Anticoagulant therapy without bleeding: A novel molybdenum‐based nanodots alleviate lethal coagulation in bacterial sepsis by inhibiting ROS‐facilitated caspase‐11 activation

Abstract

Sepsis is a leading cause of death worldwide. This syndrome is commonly accompanied by overactivation of coagulation, excessive reactive oxygen species (ROS), and inflammatory cytokine storm. Notably, disseminated intravascular coagulation (DIC) accounts for around 40% of sepsis‐associated deaths. However, anticoagulant therapy is still difficult for sepsis treatment because of the lethal bleeding side effects. Although the relationship between ROS and inflammatory cytokine storm has been described clearly, the pathogenic role of ROS in DIC, however, is still unclear, which renders novel therapeutic approaches hard to achieve bedside for inhibiting DIC. Herein, our new finding reveals that ROS greatly facilitates the entry of lipopolysaccharide (LPS) into the macrophage cytoplasm, which subsequently activates the caspase‐11/gasdermin D pathway, and finally induces DIC through phosphatidylserine exposure. Based on this finding, novel gallic acid‐modified Mo‐based polyoxometalate dots (M‐dots) with outstanding antioxidant activity are developed to provide ideal and efficient inhibition of DIC. As expected, M‐dots are capable of markedly inhibiting sepsis‐caused coagulation, organ injury, and death in sepsis. This therapeutic strategy, blocking the upstream pathway of coagulation rather than coagulation itself, can avoid the side effects of extensive bleeding caused by conventional anticoagulation therapy, and will provide a new avenue for the efficient treatment of sepsis.

INTRODUCTION

Sepsis is a life-threatening syndrome caused by disturbed host response to severe infection and progresses rapidly to multiple organ dysfunction, leading to high morbidity and mortality.^1–3 Although advancements of antibiotics and life-supporting techniques have led to a reduction in deaths caused by sepsis, the incidence of sepsis is increasing worldwide due to an increase in multidrug-resistant bacteria, explosion of viral infections (i.e., Covid-19), and an increase in aging populations.^4–6 However, effective medicines are limited for treating sepsis in clinics. As a result, sepsis is still the leading cause of mortality worldwide, accounting for approximately 11 million deaths annually.³

Sepsis-induced disseminated intravascular coagulation (DIC),⁷ a syndrome occurring in sepsis patients, accounts for around 40% of multiorgan failure and deaths caused by sepsis.^8–10 As a complex process, the coagulation cascade involves a series of cell interactions and damages. In sepsis, severe infection hyperactivates macrophages and induces phosphatidylserine (PS) exposure, thereby triggering and inflating the tissue factor (TF)/factor VIIa-dependent pathway of coagulation, which underlies the core pathogenesis of sepsis-induced DIC.¹¹ Logically, a series of anti-TF or anticoagulant drugs have been developed to relieve DIC in sepsis, such as recombinant tissue factor pathway inhibitor (rTFPI),¹² activated protein C (APC),^13,14 antithrombin (AT),^15,16 and thrombomodulin (TM).^17,18 However, use of these anti-TF or anticoagulant treatments, which focus on the coagulation cascade itself, involves high risk of extensive bleeding and can lead to death.^2,12,16,19 For example, APC was approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) two decades ago for the treatment of sepsis but was withdrawn from the market in 2011 due to bleeding side effects and lack of beneficial effects on 28-day mortality in sepsis.²⁰ There is thus an urgent need to identify the underlying DIC mechanism and develop novel medicines to improve the outcome of sepsis.

Reactive oxygen species (ROS) is essential for immune cells, in particular macrophage and neutrophils, to destroy invading microorganisms.²¹ On the other hand, ROS not only directly mediates oxidative stress damage but also generates an inflammatory cytokine storm through a vicious cycle of ROS inflammation.²² However, how ROS promotes coagulation is not fully understood. In recent decades, antioxidants have been investigated as promising therapeutic medicines for sepsis. Nevertheless, conventional antioxidants lack sufficient activity and/or sustainability.²³ Thanks to the surprising physical properties and surface chemistry of nanomedicines, nano-antioxidants have been developed for treating sepsis in animal models. Still, these nano-antioxidants have not been applied so far in clinics due to the limited biocompatibility and low reactive oxygen and nitrogen species (RONS)-eliminating activity.

