Volume 38, Issue 5, Pages 1025-1037 (May 2013) Endothelial TNF Receptor 2 Induces IRF1 Transcription Factor-Dependent Interferon-β Autocrine Signaling to Promote Monocyte Recruitment  Deepak Venkatesh, Thomas Ernandez, Florencia Rosetti, Ibrahim Batal, Xavier Cullere, Francis W. Luscinskas, Yuzhi Zhang, George Stavrakis, Guillermo García-Cardeña, Bruce H. Horwitz, Tanya N. Mayadas  Immunity  Volume 38, Issue 5, Pages 1025-1037 (May 2013) DOI: 10.1016/j.immuni.2013.01.012 Copyright © 2013 Elsevier Inc. Terms and Conditions

Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 CXCR3 Chemokine Expression in Microvascular Endothelial Cells Is Dependent on Autocrine IFN-β Production (A) Left panels show flow cytometric analysis of nonpermeabilized WT, Tnfrsf1a−/−, and Tnfrsf1b−/− MHEC stained with TNFR2 antibody. Full histogram is control IgG staining. Dark grey line histogram is TNFR2 cell-surface expression. Right panels show immunoblot analysis of protein lysates of WT MHEC treated with LPS or forskolin for the indicated times in hours. (B–D) WT and Ifnar1−/− cells were treated for 4 hr with IFN-β, TNF, or PBS and subjected to quantitative real-time PCR (qPCR) for Mx1 (related to the IFN type I response) and CXCR3 chemokines (B), adhesion molecules (C), or neutrophil chemokines (D). *p < 0.05 between Ifnar1−/− cells and similarly treated WT cells. Ns, nonsignificant. (E) qPCR analysis for mRNA expression in WT MHEC treated with TNF for 4 hr and treated with either a blocking anti-IFN-β antibody or an isotype control IgG. *p < 0.05 between TNF with IgG control and TNF with IFN-β blocking antibody. For (B)–(E), n = 3 to 5 independent experiments. qPCR data are expressed as mean mRNA fold-change compared to untreated WT. Error bars indicate SEM. Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 TNF Induces IFN-β Production, STAT1 Phosphorylation, and Mononuclear Chemoattractants in Mouse Microvascular Cells that Depend on TNFR1 and TNFR2 WT, Tnfrsf1a−/−, and Tnfrsf1b−/− MHEC treated with PBS control (−) or TNF (+) for 4 hr were subjected to qPCR for mRNA expression of (A) IFN-β, (B) Mx1 and mononuclear chemokines, (C) adhesion molecules, and (D) neutrophil chemokines and Ccl2. n = 3 to 5 independent experiments. *p < 0.05 between WT and Tnfrsf1a−/− or Tnfrsf1b−/− MHEC after TNF treatment. Ns, non-significant. Data are expressed as mean mRNA fold-change. Error bars indicate SEM. (E) Immunoblot of phospho-STAT1 after TNF treatment of WT, Tnfrsf1a−/−, Tnfrsf1b−/−, and Ifnar1−/− cells for the indicated times in hours. Tubulin (tub) is the protein loading control. One of four representative experiments is shown. Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 TNF Induces IRF1 Mediated IFN-β Autocrine Signaling (A) WT MHEC were pretreated with cycloheximide (CHX) or an NF-κB inhibitor (Bay11-7082) followed by incubation with TNF (+) or PBS (−) for 4 hr and IFN-β mRNA levels were evaluated by qPCR. (B) qPCR analysis of IRF1, IRF3, IRF5, and IRF7 following a 4 hr TNF treatment in WT cells (left panel). qPCR data are normalized to GAPDH and expressed as 2−ΔCt to allow relative comparison between the different IRF isoforms. qPCR analysis of IRF1 and IRF5 mRNA expression in WT, Tnfrsf1a−/−, and Tnfrsf1b−/− cells (right panel). (C) Time course of IFN-β and IRF1 mRNA expression measured by qPCR in WT cells following TNF treatment. (D) Indicated MHEC were treated with TNF for the times shown and samples were subjected to immunoblot analysis for IRF1 (arrowhead). Actin is a protein loading control. One representative of three experiments is shown. (E) qPCR analysis of IFN-β and target gene expression 4 hr after TNF treatment of WT and Irf1−/− MHEC. (F) WT and Irf1−/− cells were treated with TNF for the indicated times in hours and STAT1 phosphorylation (arrowhead) was evaluated by immunoblot. One representative of three experiments is shown. n = 3 independent experiments for each graph shown except (B), which is n = 2. *p < 0.05 between WT and the indicated knockout MHEC after TNF treatment. qPCR data are expressed as mean mRNA fold-change except where otherwise specified. Error bars indicate SEM. Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 TNFR2 Overexpression in Human Umbilical Vein Endothelial Cells Induces IRF1, IFN-β, and CXCR3 Chemokines and Supports Monocyte Recruitment HUVEC were transduced with lentivirus expressing control GFP alone (GFP) or Flag-tagged TNFR2 (TNFR2). (A) Left panel shows nonpermeabilized cells that were stained with anti-human TNFR2 antibody and subjected to flow cytometric analysis. Grey is control isotype IgG and black and green histograms represent GFP and TNFR2 transduced HUVEC, respectively. Right panel shows TNFR2-Flag HUVEC that were permeabilized, stained with an anti-Flag antibody, and analyzed by confocal microscopy. White arrow indicates TNFR2-Flag staining at the cell surface. (B) Cells