1. Introduction
The increasing emergence of multidrug-resistant
Staphylococcus aureus (
S. aureus; MDRSA), especially methicillin-resistant
S. aureus (MRSA), has seriously threatened public health worldwide under the global antibiotic crisis. Except for the few antimicrobial drugs under development, novel alternative therapeutic approaches are urgently needed. Among these approaches, host-directed therapy (HDT) stands out for its superiority in that it is less prone to inducing resistance. This HDT-based strategy is designed to prevent infection through blocking the interaction between bacterial factors and host factors, hindering key host mechanisms, targeting cellular pathways that are perturbed by bacterial or virulence factors, or rebalancing the disordered responses at the site of the pathology, rather than targeting bacterial viability [
1], [
2]. Although HDT drugs are considered difficult to stand alone, the combination of the HDT approach with traditional antibiotics will significantly reduce antibiotic treatment failures, which is particularly important for the treatment of antibiotic-resistant infections.
S. aureus has successfully evolved a plenty of virulence determinants to facilitate survival in the microenvironment encountered during infection and to interact with host cells or molecules, which enable the bacteria to breach host structural and immunological barriers. Among these essential virulence factors, α-toxin (Hla) represents the most pivotal and best-characterized pore-forming toxin (PFT) secreted by
S. aureus, participating in pathogenesis through multifaceted mechanisms during different diseases, including pneumonia, sepsis, and skin infection [
3]. After nearly a century of research on Hla, the first specific cellular high-affinity receptor for Hla was genetically identified [
4]. Functioning as an indispensable mediator for the pore formation of Hla, a-disintegrin and metalloproteinase 10 (ADAM10) is required for Hla to cause cytotoxicity and accelerate Hla-dependent pathogenesis of
S. aureus infection in the host. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening further determines the specificity and indispensability of ADAM10 for Hla-mediated cytotoxicity [
5]. Strikingly, Hla pore formation in turn stimulates a rapid upregulation of the metalloprotease activity of ADAM10, which triggers the proteolytic processing of classical adhesion molecules in the cadherin family, including epithelial (E)-cadherin and vascular endothelial (VE)-cadherin, thereby leading to severe barrier dysfunction that benefits bacterial expansion during
S. aureus infection [
6], [
7]. In addition, the interaction of Hla and ADAM10 alters integrin-mediated cell signaling events, causing the rapid dephosphorylation of focal adhesion kinase (FAK), paxillin, proto-oncogene tyrosine-protein kinase (Src), and 130 kDa Crk-associated substrate (p130Cas), a cellular event defined as the second molecular mechanism by which Hla further disrupts the cell layer and tissue barrier by inducing the dissolution of focal adhesions [
4]. Notably, the activation of ADAM10 by Hla also results in the cleavage of acid sphingomyelinase in internalized vesicles, which is closely connected to the degradation of tight junctions in epithelial cells or endothelial cells [
8]. Barrier breakdown in epithelial or endothelial cells is a critical pathogenesis that promotes the spread of infection during the initial state of invasive infection. Thus, ADAM10 represents a promising target for HDT-based approaches to prevent
S. aureus infections by preventing barrier disruption.
In addition to the cadherin family, zinc-dependent catalysis by ADAM10 leads to the activation of intracellular signaling pathways and the release of functional soluble ectodomains that modulate immunity and other cell events critical for developmental signaling and pathological processes [
9], [
10]. Tumor necrosis factor (TNF)-induced necroptosis, a mode of caspase-independent and highly proinflammatory programmed cell death regulated by receptor-interacting serine-threonine kinase 1 (RIP1) and RIP3 and executed by the pore-forming element known as mixed lineage kinase domain-like (MLKL) [
11], [
12]. A recent study showed that Hla-induced necroptosis is a major mechanism of
S. aureus lung damage, which leads to the impaired clearance of
S. aureus and severe inflammatory pathology during
S. aureus infection [
13]; however, the participation of ADAM10 enzymatic activity in Hla-mediated necroptosis remains unknown. Given the direct involvement of Hla in upregulating ADAM10 activity and
S. aureus-induced necroptosis, ADAM10 may also accelerate Hla-mediated cytotoxicity by modulating the TNF-induced necroptosis pathway, which should be further validated.
Kaempferol (3,4,5,7-tetrahydroxyflavone) is a natural polyphenolic that is abundant in vegetables and fruits as well as many types of herbs that have been widely used in Chinese medicines. This natural small molecule possesses a wide range of therapeutic properties, such as antioxidant, anticancer, and neuroprotective activities, and it also regulates T-cell functions [
14]. Although previous studies have extensively characterized the drug action of kaempferol in different cases, no research has reported the effect of kaempferol on the ADAM10-associated pathogenesis of
S. aureus. In the present study, we report a new role of ADAM10 enzymatic activity in
S. aureus-induced necroptosis. Moreover, we present kaempferol as a potent ADAM10 inhibitor that abrogated the dramatic upregulation of ADAM10 metalloprotease activity boosted by Hla. Accordingly, Hla/ADAM10-mediated barrier disruption was strongly suppressed. Further study demonstrated that
S. aureus-induced necroptosis and necroptosis-associated inflammatory responses were also blocked by kaempferol.
In vivo, kaempferol treatment effectively protected mice from lethal pneumonia and mastitis caused by
S. aureus. In summary, the findings in our work preliminarily elucidated the function of ADAM10 in
S. aureus-induced necroptosis and identified the natural compound kaempferol as an inhibitor of ADAM10, which proved to have great potential to hinder
S. aureus infections or perhaps other diseases related to the upregulation of ADAM10.
2. Material and methods
2.1. Reagents and antibodies
Kaempferol (> 98%) purchased from Herbpurify (China) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) to make a 20 mg·mL−1 stock solution that was stored at -20 °C. For in vitro assays, the cells were all pretreated with kaempferol 3 h prior to experimentation. For in vivo studies, 100 mg·kg−1 kaempferol was delivered to the mice via subcutaneous route 8 h prior to infection and then administered every 8 h. Fluorogenic peptide substrate (Mca-PLAQAV-Dpa-RSSSR-NH2; R&D Systems, USA) was supplied as a stock solution at a concentration of 10 mmol·L-1 in DMSO. Ionomycin purchased from Sigma-Aldrich was dissolved in sterilized water to make a 5 mmol·L-1 stock solution. Evans blue dye (Cat#E2129; Sigma-Aldrich) was dissolved in sterilized phosphate-buffered saline (PBS) to make a 2% (w/v) solution. Live/dead reagent (Invitrogen, USA) and a Cytotoxicity Detection Kit (Roche, Switzerland) were used to evaluate cell death. T-PER Tissue Protein Extraction Reagents (Thermo Scientific, USA) and a Pierce™ BCA Protein Assay Kit (Thermo Scientific) were used to extract the total protein and determine the concentration of obtained protein, respectively. DiD cell-labeling solution (Cat#C1039; Beyotime Biotechnology, China) was used to probe the cell membrane for immunofluorescence microscopy analysis.
