ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: FR-PO576

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

Session Information

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
  • Eulenberg-Gustavus, Claudia, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Jerke, Uwe, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Rousselle, Anthony, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Eckardt, Kai-Uwe, Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin, Berlin, Germany
  • Schreiber, Adrian, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Kettritz, Ralph, Experimental and Clinical Research Center, Charité – Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
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.