Journal: eBioMedicine

Article Title: Molecular imaging in experimental pulmonary fibrosis reveals that nintedanib unexpectedly modulates CCR2 immune cell infiltration

doi: 10.1016/j.ebiom.2024.105431

Figure Lengend Snippet: Protective treatment using CCR2 inhibitor propagermanium and nintedanib similarly limit lung CCR2+ cell accumulation in bleomycin-induced fibrosis . (a) Scheme of intranasal (i.n.) bleomycin (BLM, 1.6 U/kg) administration and treatment with propagermanium (PPG) or nintedanib on days 0–14 in wild type or Ccr 2-GFP mice. (b) Representative CCR2-GFP+ cells by immunohistochemistry for GFP (brown, DAB; 3,3′-diaminobenzidine) in lung sections, counterstained with haematoxylin. Box identifies region of high-power images. (c) Quantification of CCR2+ cells as in panel b. (n = 5–6 mice/condition). (d) Representative CCR2-PET/CT lung images of control, PPG, and nintedanib-treated mice at day 14. (e) Quantification of CCR2-PET lung uptake from panel d. (n = 7–8 mice/condition). (f) Representative Masson's trichrome-stained lung sections at day 14. (g) Modified Ashcroft scores from lung sections in panel g at day 14 (n = 7–8 mice/condition). (h) Quantification of CCL2 levels in BAL fluid multiplex assay. Dashed line represents the baseline level of detection. (n = 5–7 mice/condition). Data shown are the median with P values determined by the Mann–Whitney U test. Bars = 500 μm (g, f) and 100 μm (B).

Article Snippet: Nintedanib (LC laboratories, #N-9077) was dosed at approximately 60 mg/kg/day (1.5 mg to dose 60 mg/kg to a 25 g mouse), which was shown by others to decrease lung fibrosis.

Techniques: Immunohistochemistry, Positron Emission Tomography-Computed Tomography, Control, Staining, Modification, Multiplex Assay, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Molecular imaging in experimental pulmonary fibrosis reveals that nintedanib unexpectedly modulates CCR2 immune cell infiltration

doi: 10.1016/j.ebiom.2024.105431

Figure Lengend Snippet: Nintedanib inhibits CCL2-mediated chemotaxis of monocytic cells . (a) THP-1 cell viability following 2.5 h incubation using conditions indicated. Viability compared to control cells treated with media only. CCR2 antagonist (antag, BMS22). (b) Migration of THP-1 cells with CCL2 stimulation following incubation for 2.5 h using conditions indicated. (c) Effect of a 15-min pre-treatment with 100 nM nintedanib on CCL2-mediated G protein dissociation (antagonist mode) in a suspension HEK293 cell line with a heterologous CCR2 expression. (d) G protein dissociation (agonist mode) induced by the indicated ligands. In a and b, each point represents the mean of technical replicates of three independent experiments. In c and d, shown is mean ± S.D. for four independent experiments. Abbreviations: ns, not significant; S.D., standard deviation. Each point was compared to the control using the Mann–Whitney U test; ∗P < 0.05.

Article Snippet: Nintedanib (LC laboratories, #N-9077) was dosed at approximately 60 mg/kg/day (1.5 mg to dose 60 mg/kg to a 25 g mouse), which was shown by others to decrease lung fibrosis.

Techniques: Chemotaxis Assay, Incubation, Control, Migration, Suspension, Expressing, Standard Deviation, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Molecular imaging in experimental pulmonary fibrosis reveals that nintedanib unexpectedly modulates CCR2 immune cell infiltration

doi: 10.1016/j.ebiom.2024.105431

Figure Lengend Snippet: Therapeutic treatment with nintedanib decreases lung CCR2+ cells and attenuates fibrosis in the bleomycin model . (a) Scheme of intranasal (i.n.) bleomycin (BLM) administration and treatment with nintedanib on days 9–26. (b) Change in weight with bleomycin alone or with nintedanib treatment. Untreated, n = 8, Nintedanib, n = 8, mice/condition in 2 independent experiments (Untreated, Area under the curve, AUC, 309.2; Nintedanib AUC 308.7; P = 0.9868; unpaired t test). (c) Representative detection of Ccr2 RNA by fluorescent in situ hybridisation (FISH) at day 2 (red) in lung sections counterstained with DAPI (blue). Boxed region in full lung section corresponds to region in detail image (right). Scale bars, whole lung = 500 μm; detail = 100 μm. (d) Quantification of Ccr2 + cells at day 26 from panel b. (n = 4–5 mice/condition). (e) Representative images of CCR2-PET/CT lung uptake in control or nintedanib-treated mice at day 21. (f) Quantification of CCR2-PET lung uptake at day 21 (n = 8 mice/condition). (After exclusion of control animal with uptake 4.4 percent ID/gram; P = 0.014). (g) Quantification of fibrosis using modified Ashcroft scores (n = 8 mice/condition). (h) Quantification of hydroxyproline at day 26 (n = 5 mice/condition). (i) Immunodetection of collagen I expression by DAB (brown) in lung and counterstained with haematoxylin. Scale bar = 100 μm. (j) Immunoblot detection of collagen I (COL1) protein in lungs of mice at day 28. (n = 4 mice/condition). GAPDH = glyceraldehyde-3-phosphate dehydrogenase. (k) Quantification of Collagen I protein (COL1) from panel i.

Article Snippet: Nintedanib (LC laboratories, #N-9077) was dosed at approximately 60 mg/kg/day (1.5 mg to dose 60 mg/kg to a 25 g mouse), which was shown by others to decrease lung fibrosis.

