Research ArticleFibrosis

Type 2 immunity is protective in metabolic disease but exacerbates NAFLD collaboratively with TGF-β

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Science Translational Medicine  28 Jun 2017:
Vol. 9, Issue 396, eaal3694
DOI: 10.1126/scitranslmed.aal3694
  • Fig. 1. NASH and fibrosis develop late in mice fed an HFD.

    Mice were maintained on a normal diet (filled symbols; ND) or a 60% kcal from fat diet (open symbols; HFD) for 15 (circles) or 40 (squares) weeks. (A) Body weights and liver weight and liver index (% of body weight) were collected (n = 10 to 20). Characteristics of NASH were measured including isolated liver leukocyte counts (n = 9 to 12) (B), serum ALT and aspartate aminotransferase (AST) (n = 9 to 10) (C), and steatosis by Oil Red O staining (D). Scale bars, 100 μm. (E) Liver sections were stained with keratin 8/18 and ubiquitin (Ub.) to assess hepatocyte ballooning and hematoxylin and eosin (H&E) for hepatic inflammation (inset showing example of lobular inflammation). Scale bars, 40 μm. (F) Picrosirius red staining was performed to assess fibrotic collagen deposition (scale bars, 200 μm) and viewed under polarized light to enhance visualization (inset scale bars, 200 μm). Adipose inflammation was assessed from Giemsa-stained sections from 40-week–treated animals (scale bars, 150 μm) (G), crown-like structures marked with arrows, expression of tnf (H) (n = 4 to 5), and flow cytometry analysis of Siglec-F+ adipose eosinophils among CD45 leukocytes (I) (n = 5 to 12). All data points represent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

  • Fig. 2. Type 1 immunity drives metabolic disease but protects against NAFLD.

    WT (circles), IL-4−/− (squares), and IL-10/IL-4−/− (triangles) mice were maintained on a normal diet (filled symbols) or HFD (open symbols) for 15 weeks. Obesity was measured by magnetic resonance imaging (MRI) body composition (A) (n = 4 to 6), and serum leptin levels were assessed by enzyme-linked immunosorbent assay (n = 3 to 7) (B). NAFLD progression was assessed by liver weight and indices (n = 8 to 15) (C), hepatic triglyceride measurement (n = 8 to 11), serum aminotransferase levels (n = 4 to 17) (D), and Giemsa-stained liver sections (E). KO, knockout. Adipose inflammation was assessed through histological analysis of H&E-stained adipose sections (scale bars, 150 μm) (F), adipose tissue expression of tnf and ccl2 (n = 3 to 11) (G), and flow cytometry analysis of adipose eosinophils (n = 4 to 11) (H). All data points represent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

  • Fig. 3. Obese IFN-γ−/− mice develop accelerated NAFLD with fibrosis.

    (A) Activation scores of the IFN-γ pathway were assessed by Ingenuity Pathway Analysis of significantly differentially expressed genes as determined by RNA-seq from whole liver tissue from WT, IL-4−/−, and IL-10/IL-4−/− mice on HFD for 15 weeks. Expression of genes annotated as being involved in positive regulation of IFN-γ is shown in the heat map. (B) WT (circles) and IFN-γ−/− (squares) mice were maintained for 15 weeks on a normal diet (filled symbols) or HFD (open symbols) and assessed for obesity by MRI body composition measurement (n = 3 to 5). NAFLD progression was also assessed by liver weight (n = 9 to 11), liver index, hepatic triglyceride measurement (n = 3 to 6) (B), steatosis by Oil Red O staining (C) (left, normal diet; right, HFD) (scale bars, 100 μm), picrosirius red staining of collagen (scale bars, 125 μm) (C) (bottom) (visualized under polarized light; inset scale bars, 125 μm), and serum ALT levels (n = 5) (D). (D) Expression of fibrotic markers col3a1 (n = 9 to 11), postn (n = 9 to 11), and acta2 (SD in shaded regions) (n = 3 to 5) was assessed by quantitative polymerase chain reaction (qPCR). (E) Expression of tgfb1 and TGF-β–regulated genes timp1 and mmp2 were assessed (n = 9 to 11). (F) Immunofluorescence staining for phospho-Smad3 (green), keratin 8/18 (gray), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) (arrows indicate overlapping Smad3 and DAPI staining). All data points represent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

  • Fig. 4. NASH-driven fibrosis is partly TGF-β–dependent.

