Research ArticleFibrosis

Combinatorial targeting of TSLP, IL-25, and IL-33 in type 2 cytokine–driven inflammation and fibrosis

See allHide authors and affiliations

Science Translational Medicine  04 May 2016:
Vol. 8, Issue 337, pp. 337ra65
DOI: 10.1126/scitranslmed.aaf1938
  • Fig. 1. Ablating IL-25 offers no protection against type 2–mediated pathology.

    (A) Quantitative polymerase chain reaction (qPCR) analysis of gene expression in lung and liver tissue from wild-type (WT) C57BL/6 mice 7 days after hydrodynamic injection of IL-25 (n = 5 mice) or phosphate-buffered saline (PBS) (n = 2). D0, day 0. (B) Histopathology analysis of lungs from WT and IL-13Rα1−/− mice 7 days after S. mansoni egg exposure and 8 days after hydrodynamic injection of IL-25 or PBS (n = 12 to 15 per group; pooled from two independent experiments). Scale bars, 50 μm. AB-PAS, Alcian blue–periodic acid–Schiff. (C) Cytokine quantification from bronchoalveolar lavage fluid (BALF) of mice in (B) (n = 4 to 5 per group). (D) Histopathology analysis of lungs from IL-25−/− mice and littermate controls 7 days after challenge with S. mansoni eggs with (2°) or without priming (1°), with 5000 S. mansoni eggs 14 days before challenge (granuloma volume 1°, n = 18 to 23 per genotype pooled from three experiments; granuloma volume 2°, n = 9 to 10 per genotype pooled from two experiments; eosinophils, n = 5 per genotype). (E) Histopathology analysis and fibrosis quantification of livers of IL-25−/− mice and littermate controls 12 weeks after infection with S. mansoni cercariae (n = 9 per genotype). Student’s t test was used to measure all P values, and P > 0.05 except where reported. Error bars represent SEM, and each data point represents a value for an individual mouse. Data are representative of two independent experiments unless otherwise noted. Wk0, week 0.

  • Fig. 2. Ablating IL-33 offers no protection against type 2–mediated pathology.

    (A) Fibrosis quantification and histopathology analysis of lungs from WT C57BL/6 and IL-33−/− mice 7 days after challenge with S. mansoni eggs (n = 7 to 10 per genotype). (B) Fibrosis quantification and histopathology analysis of lungs of the same mouse strains 21 days after priming with S. mansoni eggs and 7 days after challenge with eggs (n = 10 per genotype). (C) Fibrosis quantification of livers from the same mouse strains infected with S. mansoni cercariae (n = 7 to 10 per genotype). (D) Micrographs of representative liver tissue sections of mice in (C) collected 9 weeks after infection and stained with picrosirius red. Scale bars, 100 μm. KO, knockout. (E) Histopathology analysis of livers from the mice in (C) (n = 7 to 10 per genotype). (F) Intracellular cytokine analysis of lymphocytes isolated from livers of mice in (C) 9 weeks after infection was measured by flow cytometry (n = 8 per genotype). Student’s t test was used to measure all P values, and P > 0.05 except where reported. Data are representative of two independent experiments for each of the models.

  • Fig. 3. Simultaneous disruption of all three mediators has a transient effect on TH2 pathology driven by S. mansoni.

    (A) Granuloma measurement (n = 14 to 19 per group pooled from two independent experiments) from livers of S. mansoni–infected WT C57BL/6 mice administered isotype control antibody and IL-33/TSLP DKO mice administered anti–IL-25. (B) Fibrosis quantification from livers of infected mice (n = 7 to 10 per group). (C) Quantification of granuloma eosinophils from livers of infected mice (n = 7 to 9 per group). (D) Quantification of CD4IL-13ST2+ICOS+ leukocytes from MLNs (n = 7 per group) and livers (week 9, n = 7 to 8 per group; week 12, n = 14 to 15 per group pooled from two independent experiments) of infected mice by flow cytometry. ICOS, inducible T cell costimulator; IP, intraperitoneal. (E) Micrographs of representative liver tissue sections of mice 12 weeks after infection and stained with picrosirius red. Scale bars, 500 μm. (F) Intracellular cytokine analysis of liver lymphocytes of infected mice by flow cytometry (n = 14 to 17 per genotype pooled from two experiments). Student’s t test was used to measure all P values, and P > 0.05 except where reported. Data are representative of two independent experiments.

  • Fig. 4. Combined TSLP, IL-25, and ST2 mAb blockade during granuloma generation diminishes type 2 immunity but not pathology.

