Nonalcoholic steatohepatitis (NASH) is the inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) and is associated with disease progression and the development of cirrhosis. Given the high attrition rates, repurposing of ‘old’ drugs is an attractive proposition, involving the use of de-risked compounds, with potentially lower overall development costs and shorter development timelines (1).
Many candidate compounds for NAFLD/NASH affect both the metabolic and immunological phases of the disease. However, most preclinical models do not distinguish between the metabolic and immunological effects (2,3). This case study presents the use of a novel model in the field of NASH with examples of previously indicated compounds targeting inflammation and fibrosis (4).
FIGURE 1. The NIF mouse shows large similarities to human NASH, involving both type I and type II inflammatory responses, mediated by the transgenic NKT cell population. Red arrows indicate pathways activated in both NIF mice and human samples of NASH.
The non-obese diabetic inflammation and fibrosis (NIF) mouse model spontaneously develops chronic liver inflammation and fibrosis, in the absence of metabolic dysfunction. This phenotype is driven by transgenic type II NKT cells generated in immunodeficient NOD.Rag mice by expressing a transgenic αβ T cell receptor isolated from a type II NKT cell line (4,5). The NIF mouse shows large similarity to human NASH, including NLRP3 inflammasome activation (Figure 1). Both type 1 and type 2 inflammatory responses have been reported to contribute to liver pathology and are present in NIF mice. The early-onset phenotype (inflammation 3 weeks, fibrosis 4 weeks) allows early treatment regime starting, as outlined below.
Rapamycin is an FDA-approved immunosuppresant drug with antifugal and antitumor effects used safely for decades. Rapamycin targets the mTOR signaling pathway, which is the master regulator of cell growth and metabolism, shown to play a role in a number of diseases (6).
Treating 8-week old NIF mice with Rapamycin (4mg/kg or vehicle control) i.p 3 times per week (4-week treatment period) leads to a decrease in previously established liver inflammation (Figure 3 C-D) in comparison to a vehicle-only treatment (Figure 3 A-B) as shown by representative H&E and Masson’s Trichrome staining of liver obtained from the NIF mice following the treatment.
FIGURE 2. NIF mice show established inflammation (A-B) which is reduced following Rapamycin treatment (C-D).
1D11 is a murine monoclonal antibody that neutralizes all three mammalian isoforms of TGF-β . Administration of 1D11 has been reported to prevent progression of kidney fibrosis in several rodent models of kidney diseases (7).
The established liver fibrosis of 6-week old NIF mice (Figure 3A) driven by TGF-β1 expression, the major driver of transition from type 1 to type 2 inflammatory response was significantly reduced following 4 weeks of 1D11 treatment (5 mg/kg of 1D11 or vehicle control) i.p. 3 times/week for a duration of 4 weeks (Figure 3A-C) (5).
FIGURE 3. NIF mice show established fibrosis (A) which is rescued following anti-TGF-β1 antibody (1D11) treatment (B-C).
Paquinimod belongs to the family of quinoline-3-carboxamide family of compounds exhibiting beneficial effects of several mouse models of inflammatory disease, such as multiple sclerosis and experimental autoimmune encephalomyelitis (8).
The established liver inflammation and fibrosis phenotype of NIF mice were reduced following the 4-week treatment with the anti-inflammatory Q compound Paquinimod (25 mg/kg body/weight/day or vehicle control in drinking water) in comparison to control NIF mice and non-treated 24αβNOD control mice (8).
FIGURE 4. NIF mice show established liver inflammation and fibrosis phenotype (A-B) in the absence of metabolic dysfunction that is reduced following Paquinimod treatment (C-D) in comparison to 24αβNOD control littermates (E-F).
The present case study shows that the NIF mouse model is a suitable model to test the efficacy of both anti-inflammatory and anti-fibrotic compounds at the same time in the same animal and in the absence of metabolic dysfunction. The early-onset, phenotype reproducibility and already established liver inflammation and fibrosis phenotype of the NIF mouse have allowed early compound administration, leading to shortened study duration.
This unique liver inflammation and fibrosis mouse model that shows large similarities with human NASH, including hepatic stellate cell and NLRP3 inflammasome activation, allows novel compounds to target these events. As shown, the NIF model can be equally used in the field of repurposing previously indicated compounds for different diseases but also novel drug therapies.
The NIF mouse has been developed & thoroughly validated by InfiCure Bio. InnoSer offers the NIF mouse as a part of a large cardio-metabolic portfolio, offering expertise in the domain of NAFLD and NASH research, including expertise in study design, compound testing in various NASH models, readouts and flexible study start times. Readouts include metabolic and liver-specific biomarkers, liver histopathology scoring, non-invasive ultrasound analysis, and behavioral analysis to assess cognitive impairments.
Contact our cardio-metabolic study experts to consult the best model and readout selection targeted to your research aims and needs.
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8. Fransén Pettersson N, Deronic A, Nilsson J, Hannibal TD, Hansen L, Schmidt-Christensen A, Ivars F, Holmberg D. The immunomodulatory quinoline-3-carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis. PLoS One. 2018 Sep 5;13(9):e0203228