Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Summary

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Abstract

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model. P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 10⁷ bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal P. gingivalis infection.

Introduction

Microvesicles (MVs), also termed microparticles or ectosomes, are plasma membrane fragments with a diameter of 0.1-1.0 µm released in body fluids and the extracellular space in response to various cell stimuli. First identified as platelet dust exposing procoagulant phospholipids, mostly phosphatidylserine (PSer), MVs constitute an additional surface for the assembly of the blood coagulation complexes^1,2. The key role of circulating MVs as procoagulant effectors has been demonstrated in patients with Scott syndrome², a rare genetic disease that leads to dysfunctional PSer exposure ....

Protocol

All experimental protocols were approved by and followed the relevant guidelines of the local Ethics Committee (APAFIS#28745-2020121815051557) and animal care of the home University and of the INSERM. Male Young C57BL/6 mice, 6-8 weeks of age, were used for the experiments. Mice were regularly examined to evaluate pain and stress, and their weights were monitored daily. Unless otherwise stated, all extracting buffers and solutions must be sterile and at room temperature.

1. Animal preparation

1. Administer the mice with six intraperitoneal injections of P. gingivalis (PG) every 2 days for 2 weeks (three in....

Results

The data provided give a full representation of the whole procedure, using two main animal conditions: control untreated young C57BL/6 mice and their littermates subjected to six intraperitoneal (IP) PG injections every 2 days, for 2 weeks. They also show the remote action of an intraperitoneal PG injection on the spleen response after 2 weeks. Splenocyte microvesicles were quantified by either prothrombinase enzymatic assay or by measuring their proteins and RNA concentration by spectrophotometric, and the proportion of.......

Discussion

The present study confirms that the spleen is a major and reliable source of MVs with physiopathological relevance compared to other sources like blood, of limited volume in mice. Provided precautions are taken, the method is easy to set up and does not require expensive equipment. Since no alternate way other than in vivo assessment is available, the current model appears to be a valuable method to study the impact of pro-drugs on MV shedding. Importantly, the standardized protocol for harvesting murine splenic.......

Materials

Name	Company	Catalog Number	Comments
2 mL tubes type Eppendorf	Dutscher	54816	Conical bottom stériel microtubes
Allegra 64 R Centrifuge	Beckman Coulter
Automatic cell counter	Biorad
Bovine serum albumin	Euromedex	04-100-812-E	Prepared, filtered with 0.22 µm sieve and stored at 4 °C under sterile conditions by using the following formulas: 2 mM EDTA, 0,5% BSA and sterile PBS
CD11 (Mac-1)	e-Biosciences	45-00112-80	Conjugated to eFluor 450; λmax excitation 405 nm λmax emission 445 nm
CD16/32	BD Biosciences	553142	unconjugated
EDTA	Calbiochem	Calbiochem	S 6381-92-6
Falcon tube	Cell star	227261	50 mL
Fetal Bovine serum	Dutscher	S1810-500	Batch number = S14028S1810
Fortessa Aria	BD Biosciences		for cell sorting
Fortessa flow cytometer	Becton-Dickinson.
Fungizone	PAN biotech	P06-01050
HBSS	Gibco	14175-053	Without phenol red, without Ca+2 and Mg+2
ICAM-1	abcam	ab171123
LYG-6 (Gr-1)	BD Biosciences	566218	Conjugated to BUV395; λmax excitation 348 nm, λmax emission 395 nm
Lysis buffer erythrocytes (ACK)	Sigma		Prepared, filtered with 0.22 µm sieve and stored at 4°C under sterile conditions by using the following formulas: NH4Cl, 0.15 M (molarity), 53.491 (mw) 4 g KHCO3 1 mM (molarity) 100.115 (mw), 50 mg EDTA  0.1 mM (molarity), 292.24 (mw), 14.6 g pH: 7.2–7.4
NanoDrop 1000 spectrophotometer	Thermoscientific
PBS	Lonza	17-516F	Without Ca+2  and Mg+2
Plastic petri dish			100 mm
Polystyren tube	Falcon	352070
q-Nano Gold	iZON science
RPMI 1640 culture medium: 2 g/L glucose	PAN biotech	p04-18047	Supplemented withsupplemented with Streptomycin (100 U/mL) /Penicillin (100 U/mL), Fungizone (250 mg/mL), L-glutamine (2 mM) and FBS 10%.
Scalpels
Sieve Nylon	Falcon USA	352360	100 µm
Streptomycin/Penicillin	PAN biotech	P06-07100
Syringe			2 mL
Trypan Blue	Biorad	1450013
VCAM1	abcam	ab215380