Cytokine Disruption Of Barrier Function Is Independent Of NF-κB

Chronic inflammation causes tissue destruction through perpetual dysregulated cytokinerelease, inflammatory cell recruitment, and vascular permeability. Cytokines such as IL-1βenact inflammatory cell recruitment through a well-defined MyD88-mediated,NF-κB-dependent pathway. How inflammatory cytokines produce a profound disruption of thevascular barrier and induce vascular permeability, and whether this pathway is NF-κBdependent, remains an important and open question.1. IL-1β-induced permeability is NF-κB independent.NF-κB activation was blocked by treatment with the IKK inhibitor, SC514. Although SC-514prevented IL-1β-induced nuclear localization of NF-κB, it was unable to rescue IL-1β-inducedpermeability. Inhibition of NF-κB activation using SC-514failed to rescue IL-1β-induceddisruption of VE-cadherin cell surface localization, a key molecular event in maintaining astabilized endothelial barrier. Small molecule inhibitors of ERK1/2, p38, and JNK were unableto rescue IL-1β-induced vascular permeability and disruption of VE-cadherin cell surfacelocalization.2. IL-1β-induced activation of MyD88-ARNO-Arf6-VE-cadherin leads to the disruptionof endothelia barrier function.Inhibition of NF-κB failed to block IL-1β-induced vascular permeability. Then which doespassway mediate it? We hypothesized that IL-1β might utilize Arf6, a known regulator ofadherens junction cell surface localization to disrupts VE-cadherin cell surface localization.Indeed, IL-1β induced rapid and sustained activation of Arf6. Using adenoviralover-expression of a constitutively active version of Arf6(Arf6-Q67L) or Arf6siRNAknockdown, we found that activation of Arf6decreases VE-cadherin cell surface localizationand inhibition of Arf6enhances VE-cadherin cell surface localization. Collectively, these datalink Arf6as a critical mediator of VE-cadherin cell surface localization and IL-1β-inducedvascular permeability.Arf6activation is inhibited by GTPase-activating proteins (GAPs) and enhanced by guaninenucleotide exchange factors (GEFs). QS11, a small molecule inhibitor of Arf-GAPs；ARNOsiRNA knockdown and SecinH3, a small molecule inhibitor of cytohesins were administratedin HMVEC-D. Inhibition of Arf-GAPs decreases VE-cadherin cell surface localization, andincreases vascular permeability. Inhibition of Arf-GEFs inhibites IL-1β-induced disruption ofVE-cadherin cell contacts as well as endothelial permeability. ARNO is necessary forIL-1β-induced activation of Arf6and subsequent induction of vascular permeability. We hypothesized that IL-1β-induced and ARNO-Arf6signaling passways converge at certainnodal points. MyD88siRNA inhibited IL-1β-induced Arf6-GTP, however IRAK siRNA failedto inhibit IL-1β-induced Arf6-GTP. These data demonstrated that the ARNO/Arf6signalingaxis is dependent on MyD88.An interaction between MyD88and ARNO was detected by co-immunoprecipitation in bothover-expression and endogenous settings. We show that ARNO binds directly to MyD88, andthus propose MyD88-ARNO-Arf6as a proximal IL-1β signaling pathway distinct from thatmediated by NF-κB.3. Inhibition of Arf-GEFs decreases CIA-induced vascular permeability and arthritis,MyD88-ARNO-Arf6as a valid target for rheumatoid arthritis.A dysregulated cytokine response causing excessive inflammation and tissue damage isclassically seen in rheumatoid arthritis. The vascular response to cytokines is implicated inarthritic pathology. A standard animal model of arthritis is collagen-induced arthritis (CIA).Targeting the IL-1β-MyD88-ARNO-Arf6pathway using SecinH3inhibited CIA-inducedvascular permeability in the joint as measured by Evans Blue leak. When SecinH3wasadministered after the onset of arthritis, a significant inhibition of arthritis was observed asassessed by scoring of inflammation, pannus development, cartilage damage, and bonedamage.