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 disease in children^6,7. Although the application of intravenous immunoglobulin has significantly improved prognosis, its etiology and pathogenesis remain unclear, which restricts the development of targeted therapeutic strategies^8,9. Therefore, establishing animal models that can accurately simulate the characteristics of human diseases has become an urgent need for current research.

Currently, a major obstacle in Kawasaki disease research is the absence of well-characterized animal models that fully recapitulate human disease pathology. Among the various models developed now, the vasculitis model induced by Lactobacillus casei cell wall extract (LCWE) is a relatively mature system, and this model can cause coronary arteritis. It is widely used to study the mechanism of immune dysregulation and specific cytokines in KD-like vasculitides^10,11. The vasculitis model induced by the water-soluble extract of Candida albicans (CAWS) has also attracted much attention due to its high similarity to the pathological features of human Kawasaki disease^12,13. After systematic optimization and improvement by multiple research teams, the CAWS-induced model has developed into an important tool for Kawasaki disease research¹⁴. Although CAWS can induce coronary artery inflammation via intraperitoneal injection, it has limitations in that it cannot fully reproduce the exact pathological process of human KD vasculitis. For example, no neutrophils were found in the late pathology of human KD¹⁵, but neutrophil infiltration still occurred in this model up to 16 weeks after CAWS injection¹⁶. Moreover, the mechanism of vasculitis caused by CAWS has not been fully clarified at present, which limits the in-depth understanding and application of the model⁹. This study aims to establish a standardized animal model of KD by optimizing the induction protocol of CAWS, elucidating the disease mechanism, and facilitating the development of targeted therapies.

This study utilized CAWS to establish a more standardized animal model of Kawasaki disease. Through systematic dose optimization experiments, it was determined that intraperitoneal injection of 8 mg daily for five consecutive days was the optimal administration regimen. This regimen can stably induce coronary artery lesions while maintaining a high survival rate in animals. In addition, we further explored the role of mitochondrial dysfunction in the formation of fibrosis during the chronic phase of KD, with a focus on the possible mechanism of voltage-dependent anion channel 1 (VDAC1), a key protein regulating mitochondrial apoptosis, during the transition from inflammation to fibrosis¹⁷. It is worth noting that, through autophagosome/lysosome co-localization analysis in this study, abnormal autophagy function was observed in this model. This optimized model provides an important tool for systematically studying the pathogenesis of coronary artery lesions in Kawasaki disease and evaluating new treatment strategies.

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, the anaerobic culture condition at 37 °C must be strictly controlled to prevent contamination by miscellaneous bacteria.
2. Rinse repeatedly with C-limiting medium and incubate at 37 °C for 48 h at a rotational speed of 4.5 x g.
3. Add an equal volume of anhydrous ethanol and let it stand overnight at 4 °C.
4. Centrifuge at 4.5 x g for 15 min at 4 °C, remove the supernatant, add an equal volume of anhydrous ethanol to the precipitate, and leave it overnight at 4 °C.
5. After centrifugation, discard the supernatant, add 50 mL of acetone to the precipitate, centrifuge, and dry the precipitate. Dissolve the precipitate in sterile normal saline for later use.

2. Constructing a mouse model of Kawasaki disease

1. Select eighty SPF-grade male C57BL/6 mice aged 4-6 weeks, and house them under controlled conditions with free access to food and water.
   NOTE: To minimize the potential interference of estrogen level fluctuations on immune and inflammatory responses, only male mice were used in this preliminary model establishment and characterization study¹⁹.
2. Divide mice into 4 groups evenly by random number table method, including 3 different dose CAWS injection groups (4 mg, 8 mg, 12 mg) and 1 normal saline control group, with 20 mice in each group.
3. Prepare CAWS powder into three concentrations of 40 mg/mL, 80 mg/mL, and 120 mg/mL using sterile normal saline (0.9% NaCl). Inject the experimental group with 0.1 mL of CAWS in the morning from the 1st to the 5th day of the experiment, and administer an equal volume of normal saline to the control group on the same schedule.
   NOTE: Intraperitoneal injection was performed using a 1 mL insulin syringe. When operating, position the mouse with its head lower and tail higher to prevent damage to organs such as the large and small intestines when the syringe is inserted.
4. Use an electronic balance at 9 a.m. every day to simultaneously weigh the remaining feed in the feeder and calculate the 24-h food consumption for each cage of mice.
5. On the 3rd, 7th, 14th, and 28th days after the last injection, randomly select and sacrifice 3 mice from each group at each time point.
6. Place mice in a closed chamber prefilled with room air. Introduce compressed CO₂ at a flow rate of 30% of the chamber volume per minute. After 5 min, cervical dislocation was performed to confirm death.
7. Collect and weigh heart specimens. Observe inflammatory lesions of the heart and blood vessels by HE staining.