    (A) WT mice were maintained for 40 weeks on a normal diet (filled symbols) or HFD (open symbols) and received 4 weeks of semiweekly control (Cont.) immunoglobulin (Ig) (squares) or anti–TGF-β (triangles) (250 μg each). Mice were assessed for obesity by MRI body composition measurement (n = 5 to 9) and hepatomegaly by liver weight and liver index scores (n = 5 to 12). (B) Hepatic triglycerides were measured, and fibrotic fraction was calculated by measuring picrosirius red–stained liver sections for characteristic NASH-associated fibrosis (n = 4 to 12). (C) Expression of four fibrosis-associated genes was measured (n = 5 to 12). Phorbol 12-myristate 13-acetate (PMA)/ionomycin–restimulated hepatic CD4 T cell production of IFN-γ, IL-4 (D), and IL-13 (left) (E) was assessed by intracellular cytokine staining (n = 5 to 10). (E) Expression of the type 2 inflammation marker chi3l3 was quantified from whole liver tissue (n = 4 to 11) (right). Freq., frequency. All data points represent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

  • Fig. 5. Accelerated NASH-driven fibrogenesis in obese IFN-γ−/− mice is characterized by severe eosinophilic inflammation during TGF-β blockade.

    (A) WT (circles) and IFN-γ−/− (squares) mice were maintained for 20 weeks on normal diet (filled symbols) or HFD (open symbols) and received 4 weeks of biweekly control Ig or anti–TGF-β (250 μg each). Liver weight and liver index were determined (n = 9 to 14), and fibrotic fraction was calculated from picrosirius red–stained liver sections (n = 3 to 12). (B) Expression of interstitial collagen genes. Periostin, IL-13, and CCL11 expression were quantified from whole liver tissue (n = 6 to 14). (C) Hepatic eosinophils were measured by flow cytometry analysis of Siglec-F+ cells among CD45 leukocytes (n = 7 to 9) (D). Eosinophils were observed in hepatic tissue by Giemsa-stained liver sections (scale bars, 50 μm) (E) and by immunofluorescence staining of tissue sections for Siglec-F+ cells (scale bars, 125 μm) (F). WT mice were maintained for 40 weeks on normal diet (filled symbols) or HFD (open symbols) and received 4 weeks of biweekly control Ig (circles), anti–TGF-β (squares), or combined anti–TGF-β and IL-13 mAbs (triangles) (250 μg each). (G) Expression of col3a1 and col6a1 from whole liver tissue were assessed by qPCR (n = 3 to 6). All data points represent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

  • Fig. 6. AMLN diet–induced NASH recapitulates IFN-γ regulation and type 2 inflammation despite modest adipose inflammation.

    (A) WT (circles) and IFN-γ−/− (squares) mice were maintained for 15 weeks on normal diet (filled symbols) or AMLN diet (open symbols) and assessed for obesity by body weight (n = 5 to 8). NAFLD progression was assessed by liver weight and liver index, (A), expression of fibrotic markers col1a1, col3a1, and acta2 as assessed by qPCR (n = 5 to 7) (B), picrosirius red– and FAST green-stained liver sections from WT (top) and IFN-γ−/− (bottom) mice on a normal diet (left) and HFD (right) (C), and serum alkaline phosphatase and ALT levels (n = 4 to 8) (D). Type 2 response was measured by intracellular staining of hepatic CD4 T cells for IL-13 after PMA/ionomycin stimulation (n = 4 to 8), postn expression (n = 5 to 8) (E), and flow cytometry analysis of hepatic eosinophils and neutrophils (n = 5 to 8) (F). (G) Adipose inflammation was measured by flow cytometry analysis of eosinophils and tnf expression (n = 5 to 8). All data points represent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

  • Fig. 7. Human NASH is characterized by type 2 eosinophilic inflammation.

