Excretory/secretory products of adult Trichinella spiralis inhibit NETosis and modulate neutrophil cytokine production | Parasites and Vectors

animals and strains

Female C57BL/6 mice (6–8 weeks old) and Wistar rats (200 ± 20 g) were purchased from the Experimental Animal Center of Basic Medical College of Jilin University (Changchun, China). All animals were maintained under pathogen-free conditions, and all experiments were performed in accordance with the guidelines of the Chinese Laboratory Animal Administration. The protocol was approved by the Institutional Animal Care and Use Committee of Jilin University (20170318).

E. coli BL21 (DE3) (Sangon, China) was transformed with PFM23 plasmid (constructed from pET28A, Novagen, USA) to promote cytoplasmic production of enhanced green fluorescent protein (EGFP) under the control of the T7 promoter to analyze bacterial phagocytosis. EGFP-E. coli The culture was maintained on LB agar supplemented with 50 μg/ml kanamycin in a 37°C incubator and stored in a 4°C refrigerator for subsequent use.

Parasites and ESP Collection

Trichinella spiralis (ISS534) were cultured and maintained in our laboratory by serial passage of Wistar rats and isolated as previously described ( 17 ).Infectious muscle larvae Trichinella spiralis Skeletal muscles from infected Wistar rats were recovered at 35 days postinfection (dpi) by manual digestion of minced muscles in a solution containing 1% pepsin-1% HCl with agitation at 37°C for 2 h ( 18).Isolation of adult stage parasites from the small intestine Trichinella spiralis– Wistar rats were infected at 3 dpi with Ad3. Trichinella spiralis Wash 3 times with physiological saline solution and then store in RPMI-1640 medium (Gibco, USA) supplemented with 100 U/ml penicillin and 100 µg/l streptomycin at 37°C, 5% CO₂ After incubation at 4 pm, the ESP-containing supernatant was concentrated using an Amicon Ultra-3 centrifugal filter unit (molecular weight cutoff (MW), 3 kDa; Millipore, USA) at 4 °C and 5000.G 30 minutes(19). Endotoxin levels in ESP (0.002 EU) were determined by Limulus amebocyte lysate (LAL, Zhanjiang A&C Biological Co., Ltd., Guangdong, China); endotoxin concentrations in ESP for further use were < 0.005 EU. The protein concentration, i.e., ESP, was determined by BCA protein assay kit (Beyotime Biotech., China).

Isolation of mouse bone marrow-derived PMNs

PMNs were isolated from C57BL/6 as previously described with modifications ( 20 ). Briefly, mice were sacrificed and immersed in 70% ethanol. Bone marrow cells were collected by flushing a syringe into the femur with phosphate-buffered saline (PBS). PMNs were subsequently isolated and purified from bone marrow cells using a mouse neutrophil isolation kit (P8550, Solarbio, China) according to the manufacturer’s instructions. PMN were collected by centrifugation at 1500 rpm for 10 min, then washed three times and resuspended in ice-cold PBS at a concentration of 5 × 10⁵

cells/ml. Cells were incubated with antibodies LY6G (1:500 dilution; Abcam Inc., USA) and CD11b (1:500 dilution; Abcam Inc., USA) for 40 min at 4°C in the dark. They were then rewashed to determine the purity of the PMN isolates before analysis by flow cytometry. Flow cytometry data were analyzed using FlowJo (Three Star Inc., USA).To confirm nuclear morphology, detached cells were plated at 2 × 10 into 24-well glass-bottomed black plates.⁵ cells/well and subsequently stained with 5 µM Hoechst 33342 (Sigma, USA). Then, the samples were observed under a laser scanning confocal microscope (Olympus FluoView FV1000, Olympus America, Inc., USA).

cell viability

Relative cell viability was tested using Cell Counting Kit 8 (CCK8; Beyotime Biotechnology, China) according to the manufacturer’s instructions. Briefly, PMNs were seeded into 96-well plates (2 × 10³ cells/well), divided into ATA treatment group (containing PMN with ATA concentrations of 5, 10, 15, 20, 25 and 30 μM), control group and blank group, with 5 wells in each group. The control group contains PMN and medium, and the blank group only contains medium. After 3 hours of incubation, add 10 μl of CCK-8 solution and incubate in the incubator for 1 hour. The absorbance at 450 nm was measured in a multimode microplate reader (Thermo Fisher Scientific, USA).

