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

  1. Srinu Tumpara
  2. Matthias Ballmaier
  3. Sabine Wrenger
  4. Mandy König
  5. Matthias Lehmann
  6. Ralf Lichtinghagen
  7. Beatriz Martinez-Delgado
  8. Elena Korenbaum
  9. David DeLuca
  10. Nils Jedicke
  11. Tobias Welte
  12. Malin Fromme
  13. Pavel Strnad
  14. Jan Stolk
  15. Sabina Janciauskiene  Is a corresponding author
  1. Department of Respiratory Medicine, Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Germany
  2. Cell Sorting Core Facility Hannover Medical School, Germany
  3. 8sens.biognostic GmbH, Germany
  4. Institute of Clinical Chemistry, Hannover Medical School, Germany
  5. Department of Molecular Genetics, Institute of Health Carlos III, Center for Biomedical Research in the Network of Rare Diseases (CIBERER), Spain
  6. Institute for Biophysical Chemistry, Hannover Medical School, Germany
  7. Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany
  8. Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Germany
  9. Department of Pulmonology, Leiden University Medical Center, Member of European Reference Network LUNG, section Alpha-1-antitrypsin Deficiency, Netherlands
4 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Relationship between CX3CR1 gene expression levels and AAT deficiency and the concentration of circulating AAT polymers.

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)…

Figure 1—source data 1

Source files, containing original data for Figure 1A and B, to document CX3CR1 expression (A), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals (B).

https://cdn.elifesciences.org/articles/64881/elife-64881-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Schematic presentation of the study design.

The German cohort comprises 41 ZZ alpha-1 antitrypsin-deficient (AATD) volunteers irrespective of clinical status and medications. For control, we collected blood from 21 non-AATD volunteers with …

Figure 2 with 5 supplements
Effects of Z alpha-1 antitrypsin (Z-AAT) and M-AAT on CX3CR1 mRNA and protein expression.

(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 …

Figure 2—source data 1

Source file, containing original data for Figure 2A, to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) (A).

https://cdn.elifesciences.org/articles/64881/elife-64881-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Quality control of isolated M- and Z-AAT (alpha-1 antitrypsin) proteins by SDS-PAGE.

(A) Plasma M- and Z-AAT was purified by affinity chromatography with AAT-specific Alpha-1 Antitrypsin Select matrix. Proteins were run on a 10% SDS-PAGE gel and stained with Coomassie Blue G250. AAT …

Figure 2—figure supplement 2
Z alpha-1 antitrypsin (Z-AAT) in a concentration-dependent manner reduces CX3CR1 mRNA expression in peripheral blood mononuclear cells (PBMCs) isolated from healthy donors.

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 …

Figure 2—figure supplement 3
Z alpha-1 antitrypsin (Z-AAT) induces association of CX3CR1 with lipid rafts.

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 …

Figure 2—figure supplement 4
CX3CR1 transcript and protein expressions in the presence of Z alpha-1 antitrypsin (Z-AAT) monomer, Z-AAT polymer, native M-AAT, and M-AAT polymer.

(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 …

Figure 2—figure supplement 5
Effect of CX3CL1 alone or in combination with Z alpha-1 antitrypsin (Z-AAT) on CX3CR1 transcript and protein expression.

Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with CX3CL1 and Z-AAT polymers separately or in combination or with RPMI medium alone (control). (A) CX3CR1 gene expression …

Figure 2—figure supplement 5—source data 1

Source file, containing original data for Figure 2—figure supplement 5A, to document CX3CR1 gene expression in peripheral blood mononuclear cell (PBMC).

https://cdn.elifesciences.org/articles/64881/elife-64881-fig2-figsupp5-data1-v2.xlsx
Figure 3 with 1 supplement
Flow cytometric analysis of CX3CR1 surface expression in peripheral blood mononuclear cells (PBMCs) after incubation with RPMI alone (control), Z alpha-1 antitrypsin (Z-AAT), or lipopolysaccharide (LPS) in the concentrations as indicated for 18 hr.

CX3CR1-expressing cells were found in the monocyte gate (A) and in the NK cell gate (CD56lo CD16+) (B). Histograms show representative results and bars represent mean (SD) of n = 4 independent …

Figure 3—source data 1

Source files, containing original data for Figure 3A,B, 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).

https://cdn.elifesciences.org/articles/64881/elife-64881-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Gating strategy: sequential gating to identify monocytes and NK cells from total peripheral blood mononuclear cells (PBMCs).

PBMCs were stained with CD14, CD16, and CD56 in the presence of 7-amino-actinomycin D (7-AAD). Dead cells were excluded by 7-AAD. From the single cells, CD14+ cells with a mid-level of granularity …

Figure 4 with 2 supplements
Z alpha-1 antitrypsin (Z-AAT) and lipopolysaccharide (LPS) induce CD14 expression.

