AXL Is a Potential Target for the Treatment of Intestinal Fibrosis

Abstract

Background: Fibrosis is the final common pathway to intestinal failure in Crohn's disease, but no medical therapies exist to treat intestinal fibrosis. Activated myofibroblasts are key effector cells of fibrosis in multiple organ systems, including the intestine. AXL is a receptor tyrosine kinase that has been implicated in fibrogenic pathways involving myofibroblast activation. We aimed to investigate the AXL pathway as a potential target for the treatment of intestinal fibrosis.

Methods: To establish proof of concept, we first analyzed AXL gene expression in 2 in vivo models of intestinal fibrosis and 3 in vitro models of intestinal fibrosis. We then tested whether pharmacological inhibition of AXL signaling could reduce fibrogenesis in 3 in vitro models of intestinal fibrosis. In vitro testing included 2 distinct cell culture models of intestinal fibrosis (matrix stiffness and TGF-β1 treatment) and a human intestinal organoid model using TGF-β1 cytokine stimulation.

Results: Our findings suggest that the AXL pathway is induced in models of intestinal fibrosis. We demonstrate that inhibition of AXL signaling with the small molecule inhibitor BGB324 abrogates both matrix-stiffness and transforming growth factor beta (TGF-β1)-induced fibrogenesis in human colonic myofibroblasts. AXL inhibition with BGB324 sensitizes myofibroblasts to apoptosis. Finally, AXL inhibition with BGB324 blocks TGF-β1-induced fibrogenic gene and protein expression in human intestinal organoids.

Conclusions: The AXL pathway is active in multiple models of intestinal fibrosis. In vitro experiments suggest that inhibiting AXL signaling could represent a novel approach to antifibrotic therapy for intestinal fibrosis such as in Crohn's disease.

Keywords: AXL; Crohn’s disease; fibrosis; inflammatory bowel disease; myofibroblast.

FIGURE 1.

Induction of AXL gene expression in multiple models of intestinal fibrosis. AXL gene expression is induced in (A) CCD-18Co stiffness model utilizing soft or pathologically stiff collagen coated acrylamide gels (n = 11 per group, P = 0.031); (B) CCD-18Co TGF-β1 model (n = 18 per group, P = 0.022); (C) mouse S. typhimurium model (n = 5 per group, P = 0.087); (D) rat TNBS model (n = 12 per group, P = 0.0015); (E) human intestinal organoids untreated vs TGF-β1 treated (n = 14 untreated, 13 treated, P = 0.077); (F) summary table of gene expression.

FIGURE 2.

BGB324 abrogates CCD18Co myofibroblast fibrogenic protein expression. A, Western blot of fibrogenic proteins in the CCD-18Co TGF-β1 model. CCD-18Co cells were treated with TGF-β1 +/- BGB324 for 48 hours. Total cell protein was extracted for Western blot analysis. Treatment with BGB324 results in the formation of an AXL cleavage product 55 kDa in size. Treatment with 0.5, 1, and 2-μM BGB324 and TGF-β1 resulted in a dose-dependent decrease in both AXL and αSMA protein expression. A longer exposure (data not shown) revealed that 2-μM BGB324 did not completely abrogate AXL and αSMA protein expression. B, Quantification of protein expression in the CCD-18Co TGF-β1 model using ImageJ. C, Western blot of fibrogenic proteins in the matrix stiffness model. CCD18Co cells were cultured for 48 hours on multiwell plates containing soft matrices or on the collagen-coated plastic well bottom. This was done in the presence or absence of BGB324 in low-serum media. Total cell protein was extracted for Western blot analysis at 48 hours. As the matrix stiffness model requires collagen coating of gels and plastic, this precludes quantitation of collagen protein in this model. Treatment with BGB324 again results in the formation of an AXL cleavage product of 55 kDa.

FIGURE 3.

Inhibition of fibrogenic gene expression by the AXL inhibitor BGB324 in the matrix stiffness model. CCD-18Co cells were cultured for 48 hours on multiwell plates containing collagen-coated acrylamide gels corresponding to soft (4.3kPa, 0.02% bisacrylamide) matrices or on the collagen-coated plastic well bottom in the presence or absence of 2-μM BGB324. Cells were harvested for gene expression analysis at 48 hours. Total cell mRNA was extracted, and gene expression of COL1A1, FN1, MYLK, and αSMA (ACTA2) were measured using QPCR analysis. Gene expression was normalized to GAPDH expression. Data are presented as random effects meta-analysis of gene expression over 5 independent experiments comparing plastic-plated vs plastic plus 2-μM BGB324. Each experiment consisted of 3 wells (technical replicates) per treatment per group.

FIGURE 4.

Inhibition of fibrogenic gene expression by the AXL inhibitor BGB324 in the TGF-β1 model. CCD-18Co cells were serum-starved for 24 hours and were treated with 0.05 ng/mL TGF-β1 +/- 2-μM BGB324 for 24 hours; then the total cell mRNA was extracted, and gene expression of COL1A1, FN1, MYLK, and αSMA (ACTA2) were measured using QPCR analysis. Data are presented as a random effects meta-analysis of gene expression over 3 independent experiments comparing TGF-β1-treated vs TGF-β1 plus 2-μM BGB324. Each experiment consisted of 2 wells (technical replicates) per treatment per group.

FIGURE 5.

BGB324 sensitizes CCD18Co cells to apoptosis. CCD18Co cells underwent induction of apoptosis with Fas ligand in the presence or absence of 2-μM BGB324. Total cell protein was extracted for Western blot analysis at 5 hours. Plating on stiff matrix (28kPa) or plastic conferred resistance to apoptosis, and this resistance was abrogated with BGB324 treatment. A, Representative gel, total n = 3. B, Quantification of protein expression using ImageJ.

FIGURE 6.

BGB324 abrogates fibrogenic gene and protein expression in TGF-β1-stimulated human intestinal organoids. A, BGB324 abrogates fibrogenic gene expression in TGF-β1-stimulated HIOs. Human intestinal organoids were cultured in the presence or absence of TGF-β1 and BGB324 followed by extraction of RNA. Total cell mRNAs were extracted and gene expression of COL1A1, FN1, MYLK, and αSMA (ACTA2) in the HIOs was measured using QPCR analysis. Gene expression was normalized to GAPDH expression. Data are presented as random effects meta-analyses of gene expression over 3 independent experiments (ORG-25, 26, 27). Each independent experiment consisted of 3 wells (technical replicates) per treatment group of a 24-well plate. Each well contained multiple (3–4) Matrigel droplets that contained 5 to 10 HIOs per drop. B, HIOs cultured in the presence or absence of TGF-β1, BGB324, or control inhibitors. Total cell protein was isolated for Western blot analysis. An αSMA Western blot which is representative of 4 separate experiments is shown. Cotreatment of HIOs with 10 μM BGB324 reduces the αSMA protein expression induced by TGF-β1. Abbreviations: Spir, spironolactone, antifibrotic control; Tofa, tofacitinib, negative control.