The antibodies used in this study were listed as follows: mouse anti-human E-cadherin antibody (Cat#610182) against the C-terminal domain of the protein was purchased from BD Biosciences (USA) for immunoprecipitation and western blot assay; rabbit anti-human E-cadherin antibody (Cat#20874-1-AP) used for immunofluorescence microscopy analysis was purchased from Proteintech (USA); a Necroptosis Antibody Sample Kit (Cat#98110T) purchased from Cell Signaling Technology (USA) was used to analyze the necroptosis pathway; rabbit anti-MLKL antibody (Cat#ab184718) used for immunofluorescence microscopy analysis was purchased from Abcam (UK); rabbit anti-c-Jun N-terminal kinase (JNK) antibody (Cat#51151-1-AP), rabbit anti-extracellular signal regulated kinase (ERK) antibody (Cat#51068-1-AP) and mouse anti-β-actin antibody (Cat#66009-1-Ig) were purchased from Proteintech; rabbit anti-phosphor-stress-activated protein kinase (SAPK)/JNK (Thr183(221) + Thr185(223)) (Cat#ARG51807), rabbit anti-phospho-ERK1/2 (Thr202/Thr204) (Cat#ARG52277), rabbit anti-phospho-nuclear factor kappa-B (NF-κB) kinase (IKK) alpha (Thr23) (Cat#ARG51630), and rabbit anti-IKK alpha (Cat#ARG65746) were purchased from Arigo (China); rabbit antibody against NF-κB p65 (Cat#8242), phospho-NF-κB p65 (Ser536) (Cat#3033S), p38 mitogen-activated protein kinase (MAPK) (Cat#8690), and phospho-p38 MAPK (Thr180/Thr182) (Cat#4511S) were purchased from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) (Cat#146 SA00001-1) and anti-rabbit (Cat#SA00001-2) antibodies were purchased from Proteintech. Alexa 488-conjugated secondary antibody (Cat#ab150077) used for immunofluorescence microscopy analysis was purchased from Abcam. Mouse interleukine (IL)-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (USA), and human IL-1β ELISA kits were purchased from Invitrogen.
2.2. Bacterial strains, cell lines, and culture conditions
MRSA USA300 was obtained from the American Type Culture Collection (ATCC; USA). Wild-type
S. aureus strain NTCC 8325-4 (wt
S. aureus) and its
hla-deficient mutant strain (
Δhla S. aureus) were a kind gift from Professor Timothy J. Foster (Department of Microbiology, Moyne Institute of Preventive Medicine Trinity College, Dublin, Ireland). All the
S. aureus strains were grown in tryptic soy broth (TSB) broth with shaking at 37 °C. Human alveolar epithelial A549 cells and murine fibroblast L929 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO
2. Human myeloid leukemia monocytes (THP-1 cells) were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. To differentiate the THP-1 cells into macrophage-like cells, the cells were cultured with 1 μmol·L
-1 phorbol 12-myristate 13-acetate (PMA) for 24 h and further grown in weaning medium without antibiotics and PMA for another 24 h. Mouse peritoneal macrophages (MPMs) and bone marrow-derived macrophages (BMDMs) from C57BL/6 mice were obtained as previously described and cultured in RPMI 1640 supplemented with 10% FBS [
15], [
16]. Unless otherwise indicated, the cells were pretreated with the indicated concentration of kaempferol or DMSO vehicle 3 h prior to experimentation throughout the study.
2.3. Susceptibility assay
The minimum inhibitory concentration (MIC) of kaempferol on
S. aureus strains was determined by the broth microdilution method [
17], and the minimum concentration that inhibited
S. aureus was defined as the MIC. The growth curves of
S. aureus were monitored by measuring the absorbances at an optical density at 600 nm (OD
600) over 30 min intervals until the cultures reached stationary phase.
2.4. Intracellular survival assay
The intracellular assay was performed as previously described [
18]. In brief, THP-1 cells were plated in 24-well plates at a density of 8 × 10
5 cells per well and differentiated with PMA 24 hours prior to infection. After pretreatment with 32 μg·mL
−1 kaempferol or DMSO vehicle, the cells were washed three times with PBS and infected with
S. aureus at a multiplicity of infection (MOI) of 20 for 1 h. Next, the cells were washed three times with PBS and then further incubated with lysostaphin (10 μg·mL
−1) and gentamicin (200 μg·mL
−1) for 1 h to kill extracellular bacteria. Then, the cells were washed again and cultured in 10% RPMI containing gentamicin. At the indicated time points, the cells were washed three times with PBS and lysed with 0.1% Triton X-100, and the bacterial count was determined by microbiological plating.
2.5. Cell transfection
The commercially available small interfering RNAs (siRNAs) used in our study were all obtained from Santa Cruz Biotechnology (USA) and transiently transfected into cells using Lipofectamine® RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions. The experiments were performed 48 h after transfection.
2.6. Cell-based metalloprotease assay
A549 cells were grown in 96-well plates at 2 × 104 cells per well 24 h prior to experimentation. Cells pretreated with the indicated concentrations of kaempferol ranging from 4 to 32 μg·mL−1 or DMSO vehicle were then washed three times with warm PBS and stimulated with 20 μg·mL−1 Hla or PBS in serum-free DMEM for 10 min at 37 °C. Following three washes with 25 mmol·L-1 Tris buffer (pH 8.0), 10 μmol·L-1 fluorogenic peptide substrate was added for a 20 min incubation. Then, the fluorescence intensity was measured on a Tecan Microplate Reader (Mannedorf, Switzerland) with excitation at 320 nm and emission at 405 nm.
2.7. Molecular docking
A molecular docking study was performed to investigate the binding mode between kaempferol and ADAM10 using AutoDock Vina 1.1.2 [
19]. The X-ray structure of the ectodomain of ADAM10 (6BE6) was downloaded from the Protein Data Bank (rcsb.org), and the three-demensional (3D) structure of kaempferol was downloaded from ChemBioDraw Ultra 14.0.