(A) Z-AAT increases CD14 gene expression. Peripheral blood mononuclear cells (PBMCs) were incubated for 18 hr with 0.5 mg/ml Z-AAT or RPMI medium alone (control). CD14 mRNA expression relative to …

Figure 4—source data 1

Source files, containing original data for Figure 4A, B to document CD14 gene expression in peripheral blood mononuclear cells (PBMCs) (A) and CD14 surface expression in monocytes (B).

https://cdn.elifesciences.org/articles/64881/elife-64881-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Inverse changes in CD14 and CX3CR1 mRNA expression in peripheral blood mononuclear cells (PBMCs) treated with different concentrations of Z alpha-1 antitrypsin (Z-AAT).

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 …

Figure 4—figure supplement 2
Z alpha-1 antitrypsin (Z-AAT) does not induce cytokine expression.

Peripheral blood mononuclear cells (PBMCs) were treated with Z-AAT or lipopolysaccharide (LPS) (used as a positive control) for 18 hr. IL1B (a), IL6 (b), and TNFA (c) mRNA expression relative to HPRT…

Figure 4—figure supplement 2—source data 1

Source files, containing original data for Figure 4—figure supplement 2 to document IL1B (a), IL6 (b), and TNFA (c) gene expression in peripheral blood mononuclear cells (PBMCs).

https://cdn.elifesciences.org/articles/64881/elife-64881-fig4-figsupp2-data1-v2.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Biological sample (Homo sapiens)PBMCsBlood samplesCollected from 41 ZZ AATD and 21 non-AATD healthy controls
Sequenced-based reagent (human)CX3CR1Thermo Fisher ScientificTaqman assay 4331182Hs00365842_m1
Sequenced-based reagent (human)TNFAThermo Fisher ScientificTaqman assay 4331182Hs01113624_g1
Sequenced-based reagent (human)IL6Thermo Fisher ScientificTaqman assay 4331182Hs00985639_m1
Sequenced-based reagent (human)IL1BThermo Fisher ScientificTaqman assay 4331182Hs01555410_m1
Sequenced-based reagent (human)HPRT1Thermo Fisher ScientificTaqman assay 4331182Hs02800695_m1
Sequenced-based reagent (human)CD14Thermo Fisher ScientificTaqman assay 4331182Hs00169122_g1
OtherNon-adherent 12-well platesGreiner Bio-One665970
OtherAlpha-1 Antitrypsin Select matrixGE Healthcare Life Sciences, Cytiva17547201
OtherPlasma purified human AATCSL BehringRespreeza
OtherLPSSigma-AldrichL2880Escherichia coli O55:B5
Commercial assay or kitPierce Chromogenic Endotoxin Quant KitThermo Fisher ScientificA39553
Commercial assay or kitUltraRIPA kitBioDynamics LaboratoryF015
AntibodyAnti-AAT polymer, LG96 (mouse monoclonal)Deposited at German Collection of Microorganisms and Cell Cultures: DSM ACC3092LG96ELISA: Coating: 1 µg/ml;
LG96-HRP conjugate: (1:2000)
Immunofluorescence: (1:5000)
AntibodyAnti-human AAT
(rabbit polyclonal)
Agilent DakoA001202-2(1:800)
AntibodyAnti-AAT polymer (mouse monoclonal)Hycult BiotechClone 2C1; HM2289(1:500)
AntibodyAnti-CX3CR1 (rabbit polyclonal)Abcamab8021(1:500)
AntibodyAnti-β-actin
(mouse monoclonal)
Sigma-AldrichClone AC-15;
A3854
HRP-conjugated; (1:20,000)
AntibodyAnti-CX3CR1 (rabbit polyclonal)Abcamab8021(1:500)
AntibodyAnti-CX3CR1 (mouse monoclonal)Invitrogen, Thermo Fisher ScientificClone 2A9-1; 12-6099-42PE-conjugated; 5 µl per test
AntibodyAnti-CD14 (mouse monoclonal)Invitrogen, Thermo Fisher ScientificClone TuK4; MHCD1401FITC-conjugated; 5 µl per test
AntibodyAnti-CD16 (mouse monoclonal)ImmunotoolsClone 3G8; 21278166APC-conjugated; 5 µl per test
AntibodyAnti-CD56 (mouse monoclonal)BD BiosciencesClone NCAM16.2; 566124BV-480 conjugated; 5 µl per test
Peptide, recombinant proteinHuman CX3CL1/FractalkineBio-Techne365-FR-025
Software, algorithmFlowJoBecton, Dickinson and Companyv10
Software, algorithmSigmaPlot 14Systat Softwarev14.0
Table 1
Primers for gene expression analysis.
PrimerAssay ID
CX3CR1Hs00365842_m1
TNFAHs01113624_m1
IL6Hs00985639_m1
IL1BHs01555410_s1
HPRT1Hs02800695_m1
CD14Hs00169122_g1

Additional files

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