Given that the link between ROS and coagulation is still unclear and nano-antioxidants that can attenuate sepsis-induced DIC have still not been reported, herein, we developed ultra-small-size gallic acid-modified Mo-based polyoxometalate dots (M-dots), which effectively reduce DIC, oxidative stress damage, and inflammatory factor storm in sepsis. The tailored M-dots demonstrated superior ability to scavenge a wide variety of ROS because of their ultra-small size and high redox activity of Mo in polyoxometalate, with redox potentials as low as 0.15 and 0.38 in aqueous solution.²⁴ Moreover, M-dots have shown one of the strongest known abilities to eliminate O₂^.− (the most predominant ROS in the sepsis), and the superoxide dismutase (SOD)-like activity reaches 858 U/mg, which achieves about 60% of the SOD activity derived from bovine serum. For the first time, we have revealed that ROS facilitates the cytosolic translocation of lipopolysaccharide (LPS) into macrophages and consequently promotes the activation of the intracellular receptor of LPS—caspase-11, thereby enhancing GSDMD-mediated pore-forming and PS exposure and boosting TF activity and coagulation cascades. Correspondingly, the protection conferred by M-dots was mainly attributed to the inhibition of ROS-facilitated LPS internalization, which consequently reduced caspase-11-dependent coagulation, organ dysfunction, and death in sepsis models. This therapeutic strategy, intervening in the upstream pathway of coagulation rather than the coagulation process itself, avoids the side effects of extensive bleeding caused by conventional anticoagulation therapy. In addition, Mo is an essential element for the human body, and the human body contains many Mo enzymes that are essential for day-to-day activities. Therefore, M-dots have excellent biocompatibility and can be completely metabolized in the body at therapeutic doses. Our study not only reveals the core mechanism of ROS in DIC and develops a high-efficient sepsis-relieving therapeutic strategy by inhibiting DIC but also provides a reference for the treatment of other types of DIC diseases.

METHODS AND REAGENTS

Reagents

Phosphomolybdic acid (CAS: 51429-74-4) and gallic acid (CAS: 149-91-7) were purchased from Shanghai Macklin Biotechnology Co., Ltd. LDH assay kits (Cat: C0017; Beyotime Biotechnology), ELISA kits, including mouse IL-1α (Cat: 88-5019; Invitrogen), mouse IL-1β (Cat: 88-7013; Invitrogen), mouse tumor necrosis factor-α (TNF-α) (Cat: 88-7324; Invitrogen), mouse interleukin-6 (IL-6) (Cat: 88-7064; Invitrogen) human IL-1α (DY200; R&D), human IL-1β (Cat: 88-7261; Invitrogen), human TNF-α (Cat: 88-7346; Invitrogen), human IL-6 (Cat: 88-7066; Invitrogen), mouse D-dimer (Cat: CEA506Mu; Cloud-Clone), mouse fibrinogen (Cat: ab108844; Abcam), mouse PAI-1 (Cat: ab197752; Abcam), mouse TAT (Cat: ab137994; Abcam) and mouse fibrin (MBS706338; MybioSource), and LEGENDplex kit (Cat: 740150; Biolegend) were obtained from commercial suppliers as indicated. The human tissue factor chromogenic activity assay kit (Cat: CT1002b) and SensoLyte internally quenched 5-FAM/QXL-520 FRET thrombin substrate (Cat: AS-72129; Anaspec) were purchased to determine TF activity and thrombin formation, respectively. Antibodies including anti-caspase-11 (Cat: C1354; Sigma), anti-mouse GSDMD (Cat: ab209845; Abcam), and anti-mouse β-actin (clone 8H10D10, Cat: 3700S; Cell Signaling Technologies) were purchased from commercial suppliers, while anti-mouse fibrin antibody (59D8) was as a gift from Prof. Nigel Mackman. LPS derived from Escherichia coli 0111:B4 for in vivo study was from Sigma (L2630), and ultra-LPS for in vitro study was from InvivoGen (tlrl-pb5lps). Outer membrane vesicles (OMV) were collected and purified as previously described.²⁵

Synthesis of M-dots

M-dots were synthesized by a redox reaction. Gallic acid (0.18 g) and phosphomolybdic acid (0.3 g) were dissolved and mixed in ultra-pure water, and then anhydrous sodium carbonate (0.675 g) was added to create an alkaline environment. The reaction was allowed to proceed under magnetic stirring at room temperature (RT) for 12 h. Thereafter, phosphomolybdic acid was reduced to a colorless solution, then the unreacted impurities were removed by dialysis, and M-dots were obtained by freeze-drying.

Characterization

Transmission electron microscope (TEM) images were taken using a TECNAI G2 high-resolution transmission electron microscope. X-ray photoelectron spectroscopy (XPS) measurements were conducted using a VG ESCALAB MKII spectrometer. XPSPEAK software (Version 4.1) was used to deconvolute the narrow-scan XPS spectra of the Mo 3d of the samples, using adventitious carbon to calibrate the C 1s binding energy (284.5 eV). Fourier-transform infrared spectroscopy was recorded on a Bruker Vertex 70 spectrometer (2 cm⁻¹). UV–vis spectra were collected using a VARIAN CARY 50 UV/vis spectrophotometer. Zeta potential and hydrodynamic size were determined using the Malvern instrument (Zatasizer Nano). The mean diameter and distribution of M-dots were detected using Particle Size Analyzer (Nicomp).

XPS measurement of M-dots

The XPS spectrum was recorded for the element/chemical state analysis of M-dots. First, we scanned the full spectra of gallic acid, phosphomolybdic acid, and M-dots. Afterward, the XPS measurement of M-dots was performed before and after incubation with H₂O₂.