2.8. Measurement of A549 monolayer transepithelial electrical resistance (TEER) and permeability
A549 cells were seeded in Corning Transwell permeable supports (24 mm Transwell with 0.4 μm pore polycarbonate membrane inserts) at 6 × 105 cells per well and cultured at 37 °C with 5% CO2 until the monolayer resistance reached approximately 600 Ω. At the day of the assay, cells pretreated with kaempferol or DMSO vehicle were washed with warm PBS and then induced with 50 μg·mL−1 Hla or PBS dispensed in serum-free DMEM in the presence of the indicated concentrations of kaempferol or DMSO vehicle. Concurrently, sodium fluorescein was added to each insert compartment at a final concentration of 10 μg·mL−1 to assess the integrity of the A549 monolayer in parallel. The TEER was measured using an epithelial volt-ohm meter (Millicell ERS-2; Millipore, USA) at one-hour intervals, and 100 μL of medium was taken from the basal compartment and deposited in a 96-well plate at each time point. Notably, equal amounts of DMEM were added back every time. The fluorescence intensity was measured using a Tecan Microplate Reader with excitation at 460 nm and emission at 520 nm.
2.9. Immunoprecipitation assay
To analyze the cleavage of E-cadherin by ADAM10 in A549 cells, the cells were grown in six-well plates at 1 × 10
6 cells per well overnight. The cells pretreated with the indicated concentrations of kaempferol or DMSO vehicle were washed three times with warm PBS and stimulated with 50 μg·mL
−1 recombinant Hla or PBS dispensed in serum-free DMEM containing kaempferol or DMSO vehicle for 1 h in a CO
2 incubator. Additionally, ionomycin, an agonist of ADAM10 [
20], was also applied to illustrate the inhibitory effect of kaempferol on the ADAM10-mediated cleavage of E-cadherin, in which the pretreated cells were incubated with 5 μmol·L
-1 ionomycin or DMSO in the presence of kaempferol or DMSO vehicle for 1 h. After induction, the cell monolayers were washed with PBS and lysed with 1.5 mL of Nonidet P-40 (NP-40) lysis buffer (150 mmol·L
-1 NaCl, 10 mmol·L
-1 Tris, 1 mmol·L
-1 ethylenediaminetetraacetic acid (EDTA), 0.5% NP-40, and 5% glycerol, pH 7.4) containing protease inhibitor (Roche). Whole-cell lysates from which cellular debris was removed by centrifugation were incubated with anti-C-terminal E-cadherin antibody with rotation for 2 h at 4 °C. Prewashed protein A/G plus agarose (Thermo Scientific) was added to each tube, and the tubes were then rotated at 4 °C overnight. Beads coupled with the immune complex were washed three times with NP-40 buffer and then resuspended in 2 × Laemmli sample buffer, followed by heating denaturation at 95 °C for 8 min. Immunoprecipitated proteins were separated by on a 4%-10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and probed with anti-C-terminal E-cadherin antibody. The image results were obtained using an enhanced chemiluminescence (ECL) Plus Western Blotting Detection System (Tanon, China).
2.10. Immunofluorescence microscopy analysis
To examine E-cadherin cleavage by immunofluorescence, A549 cells were cultured overnight in glass-bottom cell culture dishes (20 mm) at 5 × 10
5 cells per well. Following pretreatment with 32 μg·mL
−1 kaempferol or DMSO vehicle, the cells were washed three times with PBS and stimulated with 50 μg·mL
−1 Hla in serum-free DMEM with or without 32 μg·mL
−1 kaempferol for 1 h. Next, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, permeabilized with 0.2% Triton X-100 for another 10 min, washed three times with PBS and blocked with 1% bovine serum albumin in PBS containing 0.1% Tween 20 for 1 h at room temperature [
21]. Generally, the cells were stained overnight with a human E-cadherin antibody that recognized the extracellular domain (1:100 dilution) at 4 °C, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:1000 dilution). After three washes with PBS, the cells were counterstained with Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology) to probe the nuclei. Image capture was performed using a laser scanning confocal microscope (Olympus, Japan). To detect the MLKL location in THP-1 cells infected with
S. aureus at an MOI of 20 for 6 h with different treatments, live cells were stained with the DiD cell-labeling probe before fixation. The following steps were the same as those described above except for the primary antibody.
2.11. Cytotoxicity assays
To analyze the cytotoxicity caused by S. aureus, THP-1 cells were grown in 96-well plates at 1 × 105 cells per well, and MPMs and A549 cells were seeded into 96-well plates at 1 × 105 cells and 2 × 104 cells per well, respectively. On the day of the assay, the indicated concentrations of kaempferol or DMSO vehicle were added to cells 3 h prior to infection. Then, bacterial suspensions in cell medium were added to the cells at designated MOI with kaempferol for further infection. After the indicated infection duration, lactate dehydrogenase (LDH) release into the co-culture supernatant was assessed with a Cytotoxicity Detection Kit according to the manufacturer’s instructions, and the cells in the bottom were stained with live/dead reagent containing both membrane-impermeable (red) and membrane-permeable (green) fluorescent dyes. The visualized results were obtained using an inverted fluorescence microscope (Olympus).
To analyze TNF-induced necroptosis, L929 cells were cultured overnight in 96-well plates at 5 × 104 cells per well followed by pretreatment with the indicated concentrations of kaempferol or vehicle prior to induction. Necroptosis was then stimulated using a Necroptosis Inducer Kit with TSZ (Beyotime Biotechnology), which consists of TNF-α, second mitochondria-derived activator of caspase (SMAC) mimetic (SM-164), and benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), in the presence of kaempferol or vehicle. After 18 h of induction, cell death was determined by measuring the LDH release using a Cytotoxicity Detection Kit according to the manufacturer’s instructions. Subsequently, the cells in the bottom were stained with an Apoptosis and Necrosis Assay Kit containing Hoechst and Propidium Iodide (PI) dyes (Beyotime Biotechnology), and the results were visualized using a fluorescence microscope.
For analysis of lipopolysaccharide (LPS)-induced necroptosis with the Cytotoxicity Detection Kit and to counterstain with Hoechst and PI, differentiated BMDMs pretreated with kaempferol or vehicle were induced by 10 μmol·L-1 benzyloxycarbonyl-Val-Ala-Asp (Z-VAD) and designated concentrations of LPS (Escherichia coli O26:B6; Sigma-Aldrich) for 24 h. LDH release assays and fluorescence microscopy analysis were carried out as described above.
