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

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.

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.

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 suggest that a dose of 12 mg can cause excessive local inflammatory damage and systemic toxicity. Therefore, this dose was excluded from subsequent studies. The 8 mg dose was ultimately selected as the optimal dosing regimen because it could effectively induce significant coronary arteritis while maintaining an acceptable survival rate and minimal local adverse reactions. (Figure 1A). Eventually, it was determined that intraperitoneal injection of 8 mg CAWS was the optimal modeling dose. The HE staining results of the experimental group showed a typical dynamic process of inflammation and fibrosis. On the third day, focal inflammatory infiltration, mainly composed of perivascular neutrophils and monocytes, occurred. By the seventh day, the inflammatory range expanded to the myocardial interstitium, accompanied by significant myocardial cell swelling and interstitial edema. On the 14th day, the inflammation reached its peak, and myocardial fiber arrangement disorder and focal necrosis occurred. On the 28th day, although the inflammation was alleviated, early fibrosis had formed (Figure 1B and Figure 2A). In contrast, myocardial structure remained normal at all time points in the control group. All results were confirmed through a double-blind assessment, which verified that an 8 mg CAWS dose could stably induce a myocardial injury model consistent with the characteristics of Kawasaki disease.

Subsequently, the Masson tricolor staining method was employed to investigate whether CAWS-induced Kawasaki disease-like vasculitis would result in persistent fibrosis surrounding the coronary arteries (CA). The experimental results showed that in the CAWS injection group of mice, obvious collagen fiber deposits were detected around the walls of the inflamed coronary arteries and aorta, and these fibrotic areas highly coincided with the distribution locations of inflammatory cell infiltration. In contrast, mice in the PBS control group only showed weak collagen staining, which was confined within the normal structural range of the vascular wall (Figure 2A). Quantitative analysis showed that significant collagen fiber deposition occurred at 14 days, and the degree of fibrosis was statistically different from that of the control group (Figure 2C).

Given that the central pathological change in Kawasaki disease is the immune-inflammatory response of the vascular wall, which involves the coordinated action of multiple immune cells22,23, we next explored the characteristic changes of the immune microenvironment in the CAWS model through immunofluorescence (IF) staining. On the 14th day after CAWS injection, IF staining of immune cell markers was performed on the cardiac tissues of mice injected with CAWS. The experimental results showed that on the 14th day of modeling for mice in the CAWS treatment group, the infiltration of CD3+ T cells, CD8+ cytotoxic T cells, CD86+ M1 type macrophages, F4/80+ macrophages, and NK1.1+ natural killer cells in coronary arteries and myocardial tissues was significantly increased compared with the PBS control group (Figure 2B,D). By systematically analyzing the expression changes of these immune markers, it not only verified that the CAWS model could accurately simulate the immunological characteristics of human Kawasaki disease, but more importantly, it revealed the possible mechanism of different immune cell subsets in the occurrence and development of the disease, providing a theoretical basis for the subsequent development of targeted immune intervention strategies.

Research has established that mitochondrial dysfunction is a key mechanism underlying myocardial fibrosis24,25,26. It is recognized that under inflammatory stress, voltage-dependent anion channel 1 (VDAC1), a critical protein for mitochondrial quality control, can trigger abnormal opening of mitochondrial permeability transition pores (mPTP) when its expression is upregulated. This, in turn, induces apoptosis and activates fibroblasts17,27. To explore this mechanism, we assessed VDAC1 expression in the chronic phase using immunohistochemistry. Our findings revealed that, compared with the PBS control group, VDAC1 protein expression in the myocardial tissue of mice in the CAWS-treated group was significantly elevated 28 days post-modeling (Figure 3). Positive signals were predominantly localized in fibrotic areas and around blood vessels, indicating that VDAC1-mediated mitochondrial dysfunction may contribute to myocardial fibrosis in the Kawasaki disease model. These data provide experimental evidence regarding the molecular mechanisms of CAWS-induced myocardial injury.