    (A) Cytokine production from healthy (circles) (n = 2) and NASH (squares; red squares, HCV+ NASH patients) (n = 14) patient liver biopsies was measured from expanded intrahepatic lymphocytes after a CD3/CD28 restimulation for IL-4, IL-5, and IL-13. (B) Combined cytokine production stratified by METAVIR fibrosis score and representative anti–smooth muscle actin histology from F0-F2 (left) and F3-F4 (right) biopsies. (C) Eosinophils were observed in H&E-stained liver biopsies from patients (arrows). Scale bar, 25 μm. (D) Publicly available microarray data from healthy, steatotic, and NASH liver biopsies were analyzed for eosinophil-associated genes that demonstrated significantly altered expression, and hierarchical clustering by samples (left, heat map) and principal components analysis (right) was performed on the basis of this gene set. All data points represent a single patient.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/396/eaal3694/DC1

    Materials and Methods

    Fig. S1. Chronic HFD–induced NASH is associated with hepatic ballooning and activation of fibroblasts.

    Fig. S2. Analysis of adipose tissue fibrotic pathways and collagen deposition.

    Fig. S3. IL-10 in mouse and human NASH.

    Fig. S4. NAFLD/NASH in 40-week HFD-challenged WT, IL-4, and IL-10/IL-4 knockout mice.

    Fig. S5. Comparison of IL-10 versus IL-12 deletion in NAFLD progression.

    Fig. S6. Adipose tissue inflammation in IFN-γ knockouts on HFD.

    Fig. S7. Presence of cellular mediators of inflammation in NAFLD/NASH.

    Fig. S8. IL-10/IL-4 knockout animals have long-term protection from HFD-induced NASH.

    Fig. S9. Collagen- and eosinophil-associated chemoattractant expression alterations in patient biopsies.

    Fig. S10. Representative histological eosinophils in NASH patient biopsies.

    Fig. S11. Inflammatory pathways in NASH progression.

    Table S1. Primary data.

  • Supplementary Material for:

    Type 2 immunity is protective in metabolic disease but exacerbates NAFLD collaboratively with TGF-β

    Kevin M. Hart, Thomas Fabre, Joshua C. Sciurba, Richard L. Gieseck III, Lee A. Borthwick, Kevin M. Vannella, Thomas H. Acciani, Rafael de Queiroz Prado, Robert W. Thompson, Sandra White, Genevieve Soucy, Marc Bilodeau, Thirumalai R. Ramalingam, Joseph R. Arron, Naglaa H. Shoukry, Thomas A. Wynn*

    *Corresponding author. Email: twynn{at}niaid.nih.gov

    Published 28 June 2017, Sci. Transl. Med. 9, eaal3694 (2017)
    DOI: 10.1126/scitranslmed.aal3694

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Chronic HFD–induced NASH is associated with hepatic ballooning and activation of fibroblasts.
    • Fig. S2. Analysis of adipose tissue fibrotic pathways and collagen deposition.
    • Fig. S3. IL-10 in mouse and human NASH.
    • Fig. S4. NAFLD/NASH in 40-week HFD-challenged WT, IL-4, and IL-10/IL-4 knockout mice.
    • Fig. S5. Comparison of IL-10 versus IL-12 deletion in NAFLD progression.
    • Fig. S6. Adipose tissue inflammation in IFN-γ knockouts on HFD.
    • Fig. S7. Presence of cellular mediators of inflammation in NAFLD/NASH.
    • Fig. S8. IL-10/IL-4 knockout animals have long-term protection from HFD-induced NASH.
    • Fig. S9. Collagen- and eosinophil-associated chemoattractant expression alterations in patient biopsies.
    • Fig. S10. Representative histological eosinophils in NASH patient biopsies.
    • Fig. S11. Inflammatory pathways in NASH progression.
    • Legend for table S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

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