2.12. ELISA analysis
The IL-1β concentrations in the THP-1 culture supernatants were measured with human IL-1β ELISA kits according to the manufacturer’s protocol. The IL-1β, TNF-α, and IL-6 in mouse tissue homogenates were assessed using a mouse ELISA MAX Deluxe Set (Biolegend).
2.13. Immunoblot analysis
THP-1 cells infected with S. aureus at an MOI of 20 for 6 h with different treatments were washed with PBS and lysed using T-PER Tissue Protein Extraction Reagents followed by centrifugation at 10 000g for 8 min. The total protein concentration of each sample was determined using a Pierce™ BCA Protein Assay Kit. Biological samples in sodium dodecyl sulfate (SDS) buffer were separated by 4%-10% SDS-PAGE, transferred to polyvinylidene difluoride membranes using a semidry electrophoretic system (Bio-Rad, USA) and blocked with 5% skimmed milk in tris buffered saline with Tween 20 (TBST) for 2 h at room temperature. The immunoblots were detected using standard methods with primary antibodies against protein targets of interest, followed by incubation with HRP-conjugated secondary antibodies. The immunoblots were developed with the enhanced chemiluminescence ECL kit (Biosharp, China) and visualized using an ECL Plus Western Blotting Detection System. The integrated density of each western blot band was quantified using Image-Pro software and normalized to β-actin.
2.14. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA isolation was performed as described previously [
22], and the primer pairs used for qRT-PCR are listed in
Table 1. qRT-PCR was performed on an Applied Bioscience 7500 thermocycler using FastStart Universal SYBR Green Master Mix (Roche). The cycling parameters were an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. RNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (
GAPDH) as a reference gene, and the changes in gene transcription were calculated using the 2
−ΔΔCT method.
2.15. Miles assay and mouse infection analysis
C57BL/6 female mice and BALB/c female mice aged 6-8 weeks were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (China). They received humane care under standard laboratory housing conditions (specific pathogen-free, 12-h light/dark cycle, 55% humidity and (25 ± 1) °C) in individually ventilated cages (IVCs) with soft bedding. The mice were subcutaneously injected with vehicle or a single dose of 100 mg·kg−1 kaempferol at the indicated times, and drug delivery was maintained at 8 h intervals.
To assess the effect of kaempferol on Hla-induced endothelial barrier disruption in vivo, the BALB/c mice were subcutaneously injected with 7.5 μg of endotoxin-free recombinant Hla in 100 μL of PBS followed by the intravenous delivery of 100 μL of 2% Evans blue dye (w/v) in sterilized PBS 3 h later. The mice were then anesthetized with 5% isoflurane and killed by rapid cervical dislocation 30 min later. Evans blue dye extravasation from the vasculature into the excised skin was quantified by formamide extraction (65 °C, 24 h) and spectrophotometrically detected at OD620. In addition, their skin tissues were fixed in 10% formalin (w/v) and stained with hematoxylin and eosin (H&E) to assess the lesions under a microscope.
MRSA USA300, a highly virulent epidemic methicillin-resistant strain, and NTCC 8325-4, a standard laboratory strain, were used to establish lethal
S. aureus pneumonia. Fifty milliliter subculture aliquots of USA300 and NTCC 8325-4 (OD
600 = 0.6) were centrifuged (4500
g, 8 min), washed three times and resuspended in 5 mL of PBS and 1.5 mL of PBS, respectively. C57BL/6 mice pretreated with a single dose of 100 mg·kg
−1 kaempferol or vehicle were inoculated with 2 × 10
8 colony-forming unit (CFU) MRSA USA300 or 6 × 10
8 CFU NTCC 8325-4 by delivering 30 μL of those suspensions via the intranasal route. The mortality of the mice was recorded until 72 h after infection. For other analyses, the mice were challenged with 1 × 10
8 CFU USA300 bacteria and then anesthetized at 24 h post-infection as described above. Bronchoalveolar lavage (BAL) was performed according to a previous study [
23], and the BAL fluids (BALF) were subjected to an examination of total cell infiltration and protein exudation. Additionally, the concentrations of cytokines in the BALF were measured using ELISA kits according to the manufacturer’s instructions. To perform the bacterial burden assay, histopathology analysis, myeloperoxidase (MPO) detection and wet/dry weight ratio measurement, the infected mice were anesthetized at 24 h post-infection. Then, the left lungs were fixed in 10% formalin (w/v) and stained with H&E to examine the histopathologic injury under a microscope. Accordingly, the remaining lung tissues were homogenized in PBS (10% w/v) to analyze the bacterial burden by microbiological plating. In addition, the MPO activity and wet/dry weight ratios of the lungs were also assessed.
Lactating BALB/c mice were used 7 d after the birth of their offspring to establish a mouse mastitis model with an intraductal inoculation of MRSA USA300 as recently reported [
24]. In brief, on the day of the experiment, the offspring were weaned 2 h before bacterial inoculation, the mice were anesthetized with 5% isoflurane, and their teats were disinfected with 75% ethanol. The orifices of both the R4 (on the right) and L4 (on the left) glands of the fourth abdominal mammary gland pair were exposed through a slight cut near the end of the teat. Next, 50 μL of bacterial suspension containing 5 × 10
6 CFU USA300 was slowly injected through the teat canal using a syringe with a 3-gauge blunt needle. The mice were sacrificed at 24 h post-infection to harvest the mammary glands. The tissues were homogenized in sterile PBS (10% w/v) to assess the bacterial burden and inflammatory cytokines. In addition, the mammary tissues were also fixed in 10% formalin and stained with H&E for histopathological examination.
2.16. Statistical analysis
All the data analyses were performed using GraphPad Prism 5.0 (USA). The numeric data from no less than three independent experiments are presented as mean ± standard error of mean (SEM). The statistical significance between two independent groups was determined by unpaired two-tailed Student’s t test. Comparisons among more than two groups were tested using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, and the post hoc test was performed only if F achieved P < 0.05 with no significant variance in homogeneity. The mouse survival rate was analyzed using the log-rank (Mantel-Cox) test. *: P < 0.05 and **: P < 0.01.
2.17. Study approval
All the animal experiments were approved by the Animal Welfare and Research Ethics Committee at Jilin University (No. ALKT202104003) and were strictly conducted in compliance with the relevant guidelines determined by this committee.
