image_2_Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.tif

<p>Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1^−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.</p

image_1_Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.tif

<p>Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1^−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.</p

Loss of PINK1 aggravates bleomycin induced lung fibrosis.

<p><i>Pink1</i>^-/- mice and their littermate controls (male, 8–10 weeks old) were treated with 3U/kg of intratracheal bleomycin sulfate or saline on day 1 and sacrificed on Day 21. <i>Pink1</i>^-/- mice in the bleomycin group showed higher levels of hydroxyproline compared to controls (85.4 μg/mL vs. 97.0 μg/mL; *p = .05, **p<0.001).</p

TGF-β1 induces mitochondrial depolarization.

<p>A) Beas-2B cells stimulated with TGF-β1 and CCCP (positive controls) were stained with JC-1 for 15 min and analyzed by flow cytometric analysis. TGF-β1 treated cells showed decreased red fluorescence (mitochondrial depolarization). B) Quantification of relative MFI for experiment in A (p = 0.06). C) Beas-2B cells stimulated with TGF-β1 were stained with MitoSOX for 10 min and analyzed by flow cytometry. TGF-β1 treated cells showed increased red fluorescence (mitochondrial ROS production) and mitochondria-specific antioxidant (MitoTEMPO) reversed the effect of TGF-β1. D) Western blot of the mitochondrial fraction of Beas-2B cells stimulated with TGF-β1 (5ng/mL) +/- MitoTEMPO (200μM) showing decreased PINK1 expression in presence of MitoTEMPO.</p

PINK1 Expression and Mitochondrial Dysfunction in IPF Lung Tissue.

<p>A) Western blot of mitochondrial fraction of PINK1 in human lung tissue from control and IPF patients showing increased PINK1 levels in IPF samples. B) Densitometry of blot in A. C) mRNA expression of Pink1 in human lung tissue from control (n = 5) and IPF (n = 5) samples. D) Representative transmission electron micrographs from control and IPF lung tissue showing mitochondria (*). Magnif = 18500x; scale bars = 200nm. E) Quantification of total and abnormal mitochondria. Total mitochondria per μm² in control and IPF was 0.52 ± 0.39 vs. 0.46 ± 0.34. Abnormal mitochondria per μm² in control and IPF was 0.12 ± 0.19 vs. 0.32 ± 0.32, <i>p</i> = 0.003. F) Confocal immunofluorescence against PINK1(red) and LC3 (green) in control and IPF lung (magnification 10x). G) Magnified view (60x) of confocal immunofluoresence with DAPI (blue) and PINK1 (red) in control and IPF lung.</p

TGF-β1 increases PINK1 expression and induces mitochondrial fission <i>in vitro</i>.

<p>A) TGF-β1 induced PINK1 expression of Beas-2B cells in a time- and dose-dependent manner. B) Quantification by densitometry of PINK1 expression in Beas-2B cells stimulated with TGF-β1 for 6 hours. C) Confocal microscopy of Beas-2B cells stimulated with TGF-β1 (6h) showed that TGF-β1 induces formation of PINK1 puncta (red) (magnification 120x). D) Quantification of colocalization of LC3 and PINK1 punctae (* p = 0.035, ** p = 0.001). E) Confocal microscopy of Beas-2B cells transfected with vectors staining mitochondria (green) and lysosomes (red) and with TGF-β1 stimulation for 6 hours (magnification 60x). F) Western blot of Beas-2B cells stimulated with TGF-β1 (6hrs) showed increased expression of pDRP1 (ser616). G) Confocal microscopy of Beas-2B cells stimulated with TGF-β1 (5 ng/mL, 24 hrs) showed more fragmentation (fission) of mitochondria stained with MitoTracker Green (magnification 120x).</p

Loss of PINK1 augments TGF-β1 induced cell death.

<p>A) Measurement of mitochondrial ROS in murine type II alveolar epithelial cells from <i>Pink1</i>^-/- and <i>Pink1</i>^WT mice by flow cytometry and MitoSOX staining. B) Western blot against PINK1 demonstrated knockdown of expression with PINK1 siRNA. C) Beas-2B cells were treated with siRNA (50 nM, 24hrs). Then, they were stimulated with TGF-β1 (5 ng/mL, 24hrs) and analyzed by Annexin V/PI flow cytometry. D) Quantification of cell death from experiment in C (*p = 0.0005, **p = 0.0119, ***p = 0.032). Loss of PINK1 exaggerated cell death in cells treated with TGF-β1 relative to transfection with control siRNA.</p

Inflammatory cell counts in the lungs from mice challenged with OVA and DOK-1 shRNA.

<p>Inflammatory cell counts in the lungs from mice challenged with OVA and DOK-1 shRNA.</p

Effects of DOK-1 in OVA-induced inflammation and airway response.

<p>(A) The recovery of BAL cells 24 hr after OVA challenge. NEU, Neutrophil; EOS, Eosinophil; LYM, Lymphocyte; MAC, Macrophages; TOT, total cell. (B) Eosinophil peroxidase (EPO) activity in BAL fluids of OVA-sensitized and –challenged mice. (C) Airway responsiveness to aerosolized methacholine measured by non-invasive whole body plethysmography. (D) Serum IgE and IgG2a levels detected by ELISA. The values in all the panels represent means ± S.E.M. At least 5 mice were included in each group. *P<0.05, ***P<0.001 vs. OVA-challenged mice.</p

Effects of DOK-1 on STAT-6 and STAT-4 expression and nuclear translocation in airway epithelial cells.

<p>(A) Lung sections were stained with Alexa Fluor 488-conjugated DOK-1 and Alexa Fluor 568-conjugated STAT-6 antibodies and DAPI stain. (B) Lung sections were stained with Alexa Fluor 488-conjugated DOK-1 and Alexa Fluor 568-conjugated STAT-4 antibodies and DAPI stains. A representative photo of 7 similar experiments. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression.</p