Impact of the aberrant cell-matrix interaction in Non-Alcoholic fatty liver diseases progression – MATRIXNASH

The consortium will use a common multidisciplinary approach combining unbiased proteomics, transgenic mouse models, novel therapeutic approaches targeting CD44 and a large cohort of human samples to gain a comprehensive understanding of NAFLD progression into severe liver diseases.

Results at 18 months:

Study the CD44-OPN-HA-TNC axis by targeting CD44 in mice. We have already generated myeloid-, lymphoid-, endothelium- and stellate cells-CD44 KO. The specificity of the CD44 deficiency has already been evaluated and the mice will be challenged by different diets-induced NAFLD.

Study the CD44-OPN-HA-TNC axis by targeting TNC in mice. TNC-KO and inducible TNC-OE (overexpressing TNC) mice have been generated. The arising mice are currently analyzed in detail and then will be used in NAFLD protocols.

To evaluate the proteome profiling. Experiments are ongoing; a large-scale benchmarking dataset has been established, representing inter-indivual (n > 90) proteome alterations as well as defined quantitative proteome alterations. This study has led the foundation to proteomically probe from animal and human samples of the collaborative project.

Study the CD44-OPN-HA-TNC axis in cells. Preliminary results indicated that CD44 could regulate the functions of immune cells including the macrophages and neutrophils. Regarding the signaling pathway involved, CD44 could enhance the activation of the cytoplasmic non-receptor tyrosine kinase SYK in response to LPS. Since we have recently reported that this pathway plays a critical role in the onset of steatohepatitis by regulating macrophage functions, the CD44-dependent SYK pathway will be further investigated. In parallel, we demonstrated that the activation of human stellate cell-line was associated with the upregulation of CD44 and Tenascin-C. Exogenous Tenascin-C by its own enhanced the expression of fibrotic markers as well as CD44. The cross-talk between CD44 and TNC in this process will be investigated.

To evaluate in NAFLD mice the therapeutic impact of targeting CD44-OPN-HA-TNC pathway. We focused on the development of synthetic AAV capsids capable of specifically targeting the liver macrophages, T cells, endothelial cells and hepatic stellate cells. The in vivo approach coupled with ex-vivo validation already allowed to select several AAV candidates. The next steps will be the establishment of AAV vectors encoding shRNA CD44, shRNA TNC or both to down-regulate the expression of CD44 and/or TNC in specific liver cells.

To investigate the relevance of the CD44-OPN-HA-TNC pathways in human NAFLD tissue. We previously reported that CD44 is a liver marker of NASH in obese patients. Hepatic TNC expression will be evaluated according to NAFLD severity and correlated with CD44 expression. In addition, preliminary results seem to indicate that the severity of NASH is associated with the de novo expression of CD44 variants in our mouse model of diet-induced NAFLD. This event seems more related to hepatic fibrosis. The next steps will be to determine which liver cells express these CD44 variants and to evaluate the human relevance.

In the next months, our goals will be

To study the CD44-OPN-HA-TNC axis by targeting CD44 in mice. Myeloid-, lymphoid-, endothelium- and stellate cells-CD44 KO mice will be challenged by different diets-induced NAFLD. The hepatic proteome profiling will be assessed.

To study the CD44-OPN-HA-TNC axis by targeting TNC in mice. TNC-KO and inducible TNC-OE (overexpressing TNC) mice will be used in NAFLD protocols. The hepatic proteome profiling will be assessed.
Study the CD44-OPN-HA-TNC axis in cells. The role of CD44 in the regulation of macrophage and neutrophil functions will be further investigated (focusing on metabolism, cytoplasmic non-receptor tyrosine kinases, biological responses........). The cross-talk between CD44 and TNC in the regulation of the activation and functions of stellate cells will be investigated in parallel.

To evaluate in NAFLD mice the therapeutic impact of targeting CD44-OPN-HA-TNC pathway. The next steps will be the establishment of AAV vectors encoding shRNA CD44, shRNA TNC or both to down-regulate the expression of CD44 and/or TNC in specific liver cells. We will then evaluate the impact of the down regulation of CD44 and/or TNC in specific liver cells on NAFLD development and correction.

To determine the relevance of the CD44-OPN-HA-TNC pathways in human NAFLD tissue. The next steps will be to evaluate the relevance of hepatic CD44 variants and TNC according to NAFLD severity.

