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1.
Figure 9.

Figure 9. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

Elevated IL-17A and IL-1β in human IPF patient BAL fluid. BAL fluid and lung biopsies were collected from NVs and IPF patients. (A) BAL fluid was assayed for IL-17A and IL-1β by ELISA. (B) 5-µm lung sections were cut from paraffin-embedded lung biopsies and stained with Masson’s trichrome. Data shown are mean ± SEM. Images are shown at 5× (top) and dashed rectangles are magnified at 20× (bottom). Bars: (top) 60 µm; (bottom) 20 µm.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
2.
Figure 3.

Figure 3. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-10–producing CD4 cells accumulate in the lung. 0.15 U BLM was given to C57BL/6 IL-10gfp mice via intratracheal route, as in Fig. 1. One of two independent experiments shown with five animals per group. Data shown are mean ± SEM. (A) Lung, BAL, or thoracic LN cells were isolated from IL-10gfp reporter mice and stained with anti–mouse CD3 and CD4. Horizontal bars show the mean. (B) CD4+IL-10gfp− and CD4+IL-10gfp+ cells were FACS sorted (>98% pure) from the lung of BLM-treated mice at day 7. RNA was extracted and analyzed for mRNA transcripts. The dotted line refers to naive CD4+ cells.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
3.
Figure 1.

Figure 1. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-13–dependent and –independent pulmonary fibrosis. WT, il13−/−, or il13Rα2−/− mice were given either 5,000 S. mansoni eggs i.p. followed by 5,000 S. mansoni eggs i.v. 14 d later, with pulmonary fibrotic granulomas assessed 7 d later (d21; A and B), or an intratracheal delivery of 0.15 U BLM, with pulmonary fibrosis assessed on day 7 (C and D). One of two independent experiments is shown, with five animals per group. (A and C) Pulmonary collagen deposition, expressed as micromoles of hydroxyproline per lung. Data shown are mean ± SEM. (B and D) 5-µm sections of paraffin-embedded lung tissue were stained with Masson’s Trichrome. Images are shown at 10× magnification. Collagen, blue; nuclei, dark red; cytoplasm, red/pink. Bars, 60 µm.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
4.
Figure 7.

Figure 7. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-17A–dependent IL-1β–induced collagen deposition. Mice were given intratracheal 0.15 U BLM, 1 µg IL-1β, 1 µg IL-17A, or 0.5 µg each of both IL-1β and IL-17A with pulmonary collagen deposition assessed on day 7. *, P < 0.05 using a Mann-Whitney test. One of two independent experiments is shown, with five animals per group. Data shown are mean ± SEM. (A and E) 5-µm sections of paraffin-embedded lung tissue were stained with Masson’s Trichrome. Images are shown at a 20× magnification. Bars, 20 µm. (B and F) Pulmonary collagen deposition, expressed as micromoles of hydroxyproline per lung. (C and I) Thoracic LN cells were stimulated with anti-CD3ε, with IL-17A and IFN-γ measured in culture supernatants. (D) MMP2 bioactivity in BAL fluid measured by zymography. (G) Absolute counts of circulating polymorphonuclear cells were obtained from CBC counts. (H) BAL fluid TIMP-1 and IFN-γ was measured by ELISA.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
5.
Figure 8.

Figure 8. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-17A–dependent fibrosis requires TGF-β. Mice were given 0.15 U of intratracheal BLM or 1 µg IL-17A with or without 500 µg of anti–TGF-β treatment on days −1, 3, and 5. Pulmonary collagen deposition was assessed on day 7. *, P < 0.05 using a Mann-Whitney test. One of two independent experiments is shown, with five animals per group. Data shown are mean ± SEM. (A) Lung injury, measured as BAL collagen, was quantified from BAL fluid using Sircol assay. (B) Pulmonary collagen deposition, expressed as micromoles of hydroxyproline per lung. (C) Thoracic LN cells were stimulated with anti-CD3ε, with IL-17A and IFN-γ measured in culture supernatants. (D) 5-µm sections of paraffin-embedded lung tissue were stained with Masson’s Trichrome. Images are shown at a 20× magnification. Bars, 20 µm.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
6.
Figure 4.