Superoxide anion scavenging with M-dots

The reduce tetrazolium blue (NBT) method was used to detect the O₂^·− and the scavenging ability of M-dots. In the presence of methionine, riboflavin undergoes photochemical reduction and further produces O₂^·−. O₂^·− can reduce tetrazolium blue to blue methyl hydrazine, which has the maximum absorption at 560 nm. Briefly, methionine (0.1 mol/L), riboflavin (20 μmol/L), NBT (0.01 mol/L) of appropriate volume, and M-dots with different concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6 μg/mL), phosphate-buffered saline (PBS, 0.01 mol/L, pH 7.4), and deionized water were added to cuvettes and mixed. The cuvettes were then exposed to ultraviolet light for 5 min. The absorbance of blue methyl hydrazine was measured at 560 nm, and the O₂^·− scavenging ability was calculated from the intensity of M-dots-inhibited NBT photochemical reduction.

Free radical scavenging with M-dots

The ·OH scavenging efficiency of M-dots was determined by fluorescence spectrophotometry. The Fenton reaction between FeSO₄ and H₂O₂ catalyzes the formation of ·OH. The combination of nonfluorescent terephthalic acid and ·OH can produce fluorescent 2-hydroxyl terephthalic acid. The addition of M-dots can reduce the formation of the latter and the fluorescence intensity. Terephthalic acid (0.1 mmol/L), ferrous sulfate (0.05 mmol/L), H₂O₂ (1 mmol/L), and PBS (0.01 mol/L, pH 7.4) were prepared and mixed. Then, M-dots with different concentrations (0, 50, 100, 200, 400, and 800 ng/mL) were added to the reaction system. After it was allowed to stand for 6 min, the mixture was transferred to cuvettes and the corresponding fluorescence intensity was scanned under an excitation wavelength of 320 nm.

The ONOO⁻ scavenging ability of M-dots was evaluated by UV–vis spectroscopy using pyrogallol red as the indicator. Briefly, pyrogallol red (5 mmol/L), ONOO⁻, and M-dots with different concentrations (0, 8, 16, 32, and 64 μmol/L) were mixed and reacted for 15 min. Afterward, the ultraviolet absorption spectrum was scanned to measure the clearance rate of ONOO⁻.

The H₂O₂ scavenging activity of M-dots was determined by UV–vis spectrophotometry. In brief, H₂O₂ (1 mmol/L) and M-dots with different concentrations (0, 5, 10, 20, 40, and 80 mmol/L) were mixed and incubated in the dark for 12 h. The ultraviolet absorption at 425 nm was detected to measure the clearance rate of H₂O₂.

Mice and models of bacterial sepsis and DIC

C57BL/6J male mice at age of 8–10 weeks and with a body weight of 22–25 g, including Casp11^−/− (024698; Jackson Laboratory), Casp1^−/− (032662; Jackson Laboratory), Gsdmd^−/− (032662; Jackson Laboratory), Nlrp3^−/− (021302; Jackson Laboratory), and wild-type (WT) mice, were used in the present study. Saline or M-dots were intravenously administrated to mice 1 h after a challenge of cecal ligation and puncture (CLP). A DIC-like model was obtained by priming mice with 0.4 mg/kg LPS for 7 h before a challenge of 10 mg/kg LPS.^11,26,27 Interventions were intravenously administered 30 min after the challenge of 10 mg/kg LPS. To avoid complete occlusion of microvasculature such that it prevents visualization under intravital microscopy, a lower dose of LPS at 4 mg/kg after the priming process was intraperitoneally injected into mice 6 h before imaging.^11,28 All mice were allowed to access water and standard chow under standard conditions at room temperature (22–25 °C) and a 12-h light–dark cycle. Animal experiments were approved and performed on the basis of the guidelines of the Committee of Xiangya Hospital and Central South University.

CLP

In brief, mice were anesthetized using 2% isoflurance (Piramal Critical Care) in oxygen and a 1.5 cm longitudinal midline incision was subsequently made. After opening the abdomen, the cecum was ligated (75%) and a small amount of feces was extruded from a through-and-through puncture. The abdomen was closed after relocating the cecum, followed by an injection of pre-warmed saline (1 mL per mouse) for recovery. The same surgery, except for the ligation and the puncture, was conducted for sham-operated mice.

Intravital microscopy and image analysis

Using intravital microscopy, the circulation of microvasculature in the left liver was visualized in real-time. Mice, anesthetized with xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (200 mg/kg), were place on their sides with the liver exposed and externalized on a thin and long glass coverslip. After loading, AF647-conjugated anti-mouse albumin antibody (0.05 μg/mouse) was injected via the tail vein to obtain fluorescent images as previously described.^11,28

Cell culture

Mouse peritoneal macrophages and human THP-1 cells (with 20 ng/mL of PMA) were cultured in RMPI-1640 and plated overnight, followed by the treatment of siRNA (Ribobio) or material (1–10 μg/mL) and/or the challenge of OMV (10 μg/mL).