NET visualization and quantification

PMN were seeded into 24-well glass-bottomed black plates at 4 × 10⁵ cells/well and incubate at 37°C, 5% CO₂. Each experiment is divided into three groups: blank control group, experimental group, and negative control group.Previous work showed that endonuclease activity Trichinella spiralis ESP (21). In order to eliminate the DNase activity in ESP, 25 μM nuclease inhibitor gold tricarboxylic acid (ATA) and different concentrations of Ad3 ESP (2.5, 5 and 10 ng/μl) were added to the experimental group, and PMN was pretreated once. deal with. H. PMNs were then stimulated with 100 nM PMA for 3 h. After incubation, PMNs were stained with 5 µM Hoechst 33342 (Sigma, USA) and 5 µM SYTOX Green (Invitrogen, USA) and observed under a laser scanning confocal microscope (Olympus FluoView FV1000, Olympus America, Inc., USA) sample. ). In addition, changes in neutrophil morphology and nuclear size were evaluated by laser scanning confocal microscopy (Olympus FluoView FV1000, Olympus America, Inc., USA), and the nuclear expansion area of ​​500 cells in each sample well in each group was evaluated. Quantification. ImageJ software (National Institutes of Health) was used.

Active oxygen detection

The oxidation-sensitive dye 2′,7 dichlorofluorescein diacetate (DCFH-DA) (Sigma, USA) was used as a substrate to detect ROS formed in cells by activated PMN. In the presence of intracellular ROS, DCFH-DA is oxidized to form highly fluorescent dichlorofluorescein (DCF).PMN were seeded into a black-framed 96-well cell culture plate at 1 × 10⁵ cells/well. DCFH-DA was added to the culture to a final concentration of 10 µM.Samples were incubated at 37°C, 5% CO₂ 20 min; the plate was then washed twice with phenol red-free medium and different concentrations of Ad3 ESP (1, 2, 5 and 10 ng/μl) were added to each well.The experimental and control groups were then stimulated with 100 nM PMA for 3 hours at 37 °C, 5% CO₂

. Fluorescence at 485 nm excitation and 535 nm emission was detected using a fluorescence plate reader (Tecan Infinite F200).

LDH detection

Lactate dehydrogenase (LDH) released from PMA-treated PMNs was quantified to determine whether the reduction in NETs was due to ESP cytotoxicity.Briefly, PMNs were seeded into 96-well cell culture plates at 1 × 10⁵ cells/well, and different concentrations of Ad3 ESP (2, 5, and 10 ng/μl) and 25 μM ATA were added to the culture medium. After 1 hour of incubation, PMNs were stimulated with 100 nM PMA for 3 hours.All steps were maintained at 37°C, 5% CO₂.Centrifuge the plate at 400G React for 5 minutes, and transfer 120 μl of supernatant per well to a new 96-well plate. After incubation in the dark for 30 min, LDH activity in the supernatant was determined using an LDH cytotoxicity assay kit (Roche Applied Science, Germany) according to the manufacturer’s protocol. Samples included experimental groups, background controls, negative controls for unstimulated PMN (0% cell synthesis), and positive controls for maximum LDH release (100% cell synthesis).

