Fig 2: Dodecameric TNF muteins exert elevated TNF/TNFR2 cluster formation (A) BV-2 cells were incubated with the oligomerized TNF muteins (0.1, 0.3, 1.0 nM) for 15 minutes. Then cells were fixed and localization of TNFR2 (red) and oligomerized TNF muteins (green) was visualized by immunofluorescence microscopy. DAPI was used to counterstain cell nuclei. Fluorescence was analyzed using a Zeiss Axio Observer Spinning Disc microscope. Representative pictures for stimulation with 1 nM of the oligomerized TNF muteins are shown. Scale bar: 10 µm. (B) Quantification of TNF/TNFR2 clustering (n = 3 ± SEM).

Fig 3: Dodecameric TNF muteins are still selective for TNFR2 (A) HeLa cells were stimulated with oligomerized TNF muteins for 24 hours. Soluble recombinant human TNF (sTNF) was used as a positive control for activation of TNFR1. Then supernatant was collected and analyzed for presence of IL-6 by ELISA (n = 3 ± SEM). (B) L929 cells were incubated with oligomerized TNF muteins for 24 hours. Soluble recombinant mouse TNF (sTNF) was used as a positive control for activation of TNFR1. Cell viability was measured using crystal violet staining (n = 3 ± SEM).

Fig 4: Spatiotemporal expression of TNF receptor 1 and 2 after SCI. (a,b) TNFR1 protein levels in the acute (a) and delayed (b) phases after SCI. Significant indications represent comparisons to sham mice in the acute phase (a, Interaction: F5,48 = 4.52, p = 0.002; Time: F5,48 = 45.74, p < 0.0001; SCI: F1,48 = 2.19, p = 0.15) and delayed phase (b, Interaction: F4,40 = 49.51, p < 0.0001; Time: F4,40 = 52.17, p < 0.0001; SCI: F1,40 = 406.5, p < 0.0001) after SCI. (c–g) Immunofluorescent double labelling for TNFR1 (red) and neuronal MAP2 (green) within the lesion site (c), peri-lesion area (d,f,g), as well as distant from the lesion site (e) at 21 (c–e) and 28 (f,g) days after SCI. (h) Immunofluorescent double labelling for TNFR1+ (red) and CD68+ (green) cells 21 days after SCI. (i,j) TNFR2 protein levels in the acute phase (i, Interaction: F5,47 = 0.42, p = 0.83; Time: F5,47 = 3.89, p < 0.005; SCI: F1,47 = 1.75, p = 0.19) and delayed phase (j, Interaction: F4,40 = 25.04, p < 0.0001; Time: F4,40 = 36.07, p < 0.0001; SCI: F1,40 = 2702.8, p < 0.0001) after SCI. (k–n) Immunofluorescent double labelling for TNFR2 (red: k,l and green: (m,n) and GFAP+ astrocytes (green: (k,l) and red: (m,n)) and CD11b+ microglia/macrophages (green: (l)) at 21 (k) and 28 (l–n) days after SCI. (o–q) Immunofluorescent double labeling of TNFR2+ cells (arrows in (o)) and CD68+ microglia/macrophages (arrow heads in (o)). (p) Only a few CD68+ cells co-expressed TNFR2. (q) represents a high magnification image of the area squared in (p), demonstrating TNFR2 + CD68+ cells located in the peri-lesion areas 21 days after SCI (arrow heads in (q)). DAPI was used as a nuclear marker. Scale bars: (k,m) = 100 µm, (c–f,h,n–p) = 40 µm, and (g,l,q) = 20 µm. Results are expressed as mean ± SEM, n = 5/group, **** p < 0.0001. GFAP, glial fibrillary acidic protein; MAP2, microtubule associated protein 2; TNFR, tumor necrosis factor receptor.

Fig 5: Cellular and temporal expression of Tnf mRNA in the thoracic spinal cord after SCI. (a–i) In situ hybridization was used to investigate the distribution of Tnf mRNA+ cells the first 24 h after SCI. Tnf mRNA expression was undetectable in naïve mice (a). Tnf mRNA+ cells in the white matter of the posterior funiculi (arrows in b) and in neuronal-like cells in the dorsal horn (arrowhead in c), at 1 h after SCI. Tnf mRNA+ cells in the white matter of the posterior funiculi (d) and in the grey matter of the ventral horn (e). At 3 h, most cells displayed macrophage- or glial-like morphology (arrows in f). By 6 h (g), 12 h (h), and 24 h (i), Tnf mRNA+ cells were mainly located in white matter areas of the damaged spinal cord (arrows). (j) Parallel spinal cord sections that were in situ hybridized for glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA showed a largely neuronal signal and confirmed the overall suitability of the tissue for in situ hybridization. (k,l) Parallel sections hybridized with buffer alone (k) or pretreated with RNAse A before the in situ hybridization (l) were devoid of signal. (m–o) RT-qPCR analysis of Tnf mRNA (m), Tnfrsf1a mRNA (n), and Tnfrsf1b mRNA (o) levels in naïve mice and in mice allowed 1, 3, 6, and 12 h and 1, 3, 7, 14, and 28-days survival after SCI. Tnf mRNA levels were significantly increased at 1, 3, and 6 h and 7 days after SCI, compared to naïve mice (Time: p < 0.0001, F9,39 = 58.14) (m). Tnfrsf1a mRNA levels significantly increased from 6 h to 28 days after SCI, compared to naïve mice (Time: p < 0.0001, F9,39 = 20.01) (n). Tnfrsf1b mRNA levels significantly increased at 3 h after SCI, compared to naïve mice (Time: p = 0.009, F9,39 = 2.965). Results are expressed as mean ± SEM, n = 5/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bars: (a–c, f–i, k,l) = 40 µm, (d,e) = 100 µm, and (j) = 200 µm.