Platelet preparation, activation, and aggregation, and blood coagulation

Anticoagulated whole blood (sodium citrate) was centrifuged at 800 r/min for 15 min to obtain platelet-rich plasma. The plasma was subsequently centrifuged at 3000 r/min for 3 min to harvest platelets, which were then washed and resuspended in Tyrode's buffer. For platelet aggregation, 300 mL of platelets (3 × 10⁸ mL⁻¹) were stimulated with thrombin in the presence or absence of M-dots, and the aggregation levels were determined using a coagulometer (SYSMEX CS-5100). For platelet activation, the P-selectin level of the platelet was assessed using flow cytometry after a treatment with M-dots or not and incubation of FITC-conjugated P-selectin plus APC-conjugated CD41 antibodies (mouse, CD41 is a specific platelet marker). For blood coagulation, M-dots at a dose of 2 mg/kg were administrated 1 h before the harvest of anticoagulated whole blood (sodium citrate). The plasma was obtained after centrifugation at 3000 r/min for 15 min and applied to commercial kits according to the instructions of the manufacturers. Plasma-activated partial thromboplastin time (APTT) and thromboplastin time (TT) were assessed using a coagulometer (SYSMEX CS-5100).

Proximity ligation assay (PLA)

Colocalization of LPS and caspase-11 was assessed using PLA (Sigma). Briefly, macrophages on a glass slip were fixed with 4% formaldehyde and permeabilized by PBS containing 1% Triton X-100 before incubation with primary antibodies to LPS (mouse monoclonal 2D7/1, ab35654; Abcam) or caspase-11 (rat monoclonal 17D9, C1354; Sigma-Aldrich) at 4 °C overnight. PLA was conducted in situ based on the instructions of the manufacturer. The slips were imaged using a Nikon confocal laser scanning microscope and positive dots were counted from the imaged cells using ImageJ software.

Tissue histology

After the mice were killed, perfusion was carried out using cold PBS containing heparin (20 IU/mL), followed by 10% formalin. Tissues were harvested and further fixed with 10% formalin overnight. Sections of tissues were obtained after splitting for standard hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). In IHC, the primary antibody (anti-mouse fibrin antibody, 1:500), followed by the secondary antibody (anti-mouse HRP-conjugated antibody, 1:2000) were applied to the dewaxed sections, and then 3,3′-diaminobenzidine tetrahydrochloride was added for positive staining. Washes were carried out four times between steps using PBST (PBS containing 0.1% tween-20). Images were acquired using Nikon microscopy.

Western blot

Proteins, separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were transferred to polyvinylidene fluoride membranes. The membranes were blocked using 5% fat-free milk and incubated with primary antibodies at 4 °C overnight, followed by incubation of the horseradish peroxidase (HRP)-conjugated secondary antibody at RT for 2 h. The blots of the membranes were visualized using the Bio-Rad system after treatment with Western Bright ECL-Spray (Advansta, catalog number: K-12049-D50). The membranes were washed four times between steps.

TF activity and thrombin assay

After incubation with 70 μL of assay mixture (containing factor VII and factor X) at 37 °C for 30 min, human TF-positive macrophages were treated with the Factor Xa substrate. TF activity was determined by the 405 nm absorbance of samples relative to that of the standard curve. The thrombin generation assay was used to assess thrombin formation.¹¹ In brief, 50 μL of thrombin substrate was added to the mixture containing 10 μL of cell supernatant and diluted platelet-poor plasma. The fluorescence intensity was then acquired using a multifunctional fluorescence microplate reader (TECAN).

Statistical analysis

GraphPad Prism 7.0 software was used for statistical analysis in the present study. The Student t test was applied for comparison between two groups, while one- or two-way analysis of variance with post hoc tests were used for comparisons between multiple groups. The log-rank test was used to analyze the survival rates of mice. p < 0.05 was considered to indicate statistical significance.