    (A) Histopathology analysis of WT BALB/c and IL-4R−/− mice 7 days after injection with S. mansoni eggs. WT egg–injected mice were intraperitoneally administered either anti-ST2, anti-TSLP, and anti–IL-25, or corresponding isotype control antibodies (n = 8 to 9 per group). Micrographs are of representative lung sections stained with Masson’s trichrome. Scale bars, 50 μm. (B) Histopathology analysis of lungs from WT BALB/c and IL-4R−/− mice 7 days after injection with S. mansoni eggs and 21 days after priming with S. mansoni eggs (isotypes IP and triple block IP, n = 8 to 9 per group; IL-4R−/−, n = 5). WT egg–injected mice were intraperitoneally administered either anti-ST2, anti-TSLP, and anti–IL-25, or corresponding isotype control antibodies for all 3 weeks. Micrographs are of representative lung sections stained with Masson’s trichrome. Scale bars, 50 μm. (C) Quantification of gene expression in lung tissue from mice in (A) assayed by qPCR and shown relative to expression in lungs of naïve BALB/c mice (n = 3). (D) Quantification of gene expression in lung tissue from mice in (B) assayed by qPCR and compared to a different group of naïve BALB/c controls (n = 3). Student’s t test was used to measure all P values, and P > 0.05 except where reported. Data are representative of two independent experiments.

  • Fig. 5. Efficacy of TSLP, IL-25, and ST2 mAb blockade on established chronic allergy.

    WT BALB/c mice were sensitized and challenged intranasally (IN) with HDM, and starting on day 21, anti-ST2, anti-TSLP, and/or anti–IL-25 was administered in various combinations to different groups to achieve single, double, or triple blocks. Additional control groups received only isotype control antibodies with or without HDM. To properly control for the triple blockade group, groups administered single and double blocks also received immunoglobulin G1 (IgG1) in the absence of anti-ST2 or anti–IL-25, and rat IgG1 in the absence of anti-TSLP. All mice were analyzed on day 46. (A) Histopathology analysis of lung sections stained with Masson’s trichrome and scored for peribronchial and perivascular inflammation (n = 6 to 10 per group pooled from two experiments). (B) Quantification of fibrosis from lung tissue. (C) Quantification of gene expression from lung tissue measured by qPCR. (D) Quantification of leukocytes in the BALF and lung tissue. (E) Quantification of eosinophils shown as a percentage of total inflammatory cells in BALF and lung tissue. Student’s t test was used to measure all P values, and P > 0.05 except where reported.

  • Fig. 6. Disruption of all three mediators during initiation and maintenance of type 2–driven chronic allergy reduces inflammation and fibrosis.

    WT C57BL/6 and IL-33/TSLP DKO mice were sensitized and challenged intranasally with HDM over 45 days. DKO mice were intraperitoneally administered αIL-25 (DKO + αIL-25/HDM), and HDM-treated WT C57BL/6 mice were intraperitoneally administered an IgG1 isotype control (isotype/HDM). A control group of C57BL/6 mice received intranasal saline instead of HDM and the isotype (isotype/saline). All mice were analyzed on day 46. (A) Quantification of fibrosis from lung tissue (isotype/saline, n = 5; isotype/HDM, n = 9; triple block/saline, n = 8). (B) Histopathology analysis of lung sections stained with Masson’s trichrome for scoring of inflammation and AB-PAS for mucus scoring. Micrographs are of representative lung sections stained with Masson’s trichrome. Scale bars, 50 μm. (C) Quantification of leukocytes in BALF. (D) BALF leukocyte differential. (E) Quantification of eosinophils in lung tissue. (F) Intracellular cytokine quantification of lung tissue lymphocytes by flow cytometry. (G) Intracellular cytokine quantification of BALF lymphocytes by flow cytometry. Student’s t test was used to measure all P values, and P > 0.05 except where reported. Data are representative of two independent experiments.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/337/337ra65/DC1

    Fig. S1. Alarmin gene expression in the liver.

    Fig. S2. Kinetics of alarmin gene expression in chronic HDM model.

    Fig. S3. Neutralizing all three alarmins with mAbs during initiation and maintenance of type 2–driven allergy reduces inflammation and fibrosis.

    Table S1. qPCR primer sequences.

    Source data

  • Supplementary Material for:

    Combinatorial targeting of TSLP, IL-25, and IL-33 in type 2 cytokine–driven inflammation and fibrosis

    Kevin M. Vannella, Thirumalai R. Ramalingam, Lee A. Borthwick, Luke Barron, Kevin M. Hart, Robert W. Thompson, Kristen N. Kindrachuk, Allen W. Cheever, Sandra White, Alison L. Budelsky, Michael R. Comeau, Dirk E. Smith, Thomas A. Wynn*

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

    Published 4 May 2016, Sci. Transl. Med. 8, 337ra65 (2016)
    DOI: 10.1126/scitranslmed.aaf1938

    This PDF file includes:

    • Fig. S1. Alarmin gene expression in the liver.
    • Fig. S2. Kinetics of alarmin gene expression in chronic HDM model.
    • Fig. S3. Neutralizing all three alarmins with mAbs during initiation and maintenance of type 2–driven allergy reduces inflammation and fibrosis.
    • Table S1. qPCR primer sequences.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Navigate This Article