Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling that induces hepatocyte proliferation

Abstract

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1⁺ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1⁺ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Fig. 1.. Jedi T cells deplete HSCs.

(A) Representative immunohistochemical staining for GFP of Pdgfrb-GFP adult mouse liver. PV, portal vein; CV, central vein. Scale bar, 50 μm. n = 10 mice. (B) Immunofluorescence microscopy of liver section from Pdgfrb-GFP transgenic mice for GFP and desmin. Scale bar, 20 μm. n = 10 mice. (C) Experimental scheme. (D) Macroscopic liver images of control and HSC-depleted mice. Scale bar, 5 mm. (E) Liver-to-body weight ratios of control and HSC-depleted mice. Mean+SD. n=8 mice per group. (F and G) Immunofluorescence microscopy of liver sections from control and HSC-depleted mice for desmin (F) and GFP (G). Nuclei are stained with DAPI. Squares indicate areas of enlargement. Scale bars, 100 μm (lower magnification) and 20 μm (enlargements in F) and 50 μm (enlargements in G). n = 10 mice per group. (H) Quantification of desmin⁺ tissue area in control and HSC-depleted mice. Mean+SD. n=5 mice per group. (I and J) qRT-PCR for Des (I) and Pdgfrb (J) expression in whole-liver RNA. Mean+SD. n=5 mice per group. (K) H&E staining of sections of control and HSC-depleted livers. Scale bar, 50 μm. Arrows indicate examples of sinusoids. (L) Serum ALT in control and HSC-depleted mice. Mean+SD. n=10 control and 7 HSC-depleted mice. *p< 0.05, **p< 0.01, by unpaired two-tailed Mann-Whitney t-test.

Fig. 2.. HSC depletion decreases liver mass and diminishes midlobular hepatocyte numbers.

(A) Experimental scheme. (B) Immunohistochemical staining for desmin in control and HSC-depleted mice 6 weeks after adoptive transfer. Scale bar, 20 μm. n = 5. (C) Macroscopic images of control and HSC-depleted mouse livers in situ. Scale bar, 5 mm. (D) Liver-to-body weight ratios of control and HSC-depleted mice at 6 weeks. N=5 control and 4 HSC-depleted animals. *p<0.05 by unpaired two-tailed Mann-Whitney test. (E and F) High- and low-magnification of H&E staining of control and HSC-depleted livers 6 weeks after adoptive transfer. Scale bars, 50 μm (E) and 1 mm (F). n = 5 control and 5 HSC-depleted mice. (G) Immunohistochemical staining for CD31 in an HSC-depleted liver section. Arrows indicate endothelial lining of vessels. PV, portal vein; CV central vein. Stars indicate acellular cavities with absent endothelium. Square indicates area of enlargement. Scale bars, 100 μm. Image is representative of 5 mice. (H) H&E staining of HSC-depleted liver section at 6 weeks. In the annotated image, green dots indicate portal vein (PV), yellow dots central vein (CV), and blue dots acellular cavities. Scale bar, 500 μm. n = 5. (I) Immunohistochemical staining for CCND1 in control and HSC-depleted livers at 10 days and 6 weeks after adoptive transfer. Squares indicate areas of enlargement in inserts. Scale bar, 50 μm. n = 6 control and 11 HSC-depleted mice at 10 days after adoptive transfer. n=5 control and n=5 HSC-depleted mice 6 weeks after adoptive transfer. (J) Western blot showing desmin, CCND1, and IGFBP2 in whole-liver lysates from control and HSC-depleted livers 10 days after adoptive transfer. Gapdh is a loading control. Each lane represents 1 mouse. n = 3 control and 3 HSC-depleted mice. (K) Whole-liver RNA-seq analysis of control and HSC-depleted livers 10 days after adoptive transfer. n=6 controls and 4 HSC-depleted mice. #, non-differentially expressed genes (FDR>0.34). (L) Immunohistochemical staining and quantification of Ki67 and cleaved caspase 3 in control and HSC-depleted livers at 10 days and 6 weeks after adoptive transfer. Scale bar, 30 μm. Dashed rectangles indicate areas of enlargement in inserts. PV, portal vein. CV, central vein. Ki67⁺ hepatocytes per mm² in control and HSC-depleted livers at 10 days and 6 weeks after adoptive transfer. n=6 control and 11 HSC-depleted animals at 10 days after adoptive transfer. n = 5 control and 5 HSC-depleted animals at 6 weeks after adoptive transfer. Mean+SD. Kruskal-Wallis with post hoc Dunn’s test. **p<0.005.

Fig. 3.. NTF3 is an HSC-derived hepatocyte mitogen that increases CCND1.

