Introduction

Non-alcoholic fatty liver disease (NAFLD) often advances to more severe stages, namely steatohepatitis (NASH), and may further progress towards hepatocellular carcinoma (HCC). Still, the cellular and molecular mechanisms governing disease progression - and that may be ammendable to therapeutic intervention - remain elusive.

Aims & Methods

Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis.
Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n=199) or HCC (n=366 HCC and n=11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 weeks to induce NASH; and for 66 weeks to induce NASH-HCC.

Results

Results showed that hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile in obese patients. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice displayed increased peroxisome-proliferator activated receptor alpha (PPARalpha) protein levels, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis. Further, the pro-inflammatory/fibrogenic milieu was reduced. Mir21 KO mice presented with a lower number of pre-neoplastic nodules, impaired hepatocyte proliferation and downgraded oncogenic signaling, thus being protected from NASH-related carcinogenesis. The hsa-miRNA-21-5p/PPARalpha pathway was similarly deregulated in patients with HCC or NASH-related HCC, correlating with HCC markers and worse prognosis.

Conclusion

In conclusion, hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. Inhibition of hsa-miR-21-5p, leading to a pro-metabolomic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC (EXPL/MED-OUT/1317/2021, FCT, Portugal).

Introduction

Crohn’s disease (CD) is characterized by chronic uncontrolled inflammation which often leads to intestinal fibrosis. Up to 70% of patients with CD will develop strictures and will undergo surgery as the only available therapeutic option. However, the pathogenesis of CD fibrosis remains incompletely understood. Studies are mainly focused on mesenchymal cells, cytokines and growth factors, whereas the role of neutrophils remains elusive.^1–5 Previous data by our group indicated that neutrophils may acquire a pre-fibrotic role by inducing the differentiation and activation of fibroblasts.^6,7

Aims & Methods

This study aims to investigate for the first time the role of neutrophil-intestinal fibroblast interplay in the pathogenesis of CD immunofibrosis suggesting possible targets for therapeutic interventions.
Neutrophils and intestinal biopsies were obtained from treatment naive patients with moderate to severe CD (CDAI ≥220) and UC (Mayo DAI ≥6). Additionally, sex- and age-matched healthy individuals (HI), were served as controls. Intestinal tissues were characterized using immunohistochemistry and immunofluorescence. Primary intestinal fibroblasts (PIFbs) isolated from tissue specimens were also studied using real-time qPCR, confocal immunofluorescence imaging, in-cell ELISA (cytoblot) and collagen assay. Various inflammatory mediators were measured in cell cultures supernatants with ELISA and multiplex bead-based flow cytometry. The migratory capacity of neutrophils was assessed by a chemotaxis cell migration fluorometric assay.

Results

PIFbs from patients with CD, compared to UC and HI, exhibited a fibrotic phenotype that was characterized by low Krüppel-like Factor 2 (KLF2) and increased Cellular Communications Network Factor 2 (CCN2) mRNA and protein expression leading to excessive collagen production. Neutrophil-fibroblast co-existence was obvious in intestinal tissue fibrotic lesions of CD patients. In line with this, PIFbs culture supernatants from CD patients yielded high levels of IL-8 and increased chemotactic activity for HI neutrophils, which was reduced significantly upon IL-8 neutralization. Of note, compared to UC, neutrophil culture extracts from CD patients downregulated KLF2 and induced CCN2 expression in HI PIFbs acquiring a CD pre-fibrotic phenotype. This neutrophil-mediated immunofibrotic effect was abolished in-vitro by pharmaceutical inhibition of JAK/STAT signaling in PIFbs.

Conclusion

These results, provide a novel mechanistic basis for immunofibrosis in CD suggesting neutrophil/JAK/STAT/fibroblast/IL-8 molecular pathway as a candidate therapeutic target.

References

1. Zidar N, Langner C, Jerala M, Boštjančič E, Drobne D, Tomažič A. Pathology of Fibrosis in Crohn’s Disease—Contribution to Understanding Its Pathogenesis. Front Med (Lausanne). 2020;7:167. doi:10.3389/fmed.2020.00167
2. Santacroce G, Lenti MV, Di Sabatino A. Therapeutic Targeting of Intestinal Fibrosis in Crohn’s Disease. Cells. 2022;11(3):429. doi:10.3390/cells11030429
3. Wéra O, Lancellotti P, Oury C. The Dual Role of Neutrophils in Inflammatory Bowel Diseases. J Clin Med. 2016;5(12):118. doi:10.3390/jcm5120118
4. Lipson KE, Wong C, Teng Y, Spong S. CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair. 2012;5(Suppl 1):S24. doi:10.1186/1755-1536-5-S1-S24
5. Salas A, Hernandez-Rocha C, Duijvestein M, et al. JAK-STAT pathway targeting for the treatment of inflammatory bowel disease. Nat Rev Gastroenterol Hepatol. 2020;17(6):323-337. doi:10.1038/s41575-020-0273-0
6. Chrysanthopoulou A, Mitroulis I, Apostolidou E, et al. Neutrophil extracellular traps promote differentiation and function of fibroblasts. The Journal of Pathology. 2014;233(3):294-307. doi:10.1002/path.4359
7. Chrysanthopoulou A, Antoniadou C, Natsi AM, et al. Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling. Clin Immunol. 2023;247:109240. doi:10.1016/j.clim.2023.109240

