflammatory control under gluten challenge compared to worse control.
Moreover, in pre-challenge duodenal samples we identified protective genes that help maintain tolerance during gluten challenge, as reflected by a better post challenge V/C ratio. Examples are ubiquitin ligase RNF167, BAD, FOXN3, BAG1 and CLAUDIN15, whereas other genes like IRF9, CD96 and HLA-/TAP1 were predictive of a more pronounced V/C ratio decline after gluten exposure.

Conclusion

Our data derived from superbly analyzed pre- and post challenge duodenal biopsies demonstrate an active anti-inflammatory immune modulation, largely Treg cell mediated, in CeD in patients prior to and during gluten challenge. Zed1227 treatment potently activates this regulatory response. The balance of several beneficial vs detrimental predisposing Treg-related genes may play a causative role in the variable sensitivity of CeD patients to gluten exposure. Our data support future Treg based therapies to attenuate inflammation in CeD and other (intestinal) autoimmune diseases.

References

Schuppan, D., Mäki, M., Lundin, K. E. A., Isola, J., Friesing-Sosnik, T., Taavela, J., Popp, A., Koskenpato, J., Langhorst, J., Hovde, Ø., Lähdeaho, M. L., Fusco, S., Schumann, M., Török, H. P., Kupcinskas, J., Zopf, Y., Lohse, A. W., Scheinin, M., Kull, K., Biedermann, L., … CEC-3 Trial Group (2021). A Randomized Trial of a Transglutaminase 2 Inhibitor for Celiac Disease. The New England journal of medicine, 385(1), 35–45. https://doi.org/10.1056/NEJMoa2032441
Dotsenko, V., Tewes, B., Hils, M., Pasternack, R., Isola, J., Taavela, J., Popp, A., Sarin, J., Huhtala, H., Hiltunen, P., Zimmermann, T., Mohrbacher, R., Greinwald, R., Lundin, K. E. A., Schuppan, D., Mäki, M., Viiri, K., & CEC-3 Investigators (2024). Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease. Nature immunology, 25(7), 1218–1230. https://doi.org/10.1038/s41590-024-01867-0

Disclosure

V.D. and K.V. received funding from Dr. Falk Pharma to Tampere University to conduct the study. B.T., T.Z., R.M. and R.G. are employees of Dr. Falk Pharma. The data presented here are the subject of patent applications EP24173619.8 and EP24173615.6 filed by Dr. Falk Pharma, and B.T., T.Z., R.M., R.G., V.D. and K.V. are inventors on these applications. M.H. and R.P. are employees of Zedira. J.T. is a consultant for Jilab Oy and Dr. Falk Pharma. K.E.A.L. is a consultant for Amyra, Bioniz Pharmaceuticals, Chugai Pharmaceutical, Dr. Falk Pharma, Itrexon Actobios, TOPAS Therapeutics and Takeda California. D.S. is the data and safety monitor for Boehringer Ingelheim (Phil.) and is a consultant for the Dr. Falk Pharma, Takeda, Immunic, Sanofi and TOPAS Therapeutics. J.I. is the owner of Jilab Oy. M.M. is the founder, owner and Chair of the Board of Maki HealthTech (MHT). MHT receives Management/Advisory Affiliation fees from Dr. Falk Pharma and other funding not related to the research from Topas Therapeutics, Calypso Biotech, Vaccitech, ImmunogenX, Equillium and Immunic. MHT holds patents (patent number 7361480 (United States) and European Patent Office Number 1390753) licensed to Labsystems Diagnostics from where MHT receives royalties via Tampere University Hospital. All other authors declare no competing interests.

Introduction

Oncostatin M (OSM) is a pleiotropic cytokine belonging to the interleukin (IL)-6 family that acts on a large variety of cells involving two distinct heterodimeric receptor complexes: leukemia inhibitory factor receptor beta (LIFRβ) and OSM receptor beta (OSMRβ) that is the receptor able to mediate the most relevant biological effects of OSM in mice. OSM has been proposed to contribute to the progression of chronic liver diseases, hepatocellular carcinoma (HCC) development and metastasis. High levels of OSM were found in cirrhotic patients with different etiology carrying HCC. In particular we observed that OSM serum levels are significantly higher in patients carrying non-alcoholic steatohepatitis (NASH)-related HCC, as compared to those whit viral etiologies, and their increase paralleles the disease progression from simple steatosis to HCC. Noteworthy, OSM serum levels are significantly higher in patients with intermediate/advanced HCC and correlate with poor survival.

Aims & Methods

This work discusses the role of OSM in relation to the development of HCC in a NASH background. We investigated the role of OSM taking advantage of: a) cohort of NASH patients with HCC; b) human THP1 macrophage cell lines exposed to human recombinant OSM (hrOSM); c) Wild type (wt) mice fed with a control diet (CSAA) or a lipogenic diet (CDAA) for 24 weeks in order to reproduce the non-alcoholic fatty acid disease (NAFLD)/NASH pathogenic phenotype (CSAA-CDAA protocol); d) Wild type (wt) and OSMRβ knock out (OSMRβ^-/-) mice treated with a protocol of NASH-related liver carcinogenesis (DEN/CDAA protocol).

Results

In patients with NASH-related HCC, OSM is expressed in cancer cells and its expression increases with patient tumor grade. In in vitro experiments, conducted on human THP1 macrophages exposed to hrOSM, we found that OSM is able to promote an M2 pro-tumorigenic phenotype, which is due to the activation of STAT3 and PI-3K/Akt signaling pathways. Accordingly, OSM expression, which was found increased in NASH-related liver tumours of wt mice during the progression of NAFLD/NASH towards HCC, correlates with F4/80 gene expression. This data suggests an interplay between OSM and macrophages recruitment/functions in the tumor microenvironment. In particular, wt mice treated with the DEN-CDAA protocol show a stronger promotion of the M2 phenotype compared with the M1 and in these mice OSM transcript levels correlate better with M2 macrophage polarization markers. As OSMRβ is fundamental for the activation of the OSM-related STAT3 and PI-3K/Akt signaling pathways, the OSMRβ^-/- murine model was employed to exploit the role of OSM in this regard. In this respect, the data obtained in the OSMRβ^-/- murine models shows that the livers of these animals develop smaller tumours. This event appears to be due to: i) a decreased amount of M2 Tumor Associated Macrophages (TAMs) in the nodules as revealed by the reduction of transcription levels of CD163, PDL1, ARG1, CCL2 and CCR2 compared with wt mice; ii) a impairment of the angiogenic process as shown by lower transcript levels of VE-cadherin, VEGFR2 and CD105 compared with wt mice; iii) a reduction of tumor growth demonstrated by decreased levels of proliferation markers (PCNA, Ki67).

Conclusion

Experimental data highlight a pro-carcinogenic contribution for OSM in NASH, by promoting pro-tumorigenic inflammation, suggesting a possible role for the OSM/OSMRβ axis as therapeutic target for NASH-related HCC.