Article Figures & Data
- FIG 1
Dectin-1 is expressed in the human lung. (A, B) Representative paraffin sections of human lung tissues from surgical specimens from lung cancer patients were stained for Dectin-1 (clone MAB1859; R&D Systems) and photographed at an optical magnification of ×200. Boxes labeled a or b show enlargements of the respective regions in these pictures. Black arrows point to alveolar epithelium (1), bronchial epithelium (2), pleura (3), and alveolar macrophages (4).
- FIG 2
Dectin-1 is expressed in primary pulmonary epithelial cells. Flow cytometry of the expression of Dectin-1 (open histograms, GE2 antibody) on the surface of small airway epithelial cells (SAEC) and normal human bronchial epithelial (NHBE) cells. Filled histograms, control staining with PE-labeled secondary antibody. (B) Relative Dectin-1 mRNA expression levels in NHBE cells after treatment with different TLR agonists, chemokines, or cigarette smoke extract (CSE) for 24 h, measured by qPCR normalized to untreated cells. Column bar graphs show mean expressions from three independent experiments with standard errors. ns, not significant; ***, P < 0.001. (C) Regulation of Dectin-1 protein expression on NHBE cells after treatment with poly(I⋅C) or interferons for 24 h, measured by flow cytometry. Filled histograms, control staining; open histograms, Dectin-1 staining (GE2 antibody); dotted line, Dectin-1 expression of untreated cells; black line, Dectin-1 expression after the respective treatment.
- FIG 3
Dectin-1 enhances the NTHI-induced proinflammatory immune response of NHBE cells. NHBE cells were pretreated for 1 h with two different Dectin-1 inhibitors (anti-Dectin-1 antibody MAB1859 [20 µg/ml] or laminarin from Laminaria digitata [1 mg/ml]) and stimulated with 100 µg/ml zymosan, NTHI strain 2019 (MOI of 100), or NTHI strain 86-028 (MOI of 100) for 18 h. Subsequently, IL-8 (A) and IL-6 (B) cytokine levels were determined in the supernatants by ELISA. The actual level of IL-8 response of NHBE cells was in the range of 2.3 to 36.0 ng/ml for NTHI 2019, in the range of 3.0 to 49.6 ng/ml for NTHI 86-028, and in the range of 1.0 to 3.7 ng/ml for zymosan. The actual level of IL-6 induced in NHBE cells in response was in the range of 11.4 to 390.34 pg/ml for NTHI 2019, in the range of 8.9 to 513.4 pg/ml for NTHI 86-028, and in the range of 87.3 to 396.2 pg/ml for zymosan. Because of donor-specific variance in absolute cytokine levels, mean data derived from at least four independent experiments were normalized to the stimulated control (set as 100%). The error bars show the standard deviations of the normalized data. Significances were analyzed using the nonnormalized data by one-way ANOVA repeated-measures test with Dunnett’s post hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 4
The Dectin-1-mediated IL-8 response in NTHI-infected pulmonary epithelial cells depends on the Dectin-1 hemITAM domain and NF-κB. A549 cells were stably transfected with Dectin-1, a Dectin-1 hemITAM mutant (Dectin-1_Y3:15F), or the vector without Dectin-1. (A) Flow cytometry of the transfected cells after sorting. Filled histograms, control staining with PE-conjugated secondary antibody; open histograms, Dectin-1 staining (GE2 antibody). (B) Transfected cells were stimulated with NTHI strains 2019 or 86-028 for 18 h, and IL-8 and IL-6 levels were measured in the supernatants of the cells. Column bar graphs are mean intensities and standard deviations from four independent experiments. Significances were determined by one-way ANOVA test (ns, not significant; ***, P < 0.001). (C) Chromatin immune precipitation (ChIP) of polymerase II and NF-κB subunit p65 and subsequent amplification of IL-8 gene promoter. Dectin-1-expressing A549 cells and vector control were stimulated with NTHI 2019 for 60 min and subjected to ChIP and IL-8 promoter amplification. Amplicons were visualized using agarose gel electrophoresis and ethidium bromide staining. One representative experiment from three independent experiments is shown.
- FIG 5
Dectin-1 interacts with NTHI. (A) NTHI–Dectin-1 pulldown: Western blot analysis targeting human Fc after coincubation of NTHI 2019 with recombinant Fc–hDectin-1a in the presence or absence of the Dectin-1 inhibitors laminarin (1 mg/ml) or anti-Dectin-1 antibody MAB1859 (20 µg/ml). IgG1-Fc was used as a control. The image shows one representative experiment of three. (B) Adhesion of NTHI to A549 cells without Dectin-1 (vector; white bars) or A549 cells overexpressing Dectin-1 (black bars). (C) Internalization of NTHI into A549 cells overexpressing wild-type Dectin-1 (black bars) or Dectin-1 with a mutated hemITAM signaling motif (Dectin-1_Y3:15F; white bars). Bar graphs are mean CFU, given as percentages of inoculum and standard deviations from at least three independent experiments. Significant differences were calculated using a two-tailed Student t test. ns, not significant; *, P < 0.05.
