Snake Venom Disintegrin Inhibits the Activation of Toll-Like Receptors and Alleviates Sepsis through Integrin alphaVbeta3 Blockade

Bacterial infection-induced sepsis is the leading cause of septic inflammatory disease. Rhodostomin (Rn), a snake venom disintegrin, was previously reported to interact with the αVβ3 integrin and the TLR4 on phagocyte in attenuating LPS-induced endotoxemia. In this report, we further evaluated the effects of Rn on TLR2-activated monocytes and its in vivo efficacy. Rn effectively suppressed the adhesion, migration, and cytokine release of Pam3CSK4-activated THP-1 cells. Rn specifically bound to integrin αVβ3 of TLR2-activated THP-1. Integrin αV and Akt siRNA transfection both restrained Pam3CSK4-elicited cytokine release. Rn decreased the Pam3CSK4-induced phosporylation of MAPKs, degradation of IκB and activation of FAK, Akt, c-Src and Syk. The Pam3CSK4-induced translocation of MyD88, a central adaptor of TLR2, to the cell membrane was also inhibited by Rn treatment. In the polymicrobial inflammatory caecal ligation and puncture model, Rn significantly reduced pro-inflammatory cytokine and chemokine release, alleviated tissue injury and elevated survival rate in vivo. Taken together, in addition to inhibiting the activation of TLR4, Rn exhibits anti-inflammatory activity through antagonizing the activation of phagocytes and interrupting the crosstalk between αVβ3 and TLR2-dependent signaling pathways.

The Pam3CSK4-induced increase of migrated-cell number was also inhibited by Rn. These results indicate that Rn is an effective inhibitor on TLR2-mediated THP-1 adherence and transmigration through extracellular matrix.
Rn specifically binds to αVβ3 integrin to affect TLR2-activated THP-1 cell adherence and cytokine production. We previously showed that Rn specifically binds to integrin α Vβ 3 on TLR4-activated phagocytes 9 . To ask the functional roles of various integrin in TLR2 signaling, Pam3CSK4-pretreated THP-1 cells were applied into wells precoated with matrix, including bovine serum albumin (BSA), collagen, fibrinogen, fibronectin and vitronectin. The adhesion ( Fig. 2A) and TNFα release (Fig. 2B) of monocytes in response to Pam3CSK4 were raised, and especially fibronectin and vitronectin were used as matrices. Rn efficiently blocked the enhanced adhesion and TNFα release to the basal level, implying the involvement of integrin α Vβ 3.
Scientific RepoRts | 6:23387 | DOI: 10.1038/srep23387 FITC-conjugated BSA showed a very low binding activity (Fig. 2C). We also detected that Rn selectively reduced the binding fluorescence intensity of anti-α Vβ 3 mAb, with little effect on anti-β 2 mAb binding (Fig. 2D). These data confirmed that α Vβ 3 is the the specific binding site of Rn on Pam3CSK4-stimulated THP-1 cells.
We then tested the involvement of integrin α V in the TLR2-stimulated THP-1 by using a combination of two siRNA duplexes specific for integrin α V. Endogenous expression of integrin α V was reduced after transfecting with integrin α V siRNA in contrast to a negative control siRNA. TLR2 expression was not affected by transfecting with negative control or α V siRNA, displaying a specificity of integrin α V siRNA (Fig. 2E). The cell viability showed no significant difference between cells transfected with α V and negative control siRNA (data not shown).
Upon stimulation by Pam3CSK4, α V siRNA-transfected THP-1 cells exhibited reduced TNFα release as compared to negative control, whereas Rn failed to further inhibit TNFα release of the α V siRNA-transfected cells (Fig. 2E, right panel). Transfection of α M siRNA also effectively decreased Pam3CSK4-elicited cytokine production, however, Rn still showed a further inhibition on α M siRNA-transfected cells (Fig. 2E). These data reveal that Rn interacts with integrin α V, but not with integrin α M, on THP-1 cells.
