Abstract

Inflammatory bowel disease (IBD) is a chronic and relapsing disorder for many people associated with poor health. Although there are some clinical drugs for IBD treatment, the development of effective therapeutics on IBD patients has always been necessary. Here, we show that externalized phosphatidylserine (PS) is observed on the surface of colonic capillaries. Annexin A5 (ANXA5) with high affinity for PS has a good targeting to the colon and effectively alleviates experimental colitis. In contrast, ANXA5 mutant (A5m) lacking the PS-binding ability, has no accumulation in the colon and no therapeutic effects on colitis. Mechanistic investigations indicate that ANXA5 reduces the inflammatory cell infiltration by inhibiting endothelial cell activation dependent on PS-binding ability. With the increasing of PS exposure on activated HUVECs (human umbilical vein endothelial cells), ANXA5 binding induces the internalization of TLR4 via PS-dependent endocytosis. We provide new insights on the molecular mechanism of ANXA5 for its anti-inflammatory effect. Our data suggest that PS-externalization is a potential target of ANXA5 aiming at targeted drug delivery (TDD) for IBD treatment.

Highlights

  • Inflammatory bowel diseases (IBDs) are chronic relapsing disorders of the gastrointestinal tract, characterized pathologically by intestinal inflammation and epithelial injury, known as ulcerative colitis (UC), Crohn’s disease (CD), and indeterminate colitis (IC)[1]

  • PS-externalization is the most prominent characteristic of apoptotic cells, which is a well-explored phenomenon to image cell death for activated HUVECs treated with Annexin A5 (ANXA5)

  • The interaction of blood leukocytes with endothelial cells is initially induced by adhesion molecules Vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1)

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Summary

RESULTS

Exposure of phosphatidylserine on the capillaries of colonic of MECA-32 showed a network of interconnected vessels in the mucosa colonic section. Compared with A5m group, ANXA5 treatment significantly reduced the production of inflammatory cytokines, such as TNF-α, IL-6, and IL-18 (Fig. 4f) Together, these results indicated that the PS-binding ability of ANXA5 was critical for the anti-inflammatory effect in TNBS-induced colitis. FACS analysis further showed that the infiltration of EGFP+CD11b+ cells was reduced by ANXA5 treatment (vehicle group, 36.07 ± 2.313%; ANXA5 group, 18.73 ± 1.729 %, P < 0.05) (Fig. 5d) These results demonstrate that the anti-inflammatory effect of ANXA5 is due to its inhibition on inflammatory cell infiltration in colitis. When endogenous TLR4 knockdown by TLR4 siRNA (Supplementary Fig. S6), ANXA5 treatment showed no effect on NF-κB activation in HUVECs (Fig. 7i) These results suggest that the anti-inflammatory mechanism of ANXA5 is to downregulate surface TLR4 via the PSdependent endocytosis

DISCUSSION
Findings
MATERIALS AND METHODS
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