transduced with either GFP or TNFR-Flag were treated with PBS or TNF (+TNF) and subjected to flow cytometry analysis as in (A) by using anti-E-selectin, ICAM-1, or VCAM-1 antibodies. One representative of three is shown. (C–E) qPCR analysis of Cxcl9 and Cxcl10 mRNA (C), quantification of secreted chemokines by ELISA (D), and qPCR analysis of IFN-β, Mx1, IRF1, and IRF7 mRNA levels in HUVEC transduced with either GFP or TNFR2-Flag and incubated without (−) or with (+) TNF. ND, not detectable. n = 3 independent experiments. See also Figure S1. (F) Neutrophils or mononuclear cells (10%–13% were monocytes) were perfused across GFP- or TNFR2-transduced HUVEC. Cells that arrested and transmigrated were enumerated. Data is mean ± SD from three independent experiments. Left and middle panels show representative phase-contrast images. Mononuclear cells adhered (arrow) and transmigrated (arrowhead). Right panel shows immunofluorescence staining for cells positive for CD14+ (monocyte marker, arrowhead) interacting with the endothelium. qPCR data are expressed as mean mRNA fold-change. Error bars indicate SEM. *p < 0.05 compared to GFP (−) transduced samples. Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 TNFR2 Induced IFN-β Autocrine Signaling Is Required Selectively for Renal Monocyte Recruitment following Acute Systemic TNF Treatment Indicated mice were given an i.v. injection of TNF (+) or PBS (−) and kidneys were harvested 2 hr later. STAT1 phosphorylation (A) and IRF1 protein expression (B) were analyzed by immunoblot of total protein extracts from kidneys. The dashed lines indicate the removal of intervening lanes of the same gel. For (A) and (B), 1 of 3 representative experiments is shown. Bands optical density was quantified and the average fold induction ± SEM normalized to control WT PBS sample is given for each treatment. (C) Phospho-STAT1 immunohistochemistry staining of kidneys of indicated mice treated (+) or not with TNF. Phospho-STAT1-positive staining in glomerular endothelial cells is indicated (arrows) and semiquantitative scores of staining (means ± SEM) are given from three independent experiments. (D) Immunoblot analysis of Cxcl10 protein expression in kidney lysates. Quantification was performed as in (A) and (B). (E) In PBS (−) or TNF (+TNF) treated mice, renal macrophage, and neutrophil recruitment was assessed by immunohistochemistry. Representative pictures show interstitial and periglomerular macrophages (arrows) (upper panel) and glomerular neutrophils (arrows) (middle panel). Results were quantitated and expressed as mean percentage ± SEM normalized to WT + TNF (set at 100). n = 3–4 mice per group except for IRF−/− mice, which is n = 2. *p < 0.05 between WT treated with TNF (WT +) and indicated knockout mice. See also Table S1. (F) Flow cytometry analysis of membrane-bound TNF (mTNF) in human neutrophils stimulated with LPS for 30 min. Quiescent (−) or LPS-prestimulated (+) human neutrophils were incubated with MHEC isolated from indicated mice for 2 hr. Mx1 mRNA expression was then quantified by qPCR by using specific murine primers. Results are expressed as mean mRNA fold-change. Error bars indicate SEM. n = 3. Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 TNFR2 Induced IFN-β Autocrine Signaling Promotes Monocyte Recruitment following anti-GBM Nephritis (A and B) WT, Tnfrsf1a−/−, and Tnfrsf1b−/− mice reconstituted with hFcγR-Fcegr1−/− bone marrow were subjected to anti-GBM nephritis. (A) Proteinuria is graphed. Each data point represents one mouse, and the solid line is the median of the group. The dashed line indicates the mean of proteinuria in WT mice with Fcegr1−/− bone marrow at day 14. Indicated parameters were assessed following immunohistochemistry for neutrophils and macrophages, and Periodic Acid-Schiff (PAS) staining (fibrinoid necrosis) (n = 4–5 per time point). The number of neutrophils per 100 glomeruli or in 10 hpf (40×) of the interstitium was quantitated. F4/80-positive area in 5 hpf (20×) is given and at day 0 was 396 ± 150. Data are expressed as mean ± SEM; nd = not determined. *p < 0.05 compared to WT. (B) Representative pictures of F4/80 stained macrophages and Periodic Acid-Schiff (PAS) stained kidney sections of the indicated animals at day 14 (original magnification ×20). Arrow, macrophage influx; black arrow, fibrinoid necrosis; blue arrow, crescents. See also Figure S2. (C) Confocal microscopic analysis of normal kidney (top panels) and a renal specimen from a patient with anti-GBM glomerulonephritis (bottom panels). Tissue was stained with antibody to TNFR (red, left) and an endothelial marker CD31 (green, middle). The merged image (right) shows CD31 and TNFR2 colocalization (arrow) along the peripheral capillary walls. Original magnification was ×60. Immunity 2013 38, 1025-1037DOI: (10.1016/j.immuni.2013.01.012) Copyright © 2013 Elsevier Inc. Terms and Conditions