Establishment and Evaluation of a Candida albicans Water-Soluble Extract-Induced Murine Model of Kawasaki Disease-Associated Coronary Arteritis

Summary

The CAWS-induced mouse model effectively simulates the progression of Kawasaki disease from acute inflammation to chronic fibrosis, revealing key pathological and immunopathological features, and may facilitate the development of targeted therapeutic strategies for Kawasaki disease.

Abstract

Kawasaki disease (KD) is a systemic vasculitis primarily affecting children, with coronary artery lesions being its most severe complication. In this study, an optimized mouse KD model was established using the water-soluble extract of Candida albicans (CAWS). Myocardial inflammation and related pathological changes were evaluated using HE staining and Masson trichrome staining. The immunofluorescence technique detected the infiltration of immune cells in cardiac tissue. The expression and localization of VDAC1 protein in myocardial tissue were detected by immunohistochemistry. In vitro, a phagocytic model was established by co-culturing RAW264.7 macrophages with Candida albicans spores, and the formation and function of autophagolysosomes were assessed using LC3 immunofluorescence staining and a Lyso-Tracker Red probe. Through dose screening, it was determined that 8 mg was the optimal modeling dose for inducing coronary artery inflammation, with a moderate mortality rate at this dose. HE staining showed that CAWS injection stably induced coronary artery lesions consistent with the characteristics of human Kawasaki disease in mice. Masson staining confirmed that there was significant collagen fiber deposition around the coronary arteries and aorta in the CAWS group of mice, which closely coincided with the inflammatory area, and a statistically significant difference was observed from the control group at 14 days (p < 0.001). Immunofluorescence revealed that, on the 14th day of modeling, the infiltration of multiple immune cells in the cardiac tissue of the CAWS group had significantly increased (p < 0.001). The immunohistochemical results showed that, on the 28th day of modeling, the expression of VDAC1 protein in the myocardial tissue of the CAWS group was significantly upregulated (p < 0.001). In vitro experiments have shown that in macrophages infected with Candida albicans spores, the formation of autophagolysosomes increases in the early stage, while autophagic flow is blocked in the later stage, suggesting a functional disorder.

Introduction

Kawasaki disease (KD), a form of mucocutaneous lymph node syndrome, is an autoimmune disease that occurs in children under 5 years old and is accompanied by febrile vasculitis^1,2,3. Studies indicate that untreated or treatment courses exceeding 10 days of severe KD are prone to induce serious cardiovascular complications, mainly including coronary aneurysms and coronary artery stenosis^4,5. The rupture of coronary aneurysms may lead to cardiogenic shock or even sudden death, which is the main cause of acquired heart di....

Protocol

All research experiments involving animal data were approved by the ethics committee of the Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University (KY-2024-250-01). The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of CAWS

1. Inoculate Candida albicans in Sabouraud dextrose broth according to the established protocol by Duong et al.¹⁸. Then, culture the inoculated broth anaerobically at 37 °C for 48 h in a sealed anaerobic chamber with a gas mixture.
   ​NOTE: To ensure the sterility of the culture medium....

Results

Initially, we systematically evaluated the myocardial pathological changes induced by different doses of CAWS in mice. No deaths were observed in the PBS control group, the 4 mg CAWS group, and the 8 mg CAWS group (n=20 in each group). In contrast, the inflammatory response in the 4 mg group was mild and did not meet the modeling requirements. The mortality rate was higher in the 12 mg CAWS group (9/20, 45%), and deaths occurred from the 3rd to the 10th day after injection. These findings.......

Discussion

Coronary artery aneurysms (CAA) and myocardial fibrosis in KD are the main causes of long-term cardiovascular events. Despite the progress made in acute phase treatment, approximately 5% of patients still develop persistent CAA^28,29. The mechanism of this delayed pathological change is still unclear, and there is an urgent need for animal models to reveal its dynamic process³⁰. Due to the extremely limited cardiac tissue samples of human K.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
a goat secondary antibody to Rabbit IgG Alexa Fluor 488	abcam	ab15081	a goat secondary antibody to Rabbit IgG Alexa Fluor 488
Anaerobic chamber	Thermo Scientific	Thermo Scientific	Anaerobic chamber
Aniline blue	Solarbio	G3668	Aniline blue
anti-LC3	abcam	ab192890	the primary antibody against LC3
Biological safety cabinet	Thermo Scientific	1500	Biological safety cabinet
BSA	Solarbio	A8020	BSA
Candida albicans (strain)	NBRC	1385	Candida albicans (strain)
CD3e	BD Bioscience	561827	FITC Hamster Anti-Mouse CD3e(145-2C11)
CD86	BD Bioscience	105013	CD86
CD8a	BD Bioscience	100713	CD8a
Cell culture incubator	Thermo Scientific	311	Cell culture incubator
Centrifuge	Thermo Scientific	ST4R Plus	Centrifuge
Confocal microscope	Olympus	IX73	Confocal microscope
DAPI	Beyotime Biotechnology	P0131-25ml	DAPI
DMEM	Gibco	11965126	DMEM
Embedding machine	P.S.J MEDICAL	BM450A	Embedding machine
Eosin	Solarbio	G1100	Eosin
F4/80	BD Bioscience	123109	F4/80
FBS	Gibco	16000044	FBS
Formaldehyde	Solarbio	P1110	Formaldehyde
Fully automatic tissue dehydrator	Leica Biosystems	ASP3005	Fully automatic tissue dehydrator
Glass microscope slides	Citotest	250124A1	Glass microscope slides
H&E dye	Beyotime Biotechnology	C0105M	H&E dye
IHC Kit	Absin Biotechnology	abs996-5ml	IHC Kit
LC3 probe	Beyotime Biotechnology	C3018M	LC3 probe
Low Profile Microtome Blades	Thermo Fisher	3052835	Low Profile Microtome Blades
lysosome probe	Beyotime Biotechnology	C1046	lysosome probe
Marker pen	Deli	SK109	Marker pen
Masson dye	Beyotime Biotechnology	C0189M	Masson dye
Microtome	Leica Biosystems	HistoCore BIOCUT	Microtome
Neutral gum	Solarbi	G8590	Neutral gum
NK1.1	BD Bioscience	561117	NK1.1
Optical microscope	Nikon	Nikon	Optical microscope
Paraffin	Solarbio	YA0012	Paraffin
Paraffin wax	Solarbio	YA0012	Paraffin wax
PBS	Solarbio	P1020	PBS
Phosphomolybdic acid	Solarbio	G3472	Phosphomolybdic acid
VDAC1	Abcam	ab34726	Anti-VDAC1