Rn inhibits TLR2 signaling pathway. Previous study indicates that MAPKs and NFκ B signaling pathway participate in the TLR2-induced production of cytokines and play important roles in phagocytes 16 . After Pam3CSK4 (1 μg/ml) treatment, ERK, JNK, and p38 were activated obviously in THP-1 cells (Fig. 3A). As THP-1 cells were pretreated with various concentration of Rn and assayed by Western-blotting, Rn (30 μg/ml) significantly decreased the Pam3CSK4-stimulated activation of JNK, ERK and p38 (Fig. 3A). Meanwhile, Rn also reversed Pam3CSK3-induced Iκ B degradation in a concentration-dependent way (Fig. 3B). These data indicate that the inhibition of Rn on cytokine release is due to the blockade of MAPKs activation and Iκ B degradation.
Nevertheless, TLR2 signaling pathway consists of a MyD88-dependent upstream pathway and MyD88 associates to TIR domain on TLRs, resulting in the activation of MAPK and NFκ B 2 . As shown in Fig. 3C, in the absence of stimulation, only little MyD88 in membrane fraction was detected, however, an increased immunoreactivity of membranous MyD88 was observed 15 min following TLR2 stimulation. Moreover, Pam3CSK4-induced TLR2 associated MyD88 was concentration-dependently inhibited by Rn pretreatment (Fig. 3C).
Previous work has revealed that Syk is important for neutrophil integrin signaling 19 . Src-family kinases (SFKs) also are critical for the initiation of integrin signaling in macrophage 20 . As TLR2 was activated upon incubation with Pam3CSK4, Syk and c-Src phosphorylation significantly increased (Fig. 3E). Rn (30 μg/ml) also inhibited Syk and c-Src phosphorylation in response to Pam3CSK4 stimulation (Fig. 3E). Integrin αV-TLR2 complex in monocytes. To investigate the direct interaction between integrin α V and TLR2, we examined this complex formation by co-immunoprecipitation assays in Pam3CSK4-activated THP-1 cell lysates (Fig. 4A). We found that integrin α V was significantly precipitated along with TLR2, but not TLR4, in TLR2-activated THP-1 cells. It was also found that Syk was also incorporated in the complex.
The effect of integrin signaling pathway on TLR2-activated monocytes. We further investigated the role of integrin-signaling pathway molecules on TLR2-dependent activation of monocytes. As stimulated by Pam3CSK4, Akt siRNA-transfected THP-1 cells exhibited a reduced TNFα release as compared to negative control (Fig. 4B). However, FAK siRNA-transfection had no effect on TNFα release as compared to negative control.
Effect of Rn on CLP-induced cytokine production, plasma glucose concentration, mean blood pressure changes and mortality in mice. Among the experimental sepsis models, host immune responses in caecal ligation and puncture (CLP) model most mimic the course in human disease, including haemodynamic and metabolic phases of human sepsis 21 . We carried out the CLP model to examine the effect of Rn on this complex infection animal model. Since excessive elevation of pro-inflammatory cytokines in circulation is a major contributor to remote organ injury after CLP, we examined the plasma levels of pro-inflammatory cytokines. It was observed that the concentrations of cytokines were low or undetectable in sham mice (data not shown). CLP-mice exhibited elevated cytokines levels at 6 h and 24 h, including TNF-α , IL-6 and MCP-1. As Rn (10 mg/kg) was intravenously administered following CLP instantly, the increases of these cytokines were profoundly inhibited (Fig. 5A). In the sham group, plasma glucose levels were not altered during the experiment period (Fig. 5B). CLP induced biphasic changes in plasma glucose, displaying hyperglycemia at 6 hr after CLP and followed by hypoglycemia from 24 hr onwards. Pretreatment of CLP mice with Rn significantly reduced hyperglycemia at 6 hr after CLP as compared with CLP group (p = 0.0432), but had no significant effect on hypoglycemia after 24 hr (Fig. 5B). Mean blood pressure and heart rate were not affected in sham group during the experiment period, but hypotension and bradycardia occurred after CLP (Fig. 5C,D). However, Rn pretreatment improved hypotension at 6 hr after CLP (p = 0.0418) (Fig. 5C).
