MiR-690 treatment causes decreased fibrosis and steatosis and restores specific Kupffer cell functions in NASH

Abstract

Nonalcoholic steatohepatitis (NASH) is a liver disease associated with significant morbidity. Kupffer cells (KCs) produce endogenous miR-690 and, via exosome secretion, shuttle this miRNA to other liver cells, such as hepatocytes, recruited hepatic macrophages (RHMs), and hepatic stellate cells (HSCs). miR-690 directly inhibits fibrogenesis in HSCs, inflammation in RHMs, and de novo lipogenesis in hepatocytes. When an miR-690 mimic is administered to NASH mice in vivo, all the features of the NASH phenotype are robustly inhibited. During the development of NASH, KCs become miR-690 deficient, and miR-690 levels are markedly lower in mouse and human NASH livers than in controls. KC-specific KO of miR-690 promotes NASH pathogenesis. A primary target of miR-690 is NADK mRNA, and NADK levels are inversely proportional to the cellular miR-690 content. These studies show that KCs play a central role in the etiology of NASH and raise the possibility that miR-690 could emerge as a therapeutic for this condition.

Keywords: Kupffer cell; NASH; fibrosis; inflammation; liver; miR-690; steatosis.

Figure 1.. miR690 Treatment Inhibits Fibrosis in Liver Spheroids or in vivo in WD NASH Mice.

(A) Expression of fibrogenic genes in mouse liver spheroids treated with the miR-690 mimic. (B) Effects of miR-690 mimic (1nM) on fibrogenic or proinflammatory activation of human liver spheroids in the presence of a mixture of fatty acids (FAs), fructose, and lipopolysaccharide (LPS). n=4 per group. (C) Flow chart of Western diet (WD) mice injected with a scrambled RNA mimic or the miR-690 mimic. 16wks WD WT mice were intravenously injected with nanoparticles encapsulating either scrambled RNA mimic or miR-690 mimic (5nmole/mouse, 2X per week). After 4 weeks (wks) of treatment, glucose and insulin tolerance tests (GTTs and ITTs) were performed. After 8wks of treatment, livers and other tissues were collected from these mice. (D) Liver miR-690 abundance in NCD, NASH controls injected with the scrambled RNA mimic (NASH Con), and miR-690 injected NASH mice (+miR-690 mimic). (E) miR-690 expression in human livers. (F) Sirius Red staining of liver sections of NASH control and miR-690 injected NASH mice. Representative images of n=8 mice per group. (G) Liver fibrogenic gene expression in NASH control and miR-690 injected NASH mice. (H) Fibrogenic gene expression in NASH mouse HSCs treated with the miR690 mimic for 24 hours. (I) Fibrogenic gene expression in healthy human HSCs and NASH human HSCs treated with the miR-690 mimic for 24 hours. (J) mRNA levels of Tgfb in healthy human HSCs and NASH human HSCs treated with the miR-690 mimic for 24 hours. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test (A, B, E-H) or One-way ANOVA (D, I, and J). See also Figure S1.

Figure 2.. miR-690 Treatment Reduces Liver Steatosis.

(A) H&E staining of liver sections of NASH Con and miR-690 treated NASH mice. (B) Liver triglyceride (Tg) levels of NASH Con and miR-690 treated NASH mice. (C) Lipogenesis gene expression in livers of NASH Con and miR-690 treated NASH mice. (D) Lipogenesis gene expression in NAFLD mouse hepatocytes after 24hr treatment with the miR-690 mimic. (E) Lipogenesis gene expression in NAFLD human hepatocytes after 24hr treatment with the miR-690 mimic. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. See also Figure S2.

Figure 3.. miR-690 Blunts Inflammation in both In Vitro and In Vivo studies.

(A) Expression of Tgfb, Tnfa, and F4/80 in FA, fructose, and LPS cocktail treated liver spheroids in response to miR-690 mimic transfection. Immunofluorescent staining for F4/80 and Clec4f (B) and numbers of F4/80 or Clec4f positive cells (C) in the livers of NCD WT, NASH control, and miR-690 treated NASH mice. (D) Inflammatory gene expression in the livers of NASH Con and miR-690 treated NASH mice. (E) The abundance of Ccl2, Tgfb, and Il10 in mouse NASH KCs after 24hr miR-690 mimic transfection. Immunofluorescent staining for F4/80 and CD206 (F) and numbers of F4/80 or CD206 positive cells (G) in the livers of NCD WT, NASH Con, and miR-690 treated NASH mice. (H) Expression of inflammatory genes in NASH recruited hepatic macrophages (RHMs) after 24hr treatment with the miR-690 mimic. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. See also Figure S3.