Publications at 18 months related to the project (ANR-19-CE14-0044 and DFG: GR 4553/5-1)

ARTICLES

1: Luci C #, Vieira E #, Bourinet M, Rousseau D, Bonnafous S, Patouraux S, Lefevre L, Larbret F, Prod'homme V, Iannelli A, Tran A, Anty R, Bailly-Maitre B, Deckert M $, Gual P $. 3BP2-SYK pathway activity in parenchymal and myeloid cells is a key pathogenic factor in non-alcoholic steatohepatitis. Cell Mol Gastroenterol Hepatol. 2021, S2352-345X(21)00171-5. doi: 10.1016/j.jcmgh.2021.08.004. (Team 1)

2: Fahrner M, Kook L, Fröhlich K, Biniossek ML, Schilling O. A Systematic Evaluation of Semispecific Peptide Search Parameter Enables Identification of Previously Undescribed N-Terminal Peptides and Conserved Proteolytic Processing in Cancer Cell Lines. Proteomes. 2021 May 25; 9(2):26. doi: 10.3390/proteomes9020026. (Team 5)

REVIEWS

3: Luci C, Bourinet M, Leclère PS, Anty R, Gual P. Chronic inflammation in non-alcoholic steatohepatitis: molecular mechanisms and therapeutic strategies. Front Endocrinol (Lausanne). 2020;11:597648. doi: 10.3389/fendo.2020.597648. (Team 1)

Non-Alcoholic fatty liver diseases (NAFLD) with a 25% worldwide prevalence are leading causes of liver associated morbidity and mortality. No pharmacological treatment is yet available for chronic inflammation/ Non-Alcoholic SteatoHepatitis (NASH) and associated fibrosis that are the progressive forms of NAFLD. Although the mechanisms underlying NAFLD progression are not fully understood, chronic inflammation is a key player mediating dysfunction of liver, adipose tissue and the gut. Chronic inflammation modifies the physiological tolerance of the liver and promotes liver injury and fibrogenesis. The extracellular matrix (ECM) and its receptors play a key role in orchestrating chronic inflammation (liver, adipose tissue) and fibrosis leading to advanced NASH. Signaling by CD44 (cell-surface glycoprotein) is initiated by ECM molecules (osteopontin (OPN), hyaluronan (HA), and indirectly tenascin-C (TNC)) that may enhance signal intensity and duration. OPN, HA, TNC and CD44 can form complexes with cell surface receptors (e.g. c-Met, VEGFR, TLR4, EGFR, CXCR4…..) that are known to promote inflammation, angiogenesis and fibrogenesis. We propose that CD44 acts as a central integrator and potentiator by transmitting signals from the ECM into the cytoplasm thus triggering proinflammatory signaling that drives NAFLD.

Indeed, the consortium already discovered that targeting CD44 strongly alleviates liver injury, steatohepatits and fibrosis by downregulating the recruitment of macrophages and neutrophils into the liver and, by decreasing OPN levels and TNC signaling. In patients, hepatic CD44 and TNC expression strongly correlates with each other and with hepatic macrophage infiltration/abundance suggesting a combined action of both molecules in NAFLD.

The aims of MATRIXNASH are:
1.-To decipher the ECM-dependent pathways promoting onset and progression of NAFLD by proteomic and genomic approaches in murine NAFLD models with engineered levels of key candidates (e.g. CD44, OPN, TNC).
2.-To establish novel therapeutic strategies in our fibrotic-NASH models by targeting candidates (CD44, TNC) alone or in combination through general or hepatic cell type specific approaches using novel state of the art AAV delivery systems.
3.-To establish relevance for the human patient in an integrative approach. We will gain comprehensive knowledge about ECM and CD44 complex composition in human NAFLD by proteomic and genomic approaches and tissue analysis for candidates derived from the murine models (cohort of 1006 obese patients). Candidates, relevant in the human disease, will be confirmed in cell culture and our murine NAFLD models.

The consortium will employ a common multidisciplinary approach combining unbiased proteomics, transgenic mouse models, novel therapeutic targeting of CD44 and TNC and a large cohort of human tissue samples to acquire comprehensive knowledge about NAFLD onset and progression. This information may provide novel tools for diagnosis and therapy of NAFLD.