We first performed a 10-day survival study to confirm the effect of Rn on CLP-induced polymicrobial sepsis. CLP mice group showed 100% mortality on day 4, whereas Rn-treated mice group showed 41.67% mortality on day 10 (n = 12) (Fig. 5E). The mean survival time of the control septic mice was about 1.7 days, whereas that of Rn-treated septic mice was prolonged to about 5.5 days (more than 10 days was counted as 10 days). Therefore, Rn-treatment exerted a protective effect as shown by prolonging survival rate of severely septic mice. We also assayed the effect of a disintegrin-like α IIbβ 3 antagonist, tirofiban, on CLP mice, which showed 100% mortality on day 7 (n = 10) and the mean survival time about 3.5 days (Fig. 5E). Taken together, Rn treatment efficiently lowers the plasma pro-inflammatory cytokines and mortality in septic mice. Moreover, administration of tirofiban, an anti-platelet disintegrin-like drug, also improved the survival rate of CLP mice. However, Rn displayed a better efficacy than tirofiban.
Rn alleviates CLP-induced tissue injury as examined by histochemistry. In comparison with sham mice (Fig. 6A,D,G), histological examination of CLP-induction mice (Fig. 6B,E,H) showed that lung was characterized by the presence of leukocytes in the interstitium and alveoli, and thickening of the alveolar wall (Fig. 6B), while liver section exhibited leukocyte infiltration in parenchyma and showed peri-vascular infiltration (Fig. 6E). The kidney was also damaged by CLP induction with evidenced glomerular hypercellularity (Fig. 6H). We also found some occlusive vessels in liver and kidney (Fig. 6E,H). However, the treatment of Rn (Fig. 6C,F,I) protected mice from these organ damage caused by CLP.

Discussion
In this report, we observed that a snake venom disintegrin, Rn, suppresses the inflammatory responses of TLR2-stimulated monocytes. The binding of Rn towards THP-1 monocyte shows a saturated manner as its concentration increases, indicating that the interaction between Rn and monocyte is receptor-mediated. Furthermore, flow cytometry assay also showed that Rn selectively inhibited the binding of an α Vβ 3-specific mAb to THP-1 (Fig. 2). We revealed that the binding with vitronectin or fibronectin led to the increase of Pam3CSK4-elicited monocyte adherence and cytokine production; however, silencing or Rn-blockage of integrin α V attenuated these enhanced activities (Fig. 2). It is concluded that the anti-inflammatory effect of Rn depends on its blockage of the interaction between integrin α Vβ 3 and ligands.
Integrin α Vβ 3 is an important receptor of the RGD-containing ligands, including fibrinogen, vitronectin, osteopontin and disintegrins 7 . Integrins α Vβ 3 and α 4β 1 acts as the binding receptor of monocytic cells to some pro-inflammatory ligands, like monomeric C-reactive protein, fractalkine and matrices 7,22,23 . TNFα production from phorbol myristate acetate (PMA) activated THP-1 cells is also integrin α Vβ 3 and adhesion-dependent 24 . The antagonists of these integrins exert the anti-inflammatory actions through blocking ligands-integrin interaction. The effect of Rn on THP-1 in our data also indicates that integrin α Vβ 3 coordinated TLR2 responses to Pam3CSK4. Substantial literatures also support the theory that α Vβ 3-integrin plays a considerable role in the behavior of phagocyte and osteoclast 25 . These data indicate that integrin α Vβ 3 coordinates microbe-induced cytokine release and phagocyte-stimulated inflammation, and provide a potential target for clinical medication.
The signaling pathway of some TLRs, including TLR-2, -4, and -9, are regulated by MAPKs, responding to extracellular stimulation, regulating cellular activities and mediating cytokine and chemokine release 26 . Rn inhibits the Pam3CSK4 enhanced phosphorylation of MAPKs, resulting in a significant attenuation of cytokine expression at a transcription level. Besides activating MAPK pathways, Pam3CSK4 can also activate integrin signaling, e.g., phosphorylations of FAK signaling pathway. In TLR2-stimulated monocytes, we found that Rn inhibited not only phosphorylation of FAK/PI3K/Akt, but also phosphorylation of c-Src/Syk (Fig. 4). It implies that Rn blocks TLR2-activated integrin signaling to decrease phosphorylation of MAPKs.