Previous studies have found that VDAC1-mediated mitochondrial dysfunction is involved in the myocardial fibrosis process of Kawasaki disease, but its specific mechanism has not yet been clarified. Given that the maintenance of mitochondrial homeostasis depends on the normal function of the autophagy-lysosomal system. We speculate that VDAC1 may exacerbate fibrosis by influencing the formation or function of autophagolysosomes, leading to abnormal mitochondrial accumulation. To verify this hypothesis, we first established a Candida albicans spore infection model in macrophages (RAW264.7) (Figure 4) and detected the dynamic changes of autophagic flow and lysosomal activity in the model. The experimental results show that the formation of autophagy lysosomes increases in the early stage (within 2-3 h after cell infection), while autophagic flow is blocked in the later stage (3-4 h after cell infection). The change of autophagy flow can be evaluated by the change of merge staining after autophagosome and lysosome co-localization. This result confirms that dysfunction of autophagolysosomes can indeed lead to impaired cellular clearance.

Figure 1: Histopathological analysis of the heart and coronary arteries. (A) Survival curves of mice injected with PBS or different doses of CAWS (4 mg, 8 mg, and 12 mg; n = 20 per group). (B) The HE staining results of the heart in the CAWS treatment group (8 mg) at different time points (3 d, 7 d, 14 d, 28 d) were presented. Scale bars: 1500 µm. Please click here to view a larger version of this figure.

Figure 2: Analysis of collagen fibrosis and immune cell infiltration. (A) The Masson staining results of the CAWS 8 mg group and the control group. Collagen fibers present a characteristic blue color, muscle fibers and cytoplasm are red, and the cell nucleus is dark blue. Arrows indicate fungal spores. Scale bars: 1500 µm. (B) Immunofluorescence staining results of the CAWS 8 mg group and the control group(CD3 labeled CD3+ T cells, CD8 labeled CD8+ cytotoxic T cells, CD86 labeled CD86+ M1 type macrophages, F4/80 labeled F4/80+ macrophages, and NK1.1 labeled NK1.1+ natural killer cells). Scale bars: 200 µm. (C) Assess the degree of cardiac fibrosis. The degree of cardiac fibrosis was evaluated based on the percentage of trichromatic staining area in the heart tissue of the visual field at 800x magnification. The specific method is as follows: Because collagen fibers are dyed bright green with macromolecular anionic dyes, 100 visual fields at 800 times magnification (all slices with a thickness of 5 µm) were observed, and the degree of cardiac fibrosis was determined by the percentage of green area in the total area. The higher the percentage, the more severe the fibrosis. (D) The percentage of immune cells was statistically analyzed according to the results of immunofluorescence staining,***p < 0.001. Please click here to view a larger version of this figure.

Figure 3: Immunohistochemical analysis of VDAC1 expression in the PBS group and CAWS group on day 28. (A) Immunohistochemical analysis of VDAC1 expression. Scale bar: 800 µm. Brown black particles are positive results. (B) Statistical analysis of the percentage of VDAC1 immunohistochemically positive cells, ***p < 0.001. (C) Statistical analysis of the H-score of VDAC1 immunohistochemistry. The data were derived from multiple biological replicates (n = 20 mice per group), and the results were expressed as mean ± standard deviation and statistically analyzed (***p < 0.001). Please click here to view a larger version of this figure.

Figure 4: Detection of autolysosome after the phagocytosis of Candida albicans by mouse RAW264.7 cells. (A) Arrows indicate fungal spores. Scale bars: 25 µm. The change of autophagy flow can be evaluated by the change of merge staining after autophagosome and lysosome co-localization. (B) A time-course analysis of the autolysosome formation rate quantifies relevant parameters. The results were expressed as mean ± standard deviation and statistically analyzed (***p < 0.001). Please click here to view a larger version of this figure.