(A to C) Olink protein biomarker analysis (A), multidimensional scaling of Olink biomarker analysis (B), and quantification of NTF3 (C) in lysates from control and HSC-depleted livers 10 days after adoptive transfer. NPX, normalized protein expression. n=9 control and 6 HSC-depleted mice. (D) Immunofluorescence microscopy for NTF3 and desmin in wild-type adult mouse liver. Nuclei are stained with DAPI. Dashed rectangle indicates area of enlargement. Scale bar, 100 μm and 20 μm in enlargement. PV, portal vein. CV central vein. n = 5 mice. (E) Western blot of mouse and human HSC cell lines for NTF3. ACTB is a loading control. n = 3 independent experiments per cell line. (F) Proliferation of primary wild-type mouse hepatocytes after incubation with NTF3 (20 ng/ml) or vehicle in serum-free media for 5 days. Data from 3 independent experiments. Mean+SD. ***p=0.0002 by unpaired two-tailed Mann-Whitney test. (G) Cell proliferation analysis of HepG2 cells incubated with NTF3, NTF3+Loxo-195, Loxo-195, or vehicle only (control). n = 3 independent experiments. Mean+SD. (H) Proliferation of HepG2 cells treated with NTF3, NTF3+Loxo-195, Loxo-195, or vehicle only for 5 days. n = 3 independent experiments. Mean+SD. **p<0.005 by unpaired two-tailed Mann-Whitney test. (I and J) Western blot analysis and quantification of CCND1 in lysates from HepG2 cells treated with NTF3 and Loxo-195 as indicated or vehicle only. ACTB is a loading control. n = 5 independent experiments. Mean+SD. Kruskal Wallis with post hoc Dunn’s test. *p<0.05, **p<0.01.

Fig. 4.. Recombinant NTF3 increases hepatocyte CCND1 in HSC-depleted mice.

(A) Experimental scheme. (B) Macroscopic images of livers in control and HSC-depleted mice treated with vehicle or NTF3. Scale bar, 1 cm (C) Liver-to-body weight ratios of control and HSC-depleted mice treated with vehicle or the indicated concentrations of NTF3. *p<0.05 by Kruskal-Wallis, post hoc Dunn’s test. n=8–13 animals per group. (D) H&E and CCND1 staining of liver sections from control and HSC-depleted mice treated with vehicle or NTF3. Arrowheads indicate positive staining. Dashed rectangles indicate areas shown in insets. (E) Multiplex immunohistochemistry for CCND1 and HNF4α in liver sections from control and HSC-depleted mice treated with vehicle or NTF3. Dashed rectangles indicate areas of enlargement. Scale bars, 20 μm. Arrowheads indicate positive-staining nuclei. (F) Quantification of CCND1⁺HNF4α⁺ nuclei per high power field (HPF). Red bars indicate mean. n=3–5 animals per group. *p<0.05, **p<0.005, ****p<0.0001 by Kruskal-Wallis, post hoc Dunn’s test. (G) Quantification of Ki67⁺ hepatocytes in control and HSC-depleted mice treated with vehicle or NTF3. Mean+SD. *p<0.05 by Kruskal-Wallis, post hoc Dunn’s test. n=4–6 animals per group. (H) Ki67 staining of liver sections from control and HSC-depleted mice treated with vehicle or NTF3. Arrowheads indicate positive staining. Dashed rectangles indicate areas shown in insets. (I) Western blot for desmin, CCND1, IGFBP2, and PCNA in whole-liver lysates from control and HSC-depleted mice treated with vehicle or recombinant NTF3. GAPDH is a loading control. N=3 animals per group.

Fig. 5.. NTF3-induced cell proliferation is mediated by TRKB in primary mouse hepatocytes.

(A) Analysis of CCND1, NTRK1, NTRK2, and NTRK3 expression in a published mouse liver gene expression dataset (2) that enables gene expression analysis according to the hepatocyte’s position in the lobule layers. (B) Immunofluorescence microscopy for TRKA and TRKB in normal adult mouse liver at 4 months of age. Dashed rectangle indicates area of enlargement. CV, central vein. PV, portal vein. Scale bar, 100 μm. n = 3 mice. (C) Immunofluorescence microscopy for TRKB in adult mouse liver also stained with phalloidin to label F-actin. Nuclei are stained with DAPI. PV, portal vein. CV, central vein. Scale bar, 100 μm, 20 μm in enlargement. (D) Proliferation of primary wild-type mouse hepatocytes after incubation with NTF3 (20 ng/ml), TRKB antagonist ANA-12 (10 nM), NTF3+ANA-12, or ANA-12 only in serum-free medium. Mean+SD. Data from n=3 independent experiments. ****p<0.0001, **p<0.005 by Kruskal-Wallis, post hoc Dunn’s. (E) qRT-PCR for Ntrk2 expression in primary mouse hepatocytes infected with lentivirus carrying non-targeting control shRNA (shNT), or Ntrk2-targeting shRNAs (shNtrk2_1 or shNtrk2_2). Mean+SD. Data from n=3 independent experiments. *p<0.05, ****p<0.0001 by Kruskal-Wallis, post hoc Dunn’s. (F) Proliferation analysis of primary mouse hepatocytes infected with shNT, shNtrk2_1, or shNtrk2_2 lentivirus and incubated with NTF3 (20 ng/ml) or vehicle only. Analysis by two-way ANOVA, post hoc Šidak test comparing vehicle only-treated vs. NTF3-treated cells. Mean+SD. Data from n = 3 independent experiments. (G) Bulk RNA-seq analyses for Ntf3 in livers 72 hours after partial hepatectomy and controls. n=5 control and 3 hepatectomized mice per group. FC, fold change; FDR, false discovery rate. (H) Graphical summary of the main findings. The diagram depicts the metabolic zonation of hepatocytes between the central vein (CV) and the portal triad, which includes branches of the portal vein and hepatic artery and a bile duct. Under normal conditions, HSCs produce NTF3 that activates TRKB on midlobular (zone 2) hepatocytes, leading to CCND1-dependent proliferation. Upon HSC ablation by Jedi T cells, midlobular hepatocytes stop proliferating, leading to a gradual reduction in liver mass as dead hepatocytes are not replaced.