<i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor binds to elastase and impairs neutrophil function and inflammation

<div><p>Protease inhibitors have important function during homeostasis, inflammation and tissue injury. In this study, we described the role of <i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor in parasite development and as a molecule capable of regulating different models of inflammatory diseases. First, we determine that recombinant (r) <i>Sm</i>KI-1 and its Kunitz domain but not the C-terminal region possess inhibitory activity against trypsin and neutrophil elastase (NE). To better understand the molecular basis of NE inhibition by S<i>m</i>KI-1, molecular docking studies were also conducted. Docking results suggest a complete blockage of NE active site by <i>Sm</i>KI-1 Kunitz domain. Additionally, r<i>Sm</i>KI-1 markedly inhibited the capacity of NE to kill schistosomes. In order to further investigate the role of <i>Sm</i>KI-1 in the parasite, we designed specific siRNA to knockdown <i>Sm</i>KI-1 in <i>S</i>. <i>mansoni</i>. <i>SmKI-1</i> gene suppression in larval stage of <i>S</i>. <i>mansoni</i> robustly impact in parasite development <i>in vitro</i> and <i>in vivo</i>. To determine the ability of <i>Sm</i>KI-1 to interfere with neutrophil migration and function, we tested <i>Sm</i>KI-1 anti-inflammatory potential in different murine models of inflammatory diseases. Treatment with <i>Sm</i>KI-1 rescued acetaminophen (APAP)-mediated liver damage, with a significant reduction in both neutrophil recruitment and elastase activity. In the model of gout arthritis, this protein reduced neutrophil accumulation, IL-1β secretion, hypernociception, and overall pathological score. Finally, we demonstrated the ability of <i>Sm</i>KI-1 to inhibit early events that trigger neutrophil recruitment in pleural cavities of mice in response to carrageenan. In conclusion, <i>Sm</i>KI-1 is a key protein in <i>S</i>. <i>mansoni</i> survival and it has the ability to inhibit neutrophil function as a promising therapeutic molecule against inflammatory diseases.</p></div

<i>Sm</i>KI-1 treatment decreased inflammation after MSU-induced gout.

<p>Mice were treated with r<i>Sm</i>KI-1 (10 mg/kg) or PBS vehicle i.v. 15 min prior MSU injection. Then, animals were challenged with intra-articular knee injection of MSU (100μg/cavity). Mice were grouped as MSU control, PBS control, MSU+<i>Sm</i>KI-1-treatment and PBS+<i>Sm</i>KI-1-treatment. Tissue inflammation was evaluated by <b>(a)</b> relative numbers of neutrophil in periarticular tissue determined by MPO assay, <b>(b)</b> total cells and <b>(c)</b> neutrophil recruitment in the synovial cavity, <b>(d)</b> IL-1β production measured by ELISA in the periarticular knee tissue and <b>(e)</b> joint dysfunction as noted by the increase nociceptive response of mice to mechanical stimulation using an electronic paw pressure meter test 15 hrs after MSU or PBS (control vehicle) injection. <b>(f)</b> Representative photographs of hematoxylin and eosin-stained sections of knee joints of mice after 15 hrs of injection with vehicle or MSU crystals (100μg/joint). Leukocyte infiltration and hyperplasia of the synovial membrane are indicated by black arrows. (<b>g</b>) Neutrophil recruitment in the synovial cavity of mice infected with <i>S</i>. <i>mansoni</i>. Mice were grouped as PBS control, PBS infected, MSU control and MSU infected. ND = not detected. Results are the mean ± SEM of n = 6 per group. Asterisks indicate statistically significant differences of r<i>Sm</i>KI-1 compared to MSU-vehicle group *p< 0.05 or ** p< 0.005. An asterisk also indicates statistically significant differences of <i>S</i>. <i>mansoni</i> infection versus control mice that received MSU, p<0.05.</p

<i>Sm</i>KI-1 reduces hepatic APAP-induced injury.