3. Results
3.1. Kaempferol inhibited ADAM10-mediated barrier disruption by abrogating Hla-triggered rapid upregulation of ADAM10 metalloprotease activity
Given the critical role of ADAM10 in
S. aureus pathogenesis, we performed cell-based metalloprotease assays to identify the effective inhibitors of ADAM10 from a natural compound library established in our laboratory (
Fig. 1(a)). We initially screened 500 compounds to look for potential active candidates that inhibited ADAM10 activity by at least 50% at a concentration of 32 μg·mL
−1 (Fig. S1(a) in Appendix A), and the most promising compound was kaempferol (
Fig. 1(b)), a representative bioactive flavonol widely distributed in traditional Chinese herbs and dietary sources [
25]. Consistently, a sub-cytolytic concentration of Hla induced a pronounced increase in ADAM10 metalloprotease activity, yet kaempferol dose-dependently reversed the rapid upregulation of ADAM10 activity triggered by Hla, and importantly, 4 μg·mL
−1 kaempferol was sufficient to suppress ADAM10 activity to the baseline (
Fig. 1(c)). Notably, the dose-dependent inhibition of kaempferol under native ADAM10 activity without Hla stimulation was also observed (Fig. S1(b) in Appendix A). Further investigations demonstrated that kaempferol did not exert a visible influence on the transcription (Fig. S1(c) in Appendix A) or expression (Fig. S1(d) in Appendix A) of ADAM10 in the host cells. To explore the potential mechanism by which kaempferol inhibits ADAM10 activity, a molecular docking study was performed to investigate the binding mode between them. Interestingly, kaempferol engaged in the zinc-dependent metalloprotease domain of ADAM10 primarily by interacting with the residues leucine 328 (Leu328), glycine 329 (Gly329), and threonine 380 (Thr380); and the active catalysis sites Histidine 383 and glutamic acid 384 (Glu384) (Fig. S1(e) in Appendix A), indicating that kaempferol may inactivate ADAM10 through direct binding.
Hla-caused activation of ADAM10 and the resulting proteolysis of cadherins, which plays a key role in maintaining barrier integrity, is the prominent mechanism of
S. aureus dissemination [
26]. It is therefore likely that kaempferol hinders Hla/ADAM10-mediated barrier disruption by inhibiting ADAM10 activity. To confirm this hypothesis, we first evaluated the effect of kaempferol on barrier function
in vitro by measuring the TEER and permeability of the A549 epithelial monolayer exposed to Hla. Compared to PBS treatment, the addition of Hla at a concentration insufficient to cause cytotoxicity led to a rapid loss of resistance of A549 monolayer (
Fig. 1(d)), which was accompanied by a significant increase in monolayer permeability (
Fig. 1(e)), suggesting a remarkable injury to barrier integrity. As expected, kaempferol treatment reversed Hla-induced barrier dysfunction in a dose-dependent manner and achieved significance at a concentration of 8 μg·mL
−1. These data demonstrated that kaempferol could effectively suppress Hla/ADAM10-mediated barrier disruption.
The TEER drop in the A549 monolayer is a direct outcome of increased proteolysis cleavage of E-cadherin by ADAM10, a process leading to the release of the extracellular N-terminal domain from E-cadherin and the appearance of an approximately 35-kDa intracellular C-terminal fragment (CTF) [
27]. To clarify whether kaempferol abrogated Hla-mediated barrier disruption by blocking E-cadherin cleavage related to unusual ADAM10 activation, lysates from A549 cells with the indicated treatment were analyzed by immunoprecipitation and immunoblotting. Consistent with the TEER analysis, 50 μg·mL
−1 Hla triggered the obvious cleavage of E-cadherin (
Fig. 1(f)), and as expected, kaempferol completely blocked Hla-triggered E-cadherin cleavage at a concentration required to maintain barrier integrity (8 μg·mL
−1). Notably, treatment with 8 μg·mL
−1 kaempferol also attenuated E-cadherin cleavage triggered by ionomycin (
Fig. 1(g)), a calcium ionophore that enhances the cleavage of E-cadherin as an enzyme activator of ADAM10 [
20]. Immunofluorescence microscopy analysis further revealed similar surface E-cadherin in the samples with kaempferol or PBS and the visible loss of surface E-cadherin in the samples upon Hla stimulation and ionomycin induction (
Fig. 1(h)). As predicted, an attenuation of this loss was observed in cells treated with kaempferol. Taken together, our results demonstrated that kaempferol was a potent ADAM10 inhibitor that effectively prevented Hla-induced barrier disruption by suppressing the rapid upregulation of ADAM10 metalloprotease activity.
3.2. Kaempferol prevented Hla-caused barrier disruption in vivo
To investigate the
in vivo effect of kaempferol on Hla-induced endothelial barrier disruption, a Miles assay was performed. As shown in
Figs. 2(a) and
(b), the subcutaneous injection of PBS did not cause obvious dye infiltration. By contrast, visible accumulation of the nonvascular permeable dye was observed in the area surrounding the Hla injection site in vehicle-treated mice, in which the endothelial integrity was breached, with obvious erythema and induration in the central region (
Figs. 2(a) and
(b)). Similar to the
in vitro protection role in the epithelial barrier, kaempferol treatment also prevented endothelial barrier disruption by Hla in mice, as manifested by limited dye infiltration and gloss pathogenicity changes compared to the vehicle group (
Figs. 2(a) and
(b)). In addition, Hla challenge caused representative skin histological lesions in vehicle-treated mice, with a severely destroyed mastoid structure, a reduced number of sebaceous gland ducts, significantly disintegrated muscle fibers with visible necrosis, dissolved nuclei, and particularly, a loss of the granular layer under a microscope (
Fig. 2(c)). Nonetheless, kaempferol treatment markedly alleviated this injury (
Fig. 2(c)). Together, these results indicated that kaempferol could effectively hinder Hla-induced barrier disruption and tissue damage
in vivo.