RESULTS AND DISCUSSION

Synthesis and characterization of M-dots

M-dots were prepared by the reduction of phosphomolybdic acid with gallic acid under alkaline conditions (Figure 1A). In this reaction, Mo⁶⁺ is partially reduced to Mo⁵⁺ by gallic acid in phosphomolybdic acid. Correspondingly, the formed M-dots have mixed valence states of Mo⁶⁺ and Mo⁵⁺. Therefore, Mo⁶⁺ and Mo⁵⁺ form charge transfer through oxygen bridges, which results in M-dots with absorption spectra extending from the visible to near-infrared light region (Figure 1B). As measured by a TEM (Figure 1C), the M-dots have an ultra-small size (2–4 nm), with a hydrodynamic diameter of about 4–8 nm (Figure S1), which is slightly larger than that measured by TEM due to the modification of the gallic acid group. Using XPS (Figures 1D and S2), it was found that M-dots have abundant C, O, and Mo and trace amounts of P elements, and the abundant C of M-dots is mainly derived from gallic acid. This can also be further confirmed from the fine peak of C 1s (Figure S3) and the FTIR spectrum (Figure S4). In addition, the charge of M-dots was −36.6 mV, as determined by the Zeta potential, due to the modification of gallic acid (Figure S5). The Mo⁵⁺ of M-dots is as high as 75% on the basis of Mo3d XPS fine peak analysis (Figure 1E), which is very important for the elimination of various ROS because Mo⁵⁺ readily accepts free electrons in ROS because of its low redox potential.²⁴ Moreover, the ultra-small size of M-dots ensures that ROS can easily diffuse into Mo⁵⁺ in M-dots to facilitate the elimination of ROS. Correspondingly, M-dots can effectively eliminate O₂^·− (the most important primary ROS in sepsis), and its pseudo-SOD activity is as high as 858 U/mg,^29,30 which is one of the highest values reported in the current literature. In addition, M-dots can also effectively eliminate ·OH (Figure 1G), ONOO⁻ (Figure 1H), and H₂O₂ (Figure 1I). The mechanism of elimination of ROS by M-dots was also investigated. Taking ONOO⁻ and H₂O₂ as examples, it was found that both demonstrated a concentration-dependent lightening of the color of M-dots, which indicated that the oxidation of Mo⁵⁺ by ROS weakened the charge transfer between Mo⁵⁺ and Mo⁶⁺ in M-dots. The XPS Mo 3d fine peak further reveals that Mo⁵⁺/Mo⁶⁺ is significantly reduced in M-dots after reaction with H₂O₂ (Figure 1J).

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M-dots show antioxidant activity in the treatment of bacterial sepsis

To verify the therapeutic effect of M-dots in sepsis, a CLP mouse model was adopted (Figure 2A). In this model, feces are released into the peritoneal cavity to form an multi-microbial infection-induced sepsis. M-dots show a long half-life in the blood circulation system (about 8 h) thanks to their ultra-small size and negative surface charge (Figure 2B). Therefore, M-dots are effectively distributed to vital organs to exert therapeutic effects through the circulatory system, such as the liver, lungs, kidneys, and so forth. Furthermore, the distribution of M-dots in the liver, lungs, and kidneys was slightly higher in CLP-challenged mice than their sham-operated counterparts (Figure 2C). In sepsis, ROS/inflammation-induced endothelium injury increases vascular permeability, which leads to more efficient distribution of M-dots to these injured organs and improves the therapeutic efficacy of M-dots. Notably, M-dots persisted in these vital organs for an extended period (7 days), playing a role in eliminating multiple RONS, although M-dots were already eliminated from the circulation (Figure 2B,C). Therefore, the distribution characteristics of M-dots are beneficial to these vital organs and consequently reduce the mortality as a result of sepsis. As shown in Figure 2D, all CLP-challenged mice died within 5 days, while the survival rate of mice treated with M-dots (2 mg/kg) was as high as 60%. In sepsis, malondialdehyde (MDA) levels are abnormally elevated in the blood, the liver, the lung, and the kidney as ROS destroy cellular lipids to generate MDA. Previous animal and clinical studies have demonstrated the association between plasma MDA and severity of sepsis.³¹ M-dots significantly reduced MDA levels in the blood (Figure 2E), the liver (Figure 2F), the lung (Figure S6A), and the kidney (Figure S6B) due to the superior ability of M-dots to eliminate ROS. With the consideration that ROS is essential for macrophages to clear pathogens, we investigated if the anti-ROS activity of M-dots affects the bacteria-clearing ability in vitro and in vivo. We found that M-dots showed limited influence on the take-up of bacteria by macrophages (Figure S6C), and in the CLP model, administration of M-dots significantly inhibited the bacterial burden in the lung, the liver, and the kidney (Figure S6D,F). Correspondingly, dampened function of the lung (Figure S7A–D), the kidney (Figure S7E,F), and the liver (Figure S7G,H) of CLP mice were largely restored after treatment with M-dots.