3. HE staining and grading standards

1. The heart and aortic arch (about 3 mm × 3 mm) were rapidly isolated. Perform a median sternotomy according to the described method²⁰. Quickly separate the heart from the aortic arch (about 3 mm × 3 mm). The collected tissues were fixed in 10% neutral buffered formalin for 24 h.
2. After fixation, dehydrate the tissues through a graded ethanol series (70% ethanol for 1 h, 80% ethanol for 1 h, 95% ethanol for 1 h, and 100% ethanol for 1 h), clear in xylene, and embed in paraffin. Finally, section the blocks into 5 µm continuous slices.
3. For H&E staining, stain the sections with hematoxylin for 5 min, rinse in running water for 10 min, counterstain with eosin for 2 min, and then proceed with dehydration and mounting²¹.
   NOTE: According to the degree of vascular intimal injury, it is classified into three grades: Grade I is mainly characterized by endothelial swelling and neutrophil aggregation; Grade II is manifested as smooth muscle degeneration, inflammatory cell infiltration, and organelle damage; Grade III presents with severe lesions such as endothelial necrosis and shedding, and fibrin deposition.

4. Masson trichrome staining

1. After fixation, dehydrate the tissue samples through a graded ethanol series, clear in xylene, and embed in paraffin.
2. After dewaxing, hydrate the sections through a graded ethanol series to distilled water in preparation for staining.
3. Stain the nuclei with Weigert's iron hematoxylin for 5 min, rinse thoroughly under running water, and then stain the cytoplasm with Lichun Red acid fuchsin for 5 min.
4. Differentiate with 1% phosphomolybdic acid for 1 min, then directly stain the collagen fibers with aniline blue for 3 min. Rinse rapidly with 1% glacial acetic acid.
   NOTE: Strictly control the timing of the differentiation step to ensure staining specificity.
5. After dehydration with ethanol and xylene, becoming transparent, seal the neutral film.
6. Rinse the stained sections under running water to remove excess dye.
7. Dehydrate the sections through a graded ethanol series, clear in xylene, and mount with neutral gum.
8. Observe the sections under a light microscope at consistent magnification. Collagen fibers should appear blue, muscle fibers and cytoplasm red, and nuclei deep blue.

5. Immunofluorescence staining

1. Fix cell or tissue sections, and block with 5% BSA for 1 h to reduce non-specific binding.
2. Use normal serum of the same species origin as the secondary antibody, dilute it with PBS at a ratio of 1:10, and drop it onto the sample. Seal it at room temperature for 30 min. After sealing, gently rinse twice with PBS.
3. Incubate the sections with the primary antibody overnight at 4 °C. After washing with PBS, add the fluorescent secondary antibody and incubate in the dark for 1 h.
4. Staining the core with DAPI, sealing with anti-quenching mounting agent, and observing under confocal microscopy.

6. Immunohistochemistry

1. After dewaxing and hydration of paraffin sections, carry out antigen repair and block the endogenous peroxidase.
2. Antibody Incubation and Color Development: First, block with normal serum, then incubate the primary antibody and HRP-labeled secondary antibody in sequence, and finally, DAB color development. Control the reaction degree under a microscope.
3. Re-stain the cell nuclei with hematoxylin. After dehydration and transparency, seal the plates and observe the brownish-yellow positive signal under a microscope.
   NOTE: The experiment needs to set up controls and strictly control the conditions for restoration and color development.

7. Autolysosome detection

1. Co-culture RAW264.7 macrophages with Candida albicans spores in an appropriate proportion (10:1), and incubate at 37 °C and 5% CO₂ for 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, and 48 h to establish a phagocytic model.
2. Wash three times with pre-cooled PBS to remove the unphagocytic spores.
3. First, block the samples with 5% BSA for 30 min. Then, incubate successively with the primary antibody against LC3 (1:200 dilution) at room temperature for 1 h, followed by the Alexa Fluor 488-labeled secondary antibody at room temperature for 30 min.
4. Add Lyso-Tracker Red lysosome probe directly to the cells to visualize lysosomal localization. Finally, counterstain the nuclei with DAPI (1 µg/mL) for 5 min.
   NOTE: The entire operation process should be carried out in the dark to ensure that the incubation time and concentration of the antibody are accurately controlled.

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