Journal: The Journal of Experimental Medicine

Article Title: HLA-B*35-Px–mediated acceleration of HIV-1 infection by increased inhibitory immunoregulatory impulses

doi: 10.1084/jem.20091386

Figure Lengend Snippet: Preferential recognition of HIV-1 CTL epitope/HLA-B*3503 (Px) complexes by the inhibitory myelomonocytic receptor ILT4. (A and B) Binding of HLA-B*3503 (Px) and -B*3501 (PY) tetramers refolded with two different CTL epitopes to PBMCs, as determined by flow cytometry. ILT4-specific antibodies were able to fully abrogate tetramer binding to dendritic cells. (A) Histograms for one representative example are shown. (B) Cumulative flow cytometry data from HIV-1–infected patients ( n = 8) (two-tailed paired Student's t test). (C) SPR sensograms reflecting the control surface-subtracted interactions between recombinant ILT4 and immobilized HLA-B*3503 or -B*3501 tetramers refolded with the indicated epitope. ILT4 was injected in five serial twofold dilutions from a starting concentration of 1 µM. The respective apparent steady-state equilibrium constant ( K D ) is indicated. One representative experiment out of three is shown.

Article Snippet: Serial twofold dilutions of a recombinant protein dimer containing two extracellular domains of human ILT4 fused to the Fc part of human IgG (R&D Systems) were injected over the sensor chip.

Techniques: Binding Assay, Flow Cytometry, Infection, Two Tailed Test, Control, Recombinant, Injection, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: Clinical characteristics of patient samples analyzed by IL-11Rα ELISA

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: IL-11Rα is significantly increased in human vitreous samples. ( A ) Experimental setup: Following vitrectomy, undiluted vitreous and plasma samples were centrifuged and the supernatants were frozen until ELISA was performed. ( B ) Interleaved box and whiskers visualizing the protein levels of IL-11 ( N =15) and IL-11Rα ( N =16) in vitreous and corresponding plasma samples in control (macular pucker) and PDR (proliferative diabetic retinopathy) group, as measured by ELISA. The whiskers represent the minimum and maximum values while the line in between represents the median value. Statistical testing: Mann Whitney test. * p <0.001

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: In the presence of VEGF, IL-11 cis-signaling has anti-angiogenic effects on vascular endothelial cells while trans-signaling enhances their pro-angiogenic and pro-migratory potential associated with an enhanced respiratory rate. ( A ) Spheroid sprouting assay with HUVECs exposed to endothelial basal medium (EBM = negative control), VEGF (positive control), IL-11 + VEGF or IL-11 + sIL-11Rα+ VEGF for 17 h. N = 3 independent experiments with 15-22 spheroids per group and experiment. Statistical test: Kruskal-Wallis Test adjusted for multiple testing, * p <0.01. Relative sprouting length (RSL), scale bar 20 µM. ( B ) Spheroid sprouting assay with HRMVECs exposed to VEGF (positive control), IL-11 + VEGF or IL-11 + sIL-11Rα + VEGF for 17 h. N = 2 independent experiments with 10-22 spheroids per group and experiment. Statistical test: Kruskal-Wallis Test adjusted for multiple testing, * p <0.01. Relative sprouting length (RSL), scale bar 20 µM. ( C ) Scratch wound assay using HUVECs: Migratory effect of IL-11 + sIL-11Rα + VEGF and IL-11 + VEGF on HUVECs over 24 h. N = 3 independent experiments each including 6-8 technical replicates. Relative Wound Density (RWD). Exemplary analysis of the conditions 11 h after stimulation, statistical test: Kruskal-Wallis Test adjusted for multiple testing, * p <0.01. Scale bar 200 µM. ( D ) Representative graph of the oxygen consumption rate (OCR) of HUVECs after pretreatment with above mentioned cytokines for 15 h. N = 3 with each 4-8 technical replicates. Statistical test: Kruskal-Wallis Test adjusted for multiple testing, * p <0.05. ( E ) Representative graph of the normalized extracellular acidification rate (ECAR) of HUVECs after pretreatment with above mentioned cytokines for 15 h. N = 3 with each 4-8 technical replicates. Statistical test: Friedman Test adjusted for multiple testing, * p <0.05

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: Negative Control, Positive Control, Scratch Wound Assay Assay

