Kidney Week

Abstract: FR-PO576

β2 Integrin-Dependent Adhesion Is Indispensable for Inflammatory HIF1α Activation in Human Neutrophils

Session Information

• Glomerular Diseases: Lupus and Vasculitis
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1302 Glomerular Diseases: Immunology and Inflammation

Authors

• Kling, Lovis, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

Lovis Kling,

• Eulenberg-Gustavus, Claudia, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

Claudia Eulenberg-Gustavus,

• Jerke, Uwe, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

Uwe Jerke,

• Rousselle, Anthony, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

Anthony Rousselle,

• Eckardt, Kai-Uwe, Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin, Berlin, Germany

Kai-Uwe Eckardt,

• Schreiber, Adrian, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

Adrian Schreiber,

• Kettritz, Ralph, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

Ralph Kettritz,

Background

Myeloid cells migrate from blood to inflammatory sites with high cytokine but low oxygen concentrations. Hypoxic but also pharmacologic prolyl hydroxylase inhibition induces hypoxia-inducible factor 1α (HIF1α), and thereby enhances myeloid cell performance. We tested the hypothesis that cytokines and β₂-integrins co-operate in the HIF1α activation process.

Methods

We characterized HIF1α in human blood neutrophils and monocytes using immunoblotting and qPCR. Neutrophil suspension was achieved on polyhema, adhesion on fibronectin, and migration through transwells. Prolyl hydroxylases were inhibited with Roxadustat (ROX), and HIF1α translation with YC1. Signaling pathways were analyzed with β₂-integrin antibodies and the JAK2 inhibitor AZD1480.

Results

We observed HIF1α activation in ROX-, but not cytokine-treated neutrophils and monocytes after 4h incubation in tubes. A 3-fold synergistic effect occurred in neutrophils but not in monocytes with combined GMCSF and ROX treatment. Neutrophils that interacted with fibronectin showed high HIF1α protein abundance, whereas HIF1α protein was completely absent under stringent suspension conditions on polyhema that excluded cell-cell and matrix contacts. Consequently, pre-incubation with blocking β₂-integrin antibodies prevented neutrophil HIF1α protein in both tubes and on fibronectin. GMCSF induced strong HIF1α mRNA via JAK2/STAT but independent of adhesion. However, an additional β₂-integrin signal was required for HIF1α protein upregulation. Adhesion did not accelerate transcription but rather involved post-transcriptional mechanisms stabilizing HIF1α protein as shown by YC1-mediated inhibitory effects. These novel synergistic mechanisms led to strong HIF1α protein in neutrophils that migrated towards GMCSF and ROX in transwell experiments.

Conclusion

Human neutrophils are unable of activating HIF1α in suspension. In contrast, cytokine, β₂-integrin, and prolyl hydroxylase co-operation at inflammatory sites enables strong HIF1α activation.

Funding

• Government Support – Non-U.S.