Journal: JCI Insight

Article Title: Interleukin-17 limits hypoxia-inducible factor 1 α and development of hypoxic granulomas during tuberculosis

doi: 10.1172/jci.insight.92973

Figure Lengend Snippet: (A and B) FeJ bone marrow–derived macrophages (BMDMs) were infected with Mycobacterium tuberculosis (Mtb) clinical strain HN878 (MOI 1) and treated with rIL-17 (100 ng/ml) for 48 hours (n = 7 biological replicates) or left uninfected (n = 4 biological replicates) and treated with rIL-17 (n = 4 biological replicates), or untreated (n = 5 biological replicates), and (A) lactate accumulation was measured by lactate assay. (B) HIF1α expression was analyzed by Western blots (representative blot on left), which were quantified by densitometry (right, n = 3 biological replicates/group). Dashed line represents uninfected control. (C–K) FeJ mice were aerosol infected with approximately 100 CFU HN878 and received IL-17–blocking antibody (100 μg) between 10 and 30 (days postinfection [d.p.i.]), and lungs were harvested at 37 d.p.i. (C) Lung bacterial burden was assessed by plating from HN878-infected isotype (n = 5 mice) and anti–IL-17–treated mice (n = 5 mice). (D) Lung area per lobe covered by inflammation on H&E–stained formalin-fixed paraffin-embedded (FFPE) sections from HN878-infected isotype-antibody-treated (n = 4 mice) and anti–IL-17–treated mice (n = 3 mice) was quantified using the morphometric tool of the Zeiss Axioplan microscope. (E) Representative H&E images shown (×50 magnification). (F) Fibrosis was assessed using Masson’s trichrome staining (×50 magnification, arrowheads depict collagen deposition). (G) Immunofluorescence staining using antibodies specific for HIF1α (red) and F4/80 (green) was carried out on FFPE sections (arrows indicate HIF1α-expressing macrophages) and (H) HIF1α staining was quantified (isotype-treated, n = 5 mice; anti–IL-17–treated, n = 10 mice). (I) Hypoxia was determined using pimonidazole (PIMO, red) and F4/80 (green) staining by immunofluorescence, ×200 magnification (arrowheads depict areas of hypoxia) and (J) PIMO staining was quantified (isotype-treated, n = 5 mice; anti–IL-17–treated, n = 10 mice). All data shown as mean ± SD. **P < 0.01, ***P < 0.001 by 1-way ANOVA (A) or Student’s t test (B–D, H, and J). ns, not significant.

Article Snippet: ]), and lungs were harvested at 37 d.p.i. ( C ) Lung bacterial burden was assessed by plating from HN878-infected isotype ( n = 5 mice) and anti–IL-17–treated mice ( n = 5 mice). ( D ) Lung area per lobe covered by inflammation on H&E–stained formalin-fixed paraffin-embedded (FFPE) sections from HN878-infected isotype-antibody-treated ( n = 4 mice) and anti–IL-17–treated mice ( n = 3 mice) was quantified using the morphometric tool of the Zeiss Axioplan microscope. ( E ) Representative H&E images shown (×50 magnification). ( F ) Fibrosis was assessed using Masson’s trichrome staining (×50 magnification, arrowheads depict collagen deposition). ( G ) Immunofluorescence staining using antibodies specific for HIF1α (red) and F4/80 (green) was carried out on FFPE sections (arrows indicate HIF1α-expressing macrophages) and ( H ) HIF1α staining was quantified (isotype-treated, n = 5 mice; anti–IL-17–treated, n = 10 mice). ( I ) Hypoxia was determined using pimonidazole (PIMO, red) and F4/80 (green) staining by immunofluorescence, ×200 magnification (arrowheads depict areas of hypoxia) and ( J ) PIMO staining was quantified (isotype-treated, n = 5 mice; anti–IL-17–treated, n = 10 mice).

Techniques: Derivative Assay, Infection, Lactate Assay, Expressing, Western Blot, Blocking Assay, Staining, Formalin-fixed Paraffin-Embedded, Microscopy, Immunofluorescence

Journal: JCI Insight

Article Title: Interleukin-17 limits hypoxia-inducible factor 1 α and development of hypoxic granulomas during tuberculosis

doi: 10.1172/jci.insight.92973

Figure Lengend Snippet: FeJ mice were aerosol infected with approximately 100 CFU Mycobacterium tuberculosis (Mtb) clinical strain HN878. IL-17 was neutralized (A–E) early (10–24 days postinfection [d.p.i]) or (A–D) late (24–31 d.p.i) and mice were harvested at 37 d.p.i. (A) Representative images from formalin-fixed, paraffin-embedded (FFPE) lung sections stained using H&E, ×50 magnification. (B) Lung inflammation in H&E–stained FFPE sections was quantified using the morphometric tool of the Zeiss Axioplan microscope (n = 5 mice/group). (C) Lung bacterial burden was assessed by plating lung homogenates of both isotype-antibody-treated (n = 5 mice) and IL-17–neutralized mice (n = 4 mice/early anti–IL-17; n = 5 mice/late anti–IL-17). (D) Lungs were processed to single-cell suspensions and flow cytometry was used to determine the lymphocyte populations producing IL-17 in the lungs of HN878-infected FeJ mice (n = 5 mice/group). (E) At 37 d.p.i., bacterial burden was plated and the percentage of isoniazid-resistant HN878 was measured by normalizing growth on isoniazid plates to total HN878 growth on non-isoniazid plates (n = 4 mice/isotype, n = 7 mice/anti–IL-17). All data shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA (B–D) or Student’s t test (E).

Article Snippet: ]), and lungs were harvested at 37 d.p.i. ( C ) Lung bacterial burden was assessed by plating from HN878-infected isotype ( n = 5 mice) and anti–IL-17–treated mice ( n = 5 mice). ( D ) Lung area per lobe covered by inflammation on H&E–stained formalin-fixed paraffin-embedded (FFPE) sections from HN878-infected isotype-antibody-treated ( n = 4 mice) and anti–IL-17–treated mice ( n = 3 mice) was quantified using the morphometric tool of the Zeiss Axioplan microscope. ( E ) Representative H&E images shown (×50 magnification). ( F ) Fibrosis was assessed using Masson’s trichrome staining (×50 magnification, arrowheads depict collagen deposition). ( G ) Immunofluorescence staining using antibodies specific for HIF1α (red) and F4/80 (green) was carried out on FFPE sections (arrows indicate HIF1α-expressing macrophages) and ( H ) HIF1α staining was quantified (isotype-treated, n = 5 mice; anti–IL-17–treated, n = 10 mice). ( I ) Hypoxia was determined using pimonidazole (PIMO, red) and F4/80 (green) staining by immunofluorescence, ×200 magnification (arrowheads depict areas of hypoxia) and ( J ) PIMO staining was quantified (isotype-treated, n = 5 mice; anti–IL-17–treated, n = 10 mice).

Techniques: Infection, Formalin-fixed Paraffin-Embedded, Staining, Microscopy, Flow Cytometry