3.3. ADAM10 was involved in S. aureus-induced necroptosis
In addition to breaching the barrier integrity via ADAM10 interaction, Hla also enables
S. aureus to break through the host immune system by inducing necroptosis, a newly identified pathogenic mechanism of
S. aureus pneumonia [
28]. A previous study showed that the ADAM10-mediated ectodomain shedding of cell-surface proteins could accelerate necroptosis downstream of TNF receptor 1 (TNFR1) and trigger a necroptosis-associated inflammatory response [
12]. Given the rapid upregulation of ADAM10 by Hla, we asked whether ADAM10 was also required for
S. aureus-mediated necroptosis, which is still unknown. To investigate this possibility, differentiated THP-1 cells were used in the current study. Consistently, challenges with wt
S. aureus markedly increased the phosphorylation of RIP1/RIP3/MLKL (P-RIP1/P-RIP3, P-MLKL) mediators and caused significant cytotoxicity in cells transfected with irrelevant siRNA, while gene silencing using siRNA targeting
ADAM10 almost completely blunted these observations (
Figs. 3(a)-(c)). Notably,
Δhla S. aureus induced only a slight activation of the necroptosis pathway and significantly reduced cytotoxicity in both control cells and ADAM10-knockdown cells (
Figs. 3(b) and
(c)), which confirmed the indispensable role of both ADMA10 and Hla in
S. aureus-induced necroptosis.
Apart from the phosphorylation of RIP1/RIP3/MLKL, the membrane translocation of MLKL is another critical hallmark of necroptosis, followed by the formation of clusters and hotspots (pore-like structures) on cell membranes and consequently of necrotic cell death [
29]. Thus, we examined the localization of MLKL by immunofluorescence microscopy analysis. Consistent with the pathway analysis, the apparent translocation of MLKL was observed in THP-1 cells transfected with irrelevant siRNA and infected with wt
S. aureus but not
Δhla S. aureus, along with the obvious aggregation of MLKL at the plasma membrane and typical pore-like structures (
Fig. 3(d)). By contrast, MLKL translocation was not observed in siADAM10-transfected cells (
Fig. 3(d)). Collectively, these results demonstrated the indispensable role of Hla/ADAM10 in
S. aureus-induced necroptosis for the first time and uncovered ADAM10 as a new potential host target for
S. aureus infection based on necroptosis inhibition.
3.4. Kaempferol suppressed S. aureus-induced necroptosis via ADAM10 inhibition
Based on the above findings, we next verified whether kaempferol could block
S. aureus-caused necroptosis through inhibiting ADAM10. As shown in
Fig. 4(a), challenges with wt
S. aureus led to the obvious phosphorylation of RIP1/RIP3/MLKL in THP-1 cells, consequently leading to high cytotoxicity (
Fig. 4(b)). As speculated, kaempferol treatment (32 μg·mL
−1) almost blunted the activation of the necroptosis pathway triggered by wt
S. aureus (
Fig. 4(a)) as well as cell death (
Fig. 4(b)). Intriguingly, although kaempferol did not exert an inhibitory effect on the minimal cytotoxicity conferred by
Δhla S. aureus (
Fig. 4(b)), the slight phosphorylation of MLKL induced by
Δhla S. aureus was abrogated by adding kaempferol (
Fig. 4(a)), indicating a potential role for kaempferol in the pathway irrelevant to Hla. MLKL pore formation could also reportedly activate the inflammasomes, therefore boosting the production of IL-1β [
30]. Thus, we further measured IL-1β production in cells infected with
S. aureus. Consistent with the analysis of MLKL activation, the IL-1β production elicited by both wt
S. aureus and
Δhla S. aureus was significantly suppressed in the presence of kaempferol (
Fig. 4(c)).
To verify the effect of kaempferol on this mode of programmed cell death, the cells were stained with live/dead reagent containing both membrane-impermeable (red) and membrane-permeable (green) fluorescent dyes. Consistent with the results of the LDH release assay, a considerable proportion of cells infected with wt
S. aureus were stained with membrane-impermeable red fluorescent dye, indicating perforation of the cell membrane by MLKL pore formation (
Fig. 4(d) and Fig. S2(a) in Appendix A). By contrast, the percentage of dead cells with red fluorescence was significantly reduced by kaempferol treatment (32 μg·mL
−1). Conversely, most cells without bacterial stimulation or those infected with
Δhla S. aureus were stained with membrane-permeable green fluorescent dye. Furthermore, kaempferol treatment exerted no toxicity on the cells, given that almost no red fluorescence was observed in the uninfected or
Δhla S. aureus-infected samples. Based on the above results, we further examined the effect of kaempferol on the membrane translocation of MLKL. As shown in
Fig. 4(e), challenges with wt
S. aureus, but not
Δhla S. aureus, resulted in the obvious aggregation of MLKL at the plasma membrane, which was also suppressed by kaempferol treatment (Fig. S2(b) in Appendix A). Promisingly, the dose-dependent inhibition of kaempferol on wt
S. aureus-induced cytotoxicity was observed in THP-1 cells (Fig. S2(c) in Appendix A) and other host cells, including MPMs (Fig. S2(d) in Appendix A) and A549 cells (Fig. S2(e) in Appendix A).
Then, we further determined whether necroptosis inhibition by kaempferol relied on ADAM10. Consistent with our hypothesis, kaempferol treatment significantly inhibited wt
S. aureus-induced necroptosis and cytotoxicity in the control cells (
Figs. 4(f) and
(g)). However, an
ADAM10 knockdown using siRNA eliminated necroptosis activation by wt
S. aureus infection as well as the inhibitory effect of kaempferol. Interestingly, the wt
S. aureus-induced activation of the necroptosis pathway was also remarkably impaired by EDTA, a broad-spectrum inhibitor of metalloproteinase, indicating that both the expression and activity of ADAM10 were required for
S. aureus-induced necroptosis. As gene silence did, EDTA treatment abrogated the inhibition of necroptosis by kaempferol (
Fig. 4(h)), suggesting that kaempferol could effectively block
S. aureus-induced necroptosis by inhibiting ADAM10 activity.
Additionally, we evaluated the potential antibacterial effect of kaempferol on
S. aureus. Consistent with previous research [
31], the MIC of kaempferol against
S. aureus strains was greater than 1024 μg·mL
−1. Consistently, bacteria treated with the indicated concentrations of kaempferol exhibited similar growth trends compared with the control group (Figs. S3(a)-(c) in Appendix A). Importantly, we further analyzed the intracellular survival of
S. aureus in differentiated THP-1 cells. Consistent with a previous study that ruled out the participation of Hla in the escape of
S. aureus from human macrophages [
18],
Hla deficiency had no significant effect on the intracellular survival of
S. aureus within THP-1 macrophages, and kaempferol treatment (32 μg·mL
−1) did not exhibit a visible impact on
S. aureus intracellular infection in THP-1 cells (Fig. S3(d) in Appendix A).
Taken together, our results established that kaempferol prevented cells from Hla-mediated cytotoxicity through the ADAM10-dependent blockage of the necroptosis pathway, rather than the action against S. aureus viability or intracellular survival.