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Besides organ dysfunction, the most striking feature of sepsis is a systemic storm of inflammatory cytokines. Inflammation and ROS can form a vicious cycle in sepsis and accelerate the progression of sepsis.²² Numerous metal-based nanomaterials, such as iron oxide nanoparticles, manganese-based nanoadjuvants, nanoscale titanium, and so on, have been reported to generate ROS and/or promote inflammatory responses, thereby inhibiting cancer and other diseases involving hypoactivation of the immune system.³² In contrast, a series of inorganic nanomaterials protect against diseases caused by immune hyperactivation due to their antioxidant activity.³² Correspondingly, M-dots with strong ROS scavenging ability would reduce the inflammatory cytokine storm by breaking this vicious cycle in sepsis. As a predominant pathogen derived from bacteria, LPS is generally involved in two signaling pathways: toll-like receptor 4 (TLR4, signal 1 for priming) and caspase-11 (signal 2 for activation), which stimulate inflammatory cells to release different types of inflammatory factors (Figure 2I). In the signal 1 pathway, LPS activates TLR4 on the surface of inflammatory cells such as macrophages to secrete TNF-α and IL-6 and upregulate the expression of inflammasome components. In the signal 2 pathway, bacteria release OMV, which transfers LPS into the cytosol by macrophage scavenger receptor 1 (MSR1).³³ Once accessed by cytosolic LPS, caspase-11 is self-cleaved and activates the cleavage of GSDMD (downstream of caspase-11). N-terminal GSDMD is consequently released and forms pores on the membrane, resulting in the release of IL-1α and efflux of K⁺. The efflux of K⁺ reinforces the assembly of inflammasome containing NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), and caspase-1, and activates caspase-1, thereby inducing the cleavage of GSDMD and the development and release of IL-1β and IL-18. As predicted, the challenge of CLP led to an increase in a series of cytokines (Figures 2G, and S6H). Among the 13 cytokines, sepsis-augmented IL-1α and IL-1β (the two representative inflammatory factors for the signal 2 pathway) were inhibited to the highest extent by M-dots (Figure 2G,H). In contrast, TNF-α, a representative inflammatory factor for the signal 1 pathway, was only slightly reduced. Interferon-γ (IFN-γ), mainly an inflammatory factor released by T cells, was also considerably inhibited. To test whether IFN-γ reduction by M-dots increases survival in sepsis, a specific CD3 antibody was used as a control. As shown in Figures 2D and S7 and S8, CD3 antibody treatment only slightly reduced organ injury and increased the survival rate in CLP mice, and administration of M-dots further improved the effects on the basis of the CD3 antibody, suggesting that IFN-γ reduction is not the core mechanism underlying the effects of treatment with M-dots. Therefore, the above evidence fully indicated that M-dots exert therapeutic effects mainly by inhibiting caspase-1 or caspase-11 activation.

M-dots exert therapeutic effects through the caspase-11 pathway

Caspase-1 and caspase-11 knockout mice (Casp1^−/− and Casp11^−/−) were used to further clarify the mechanism by which M-dots reduce sepsis-associated organ dysfunction and death (Figure 3A). As shown in Figure 3B, caspase 1 knockout did not affect the survival rate of CLP mice, while M-dots significantly improved the survival rate of Casp1^−/− mice in CLP model. Functions of the lung (Figures 3C and S9A,B), the liver (Figures 3D and S9C), and the kidney (Figures 3E and S9D), and H&E staining of corresponding tissue sections (Figure S10) phenocopied the results in which M-dots but not caspase-1 deficiency did alleviate sepsis-induced organ damage. Therefore, the effectiveness of M-dots does not depend on the caspase-1 pathway.

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To identify whether the protective effect of M-dots on sepsis extends to inhibiting caspase-11 signaling in vivo, we determined the plasma levels of caspase-11-related cytokines, such as IL-1α and IL-1β, and hepatic levels of caspase-11 expression and GSDMD cleavage of the mice in the CLP model. To our delight, M-dots robustly blocked the augmented IL-1α and IL-1β (Figure 3F,G) and the cleavage of GSDMD, but did not affect caspase-11 expression (Figures 3H and S11). In addition, both administration of M-dots and deletion of caspase-11 dramatically alleviated mice death in the sepsis model, and M-dots did not exert additional effects on the basis of caspase-11 deficiency (Figure 3B). Given that the majority of deaths in sepsis is attributed to organ dysfunction, we measured key indicators of liver, kidney, and lung function (Figures 3C–E, and S9) in WT or Casp11^−/− mice with or without treatment with M-dots after a challenge of CLP. The results indicated that CLP-induced dysfunction of the liver, the kidney, and the lung was dramatically attenuated by administration of M-dots or deletion of caspase-11, and intervention with M-dots did not lead to further improvement in Casp11^−/− mice. A similar trend was also observed in lung, liver, and kidney injury, as indicated by H&E staining (Figure 3I). Thus, M-dots prevent sepsis-induced organ dysfunction and lethality by inhibiting caspase-11 signaling.