Disclosure

This study was supported by the Greek General Secretariat for Research and Technology (GSRT), Research & Innovation Programme CytoNET, grant MIS-5048548 and by GSRT, Regional Excellence Programme InTechThrace, grant MIS-5047285.

Introduction

The ongoing phase 3 ESSENCE trial (NCT04822181) reported positive results for histological and non-invasive test (NIT) endpoints in an interim analysis of the first 800 randomised participants with biopsy-defined metabolic dysfunction-associated steatohepatitis (MASH) and F2/F3 fibrosis receiving once-weekly subcutaneous semaglutide 2.4 mg vs placebo. Participants receiving semaglutide achieved a mean weight loss of 10.5% vs 2.0% with placebo. In this post hoc analysis, we assessed the weight dependency of the effects of semaglutide 2.4 mg on study endpoints (NITs and histology) after 72 weeks of treatment, using weight loss-independent and weight loss-dependent pathways as covariates.

Aims & Methods

NITs and biopsies were assessed at baseline and week 72. MASH-related NIT responder endpoints were change in alanine aminotransferase (ALT; ≥17-unit reduction) and FibroScan-aspartate aminotransferase (FAST) score (≥0.22 reduction). Fibrosis-related NIT responder endpoints were change in vibration-controlled transient elastography (VCTE) liver stiffness measurement (30% reduction) and Enhanced Liver Fibrosis (ELF) score (≥0.5-unit reduction). Histological endpoints included resolution of MASH and improvement in fibrosis. All endpoints were assessed using logistic regression at week 72 with treatment as exposure, percentage weight loss from baseline to week 72 as mediator, and baseline type 2 diabetes status, fibrosis stage and body weight as covariates. The total and weight loss-independent and weight loss-dependent effect sizes were calculated as odds ratios (ORs), and missing data were omitted. All data are based on the full analysis set from the on-treatment observation period.

Results

For MASH-related endpoints, the total effect (OR [95% confidence interval (CI)]) for ALT, FAST score and resolution of MASH without worsening of fibrosis was 4.7 (3.3, 6.6), 6.9 (4.3, 10.9) and 3.9 (2.8, 5.5), respectively. ORs (95% CI) for the weight loss-independent effect were 3.0 (2.0, 4.6), 2.8 (1.7, 4.7) and 2.0 (1.4, 3.0), respectively; for the weight loss-dependent effect, ORs (95% CI) were 1.5 (1.2, 2.0), 2.5 (1.8, 3.4) and 1.9 (1.6, 2.4), respectively. Overall, 71.9%, 53.3% and 51.9% of the total effect for ALT, FAST score and resolution of MASH, respectively, were not mediated by weight loss. For the fibrosis-related endpoints, the total effect (OR [95% CI]) for VCTE, ELF score and improvement in fibrosis without worsening of MASH were 3.0 (2.0, 4.4), 4.5 (3.1, 6.4) and 2.1 (1.5, 3.1), respectively. ORs (95% CI) for the weight loss-independent effect were 1.7 (1.1, 2.7), 2.4 (1.6, 3.7) and 1.5 (1.0, 2.4), respectively; for the weight loss-dependent effect, ORs (95% CI) were 1.7 (1.4, 2.2), 1.9 (1.5, 2.3) and 1.4 (1.1, 1.8), respectively. This shows that 48.9%, 58.5% and 55.5% of the total effect for VCTE, ELF score and fibrosis improvement, respectively, were not mediated by weight loss.

Conclusion

Semaglutide 2.4 mg improved MASH-related histological and NIT endpoints and fibrosis-related NIT endpoints through equal contributions of weight loss-independent and weight loss-dependent metabolic mechanisms, with effects beyond weight loss.