Phagocytosis assay

Neutrophil phagocytosis was assessed using the following two experimental methods: First, the phagocytic capacity of PMNs was quantitatively assessed by measuring the phagocytic index using the Vybrant Phagocytosis Assay Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Briefly, PMN (1 × 10⁵ cells/well) in 96-well plates were treated or untreated with different concentrations (1, 2, 5, and 10 ng/μl) of ESP and incubated at 37 °C for 2 h. After incubation, the supernatant was replaced with 100 µl of fluorescent particles provided by the Vybrant Phagocytosis Assay Kit (in 100 µl PBS) and further incubated in the dark at 37 °C for 2 h. Stop the reaction by adding 100 μl of ice-cold trypan blue suspension. Phagocytosis was quantified by measuring the fluorescence of each sample using a microplate-reading fluorometer at 494 nm excitation and 518 nm emission (Biotek, USA). Calculate the percentage of phagocytosis using the following formula: % effect (phagocytosis) = (absorbance value of experimental group – background absorbance) / (absorbance value of positive group – absorbance value of negative group) × 100%.

The second method involves three groups: blank control group (PMN only), negative control group (only 10 ng/μl ESP added), and experimental group (25 µM ATA and 10 ng/μl ESP added). µl ESP). After pretreatment, each group (5 × 10⁵ cells/well in a 24-well plate) combined with EGFP-E. coli (Initial concentration is 1×10⁷ CFU/ml) and incubated at 37°C for 2 hours, and the multiplicity of infection (MOI) was adjusted to MOI 25.Next, PMNs were co-cultured with EGFP-E. coli Centrifuge at 2500 rpm for 10 minutes to remove suspended EGFP-E. coli and obtain PMN. After resuspending in PBS, Hoechst 33342 (1:1000) dye was added, and the samples were observed using a laser scanning confocal microscope (Olympus FluoView FV1000, Olympus America, Inc., USA). The supernatants of each group were collected and centrifuged at 12,000 rpm for 1 min. The obtained pellet was resuspended in pre-chilled PBS and then serially diluted in a ratio of 10. Plate 100 µl aliquots of each dilution onto LB agar plates, then invert and incubate or incubate at 37°C for 14-18 hours. Count the number of colonies for each dilution to determine CFU/ml, which corresponds to the reduction in the number of viable bacteria in the supernatant, which is an indirect measure of phagocytosis.

Quantitative PCR (qPCR)

Total RNA was extracted from Ad3 ESP-treated or untreated PMNs using RNAprep Pure Cell Kit (Tiangen, China) according to the manufacturer’s instructions. cDNA was generated using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to the manufacturer’s recommendations for oligonucleotide (dT) primer cDNA synthesis on 1 μg of RNA. Finally, qPCR was performed using SYBR Green qPCR Master Mix (TaKaRa, Japan) according to the manufacturer’s instructions. The primers used in qPCR are shown in Table 1. All F/R primer pairs were synthesized by Sangon Biotechnology. The total volume of the qPCR reaction mixture is 25 μl: 12.5 μl SYBR Green qPCR Master Mix (2 ×), 1 μl forward primer (10 μmol/l), 1 μl reverse primer (10 μmol/l), 2 μl template and 8.5 μl ddH₂O. The qPCR curve used is as follows: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of amplification at 95°C for 15 seconds, 56°C for 30 seconds, and 72°C for 15 seconds. All steps were performed using the Step One Plus real-time PCR system (Applied Biosystems, USA).The relative mRNA level of the target gene is calculated by 2^-ΔΔCT.

ELISA analysis of cytokine expression

PMNs were incubated with or without Ad3 ESP (10 ng/μl) for 3 h, and supernatants were harvested for cytokine analysis. IL-1β, IFN-γ, IL-10, IL-4 and TNF-α levels were measured using corresponding mouse ELISA kits (Invitrogen, USA) according to the manufacturer’s instructions.

Statistical Analysis

All experiments were performed in five replicates (n = 5) or sixfold (n= 6) and repeat 3 times. All results are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, USA). One-way analysis of variance (ANOVA) was used to compare differences between conditions. ask< 0.05 was considered statistically significant. askValues ​​are expressed as *ask< 0.05, **ask< 0.01, ***ask< 0.001.