NF-κB inhibitor targeted to activated endothelium demonstrates a critical role of endothelial NF-κB in immune-mediated diseases

Abstract

Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inflammatory genes crucially involved in the pathogenesis of inflammatory diseases. NF-κB governs the expression of adhesion molecules that play a pivotal role in leukocyte-endothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a "sneaking ligand construct" (SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i) an E-selectin targeting domain, (ii) a Pseudomonas exotoxin A translocation domain, and (iii) a NF-κB Essential Modifier-binding effector domain interfering with NF-κB activation. The E-selectin-specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inflammatory cells. Furthermore, SLC1 treatment significantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the pathogenesis of arthritis and can be selectively inhibited in a cell type- and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specific manner and enables selective targeting of distinct cell populations to improve effectiveness and risk-benefit ratios of therapeutic interventions.

Keywords: autoimmune disorders; cell targeting; inhibit inflammation; intracellular signaling; mouse models.

Fig. 1.

Schematic representation of functional and nonfunctional SLCs. The multimodular synthetic gene was ligated into the pAK400 plasmid using SfiI restriction sites. EBL, three repeats of E-selectin–specific peptide (DITWDQLWDLMK) connected with a S₄G linker. MutEBL, WKLDTLDMIQD (22). ETA II, translocation domain of Pseudomonas exotoxin A domain II (23). NBP, NEMO-binding peptide encompassing amino acids 644–756 from IKK2 (24). Amino acids that were identified for NEMO interaction were mutated: MutNBP1 FTALDASALQTE. MutNBP2 (scrambled peptide): DLAWQTFLTES.

Fig. 2.

Internalized SLC1 selectively inhibits NF-κB activation in vitro. (A) Immunofluorescence microscopy depicts CHO_E cells stained with SLC1-FP635 at 37 °C. Plasma membrane was stained with AlexaFluor488-labeled ConA. (B) CHO_E cells were stained with SLC1-FP635 at 4 °C. Experiments were performed at least five times. (C) NF-κB–dependent luciferase expression is reduced in SLC1-pretreated CHO_E cells in a dose-dependent manner. Error bars represent the ±SD obtained from five independent experiments. Each experiment was done in triplicate. (D) Transcriptional NF-κB activity is not reduced by SLC1 in CHO_wt cells. (E) NF-κB EMSA in SLC1-pretreated and IL-1β–stimulated CHO_E cells demonstrates inhibition of NF-κB nuclear DNA-binding activity. Representative independent experiments are shown. Experiments were performed five times. Densitometric analysis shows inhibition of NF-κB DNA-binding activity by ∼35% upon SLC1 treatment. Densitometric measurements were performed from five separate experiments. P values were calculated by one-way ANOVA followed by Bonferroni’s multiple-comparison post test: ****P < 0.0001; ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 3.

SLC1 reduces leukocyte–endothelium interactions in vitro and in vivo and prevents vascular leakage. (A) Granulocyte adhesion was reduced in SLC1-treated and cytokine-stimulated mouse endothelial cells (mlEND). Experiments were performed five times in triplicate. (B) In vitro transmigration of leukocytes through cytokine-activated mlENDs is inhibited by SLC1 application as well as by Antp-NBP. Experiments were performed five times in triplicate. (C) SLC1 application inhibited neutrophil migration in vivo in murine peritonitis. PBS, n = 9; SLC1, n = 10; MutNBP2, n = 5; MutEBL, n = 5. (D) Extravasation of Evans blue is prevented in cytokine-induced skin vessels in SLC1-treated mice. PBS, n = 8; SLC1, n = 10; MutNBP2, n = 5; MutEBL, n = 6. P values were calculated by one-way ANOVA followed by Bonferroni’s multiple-comparison posttest: ****P < 0.0001; ***P < 0.001, **P < 0.01, *P < 0.05. Error bars represent the ±SD.

Fig. 4.

SLC1 binding to the vascular endothelium in vivo is dependent on its cytokine-activation status: inhibition of NF-κB activation. (A) Visualization of vessels in a nonactivated state (Top) exhibits the GFP-background fluorescence but no staining by the systemically administered red-fluorescent SLC1-FP635. Upon cytokine activation, the red fluorescent SLC1-FP635 accumulates in the vessel wall, leading to an intense orange staining (arrow, Middle) that is not detectable when the deletion mutant DelEBL-FP635 is administered (Bottom). Experiments were performed three times. (B) Representative images of whole-mount immunofluorescence skin sections stained for activated NF-κB p65 and VCAM-1. Experiments were performed three times.

Fig. 5.

Clinical and histological manifestations are ameliorated in the K/BxN serum transfer model after SLC1 treatment. (A) Mice were three times treated with 100 µg SLCs or 200 µg Antp-NBP as indicated by arrows. PBS, n = 19; SLC1, n = 13; MutNBP2, n = 9; DelEBL, n = 5; Antp-NBP, n = 5. (B) Treatments with the indicated SLCs or Antp-NBP were continued on days 2, 4, and 5. SLC1, n = 10; MutNBP2, n = 10; DelEBL, n = 10; Antp-NBP, n = 10. (C) On day 8 diameters of paws (paw and ankle) were measured. (D) Representative microphotographs of H&E-stained sections at day 8 after K/BxN serum transfer. (E) Histological analyses of synovitis, cartilage, and bone erosions. P values were calculated by one-way ANOVA followed by Bonferroni’s multiple-comparison post test: ****P < 0.0001; ***P < 0.001, **P < 0.01, *P < 0.05.