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 KD patients and the difficulty in clinical research to dynamically track the long-term changes of coronary arteries and myocardium, there is an urgent need for an animal model that can simulate the entire process of KD from acute vasculitis to chronic fibrosis to reveal its underlying mechanisms and explore intervention strategies^16,30.

In this study, a mouse vasculitis model induced by CAWS was adopted to systematically simulate the dynamic evolution process of the acute inflammatory phase and chronic fibrotic phase of KD. The establishment of this model provides a new perspective for studying the immunopathological mechanism of Kawasaki disease. Through immunofluorescence staining, we observed that in the CAWS-induced model, the infiltration of various immune cells in coronary arteries and myocardial tissues significantly increased, which was highly consistent with the pathological characteristics of human KD^22,31. Furthermore, our research indicates that there is significant perivascular collagen deposition during the chronic phase, suggesting progressive fibrosis. This fibrotic remodeling is a key long-term complication of Kawasaki disease, mainly caused by excessive activation of myofibroblasts. Myofibroblasts play a crucial role in coronary artery remodeling in Kawasaki disease. Targeting the activation, proliferation, or induction of apoptosis of myofibroblasts may become a highly promising new therapeutic strategy for alleviating the long-term fibrotic sequelae of Kawasaki disease.

This research further revealed that CAWS-induced coronary arteritis is accompanied by upregulation of VDAC1 expression. VDAC1 is a core regulatory molecule of mitochondrial apoptosis. Previous studies have shown that overexpression and oligomerization of VDAC1 can promote the collapse of mitochondrial membrane potential and the release of cytochrome C, thereby initiating a caspase-dependent apoptotic cascade^32,33,34,35. The driving role of apoptotic cells in the process of fibrosis has been fully confirmed. Apoptotic cardiomyocytes release pro-fibrotic signals such as damage-associated molecular patterns (DAMPs) and TGF-β, continuously activating fibroblasts and leading to excessive extracellular matrix deposition and pathological scar formation³⁶. Therefore, it is necessary to deeply explore the causal relationship between cardiomyocyte apoptosis and VDAC1 overexpression in the future. By leveraging cardiomyocyte-specific VDAC1 knockout models, new intervention targets can be sought to delay KD fibrosis. Subsequently, a Candida albicans spore infection model was established in the macrophage cell line RAW264.7. By detecting the dynamic changes of autophagy flow and lysosomal activity, it was found that the formation of autophagy lysosomes increased in the early stage, while autophagy flow was blocked in the later stage. This suggests that the dysfunction of the autophagy-lysosomal system in macrophages may lead to abnormal mitochondrial clearance disorders³⁷. The obstruction of autophagy flow occurs earlier than the peak expression of VDAC1, suggesting that autophagy deficiency may be a driving factor for mitochondrial damage. These findings contribute to the understanding of the fibrotic mechanisms in diseases such as Kawasaki disease, suggesting that targeting VDAC1 or the autophagy process may become a potential therapeutic strategy.

This study standardizes the preparation process of CAWS and optimizes the administration regimen. The dose selection of 8 mg per day balanced the pathological changes and survival rate. Despite these advancements, several limitations of our study should be acknowledged. First, although this model can simulate coronary artery lesions in human KD, it lacks common systemic symptoms such as rashes and mucosal lesions in human KD. Second, the connection between VDAC1 upregulation and fibrosis, although supported by indirect evidence, still requires further direct experimental verification. Future research will quantify apoptosis through TUNEL staining and combine it with co-localization analysis of fibroblast markers to reveal the key role of apoptotic events in the initiation and evolution of fibrosis. Third, key mechanistic aspects remain unresolved and require further investigation. The precise role of VDAC1 in cardiomyocytes needs to be verified using myocardial-specific knockout models; likewise, the causal relationship between autophagy deficiency and mitochondrial damage warrants additional experimental clarification. Future research can further explore other potential biomarkers and therapeutic targets to deepen the understanding of the pathological changes and intervention strategies in the chronic phase of KD.