- TABLE 1
Patient characteristics
Characteristic Value(s) No. of males/females 9/10 Mean age (±SD) 63 (±11) No. of current smokers/ex-smokers/nonsmokers 5/11/3 Mean no. of pack-years (±SD) for current smokers/ex-smokers/nonsmokers 61 (±22)/33 (±22)/0 No. of patients with COPD/without COPD 8/11 - TABLE 2
Analysis of the immunohistochemistry of human lung sections (n = 19)a
Lung section No. of Dectin-1-positive specimens/total no. of specimens No. of specimens (/total specimens) with a staining score of: 0 1 2 3 Alveolar epithelium 17/19 2/19 7/19 10/19 0/19 Bronchial epithelium 14/16 2/16 1/16 10/16 3/16 Pleura 19/19 0/19 2/19 6/19 11/19 ↵a Since not all tissue types were found in all specimens, the number of positive specimens and the number of total specimens, including the respective tissue, are given. The intensity of Dectin-1 staining of the respective tissues was evaluated in the range from 0 (no staining) to 3 (strong staining) relative to the IgG control staining.
Supplemental Material
Figure S1
IgG control staining of paraffin sections. (A, B) Representative paraffin sections of human lung tissues from surgical specimens from lung cancer patients were stained with a control antibody and photographed at an optical magnification of ×200. Boxes labeled a or b show enlargements of the respective regions in these pictures. Black arrows point to alveolar epithelium (1), bronchial epithelium (2), and pleura (3). Download Figure S1, TIF file, 7 MB.
Copyright © 2014 Heyl et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
No association of Dectin-1 expression with smoking status or COPD in human lung. Intensity of Dectin-1 staining of respective tissues was evaluated within a range of 0 (no staining) to 3 (strong staining) relative to IgG control staining. No differences were found between COPD patients and non-COPD patients (A) or between nonsmokers, ex-smokers, and current smokers (B). There was also no relationship found between intensity of staining and pack-years (C) or the level of C-reactive protein (CRP), given in mg/dl. (A, B) Column bar graphs are mean intensities and standard deviations; (C, D) scatter plots are mean pack-years or mean CRP levels and standard deviations. Download Figure S2, TIF file, 4.1 MB.
Copyright © 2014 Heyl et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Dectin-1 inhibition has no effect on IL-8 secretion of NHBE cells that are Dectin-1 negative. NHBE cells negative for Dectin-1 staining were treated with Dectin-1 inhibitors (20 µg/ml MAb anti-Dectin-1 clone MAB1859 or 1 mg/ml laminarin) and stimulated with 100 µg/ml zymosan, NTHI strain 2019 (MOI of 100), or NTHI strain 86-028 (MOI of 100) for 18 h. Afterward, IL-8 cytokine levels were measured in the supernatants by ELISA. Column bar graphs represent mean cytokine concentrations and standard deviations from 3 independent experiments. Download Figure S3, TIF file, 1.6 MB.
Copyright © 2014 Heyl et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
NTHI expresses no β-(1,3)-glucan at detectable levels. Fluorescence microscopy of heat-killed (positive-control) or UV-killed (negative-control) Candida albicans or paraformaldehyde (PFA)-fixed NTHI (GFP-expressing NTHI 86-028pRSM2211), stained with a specific antibody targeting β-(1,3)-glucan [monoclonal mouse anti-β-(1,3)-glucan (Biosupplies Australia); secondary antibody, Alexa Fluor 546 goat anti-mouse IgG (Invitrogen)]. Images were taken with a 63× objective (Axio Observer Z1 microscope; Zeiss). (A) Transmission light microscopy of hkCa. (B, C) Fluorescence microscopy of hkCa. B, only secondary antibody; C, β-(1,3)-glucan staining. Note that the inner beta-glucan layer of the cell wall is accessible due to the heat killing. (D) Fluorescence microscopy of β-(1,3)-glucan staining of UVkCa, where the beta-glucans of the inner cell wall layer are covered by the outer cell wall layer. (E, F) Fluorescence microscopy of GFP expression (E) and β-(1,3)-glucan staining (F) of NTHI 86-028pRSM2211. hkCa, heat-killed Candida albicans; UVkCa, UV-killed Candida albicans; β(1,3) Glu, β-(1,3)-glucan. Download Figure S4, TIF file, 1.3 MB.
Copyright © 2014 Heyl et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
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