3.5. Kaempferol suppressed S. aureus-induced necroptosis through interfering with the interplay between ADAM10 and TNF necroptosis signaling
We next addressed the potential mechanism by which kaempferol inhibited necroptosis. Necroptosis can be induced by several exogenous or endogenous biological stimuli, such as the toll-like receptor (TLR) ligands LPS (TLR4) and poly(I:C) (TLR3) and TNF. In contrast to TNF-α, which activates RIP1 via TNFR1, LPS-induced RIP1 activation and necroptosis are dependent on Toll-interleukin-1 receptor domain-containing adapter-inducing interferon-beta (TRIF). Despite this characteristic, LPS-induced necroptosis follows the same pathway downstream of TNF death receptor activation [
32]. Using L929 cells, we observed that TNF-induced necroptosis was attenuated by kaempferol in a dose-dependent manner, as manifested by reduced cytotoxicity and PI uptake (
Figs. 5(a) and
(b)). By contrast, kaempferol did not exert a similar suppressive effect on LPS-induced necroptosis in BMDMs in the absence of caspase activity (
Figs. 5(c) and
(d)). The differential effect of kaempferol on TNF-induced necroptosis and LPS-induced necroptosis excluded the possibility that kaempferol blocked necroptosis via direct inhibition of the mediators in the necroptosis pathway. Thus, kaempferol suppressed TNF-induced necroptosis by targeting ADAM10 because ADAM10 activation was required to accelerate necroptosis upon TNF-α stimulation [
12]. Together, our results indicated that kaempferol suppressed
S. aureus-induced necroptosis via blocking the interplay between ADAM10 metalloproteinase activity and TNF necroptosis signaling.
3.6. Kaempferol reduced S. aureus-evoked inflammatory response via inhibition of ADAM10
Necroptosis is known as highly proinflammatory lytic cell death because the pore formation of MLKL can directly cause membrane rupture with a massive leakage of danger-associated molecular patterns (DAMPs) to induce a strong inflammatory response and consequently boost proinflammatory genes [
33]. Therefore, we also explored whether kaempferol could suppress
S. aureus-evoked inflammatory response. As expected, the dramatic activation of the MAPK and NF-κB signaling pathways upon
S. aureus stimulation was almost completely blocked by kaempferol treatment (
Figs. 6(a) and
(b)), with attenuated phosphorylation levels of inflammatory mediators, including p38, ERK, JNK, IKK, and p65. As a result, the transcription levels of the proinflammatory genes
IL-1β (
Fig. 6(c)),
TNF-α (
Fig. 6(d)),
IL-6 (
Fig. 6(e)), and CXCL1 (
Fig. 6(f)) were all significantly reduced following kaempferol treatment. In fact, ADAM10 can also directly control these two signaling cascades in a manner dependent on its metalloproteinase activity [
34], [
35]. Therefore, kaempferol-mediated inhibition of the inflammatory response and proinflammatory gene transcription was also observed in
Δhla S. aureus-infected cells (
Fig. 6). We then found that the activation of NF-κB and MAPK pathway in response to
S. aureus infection was remarkably attenuated by
ADAM10 silencing using siRNA (Fig. S4 in Appendix A). Consistently, the extraordinary inhibitory effect of kaempferol was also impaired in
ADAM10-knockdown cells (Fig. S4). Taken together, these data demonstrated that kaempferol effectively reduced
S. aureus-evoked inflammation response through inhibiting ADAM10, which was potentially related to necroptosis inhibition as well as the direct contribution of ADAM10 to inflammation.
3.7. Kaempferol was protective against S. aureus pneumonia and mastitis in mice
Next, we investigated the
in vivo effect of kaempferol on
S. aureus pneumonia and a mastitis model. MRSA strain USA300 and methicillin-susceptible
Staphylococcus aureus (MSSA) strain NTCC 8325-4, which represented the methicillin-resistant clinical strain and standard laboratory strain, respectively, were used to establish lethal pneumonia infections. As expected, pretreatment with kaempferol (100 mg·kg
−1) for 8 h before
S. aureus challenge effectively protected mice from lethal
S. aureus pneumonia. The survival rate of mice challenged with 6 × 10
8 CFUs of NTCC 8325-4 was significantly enhanced by kaempferol treatment (
P = 0.0488), with an increase of 38.46% (
Fig. 7(a)). In the mice challenged with 2 × 10
8 CFUs USA300, the survival rate was increased from 38.46% to 84.62%, with an increase of 46.16% (
P = 0.0119) (
Fig. 7(b)). To better mimic the clinical situation in which pneumonia often occurs only after the infection has already been established, kaempferol administration was deferred until just after MRSA USA300 challenge. Promisingly, kaempferol significantly delayed death and increased the survival rate by 33.33% when administered following
S. aureus infection (Fig. S5 in Appendix A), confirming that kaempferol was effective as a treatment.
Sublethal infection with USA300 caused dark red lesions with extravasated blood in the lungs derived from mice that received vehicle treatment (
Fig. 7(c)), and histopathological examination revealed severe edema with visible swelling and congestion. Conversely, both gross and histopathologic lesions were greatly alleviated in the infected mice treated with kaempferol (
Fig. 7(c)). Additionally, kaempferol treatment also markedly decreased the bacterial load (
Fig. 7(d)), wet/dry weight ratio (
Fig. 7(e)), and MPO activity (
Fig. 7(f)) in the lung tissues of infected mice. Acute lung injury caused by
S. aureus infection is often accompanied by the loss of alveolar barrier function and the excessive secretion of cytokines. Consistent with the Miles assay results, kaempferol administration effectively preserved the integrity of the alveolar barrier, which was evidenced by significantly reduced cell infiltration and protein leakage in the BALF (
Figs. 7(g) and
(h)). Furthermore, the levels of IL-1β, TNF-α and IL-6 in the BALF were also strongly reduced (
Fig. 7(i)). In conclusion, these results demonstrated that kaempferol treatment provided effective protection against
S. aureus pneumonia.
For
S. aureus mastitis infection, the histopathology examination revealed that kaempferol treatment visibly reduced mammary gland injury and inflammatory cell infiltration caused by
S. aureus (
Fig. 8(a)). In addition, the bacterial loads in mice mammary gland tissues derived from kaempferol-treated mice were much lower than those from mice that received vehicle (
P < 0.01) (
Fig. 8(b)). Moreover, the production of proinflammatory cytokines in the mammary gland tissues of infected mice was also significantly reduced following kaempferol treatment (
Figs. 8(c)-(e)). Taken together, our results established that kaempferol is a potential treatment against