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: IL-11 trans-signaling activates intracellular signaling pathways beyond STAT3. ( A ) Scatter plots with bar visualizing the JAK activation in HUVECs after stimulation with VEGF, IL-11+VEGF, sIL-11Rα+VEGF or IL-11+sIL-11Rα+VEGF for 15 min. Representative images of N = 3 independent experiments. ( B ) Scatter plots with bar visualizing activated signaling pathways in HUVECs after stimulation with VEGF, IL-11+VEGF, sIL-11Rα+VEGF or IL-11+sIL-11Rα+VEGF for 15 min. Representative images of N = 4 independent experiments. ( C ) Graphical summary of the activated pathways by IL-11 or IL-11+sIL-11Rα and their angiogenic effects in HUVECs. ( D ) Western blot of activated signaling pathways in HRMVECs after stimulation with VEGF, IL-11+VEGF, sIL-11Rα+VEGF or IL-11+sIL-11Rα+VEGF for 15 min. N = 1 experiment

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: Activation Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: Transcriptional analysis of IL-11 + VEGF and IL-11 + sIL-11Rα + VEGF. ( A ) Experimental setup for RNA Seq analysis from 3D cell culture setting. N = 3 independent experiments using HUVECs with 14-22 spheroids per group and experiment. ( B ) PCA analysis of all samples. ( C ) Total number of up- and downregulated DEGs compared to VEGF (control). ( D ) Scatter blot visualizing all genes with at least one count when comparing IL-11 + sIL-11Rα + VEGF against IL-11 + VEGF. Differentially expressed genes (DEGs) were defined by padj <0.05 and abs(log2 foldchange) >1. The five most expressed up- and downregulated DEGs are labeled. All following analyzes were conducted by comparing IL-11 + sIL-11Rα + VEGF against IL-11 + VEGF. ( E ) The five most significantly enriched and depleted biological processes according to Gene Ontology (GO) terms. Positive normalized enrichment score (NES) refers to enrichment in the IL-11 + sIL-11Rα + VEGF treatment and negative NES to depletion and consequently enrichment in IL-11+VEGF. ( F ) Gene Set Enrichment Analysis (GSEA) for the GO term “angiogenesis” (GO:0048514). ( G ) Heatmap illustrating the 20 leading edge genes of the GSEA from F

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: RNA Sequencing Assay, Cell Culture, Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: Angiogenic effects and altered signaling patterns of IL-11 cis- and trans-signaling following STAT3 knockdown. ( A ) Spheroid sprouting assay using HUVECs: Cells transfected with control siRNA or STAT3 siRNA were treated under following conditions: EBM (negative control), IL-11, VEGF (positive control), IL-11 + VEGF. RSL = Relative Sprouting Length. N = 4 independent experiments each consisting of 12-23 spheroids per group and experiment, statistical testing: Kruskal-Wallis Test adjusted for multiple testing, * p <0.05. ( B ) Spheroid sprouting assay using HUVECs: Cells transfected with control siRNA or STAT3 siRNA were treated under following conditions: EBM (negative control), IL-11 + sIL-11Rα, VEGF (positive control), IL-11 + sIL-11Rα + VEGF. RSL = Relative Sprouting Length. N = 4 independent experiments each consisting of 9-22 spheroids per group and experiment, statistical testing: Kruskal-Wallis Test adjusted for multiple testing, * p <0.01. ( C ) Semi-quantitative Western blot analysis of signaling molecules in HUVECs after STAT3 knockdown and treatment under the conditions mentioned in ( A ) for 15 min. Interleaved box and whiskers: the whiskers represent the minimum and maximum values while the line in between represents the median value. N = 3 independent experiments. ( D ) Semi-quantitative Western blot analysis of signaling molecules in HUVECs after STAT3 knockdown and treatment under the conditions mentioned in ( B ) for 15 min. Interleaved box and whiskers: the whiskers represent the minimum and maximum values while the line in between represents the median value. N = 3 independent experiments