Figure 4. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-10 restricts IL-17A and IFN-γ and the extent of pulmonary fibrosis. 0.15 U BLM was given to WT, IL-10gfp, or il10−/− mice, via the intratracheal route, as in Fig. 1. *, P < 0.05 using a Mann-Whitney test. One of three independent experiments is shown with five animals per group. Data shown are mean ± SEM. (A) Pulmonary collagen deposition, expressed as micromoles of hydroxyproline per lung. (B) Thoracic LN cells were stimulated with anti-CD3ε, with IL-17A or IFN-γ measured in culture supernatants. BAL collagen was quantified from BAL fluid using Sircol assay. (C) Lung cells, isolated at day 7 after BLM, were stained with anti–mouse, CD4, CD8, B220, γδTCR, NK1.1, IL-17A, and IFN-γ, after a brief stimulation with PMA and ionomycin in the presence of BFA. The percentage of CD4+ or γδ+ cells producing IL-17A, in addition to the total number of cells recovered from the lungs, is enumerated in the table. (D) Homogenized lung supernatant was assayed by ELISA for the indicated cytokines/chemokines.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
7.
Figure 2.

Figure 2. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-17A and IFN-γ production during BLM-induced fibrosis. 0.15 U BLM was given to WT mice via intratracheal route, as in Fig. 1, with local immune profiling and assessment of tissue pathology analyzed from days 0 to 21, as indicated. One of three independent experiments is shown, with five animals per group. Data shown are mean ± SEM. (A) Weight loss, assessed at several time points after BLM, as indicated. (B) Pulmonary collagen score from histology sections. (C) RNA was extracted from lung tissue, with timp-1, mmp12, and pro–COL-3 mRNA quantified by quantitative RT-PCR. (D) 5-µm sections of paraffin-embedded lung tissue obtained from mice at the indicated day after BLM and stained with Masson’s Trichrome. Images are shown at a 5× magnification. Bars, 60 µm. (E) BAL, lung, and lung-draining thoracic LN (t:LN) cells were isolated and stimulated with anti-CD3ε for 4 d. IL-17A, IFN-γ, and IL-13 were measured in culture supernatants by ELISA. (F) 5-µm sections of paraffin-embedded lung tissue obtained from mice at day 7 after BLM stained with Giemsa or Picrosirius red (inset) shown under polarized light. Images are shown at 10× magnification. Bars, 60 µm.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
8.
Figure 6.

Figure 6. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

Attenuated pulmonary fibrosis in il17a−/− mice. 0.15 U BLM was given to WT, il10−/−, il17a−/−, or il10−/−il17a−/− mice, via the intratracheal route, as in Fig. 1. *, P < 0.05 using a Mann-Whitney test. One of two independent experiments is shown, with five animals per group. Data shown are mean ± SEM. (A) Lung injury, measured as BAL collagen, was quantified from BAL fluid using Sircol assay. (B) Pulmonary collagen deposition, expressed as micromoles of hydroxyproline per lung. (C) Absolute counts of circulating polymorphonuclear cells were obtained from CBC counts. (D) Percentage of weight change, 7 d after BLM. (E) 5-µm sections of paraffin-embedded lung tissue taken from WT or il10−/− mice at day 7 or 21 after BLM and stained with Masson’s Trichrome. Images are shown at a 40× magnification. Bar, 7 µm. (F) RNA was extracted from lung tissue, with indicated mRNA quantified by quantitative RT-PCR. (G and H) Thoracic LN cells were stimulated with anti-CD3ε, with IL-17A (G) and IFN-γ (H) measured in culture supernatants. (I) MMP2 and MMP9 bioactivity measured in BAL by zymography.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.
9.
Figure 5.

Figure 5. From: Bleomycin and IL-1?-mediated pulmonary fibrosis is IL-17A dependent.

IL-12/23p40 deficiency significantly impacts IL-17A and IFN-γ and curtails pulmonary fibrosis. 0.15 U BLM was given to WT, il10−/−, il12/23p40−/−, or il10−/−il12/23p40−/− mice via the intratracheal route as in Fig. 1. *, P < 0.05 using a Mann-Whitney test. One of two independent experiments is shown, with five animals per group. Data shown are mean ± SEM. (A) 5-µm sections of paraffin-embedded lung tissue taken from WT or il10−/− mice at day 7 or 21 after BLM and stained with Masson’s Trichrome. Images are shown at 5× magnification with dotted squares magnified at 40× in the insets. Bars: 60 µm; (inset) 7 µm. (B) Lung injury, measured as BAL collagen, was quantified from BAL fluid using Sircol assay. (C) Pulmonary collagen deposition, expressed as micromoles of hydroxyproline per lung. (D) Absolute counts of circulating poly morphonuclear cells (PMNs) were obtained from CBC counts. (E and F) Thoracic LN cells were stimulated with anti-CD3ε, with IL-17A (E) and IFN-γ (F) measured in cultures supernatants. (G) Thoracic LN cells were stained with anti–mouse CD4, CD8, B220, γδTCR, NK1.1, IL-17A, and IFN-γ after a brief stimulation with PMA and ionomycin in the presence of BFA. Data shown is gated on CD4+ cells.

Mark S. Wilson, et al. J Exp Med. 2010 March 15;207(3):535-552.

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