Techniques: In Situ, Hybridization, Positron Emission Tomography-Computed Tomography, Control, Modification, Immunodetection, Expressing, Western Blot

Journal: eBioMedicine

Article Title: Molecular imaging in experimental pulmonary fibrosis reveals that nintedanib unexpectedly modulates CCR2 immune cell infiltration

doi: 10.1016/j.ebiom.2024.105431

Figure Lengend Snippet: Nintedanib modulates CCR2+ cells and fibrosis in a silica-induced fibrosis model . (a) Scheme of oropharyngeal (o.p.) silica (20 mg/kg) administration and treatment with nintedanib days 5–21. (b) Representative detection of CCR2-GFP+ cells by immunohistochemistry for GFP (brown) in lung sections, counterstained with haematoxylin. Boxed region in full lung section corresponds to high power image (right). (c) Quantification of CCR2-GFP+ cells from panel b. (n = 6–7 mice/condition). (d) Representative CCR2-PET/CT lung images of uptake in control and nintedanib-treated mice at day 14. (e) Quantification of CCR2-PET lung uptake at day 14 from panel d. (n = 14 control; n = 7 nintedanib). (f) Representative images of Masson's trichrome stained lung sections at day 21 exhibiting two different patterns of fibrotic nodules in lungs. (g) Quantification of fibrosis expressed as percentage of fibrotic area per total area of the lung at day 21. (h) Immunoblot detection for Collagen I (COL1) protein content in lungs of control and nintedanib-treated mice at day 21. (n = 3–4 mice/condition). Data are shown as the median with P values determined by the Mann–Whitney U test. Bars = 500 mm (b, f) and 100 mm (b).

Article Snippet: Nintedanib (LC laboratories, #N-9077) was dosed at approximately 60 mg/kg/day (1.5 mg to dose 60 mg/kg to a 25 g mouse), which was shown by others to decrease lung fibrosis.

Techniques: Immunohistochemistry, Positron Emission Tomography-Computed Tomography, Control, Staining, Western Blot, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Molecular imaging in experimental pulmonary fibrosis reveals that nintedanib unexpectedly modulates CCR2 immune cell infiltration

doi: 10.1016/j.ebiom.2024.105431

Figure Lengend Snippet: CCR2 cell lung burden monitored by CCR2-PET predicts the development of fibrosis . (a) Scheme of intranasal (i.n.) bleomycin (BLM) administration and treatment with nintedanib on days 9–26. (b) Relationship between CCR2-PET lung uptake at day 14 and the associated fibrosis quantified by modified Ashcroft score at day 26 in control and nintedanib-treated mice. (n = 4–5 mice/group). (c) Simple linear regression curves representing the relationship between CCR2-PET lung uptake and Ashcroft scores for bleomycin model control and nintedanib groups. A single treatment point was excluded as an outlier analysed by the Grubbs test (G = 1.779). (d) Scheme of oropharyngeal (o.p.) silica administration and treatment with nintedanib days 6–21. (e) Relationship between CCR2-PET lung uptake at day 14 and fibrosis at day 21 in control and nintedanib-treated mice. (n = 5–6 mice/group). (f) Linear regression of the relationship between CCR2-PET lung uptake and percentage fibrotic area for silica model control and nintedanib groups.

Article Snippet: Nintedanib (LC laboratories, #N-9077) was dosed at approximately 60 mg/kg/day (1.5 mg to dose 60 mg/kg to a 25 g mouse), which was shown by others to decrease lung fibrosis.

Techniques: Modification, Control

Journal: EMBO Molecular Medicine

Article Title: The ASC inflammasome adapter governs SAA-derived protein aggregation in inflammatory amyloidosis

doi: 10.1038/s44321-024-00107-0

Figure Lengend Snippet: ( A , B ) ASC and Congo-red-stained myocard from patients without ( A ) and with AA amyloidosis ( B ; 68 and 82-year old males, respectively). ( C – E ) Individual and merged confocal images of cardiac blood vessels (labeled with PAI1) from post-mortem tissue of a patient with AA amyloidosis, presenting high level of SAA (labeled with MAS41676 and DM003) and ASC (labeled with AL177 and MAB/MY6745). ( F , G ) Individual and merged confocal images of cardiac blood vessels (labeled with PAI1) from post-mortem tissue, presenting low level of SAA (labeled with MAS41676 and DM003) and ASC (labeled with AL177). ( H ) Graphical representation of the quantification of 100X confocal images for SAA and ASC. Each point represents one field of view, bars represent median. Integrated density was normalized by the mean value control. Data from one patient in each group are shown. ( I ) Confocal images from post-mortem tissue of a patient with AA amyloidosis showing accumulation of both SAA (MAS41676, magenta) and ASC (AL177, yellow) within the blood vessel border (labeled with PAI1) and in adjacent muscle cells. ( J ) STED imaging of co-aggregation SAA (magenta) and ASC (yellow). Imaris 3D model shows the intricacy of SAA and ASC within these aggregates. ( K ) Confocal images from post-mortem tissue of patient with AA amyloidosis showing accumulation of both SAA (DM003, magenta) and ASC (MAB-ASC (MY6745), yellow) within the blood vessel border (labeled with PAI1) and in adjacent muscle cells. ( L ) STED imaging of co-aggregation of SAA (magenta) and ASC (yellow). Imaris 3D model shows the intricacy of SAA and ASC within these aggregates. .

Article Snippet: PAI1 (PA1-9077, ThermoFisher), SAA (MAS41676, Invitrogen), ASC (clone AL177, AdipoGen) and ASC (MY6745, Mabylon) were used at 1:200 dilution, SAA (DM003, OriGene Technologies) was used at 1:20 dilution, all in saturating buffer, for 48 h at 4 °C.

Techniques: Staining, Labeling, Control, Imaging