M-dots suppress caspase-11 signaling in mouse macrophages and human THP-1 cells

Caspase-11, the intracellular receptor of LPS capable of inducing pyroptosis and the release of IL-1α and IL-1β, is implicated in the pathogenesis of sepsis.^11,34,35 To verify whether M-dots inhibit caspase-11 signaling in vitro, we treated WT and gene-modified mice peritoneal macrophages with M-dots before a challenge of E. coli-derived OMV—the major vehicle used to transport LPS into the cytosol, or E. coli itself (Figure 4A). It was observed that deletion of caspase-11 or GSDMD, but not Nlrp3 (upstream of caspase-1), significantly blocked OMV- or E. coli-mediated cytotoxicity (Figure 4B) and IL-1α release (Figure 4C), suggesting dependence on caspase-11 signaling. Similarly, treatment with M-dots robustly inhibited OMV-augmented medium levels of lactic dehydrogenase (LDH) and IL-1α in WT and Nlrp3^−/− macrophages, but did not induce further inhibition in Casp11^−/− or Gsdmd^−/− macrophages (Figure 4B,C). The inhibitory effects of M-dots were dose-dependent, that is, a dose as low as 0.1 μg/mL significantly reduced OMV-mediated cytotoxicity and release of IL-1α as well as IL-1β, while a dose at 2 μg/mL almost completely blocked OMV-mediated cytotoxicity and release of IL-1α as well as IL-1β (Figure 4D–F). To verify application in human samples, we subjected human THP-1 to the same treatment and challenge, and observed a similar dose-dependent inhibition on OMV-mediated pyroptosis (Figure 4G–I). These results suggested the outstanding inhibitory effect of M-dots on caspase-11 signaling. Still, it is possible that M-dots inhibit caspase-11 signaling via TLR4, an extracellular LPS receptor that increases the expression of caspase-11 in the priming process and facilitates caspase-11-dependent pytoptosis. Clinical trials have demonstrated that antagonists of TLR4 or neutralizing antibody of TNF-α fail to improve the prognosis of sepsis.^36–38 We further determined TLR4-related cytokines, such as TNF-α and IL-6, to assess the potential effect of M-dots on TLR4. We found that M-dots did not significantly alter augmented TNF-α and IL-6 in either mouse macrophages (Figure 4J) or human THP-1 cells (Figure 4K) after a challenge of OMV. Moreover, western blot analysis indicated that the treatment of M-dots significantly suppressed OMV-mediated GSDMD cleavage but not caspase-4/11 expression (Figures 4L and S12). Collectively, M-dots effectively prevent OMV-mediated pyroptosis by inhibiting caspase-11 signaling independent of the TLR4 pathway.

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M-dots attenuate coagulation by inhibiting caspase-11 signaling

We have previously demonstrated that genetic deletion of caspase-11 prevents coagulation and consequently reduces organ dysfunction and lethality in bacterial sepsis.^11,33 To assess the effects of M-dots on coagulation, herein, we visualized the microcirculation of hepatic sinusoid in real-time with intravital microscopy (Figure 5A). We observed that challenge with LPS (DIC model) robustly induced the formation of thrombosis and resulted in vessel occlusion, and administration of M-dots markedly reduced the occlusion in line with the level of Casp-11-deficient mice (Figure 5B). Besides, DIC markers,^28,33 such the thrombin–antithrombin (TAT) complex, plasminogen activator inhibitor type 1 (PAI-1) and D-dimer, and the consumption of fibrinogen, which indicate systematic coagulation activation, were remarkably inhibited on administration of M-dots to an equivalent level of caspase-11 deletion (Figure 5C,D). Further reduction was not observed in Casp-11-deficient mice on administering M-dots (Figure 5C,D). In addition, as the terminal executor of thrombosis in the coagulation cascade, fibrin was markedly boosted in the lung and the liver after a challenge of LPS (Figure 5E,F), and was significantly inhibited after administration of M-dots and deficiency of caspase-11 (Figure 5E,F). Taken together, these results suggest that M-dots can prevent coagulation in sepsis by inhibiting the caspase-11 pathway.

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Given that the small size of nanomaterials may facilitate platelet aggregation and directly affect the coagulation cascade,³⁹ we determined the expression of P-selectin, the activation marker, and the aggregation level of the platelet treated with M-dots or not. We found that M-dots did not induce the expression of P-selectin or facilitate the aggregation of platelets (Figure S13A). In addition, administration of M-dots did not affect the time of blood coagulation (Figure S13B,C). These results indicated that M-dots do not activate platelet and affect blood coagulation directly. Moreover, M-dots, unlike APC, did not facilitate but protected against sepsis-induced bleeding in the lung (Figure S13D,E). Thus, M-dots suppress sepsis-associated coagulation independent of the coagulation cascade, and will not induce bleeding.

M-dots inhibiting ROS-facilitated LPS internalization

Accessing and binding of LPS are required for the oligomerization and activation of caspase-11.³⁴ Herein, we used PLA to detect the colocalization of LPS and caspase-11, to determine the mechanism by which M-dots inhibit caspase-11 signaling. As shown in Figure 6A, LPS enters the cytosol and binds to intracellular caspase-11, which leads to production of red fluorescence by the Förster resonance energy transfer (FRET) effect. When Casp11-deficient macrophages were used as the negative control, OMV induced strong red fluorescence in wild-type macrophages, which was significantly inhibited by M-dots, indicating the inhibitory effect of M-dots on the colocalization of LPS and caspase-11 (Figure 6B,C). To uncover which process in caspase-11 activation is disturbed by M-dots, we used electroporation to physically transfer LPS into the cytosol of macrophages. It is noteworthy that M-dots lost the inhibitory effect on electroporated LPS-induced cell death (Figure 6D) and release of cytokines (Figure 6E,F). In addition, limulus amebocyte lysate (LAL) assay revealed that OMV-augmented intracellular LPS levels were significantly dampened by addition of M-dots (Figure 6G). These results suggested that M-dots inhibit caspase-11 signaling by preventing LPS internalization. In view of the excellent antioxidant activity of M-dots, we stimulated macrophages with H₂O₂ and OMV in the presence or absence of M-dots. Interestingly, H₂O₂ facilitated OMV-induced LPS endocytosis (Figure 6G), which was significantly inhibited by the treatment of M-dots. In addition, we have previously demonstrated that activated caspase-11 cleaves GSDMD and induces membrane pores that lead to PS exposure and increase TF activity, triggering the extrinsic coagulation cascade and eventually forming thrombin. Consistently, we found that H₂O₂, by facilitating LPS internalization and caspase-11 signaling, significantly enhanced OMV-inflated TF activity and thrombin formation, which was remarkedly blocked by the treatment of M-dots (Figure 6J,K). Thus, oxidative stress promotes LPS internalization in the activation of caspase-11 signaling and consequently enhances TF activity in coagulation cascades, and M-dots, by scavenging ROS, prevent caspase-11-dependent coagulation in sepsis (Figure 6L).

