Adenosine deaminase mediates endothelial inflammation via an ADA1-CD26 interaction in post-COVID

Figures

• 

  FIGURE 1. Total adenosine deaminase (tADA) activity in peripheral blood mononuclear cells is higher in post-COVID patients than in healthy controls, and it is enhanced in monocytes/macrophages after incubation with post-COVID serum. (A) tADA activity in the homogenates of peripheral blood mononuclear cells (PBMC) isolated from post-COVID patients (n = 5) and healthy controls (n = 5). (B) tADA activity in the homogenates of monocytes/macrophages (THP-1) treated for 48 h with. control (Control-S, N = 5 independent experiments with five different patients’ sera, n = 3 biological repetitions for each patient) and post-COVID patients’ (postCOVID-S, N = 3 independent experiments with three different patients’ sera, n = 3 biological repetitions for each patient) sera or vehicle (control, n = 4) and 10 ng/mL TNFα (n = 4). Results are shown as mean ± SEM. *p < 0.05, **p < 0.01.

• 

  FIGURE 2. Adhesion of monocytes/macrophages to the lung microvascular endothelial cells is increased in the presence of post-COVID patient sera. (A) Representative images and (B) quantitative analysis of the adhesion of monocytes/macrophages (THP-1) to primary lung microvascular endothelium (HULEC) pre-treated for 48h with control (Control-S, N = 5 independent experiments with five different patients’ sera, n = 3 biological repetitions for each patient) and post-COVID patient serum (postCOVID-S, N = 5 independent experiments with five different patients’ sera, n = 3 biological repetitions for each patient) or vehicle (control, n = 6) and 10 ng/mL TNFα (n = 6). Results are shown as mean ± SEM, **p < 0.01; Each image includes a scale bar (bottom right) indicating 100 µm.

• 

  FIGURE 3. ADA1-CD26 interaction inhibitor, glycoprotein 120 (gp120), prevented the increase in tADA activity on the surface of activated lung microvascular endothelial cells. Mean fluorescence intensity (MFI) for green (AF488) and red (AF594) fluorescence that corresponded to ADA1 (AF488) and CD26 (AF594) in primary lung microvascular endothelial cells (HULEC) treated for 48h with (A) control (Control-S, N = 5 independent experiments with five different patients’ sera, n = 3 biological repetitions for each patient) and post-COVID patients’ sera (postCOVID, N = 5 independent experiments with five different patients’ sera, n = 3 biological repetitions for each patient), or with (B) vehicle (Control, n = 5) and 10 ng/mL TNFα (n = 5). (C) Cell-surface total ADA (tADA) activities in HULEC pre-treated for 30 min with 0, 1.2, or 6 μg/mL gp120 and then treated for 47.5h with (C) control (Control-S, N = 5) and post-COVID patients’ (postCOVID-S, N = 5) sera, or with (D) vehicle (Control, n = 5) and 10 ng/mL TNFα (n = 5). (E) Representative images for immunofluorescence staining of CD26 (AF488, green), SARS-CoV-2 spike protein 1 (SP, AF647, red), cell nuclei (DAPI, blue) in HULEC pre-treated for 30…

• 

  FIGURE 4. Glycoprotein 120 (gp120), counteracted the increased adhesion of monocytes/macrophages to activated lung microvascular endothelial cells. (A) Representative images and (B) quantitative analysis of the adhesion of monocytes/macrophages (THP-1) to primary lung microvascular endothelial cells (HULEC) pre-treated for 30 min with 1.2 μg/mL gp120 and then treated for 47.5h with control (Control-S) and post-COVID patients’ (postCOVID-S) sera, or vehicle (Control) and 10 ng/mL TNFα. N = 5 independent experiments with five different patients’ sera, n = three to four biological repetitions for each patient (Control-S, post-COVID-S), n = 6–10 (control, TNFα). Results are shown as mean ± SEM. *p < 0.05, **p < 0.01. Each image includes a scale bar (bottom right) indicating 100 µm.

• 

  FIGURE 5. Explanatory figure. ADA1 competed with SARS-CoV-2 spike protein for CD26 binding. Activated adenosine deaminase silenced the protective effects of adenosine on the endothelium. AR–adenosine receptors, ADA1 – adenosine deaminase 1.