Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: CX3CR1 gene expression levels in peripheral blood mononuclear cells (PBMCs) related to alpha-1 antitrypsin-deficiency (AATD) and plasma concentrations of CX3CR1 ligand (CX3CL1) and Z-AAT polymer. ( A )PBMCs were isolated from AATD subjects and non-AATD controls. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Measurements were carried out in duplicates. Data are presented as median (IQR) in boxplots, lines represent medians. Outliers are defined as data points located outside the whiskers. p-Value was calculated by Mann-Whitney U test. ( B ) Plasma levels of CX3CL1 in AATD (plasma available for n = 38 AATD) and non-AATD individuals measured by ELISA. Measurements were carried out in triplicates. Data are presented as median (IQR) in boxplots with whiskers. Outliers are defined as data points located outside the whiskers. ( C ) Negative correlation of CX3CR1 mRNA in PBMCs and plasma Z-AAT polymer levels from ZZ AATD individuals from graph ( B ). Pearson’s correlation test, r 2 = −0.313, p=0.055, n = 38. Figure 1—source data 1. Source files, containing original data for , to document CX3CR1 expression ( A ), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals ( B ).

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Curves show results from two independent experiments. Each point represents mean of two repeats.

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: Incubation, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: ( A ) CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with plasma-derived Z-AAT, lipopolysaccharide (LPS), or M-AAT in the concentrations as indicated, or with RPMI medium alone (control). The data from n = 6 independent experiments are presented as median (IQR) in box and whisker plot format; lines represent medians in each box. Measurements were carried out in duplicates. p-Value was calculated by nonparametric Kruskal-Wallis test. ( B ) Representative uncut Western blot (n = 3 independent experiments) of CX3CR1 in RIPA lysates prepared from PBMCs incubated for 18 hr alone or with LPS (1 µg/ml), M-AAT (1 mg/ml), or Z-AAT (0.5 mg/ml). For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. Relative intensities were calculated for each band using the ratio relative to β-actin, as a loading control, and then normalized by the experimental control. ( C ) For analysis of cellular AAT, the same lysates were separated under non-reducing conditions. Western blots were probed with polyclonal rabbit anti-human AAT recognizing monomeric, polymeric, or truncated forms of AAT. One representative blot from n = 3 independent experiments is shown. β-Actin was used for a loading control. ( D ) and ( E ) Co-distribution of Z-AAT polymers with CX3CR1 in human total PBMCs incubated with 0.5 mg/ml Z-AAT polymers for 18 hr. ( D ) Immunofluorescence microscopy revealed co-localization of Z-AAT polymers ( red ) with CX3CR1-positive structures ( green ). Arrows point areas of co-localization. Scale bar, 10 µm. ( E ) Confocal microscopy 3D stack with orthogonal reconstruction shows an aggregate of Z-AAT polymers ( red ) surrounded by CX3CR1-positive ( green ) cellular extensions forming a cap-like structure (arrowhead). Scale bar, 5 µm. The images with indicated channels merged and the corresponding differential interference contrast (DIC) image are presented. 4', 6- Diamidino 2-phenylindole (DAPI) was used for nuclei staining ( blue ). Figure 2—source data 1. Source file, containing original data for , to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Derivative Assay, Whisker Assay, Western Blot, SDS Page, Immunofluorescence, Microscopy, Confocal Microscopy, Staining

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: CX3CR1-expressing cells were found in the monocyte gate ( A ) and in the NK cell gate (CD56 lo CD16 + ) ( B ). Histograms show representative results and bars represent mean (SD) of n = 4 independent biological repeats each measured one time. After incubation with Z-AAT or LPS, monocytes and NK cells show significantly reduced CX3CR1 surface expression in comparison to untreated control cells. p-Values were calculated by one-way ANOVA. Figure 3—source data 1. Source files, containing original data for , to document reduced CX3CR1 surface expression in monocytes ( A ) and NK cells ( B ) after treatment with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) ( A ).

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: Expressing, Incubation

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: Lipid rafts were solubilized from membrane fractions with UltraRIPA kit. ( a ) For analysis of CX3CR1, equal amounts of protein were separated by SDS-PAGE under reducing conditions. One representative blot from n = 3 independent experiments is shown. ( b ) For analysis of lipid raft associated AAT polymers, the same samples were separated under non-reducing conditions. The Western blot was probed with monoclonal antibody (2C1) recognizing polymeric AAT. One representative blot from n = 3 independent experiments is shown.

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: SDS Page, Western Blot

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: ( A ) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr in RPMI medium alone or with addition of Z-AAT monomer or Z-AAT polymer (each 0.5 mg/ml), or lipopolysaccharide (LPS) (1 µg/ml) or native M-AAT (0.5 mg/ml). Measurements were carried out in duplicates. Data are represented as bars from four independent experiments. ( B ) Levels of CX3CR1 lysates prepared in RIPA buffer from PBMCs incubated for 18 hr with RPMI (control) and with Z-AAT monomer (0.5 mg/ml) or LPS (1 µg/ml) (used as a positive control). For analysis of CX3CR1, equal amounts of protein were separated by 10% SDS-PAGE under reducing conditions followed by Western blotting. Representative uncut Western blot (n = 2 independent experiments) is shown. β-Actin was used for a loading control. ( C ) CX3CR1 mRNA expression relative to HPRT1 was determined by real-time PCR using Taqman gene expression assays. PBMCs were incubated for 18 hr in RPMI medium alone or with different concentrations of heat-induced M-AAT polymers (60°C, for 3 hr). Measurements were carried out in duplicates. Data are represented as curves from two independent donors.

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Positive Control, SDS Page, Western Blot

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet: PBMCs were incubated for 18 hr with plasma-derived Z-AAT in the concentrations as indicated, or with RPMI medium alone (control). CX3CR1 and CD14 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Curves represent two independent experiments.

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: Incubation, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

doi: 10.7554/eLife.64881

Figure Lengend Snippet:

Article Snippet: Membranes were blocked for 1 hr with 5% low fat milk (Carl Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) (DAKO A/S, Glostrup, Denmark), mouse monoclonal anti-AAT polymer antibody (clone 2C1, 1:500, Hycult Biotech, Uden, The Netherlands), rabbit polyclonal anti-CX3CR1 (1:500, Abcam, Cambridge, UK), or HRP-conjugated monoclonal anti-β-actin antibody (1:20,000, Sigma-Aldrich, Merck, St. Louis, MO) for a loading control.

Techniques: TaqMan Assay, Purification, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Recombinant, Software