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: Knockdown, Transfection, Control, Negative Control, Positive Control, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: Characterization of angiogenic effects and compensatory changes of signaling pathways after STAT1 knockdown. ( A ) Spheroid sprouting assay using HUVECs: Cells transfected with control siRNA or STAT1 siRNA were treated under following conditions: EBM (negative control), IL-11, VEGF (positive control), IL-11+VEGF. RSL = Relative Sprouting Length. N = 4 independent experiments each consisting of 14-22 spheroids per group and experiment, statistical testing: Kruskal-Wallis Test adjusted for multiple testing, * p <0.05. Scale bar 20 µM. ( B ) Spheroid sprouting assay using HUVECs: Cells transfected with control siRNA or STAT1 siRNA were treated under following conditions: EBM (negative control), IL-11+sIL-11Rα, VEGF (positive control), IL-11+sIL-11Rα+VEGF. RSL = Relative Sprouting Length. N = 3 independent experiments each consisting of 11-20 spheroids per group and experiment, statistical testing: Kruskal-Wallis Test adjusted for multiple testing, * p <0.01. Scale bar 20 µM. ( C ) Semi-quantitative Western blot analysis of signaling molecules in HUVECs after STAT1 knockdown and treatment under the conditions mentioned in ( A ) for 15 min. Interleaved box and whiskers: the whiskers represent the minimum and maximum values while the line in between represents the median value. N = 3 independent experiments. ( D ) Semi-quantitative Western blot analysis of signaling molecules in HUVECs after STAT1 knockdown and treatment under the conditions mentioned in ( B ) for 15 min. Interleaved box and whiskers: the whiskers represent the minimum and maximum values while the line in between represents the median value. N = 3 independent experiments

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: Knockdown, Transfection, Control, Negative Control, Positive Control, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: IL-11 and IL-11 + sIL-11Rα decrease the retinal neovascularization in vivo. ( A ) Representative images of retinal flatmounts with quantification of NV and VO in OIR P17 mice after intravitreal IL-11 or PBS control injection. N = 12 mice per condition from three independent OIR experiments, statistical testing: Mann–Whitney Test, * p <0.01. ( B ) Representative images of retinal flatmounts with quantification of NV and VO in OIR P17 mice following intravitreal IL-11+sIL-11Rα or PBS control injection. N = 16 mice per condition from three independent OIR experiments, statistical testing: Mann–Whitney Test. ( C ) Western blot of whole retina lysates 12 h after intravitreal injection of IL-11 or PBS control. N = 4 biological replicates representing four mice. ( D ) Western blot of whole retina lysates 12 h after intravitreal injection of IL-11+sIL-11Rα or PBS control. N = 3 biological replicates representing three mice

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: In Vivo, Control, Injection, MANN-WHITNEY, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: IL-11 and IL-11 + sIL-11Rα activate Müller cells in vivo. ( A ) Representative images of retinal cryosections of C57BL/6J mice 12 h after injection with IL-11, IL-11+sIL-11Rα or PBS control at OIR P12. Scale bar: 25 μm. ( B ) Representative higher magnification images of the ganglion cell layer, inner plexiform layer and inner nuclear layer after injection with IL-11 or IL-11+sIL-11Rα for better visualization of pSTAT3 Tyr705 and Isolectin co-staining. Scale bar: 25 μm. ( C ) Representative images retinal cryosections and of higher magnification images of C57BL/6J mice for GFAP 12 h after injection with IL-11, IL-11+sIL-11Rα or PBS control at OIR P12. Scale bar: 25 μm. ( D ) Representative images of retinal cryosections of ALDH1L1-GFP + transgenic mice 12 h after injection with IL-11, IL-11+sIL-11Rα or PBS control at OIR P12. GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. Scale bar: 25 μm

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: In Vivo, Injection, Control, Staining, Transgenic Assay

Journal: Journal of Neuroinflammation

Article Title: Characterization of the angiomodulatory effects of Interleukin 11 cis- and trans-signaling in the retina

doi: 10.1186/s12974-024-03223-3

Figure Lengend Snippet: In Müller cells, IL-11 cis- and trans-signaling activate similar intracellular signaling pathways in vitro. ( A ) Western blot analysis for activated intracellular signaling molecules in Müller cells stimulated with VEGF, mIL-11+VEGF, sIL-11Rα+VEGF or mIL-11+sIL-11Rα+VEGF for 15 min. N = 4 independent experiments. ( B ) Graphical summary of the activated pathways by IL-11 and IL-11+sIL-11Rα in Müller cells. ( C ) Immunocytochemistry of primary Müller cells expressing the Müller glia-specific markers glutamine synthetase, K ir 4.1 and nestin. N = 3 independent experiments, scale bar: 50 μm. ( D ) Immunocytochemistry of primary Müller cells after stimulation with control medium (Diff), mIL-11, sIL-11Rα or mIL-11+sIL-11Rα for 15 min. N =3 independent experiments, scale bar: 50 μm

Article Snippet: Murine Müller cells were stimulated with murine IL-11 (mIL-11, 100 ng/mL, #220 − 11, Peprotech), soluble mIL-11Rα (600 ng/mL, #7405-MR-050, R&D Systems) or mIL-11 + sIL-11Rα.

Techniques: In Vitro, Western Blot, Immunocytochemistry, Expressing, Control