Figure 4.. Functional switch in KCs in response to NASH development.

(A) Phagocytosis of senescent RBCs by NCD WT, NASH WT, or miR-690 treated NASH WT KCs. (B) Flow cytometry analysis of the engulfment of PKH26 labeled senescent RBCs by NCD WT, NASH WT, or miR-690 treated NASH WT KCs. (C) Fibrogenic gene expression in NCD mouse HSCs co-cultured with NCD WT KCs, NASH WT KCs, or NASH WT KCs transfected with the miR690 mimic. (D) AKT phosphorylation levels in NCD mouse hepatocytes co-cultured with NCD WT KCs, NASH WT KCs, or NASH WT KCs transfected with the miR690 mimic. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA (B-D).

Figure 5.. miR-690 Modulates KC Function.

(A) miR-690 abundance in Clec4f+F4/80+ KCs of NCD WT and NASH WT mice. (B) miR-690 abundance in Clec4f+F4/80+ KCs of NCD WT and Clec4f+ cell-specific miR-690 knock out (KC-miR-690KO) mice. (C) Proportion of Clec4f+F4/80+ KCs in NCD WT and KC-miR-690KO mice. (D) Inflammatory gene expression in Clec4f+F4/80+ KCs from NCD WT and KC-miR-690KO mice. (E) Expression of Arg1, Il10, and Tgfb in NCD WT Clec4f+F4/80+ KCs after 24hr treatment with a miR-690 antagomir. (F) Nadk expression in Clec4f+F4/80+ KCs of NCD WT and NASH WT mice. (G) NADK abundance in the livers of NASH WT mice after 8wks of miR-690 treatment. (H) Fibrogenic gene expression in NCD WT HSCs after co-culture with NASH KCs or NASH WT KCs transfected with siRNA-Nadk. (I) NAD+ levels in Clec4f+F4/80+ KCs isolated from NCD WT and NASH WT mice. (J) Inflammatory gene abundance in NASH WT KCs after 24hr treatment with nicotinamide mononucleotide (NMN). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. See also Figure S4.

Figure 6.. KCs are the main cells secreting miR-690 into neighboring hepatic cells.

(A) miR-690 abundance in KCs, hepatocytes, RHMs, and HSCs in NCD WT livers. miR-690 abundance in hepatocytes (B) and HSCs (C) isolated from NCD WT and NASH WT mice. miR-690 abundance in hepatocytes (D) and HSCs (E) of NCD WT, Clodronate liposome-treated, or diphtheria toxin (DT)-treated Clec4f+ cell-specific DTR+ mice. miR-690 abundance in hepatocytes (F) and HSCs (G) from NCD WT and KC-miR-690KO mice miR-690 abundance in hepatocytes (H) and HSCs (I) from NCD WT and KC-Rab27KO mice Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test (B-D, and F-I) or One-way ANOVA (A, D, and E). See also Figure S5.

Figure 7.. miR-690 regulates hepatocyte lipogenesis and HSC fibrogenic activation through NAD abundance.

(A) Lipogenic gene abundance in NCD WT hepatocytes after 24hr treatment with a miR-690 antagomir. (B) Expression of fibrogenic genes in NCD WT HSCs after 24hr treatment with a miR-690 antagomir. (C) Lipogenic gene abundance in hepatocytes isolated from NCD WT and KC-miR-690KO mice. (D) Expression of fibrogenic genes in HSCs isolated from NCD WT and KC-miR-690KO mice. Triglyceride levels (E) and Sirius Red staining (F) in the livers of 16wks WD WT or KC-miR-690KO mice. Representative images of n=3–4 mice per group. (G) Nadk abundance in NCD WT hepatocytes and HSCs after 24hr treatment with a miR-690 antagomir. (H) Tg levels in human NAFLD hepatocytes treated with siRNA-Nadk. (I) Fibrogenic gene expression in human NASH HSCs transfected with siRNA-Nadk. (J) NAD+ levels in hepatocytes and HSCs isolated from NCD WT and NASH WT mice. (K) NAD+ abundance in hepatocytes and HSCs isolated from NASH mice after 8wks miR-690 treatment. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. See also Figure S6.