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Biocompatibility of M-dots

To assess the druggability of M-dots, the cytotoxicity of M-dots was first investigated. As shown in Figure S14, M-dots also did not have any effect on the viability of macrophages, which was phenocopied in other cell types, including HL-1 (the heart), TC-1 (the lung), TCMK1 (the kidney), and primary hepatocytes (the liver). The effect of M-dots on the inflammatory secretion of macrophages was also further studied. As shown in Figure S15, M-dots do not induce macrophages to produce inflammatory cytokines under normal conditions. The above evidence indicated that M-dots showed excellent biocompatibility in cell-based in vitro experiments. The biocompatibility of M-dots was further investigated in vivo. An intravenous injection of M-dots up to 2.5 times the therapeutic dose did not have any adverse effects on lung, liver, and kidney function (Figure S16), and did not alter the levels of blood indicators (Figure S17). Moreover, M-dots did not induce histopathological changes in the heart, the liver, the spleen, the lung, the kidney, and the small intestine as shown by H&E staining (Figure S18).

The metabolism of M-dots was also further analyzed in vivo. M-dots can be excreted through the urinary system because of their small size (smaller than the glomerular filtration barrier, about 10 nm). As shown in Figure S19A, Mo was detected in urine in both CLP and healthy mice, and the concentration decreased with time. In addition, Mo element was also present in the feces of CLP and healthy mice (Figure S19B), suggesting that M-dots can also be metabolized by the liver and excreted from the bile duct into the digestive system. Given that Mo is an essential trace element for mammals, they have corresponding pathways to metabolize molybdenum out of the body.⁴⁰ As shown in Figure 2C, the content of Mo element in major organs gradually decreased over time in CLP and healthy mice after injection. Moreover, almost the entire concentration of Mo elements in these organs was metabolized and eliminated within 30 days after injection of M-dots (Figure S20).

CONCLUSION

DIC is a major cause of multiorgan dysfunction and death in sepsis and there is dissatisfied treatment due to the limited understanding of the pathogenic mechanisms. Herein, we revealed a new link between ROS and DIC in which ROS facilitates cytosolic translocation of LPS and activation of caspase-11 signaling, thereby promoting caspase-11/GSDMD-inflated TF activity and coagulation cascade. In view of the fact that excessive oxidative stress and DIC are highly associated with Covid-19 infection,^22,41–43 and silencing caspase-4/11 attenuates Covid-19-induced thrombosis,⁴⁴ our finding may also provide an in-depth understanding of Covid-19-induced DIC. Based on the new finding, we discovered a novel molybdenum (Mo)-based nanodot, with excellent capacity to scavenge ROS, capable of protecting against coagulation activation and thus organ dysfunction and death in bacterial sepsis. The strategy inhibits upstream coagulation and leaves the cascade itself intact, which may avoid the lethal bleeding side effect and be beneficial for the treatment of sepsis. Given that TLR-4-related cytokines, such as TNF-α and IL-6, are essential for global inflammation against infection defense, M-dots do not significantly affect the systemic inflammation, which may avoid immunodepletion/immunosuppression and retain host response to secondary infection. Moreover, the good distribution, long-term maintenance, and excellent biocompatibility of M-dots allow timely scavenging of ROS and improve the effectiveness sepsis treatment. Taken together, this study yields a new finding that ROS facilitates LPS internalization and caspase-11-dependent coagulation, which provides a bridge to link oxidative stress and lethal coagulation. Thus, scavenging ROS is a promising strategy for preventing sepsis-associated coagulation and death, and M-dots are effective therapeutic agents in the treatment of sepsis.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (Nos. 82373871, 21974134, 82072152, 82270137, and 82370140), the Hunan Science Fund for Distinguished Young Scholar of China (Nos. 2021JJ10067 and 2021JJ10072), the National Key Research and Development Program of China (No. 2022YFC2304600), the Innovation-Driven Project of Central South University (No. 202045005), the Hunan Provincial Natural Science Foundation of China (Nos. 2022JJ30793 and 2021JJ31066), and the Key Program of Ningxia Hui Autonomous Region Natural Science Foundation of China (No. 2022JJ21059).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT

All animal experiments were approved and performed on the basis of the guidelines of the committee of Xiangya Hospital and Central South University (XMSB-2022-0244).

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