S. aureus infection, such as pneumonia and mastitis, in mice.
4. Discussion
The key role of Hla in
S. aureus pathogenicity renders it as a promising therapeutic target based on an anti-virulence strategy. Several inhibitors and antibodies targeting Hla have been reported in the past few years [
36]. However, a host-directed antibacterial strategy of targeting Hla’s specific cellular receptor ADAM10 has not been characterized. ADAM10 stands out for its indispensable role in mediating Hla pore formation in
S. aureus pathogenesis. Dramatically, Hla pore formation in turn leads to the Ca
2+-dependent activation of ADAM10 metalloproteinase activity, resulting in severe barrier disruption by cleaving cell surface adhesins and initiating the intracellular signaling events that contribute to the dissolution of focal adhesions, which facilitates bacterial spreading and maximally determines the outcome of
S. aureus infections [
37]. As such, the inhibition of ADAM10 activity or interfering with the formation of Hla/ADAM10 complex is a developable strategy to curb
S. aureus infection. In addition to causing cadherin disruption in barrier-forming cells and therefore accelerating invasive infection through upregulating ADAM10, Hla also triggers RIP1/RIP3/MLKL-mediated necroptosis, a newly identified mechanism of
S. aureus infection representing another novel target for drug design [
38].
Our striking observation was that ADAM10 was also involved in S. aureus-mediated necroptosis. Although the role and mechanism of ADAM10 and necroptosis in S. aureus pathogenesis as well as the interplay between ADAM10- and TNF-induced necroptosis has been extensively investigated in recent years, the potential participation of ADAM10 in S. aureus-mediated necroptosis remains unclear. In the present study, we were the first to find that ADAM10 is required for S. aureus to activate the necroptosis pathway, as evidenced by the silencing of ADAM10, eliminating the activation of necroptosis and cytotoxicity upon S. aureus stimulation. These findings enhance our knowledge of ADAM10 in S. aureus infection, uncovering a new contribution of ADAM10 to S. aureus-induced necroptosis.
More importantly, we have successfully identified kaempferol, a natural small molecule, as an effective inhibitor of the enzymatic activity of ADAM10 without affecting ADAM10 expression and activation processes. In the present study, the molecular docking experiment revealed that kaempferol potentially inhibited ADAM10 activity by directly interacting with the residues Leu328, Gly329, and Thr380 and especially the active catalysis sites Hid383 and Glu384 in the zinc-dependent metalloprotease domain. Our data indicated that kaempferol could efficiently restrain Hla-induced barrier disruption by suppressing ADAM10-mediated cleavage of cadherins (
Fig. 9). In this way, kaempferol might also function against other bacterial infections because, except for Hla, several PFTs derived from both Gram-negative and -positive bacteria share the ability to cause Ca
2+ influx and the subsequent ADAM10 activation and cadherin cleavage, including Streptococcus pneumoniae pneumolysin [
6], Pseudomonas aeruginosa ExlA, and Serratia marcescens ShlA [
39]. Somewhat surprisingly, by functioning as an inhibitor of ADAM10 enzymatic activity rather than a signaling component of the necroptosis cascade, kaempferol also exhibited a significant suppressive effect on
S. aureus-induced necroptosis and the inflammatory response, which might be associated with the interplay between ADAM10 metalloproteinase activity and TNF necroptosis signaling (
Fig. 9). It is worth noting that necroptosis is also a common consequence of PFTs. In addition to
S. aureus, several PFT-producing pathogens, including Streptococcus pneumoniae, Serratia marcescens
, Listeria monocytogenes, and uropathogenic
Escherichia coli [
40], could all trigger necroptosis depending on the pore formation of those cytotoxins. The findings in our study have indicated that ADAM10 activation might also be involved in the necroptosis triggered by these pathogens. Additionally, our findings further shed light on the potential therapeutics power of kaempferol for treating PFT-producing bacteria due to its inhibition on ADAM10 activity and ADAM10-modulated necroptosis.
In vivo, kaempferol treatment conferred effective protection against MRSA and MSSA infection, confirming that ADAM10 inhibition is a viable strategy for curbing
S. aureus infection. With the declining effectiveness of broad-spectrum antibiotics and the increasing cases of resistant
S. aureus infections, the combination of ADAM10 inhibitors, including kaempferol and antibiotics, may greatly improve the clinical outcome by simultaneously acting directly against the pathogen and the host target that accelerate bacterial pathogenesis. In addition, ADAM10 has been implicated as a promising therapeutic target for multiple pathological processes in other diseases, ranging from breast cancer and chronic liver inflammation to Huntington’s disease [
41]. Our findings suggest the possibility of developing kaempferol as a drug candidate for diseases associated with ADAM10 upregulation. The most recent research reported that kaempferol prevents dopaminergic neuronal degeneration in Parkinson's disease and other neurodegenerative disorders (NDs) by promoting autophagy [
14], [
42]. Here, we emphasized that the inhibitory effect of kaempferol on ADAM10 should be carefully considered when applying kaempferol to ND treatment because the upregulation of ADAM10 is in fact beneficial to NDs by promoting the clearance of amyloid deposits [
43].
In conclusion, this study is the first to provide insights into the contribution of ADAM10 to S. aureus-induced necroptosis, which enhances our knowledge of ADAM10 in S. aureus infection. More importantly, we also identified kaempferol as a novel ADAM10 inhibitor that effectively eliminated the Hla-induced rapid upregulation of ADAM10 activity and thereby abrogated the consequent barrier disruption and necroptosis during S. aureus infection, which paved the way for the development of therapeutic strategy for diseases dependent on ADAM10 upregulation. In view of the broad participation of ADAM10 in both infectious and noninfectious diseases, kaempferol-driven ADAM10 inhibition may have important implications for drug development.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (U22A20523, 32172912, and 32102722) and the Interdisciplinary Integration and Innovation Project of Jilin University (JLUXKJC2021QZ04).
Authors’ contribution
Xuming Deng and Qianghua Lv designed the study, conceived and supervised the experiments, and reviewed and edited the manuscript. Tingting Wang and Jianfeng Wang performed most of the experiments and wrote the manuscript with the input from all the authors. Xiangzhu Xu, Fan Jiang, Hongfa Lv, Qinghui Qi, and Can Zhang performed some of the experiments and analyzed the data. Jianfeng Wang analyzed the data and reviewed the manuscript.
Compliance with ethics guidelines
Tingting Wang, Jianfeng Wang, Xiangzhu Xu, Fan Jiang, Hongfa Lv, Qinghui Qi, Can Zhang, Qianghua Lv, and Xuming Deng declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2023.03.006.
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