<p><b>(a)</b> MPO and <b>(b)</b> Elastase activities were measured in r<i>Sm</i>KI-1 treated mice during liver APAP-induced hepatotoxicity. Treated-mice received <i>Sm</i>KI-1 (10 mg/kg) or PBS vehicle i.v. 15 min prior APAP administration (600 mg/kg). <b>(c)</b> Number of neutrophils per field of view (FOV) in the liver of r<i>Sm</i>KI-1 treated animals. <b>(d)</b> Liver confocal intravital microscopy showing neutrophil (anti-GR1 PE in red) migration into necrotic sites (Sytox green staining) following 24 hours of APAP challenge. Scale bar = 100 μm. <b>(e)</b> Left panels represent histology of hematoxylin and eosin-stained liver sections, scale bar = 300μm. In right panels, liver damage is highlighted. <b>(f)</b> serum ALT levels confirmed severe liver damage in APAP group and reduction of liver necrosis in mice treated with APAP+<i>Sm</i>KI-1. Results are the mean ± SEM of n = 6 per group. An asterisk indicate statistically significant differences of r<i>Sm</i>KI-1 compared to APAP group (p< 0.05) or ** p< 0.005.</p

<i>Sm</i>KI-1 Kunitz type domain sequence and structure.

<p>(<b>a</b>) Multiple Sequence Alignment between the SmKI-1 protein from <i>Schistosoma mansoni</i>, and its homologous proteins performed using Clustal Omega, refined using BoxShade and then manually. The residues that are similar are shaded in gray, identical in dark-black and in yellow absolutely conserved cysteine residues. (<b>b</b>) Disordered Probability Prediction showing the structured Kunitz domain and unstructured C-terminal region. Analysis performed using COILS algorithms available at the Expasy website. (<b>c</b>) Schematic representation and linear view of the domains of the full-length SmKI-1 protein showing the Kunitz domain with the three disulfide bounds arrangements. (<b>d</b>) 3D protein structure of the <i>Sm</i>KI-1 Kunitz domain modeled using MODELLER v9.17.</p

Recombinant <i>Sm</i>KI-1 and its Kunitz domain inhibit serine proteases and protect <i>S</i>. <i>mansoni</i> against neutrophil elastase.

<p>Recombinant <i>Sm</i>KI-1, its Kunitz or C-terminal domains (100 nM) were tested as inhibitor of serino proteases: (<b>a</b>) Bovine Trypsin activity (100nM), (<b>b</b>) Human Neutrophil Elastase activity (300nM) and (<b>c</b>) Neutrophil-secreted elastase activity. In all experiments, bovine serum albumin (BSA, 300nM) was used as a negative control. Enzyme inhibition was detected over two-hour incubation with r<i>Sm</i>KI-1 or its Kunitz domain. Bars indicate each enzyme activity mean ± standard deviation. (<b>d</b>) Protective effect of rSmKI-1 (0.15 mg/mL) in cultured schistosomula treated with purified elastase (0.05 mg/mL). Bars represent live parasites ± standard deviation. Data are representative of at least three independent experiments. For (<b>a</b>) and (<b>b</b>), an asterisk indicate statistically significant differences of r<i>Sm</i>KI-1 or Kunitz domain compared to control group p< 0.05. For (<b>c</b>) and (<b>d</b>), ** asterisks indicate statistically significant differences of r<i>Sm</i>KI-1, compared to control group or elastase group p< 0.005. <b>(e)</b> Binding mode of <i>Sm</i>KI-1 Kunitz domain (purple) to neutrophil elastase (gray) predicted by docking with CLUSPRO 2.0. Residues from elastase catalytic triad (His⁷⁰, Asp¹¹⁷ and Ser²⁰²) are highlighted in orange sticks. (<b>f</b>) Detailed analysis of the docking predicted interface reveals residues involved in hydrogen bonds, a salt bridge and a π-stacking interaction (all interactions shown as green dashes). <i>Sm</i>KI-1 residues are represented and labeled in purple, NE residues in gray.</p

<i>Sm</i>KI-1 treatment reduces neutrophil migration into pleural cavity in response to carrageenan injection.

<p>After carrageenan injection (2mg/mL) into pleural cavity, animals received an intravenous dose of <i>Sm</i>KI-1 (10 mg/kg) or PBS (vehicle). Four hours later, we recovered cells by washing pleural cavity with PBS. Counting of (<b>a</b>) total cells and (<b>b</b>) neutrophils were performed by cytospin preparations. Specific cell populations in pleural fluid were also evaluated by flow cytometry, being the percentage of (<b>c</b>) neutrophils (Ly6G⁺CD11b⁺), (<b>d</b>) macrophages (F4/80⁺CD11b⁺), and (<b>e</b>) T lymphocytes (CD3⁺ cells) calculated from the total cell numbers. Results are expressed as the number of cells per cavity or percentage of cell subpopulations (mean ± SD) for each treated group (5–6 mice each). An Asterisk indicates statistically significant differences of carrageenan+<i>Sm</i>KI-1 compared to carrageenan vehicle group (p< 0.05).</p