Interleukin receptor activates a MYD88-ARNO-ARF6 cascade to disrupt vascular stability

Abstract

The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1β (IL-1β), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1β on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1β signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.

Figure 1. Immediate effects of IL-1β are NF-κB independent and ARF6 dependent

Monolayers of HMVEC-D stimulated with IL-1β assayed for: (a) permeability to horse-radish peroxidase (HRP) over time; (b) permeability to HRP following two hours treatment with SC514; (c) immunofluorescent localization of VE-cadherin; (d) permeability to HRP after thirty minutes treatment with Actinomycin-D, or Cycloheximide. (e) IL-1β-stimulated HMVEC-D lysates precipitated with GST-GGA3 and immunoblotted for ARF6. HMVEC-D monolayers infected with Ad-GFP or Ad-ARF6-Q67L: (f) permeability; (g) VE-cadherin (red) localization, ARF6 expression (green). Membrane fractions from (h) adenoviral-vector infected or (i) ARF6 siRNA-treated HMVEC-D immunoblotted for VE-cadherin. ARF6 siRNA-treated HMVEC-D stimulated with IL-1β: (j) VE-cadherin (arrows=disrupted VE-cadherin cell-surface localization) (k) permeability. N≥3; error bars=SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Inhibition of ARF-GAPs and ARF-GEFs affect ARF6 activation and VE-cadherin localization

HMVEC-D treated with QS11: (a) whole-cell lysates precipitated with GST-GGA3 and immunoblotted for ARF6; b) membrane fractions immunoblotted for VE-cadherin. (c) immunofluorescence for VE-cadherin (red); (d) permeability to HRP. HMVEC-D treated with IL-1β and SecinH3: (e) immunoblotting for ARF6; (f) Immunofluorescence for VE-cadherin (red); (g) permeability to HRP. HMVEC-D treated with anti-ARNO siRNA and stimulated with IL-1β: (h) permeability to HRP; (i) GST-GGA3 precipitation and ARF6 immunoblotting; (j) immunofluorescence of VE-cadherin (red) (Arrow=less cell-surface VE-cadherin. N≥3; error bars=SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. The immediate IL-1β-induced permeability pathway diverges at MYD88

(a) IRAK1 siRNA treated HMVEC-D, stimulated with IL-1β, subjected to GTP-ARF6 pull down and immunoblotted for ARF6. MYD88 siRNA-treated HMVEC-D stimulated with IL-1β: (b) permeability; (c) ARF6 activation. (d) NF-κB p65 immunofluorescence of HMVEC-D stimulated with IL-1β and SecinH3. (Arrow=nuclear localization). (e) Cell lysates from Ad ARNO-Myc infected HMVEC-D immunoprecipitated with anti-Myc antibodies and immunoblotted with anti-MYD88 antibodies. (f) Lysates from HMVEC-D immunoprecipitated with anti-MYD88 antibodies and immunoblotted with anti-ARNO antibodies. N≥3, error bars=SEM, * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. Inhibition of ARF-GEFs decreases CIA-induced vascular permeability and arthritis

(a) Arthritis-induced vascular permeability in the joint measured by Evans Blue leak seven days after treatment initiation in the presence of SecinH3 or Enbrel. N=14 per group. (b) Chronological arthritic assessment. N=10/group. (c) H&E staining of sections through joints; Bn=bone, HC=hyaline cartilage, JS=joint space, Pn=inflammed pannus, *cartilage & bone loss, arrows=eroded cartilage. (d) Summary of histological changes of inflammation, pannus, cartilage, and bone damage of indicated treatment. Control, N=5; Enbrel and SecinH3, N=10; error bars=SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs disease group.