Disclosure

Philip N. Newsome reports grants from Novo Nordisk, and has received consulting fees from Boehringer Ingelheim, Madrigal and Novo Nordisk. PNN also reports honoraria as a speaker from AiCME, Echosens and Novo Nordisk; support for attending meetings for Novo Nordisk; and participation on an advisory board for Boehringer Ingelheim, GSK, Madrigal, Novo Nordisk and Sagimet.
Elisabetta Bugianesi served as a consultant or advisory board member for Boehringer Ingelheim, Gilead, Intercept, Merck, Novo Nordisk, Pfizer and ProSciento, and as a speaker for Gilead, Intercept, Merck, Novo Nordisk and Pfizer. EB has also received a research grant from Gilead for fatty liver research.
Vlad Ratziu received consulting fees from Boehringer Ingelheim, GSK, Madrigal, Novo Nordisk, ProSciento and Sagimet, and research grants (to institution) from MSD.
Mary E. Rinella consults for 89bio, Akero, Boehringer Ingelheim, CytoDyn, GSK, HistoIndex, Intercept, Madrigal, NGM Bio, Novo Nordisk, Sagimet and Sonic Incytes. MER has received fees for consulting or participation in advisory boards for Boehringer Ingelheim, Echosens, Eli Lilly and Novo Nordisk. MER also reports honoraria as a speaker for CME events sponsored by Boehringer Ingelheim, Madrigal and Novo Nordisk.
Michael Roden received lecture fees or served on advisory boards for AstraZeneca, Echosens, Eli Lilly, Madrigal, Merck-MSD, Novo Nordisk and Target RWE, and performed investigator-initiated research with support from Boehringer Ingelheim, Novo Nordisk and Nutricia/Danone to the German Diabetes Center.
Arun J. Sanyal consults for and advises AstraZeneca and Avant Santé. AJS also consults for and has received grants from Akero, BMS, Eli Lilly, Intercept, Madrigal and Novo Nordisk. AJS consults for and owns stock in Rivus, and consults for 89bio, AGED Diagnostics, Albireo, Alnylam, Altimmune, Boehringer Ingelheim, Echosens, Genentech, Gilead, GSK, HistoIndex, Mallinckrodt, Merck, NGM Bio, Novartis, PathAI, Pfizer, Poxel, Regeneron Pharmaceuticals, Inc., Salix, Siemens, Surrozen, Takeda, Terns and Zydus. AJS owns stock in Durect, Exalenz, Genfit, Indalo, Inversago and Tiziana, and has received royalties from Elsevier and Wolters Kluwer.
Adel Belloum, Niels Krarup, Anna Cali, Mohamed Tawfik and Thea Vestergaard are employees and stockholders of Novo Nordisk A/S.
Jörn M. Schattenberg declares consultant honoraria from 89bio, Alentis, Alexion, Altimmune, AstraZeneca, Bionorica, Boehringer Ingelheim, Gilead Sciences, GSK, Inventiva Pharma, Ipsen, Eli Lilly, Madrigal Pharmaceuticals, MSD, Northsea Therapeutics, Novartis, Novo Nordisk, Pfizer, Roche, Sanofi and Siemens Healthineers; speaker honoraria from AbbVie, Academic Medical Education, Boehringer Ingelheim, Echosens, Forum für Medizinische Fortbildung, Gilead Sciences, Madrigal Pharmaceuticals, MedicalTribune, MedPublico GmbH, MedScape and Novo Nordisk; and stockholder options in AGED Diagnostics and Hepta Bio.
Matthew J. Armstrong reports investigator fees from Novo Nordisk.
Anja Geerts reports no conflicts of interest.
Jacob George is supported by the Robert W. Storr Bequest to the Sydney Medical Foundation, University of Sydney; a National Health and Medical Research Council of Australia Program Grant (APP1053206), Project, Ideas and Investigator grants (APP2001692, APP1107178, APP1108422, APP1196492) and a Cancer Institute, NSW grant (2021/ATRG2028). He serves on advisory boards for AbbVie, AstraZeneca, Boehringer Ingelheim, Novo Nordisk and Roche.
Chun-Jen Liu reports no conflicts of interest.
Ewa Janczewska was involved in clinical trials and lectures with Novo Nordisk, was involved in clinical trials with Inventiva Pharma, MSD, Galectin, Dr Falk and Sanofi and reports travel grants from Novo Nordisk and Eli Lilly.
Håvard Midgard is involved in lectures with Novo Nordisk, AbbVie and Gilead, and reports ad boards for Boeringer Ingelheim, AbbVie and Gilead.
Igor Bakulin reports involvement in clinical trials for Novo Nordisk and Eli Lilly and lectures for AbbVie and Johnson & Johnson.