Fig 2: Patients with diabetic retinopathy exhibit increased expression of TRAF2 and TRAF6 in Müller cells. Posterior poles from patients with DR, PDR, and non-diabetic controls were incubated with anti-TRAF2 or anti-TRAF6 Abs plus anti-vimentin antibody. Arrowheads show some of the areas where vimentin is co-expressed with CD40 and TRAF2 or TRAF6. Graphs show signal intensity for TRAF 2 or TRAF6 in vimentin+ cells. Original magnification times 400. Scale bar, 50 µm. Graphs show pixel intensity (AU) for TRAF 2 or TRAF6 in vimentin+ cells. Six to eight fields per subject were analyzed. Statistical comparison among patient samples was done using the mean values for these measurements. ***P < 0.001 by ANOVA.

Fig 3: Model of the changes in expression of CD40, TRAF2, TRAF6, and inflammatory molecules in diabetic retinopathy in humans. Expression of CD40 and its downstream signaling molecules TRAF2 and TRAF6 are increased in retinal Müller cells and endothelial cells from patients with diabetic retinopathy. This is accompanied by increased expression of ICAM-1 in retinal endothelial cells, upregulation of CCL2 in Müller cells, and expression of activated PLCγ1 in Müller cells. TNF-α does not appear to be expressed in Müller or endothelial cells whereas it is detected in cells that likely represent microglia/macrophages. Prior in vitro studies revealed that CD40-TRAF signaling upregulates ICAM-1 in retinal endothelial cells and CCL2 production by Müller cells (Ref. 27), as well as activates PLCγ1 in Müller cells, triggering release of extracellular ATP that in turn promotes TNF-α production by myeloid cells (Ref. 28). Created with BioRender.

Fig 4: Patients with diabetic retinopathy exhibit increased expression of TRAF2 and TRAF6 in retinal endothelial cells. Posterior poles from patients with DR, PDR, and non-diabetic controls were incubated with anti-TRAF2 or anti-TRAF6 Abs plus anti-von Willebrand factor antibody. Arrowheads show areas where von Willebrand factor is co-expressed with CD40 and TRAF2 or TRAF6. Original magnification times 400. Scale bar, 20 µm. Graphs show pixel intensity (AU) for TRAF 2 or TRAF6 in von Willebrand factor+ cells. Six to eight fields per subject were analyzed. Statistical comparison among patient samples was done using the mean values for these measurements. **P < 0.01 by ANOVA.

Fig 5: Co-expression of CD40 and pro-inflammatory molecules in the retinas of patients with diabetic retinopathy. Posterior poles from patients with DR, PDR, and non-diabetic controls were incubated with anti-CD40 mAb with either anti-von Willebrand factor plus anti-ICAM-1 antibodies (A) or anti-CRALBP plus anti-CCL2 antibodies (B). Arrowheads show some of the areas where von Willebrand factor or CRALBP co-express with CD40 and ICAM-1 or CCL2. Original magnification times 400. Scale bar, 20 µm (A) or 50 µm (B).