To further clarify the downstream signaling pathway crosstalk of TLR2 and integrin α Vβ 3, we considered FAK and Akt, two typical molecules activated in the integrin signaling pathway 19 . Though Pam3CSK4 activated FAK (Fig. 3D), our data revealed that FAK siRNA transfection has no effect on TLR2-dependent cytokine release (Fig. 4B). Besides being the downstream of integrin, Akt also involves in several signaling pathways 19,27,28 . Binding of TLRs turns on intracellular signal-transduction pathways, which leading to activate transcriptional factors such as interferon regulator factors (IRFs), PI3K/Akt, AP-1, and NFκ B. In response to viral and bacterial components, Akt serves as a hub in the α Vβ 3-integrin-promoted TLR2 signaling 8 . We also observed that Akt siRNA transfection reduced TLR2-dependent cytokine release from Pam3CSK4-stimulated monocytes (Fig. 4B). Akt plays a key role in the cross-talk route between integrin α Vβ 3 and TLR2 (Fig. 7).
The inflammatory phenotype in motheaten mice primarily results from dysregulated neutrophil integrin signal transduction due to increased SFK and Syk activity, but deletion of Syk or SFK in neutrophils would reverse the inflammatory disease 29 . Src, together with Syk, are also important molecules activated in the integrin signaling pathway 16 . The activation of TLR2 also stimulated the phosporylation of Src and Syk and Rn concentration-dependently attenuated these activation (Fig. 3E). TLR2 forms complex with integrin α Vβ 3 in stimulated-monocytes, and Syk also can be found in the complex (Fig. 4A). From these results, it is suggested that Rn attenuates TLR2 signaling through α Vβ 3 and c-Src/Syk-dependent signaling pathway as illustrated in Fig. 7.
It is critical that innate immune system senses the microbes and turns on the immune responses. TLRs cooperate with other microbe-identification protein to take efficient responses against pathogens 2 , but chronic or excessive response may injure tissue and organ 30 . For instance, TLR2 and TLR4 participate in the pathogenesis of atherosclerosis, autoimmune colitis, systemic lupus erythematosus (SLE), diabetes and Alzheimer's disease 31 . Although several TLR blockers presently are under ongoing clinical trials, but none was accepted for clinic medication 32,33 . Thus, inhibition of immoderate TLR activation is a therapeutic target being enthusiastically investigated on these sickness 34,35 . The potential application of Rn as an inhibitor of TLR-2 and TLR-4 activation provides a promising lead for drug development in infectious diseases induced by complicated microbial patterns.
Sepsis is caused by systemic reactions to severe inflammation. The leading cause of sepsis is bacterial infections, including both Gram-positive and Gram-negative bacteria. Though sepsis and septic shock cause million death annually worldwide, the only antisepsis agent, Xigris (human recombinant activated protein C/Drotrecogin alfa), was withdrawn from the market after a follow-up placebo-controlled trial due to lack of beneficial effect on mortality 36 . Polymicrobial sepsis elicited by CLP is the most often utilized model for researching complex inflammatory pathology and pharmacology 29 . Since Rn is an inhibitor of both TLR-2 and -4 signaling, it effectively attenuated LPS-endotoxemia syndromes 9 and reduced mortality in CLP model. Our data showed that the crosstalk between integrin α Vβ 3 and TLRs signaling pathways is important and worthy to be further investigated.
Activation of the coagulation system leading to microvascular thrombi is the cause of multiple organ failure, and correlates with mortality in severe sepsis 3 . In LPS-induced endotoxemia model, a moderate inhibitory effect of heparin on cytokine release was also observed 9 . Tirofiban, a specific α IIbβ 3 antagonist, also showed a significant, but less-pronounced beneficial effect on the survival rate of septic mice (Fig. 5B). These anti-coagulation or anti-platelet drugs may improve organ failure in the CLP-challenged host. Rn effectively blocks platelet aggregation and adherence to various matrices, preventing platelet activation and thrombus formation. Therefore, we would not exclude the possible contribution of its anti-thrombotic activity through platelet α IIbβ 3 antagonism in vivo.
Rn markedly blocked phagocyte activity and reduced cytokine production of THP-1 cells stimulated by both Gram positive and negative bacteria, indicating that integrin α Vβ 3 on phagocytes not only promotes cell adherence but also assists the innate inflammatory activities induced by TLR2. Moreover, the blockade of integrin α Vβ 3 effectively protects the animal afflicted by complex bacterial-infected inflammatory diseases. However, the detail mechanism regarding how Rn interferes with the TLRs-elicited signaling is under exploration.

