Longistatin in tick saliva blocks advanced glycation end-product receptor activation
Anisuzzaman, … , Kozo Fujisaki, Naotoshi Tsuji
Anisuzzaman, … , Kozo Fujisaki, Naotoshi Tsuji
Published October 1, 2014; First published September 2, 2014
Citation Information: J Clin Invest. 2014;124(10):4429-4444. https://doi.org/10.1172/JCI74917.
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Categories: Research Article Infectious disease

Longistatin in tick saliva blocks advanced glycation end-product receptor activation

Abstract

Ticks are notorious hematophagous ectoparasites and vectors of many deadly pathogens. As an effective vector, ticks must break the strong barrier provided by the skin of their host during feeding, and their saliva contains a complex mixture of bioactive molecules that paralyze host defenses. The receptor for advanced glycation end products (RAGE) mediates immune cell activation at inflammatory sites and is constitutively and highly expressed in skin. Here, we demonstrate that longistatin secreted with saliva of the tick Haemaphysalis longicornis binds RAGE and modulates the host immune response. Similar to other RAGE ligands, longistatin specifically bound the RAGE V domain, and stimulated cultured HUVECs adhered to a longistatin-coated surface; this binding was dramatically inhibited by soluble RAGE or RAGE siRNA. Treatment of HUVECs with longistatin prior to stimulation substantially attenuated cellular oxidative stress and prevented NF-κB translocation, thereby reducing adhesion molecule and cytokine production. Recombinant longistatin inhibited RAGE-mediated migration of mouse peritoneal resident cells (mPRCs) and ameliorated inflammation in mouse footpad edema and pneumonia models. Importantly, tick bite upregulated RAGE ligands in skin, and endogenous longistatin attenuated RAGE-mediated inflammation during tick feeding. Our results suggest that longistatin is a RAGE antagonist that suppresses tick bite–associated inflammation, allowing successful blood-meal acquisition from hosts.

Authors

Anisuzzaman, Takeshi Hatta, Takeharu Miyoshi, Makoto Matsubayashi, M. Khyrul Islam, M. Abdul Alim, M. Abu Anas, M. Mehedi Hasan, Yasunobu Matsumoto, Yasuhiko Yamamoto, Hiroshi Yamamoto, Kozo Fujisaki, Naotoshi Tsuji

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Figure 2

Longistatin binds with the membrane of HUVECs through RAGE.

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Longistatin binds with the membrane of HUVECs through RAGE. (A) Colocali...
(A) Colocalization of longistatin and RAGE. Serum-starved cells were stimulated with TNF-α (1 ng/ml), fixed, and treated with longistatin (5 μg/ml), followed by the treatment with anti-longistatin (1:1000) and anti-RAGE (1:1000). Bound antibodies were probed, and images were merged. Scale bar: 20 μm. (B) Binding of longistatin with RAGE present in HUVEC lysate. Longistatin (5 μg/ml) was coated and reacted with whole-cell lysate. Bound protein was detected with anti-RAGE (1:1600 to 1:100). (C) Pull-down analysis. His-tagged longistatin (10 μg) was trapped with Talon Metal Affinity Resin and incubated with the membrane fraction of HUVECs (100 μg) in buffer A. Proteins were eluted and analyzed by WB using anti-longistatin (1:1000) or anti-RAGE (1:1000). (D) Ex vivo binding of HUVECs with longistatin through RAGE. Ninety-six–well cell-culture plates were coated with BSA, longistatin, or CML-BSA (5 μg/ml) and treated without or with sRAGE (5 μg/ml) for 1 hour at RT. Then stimulated normal or siRNA-treated HUVECs (2 × 103 cells/well) were seeded and incubated for 2 hours, and adhered cells were counted. Scale bar: 100 μm. n = 4. *P < 0.05; **P < 0.01.