Materials and Methods
Materials. Enzyme-linked immunosorbent assays (ELISA) kit for IL-1β , IL-6, IL-8 and TNFα were pur- Cell culture. The human monocyte, THP-1, was acquired from the American Type Culture Collection and was grown in RPMI-1640 media with 10% FBS. The cell was maintained at 37 o C in an atmosphere containing 5% CO 2 .
Cytokine production by TLR2-activated phagocytes. After monocytes were incubated with Pam3CSK4 (1 μg/ml) and various concentrations of Rn for 24 h, media were collected by centrifugation. The concentration of cytokines was analyzed with ELISA kit (eBioscience, USA).
Adhesion assay. THP-1 monocytes were labeled with BCECF/AM and resuspended in RPMI-1640 medium to a density of 1 × 10 6 cells/ml. The resuspended cells were activated with Pam3CSK4 (1 μg/ml) or not, then incubated with PBS or various concentrations of Rn for 30 min at 37 °C. Cells were subjected to adhesion as previously described 9 .
Migration assay. Costar Transwell (polycarbonate filter, 5 μm pore size) was coated with gelatin (2%) and dried for 2 h. THP-1 (5 × 10 5 cells) incubated with Pam3CSK4 (1 μg/ml) or PBS was treated with various concentration of Rn at 37 °C for 30 min, and then cells were seeded onto the upper chamber. The bottom chamber was only added RPMI-1640 medium. Migration assay of THP-1 monocyte was measured as described before 37 .
Western blotting of protein kinases. In the presence or absence of 1 μg/ml Pam3CSK4, THP-1 cells were treated with various concentration of Rn at 37 °C for 30 min. After incubation, cells were harvested and subjected to Western blotting assay as previously described 9 . Subcellular fractionation. Membrane and cytoplasmic protein were obtained with Proteo JET TM membrane protein extraction kit (Thermo Fermentas, EU) according to manufacturer's protocol.  Resources, 1996) and were treated ethically. The mice were maintained on a 12-hr light/dark cycle under controlled temperature (20 ± 1 °C) and humidity (55 ± 5%). Animals were given continuous access to food and water. The protocol of animal study was approved by Laboratory Animal Center, College of medicine, National Taiwan University.
CLP-induced sepsis was induced with modification according to previously described method 38 . After mouse was anesthetized with pentobarbital (50 mg/kg, i.p.), cecum and adjoining intestine were taken out through a Scientific RepoRts | 6:23387 | DOI: 10.1038/srep23387 20 mm incision. The cecum was tightly ligated with 3-0 silk suture 1 cm from the cecal tip and punctured through once with a 23-gauge needle. The cecum was then returned to the abdominal cavity and the laparotomy site was sutured. In sham controls, the cecum was taken out and returned without ligation or puncture.
Mice whole blood and serum collection. Whole blood of mice was obtained from orbital venous sinus and collected at different time (6 h or 24 h after surgery). After centrifugating at 1000 × g for 10 min, plasma was separated and collected. The plasma concentration of cytokines, IL-6, TNF-α , IL-1β and MCP-1, were measured with ELISA kit following the manufacturer's protocol (eBioscience, USA). Plasma glucose Measurement. Blood was obtained from eyes by puncture, and plasma glucose levels were measured at 6, 24 and 48 hr after sham or CLP with glucose liquicolor kit following the manufacturers protocol (Human, Germany).
Measurement of vital signs using the tail-cuff method. A thermostatically regulated, heating platform was used to maintain body temperature at 37 °C. During the experiment, conventional non-invasive blood pressure and heart rate in conscious mice was measured using the tail-cuff method (BP-2000; Visitech Syatems, Inc., Apex, NC, USA), for the measurement of blood pressure.
Histological examination. Lung, liver and kidney segments were fixed in 10% v/v phosphate-buffered formalin for 48 ~ 72 hr and then embedded in paraffin. Next, the samples were sectioned (5 μm) using a microtome, stained with H&E, and examined with light microscopy at × 200 magnifications.