Characterization of Inflammatory Factors and T Cell Subpopulations in a Murine Model of Kawasaki Disease Induced by Candida albicans Cell Wall Extracts (CAWS)

Abstract

Background: Kawasaki disease (KD) is an acute vasculitis in children, which ranks as the main cause of acquired heart disease in children in developed countries. The etiopathogenesis of KD remains to be clarified. Our study constructed a KD murine model and monitored the alterations of inflammatory factors and T cell subpopulations.

Material/Methods: Candida albicans cell wall extracts (CAWS) were utilized for inducing KD murine models. After 5 days, the mice were subcutaneously injected with rhG-CSF lasting 5 consecutive days. At 4 weeks, histopathology of hearts and arteries was observed via H&E staining. Inflammatory cytokine, chemokine, and adhesion molecule expression in serum specimens was detected via RT-qPCR and ELISA. T cell subpopulations in cardiac tissues were labeled through immunofluorescence and flow cytometry.

Results: CAWS induced cardiac dysfunction (reduced fraction shortening, increased left ventricle end-diastolic diameter and heart weight/body weight ratio) and cardiac and vascular inflammation, which were ameliorated by rhG-CSF treatment. CAWS-induced mice had increased levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), chemokine (RANTES), and adhesion molecules (MCP-1, VCAM-1, ICAM-1, E-selectin), as well as the decreased levels of anti-inflammatory cytokines (IL-10, G-CSF). Their levels were decreased following rhG-CSF administration. There were decreased CD3+, CD4+, CD8+, CD8+CD45+, Foxp3+, CD25+ and Foxp3+CD25+ T lymphocyte subpopulations and an increased CD45+ T lymphocyte subpopulation in cardiac tissues of CAWS-induced mice, which were ameliorated by rhG-CSF administration.

Conclusions: Our findings reveal the characteristics of inflammatory factors and T cell subpopulations in CAWS-induced KD. Moreover, rhG-CSF might be an effective therapeutic regimen against KD.

Figure 1

The CAWS-induced murine model of KD with cardiac dysfunction, cardiac and vascular inflammation. (A) Fraction shortening, left ventricle end-diastolic diameter (LVEDD) and heart weight/body weight ratio were examined at 4 weeks after rhG-CSF treatment in CAWS-induced mice. **** p<0.0001. (B, C) The sections of hearts and arteries were stained at 4 weeks following rhG-CSF administration in CAWS-induced mice. Black arrow points to the indicated lesion location. Bar=50, 20 or 200 μm. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) and Olympus software (version 2.2; Olympus, Japan) were used to create the pictures.

Figure 2

The CAWS-induced murine model of KD with the activation of pro-inflammatory cytokines and the inactivation of anti-inflammatory cytokines. (A) ELISA and (B) RT-qPCR were utilized for quantifying the serum levels of inflammatory cytokines IL-1β, IL-6, IL-10, G-CSF and TNF-α at 4 weeks following rhG-CSF administration in CAWS-induced mice. * p<0.05; *** p<0.001; and **** p<0.0001. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) was used to create the pictures.

Figure 3

The CAWS-triggered murine model of KD with the activation of chemokines and adhesion molecules. (A) ELISA and (B) RT-qPCR were utilized for quantifying the serum levels of MIP-1α, RANTES and MCP-1 at 4 weeks after rhG-CSF administration in CAWS-induced mice. VCAM-1, ICAM-1 and E-selectin expressions were examined in serum specimens from CAWS-induced mice at 4 weeks after rhG-CSF administration. *** p<0.001; and **** p<0.0001. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) was used to create the pictures.

Figure 4

The CAWS-induced KD mice with reduced CD4+, CD8+, and Foxp3+ T lymphocyte subpopulations in cardiac tissues. (A–C) Cardiac tissues were stained by (A) CD4+, (B) CD8+, and (C) Foxp3+ T lymphocyte subpopulations at 4 weeks after rhG-CSF administration in CAWS-induced mice. Bar=20 μm. * p<0.05; *** p<0.001; and **** p<0.0001. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) and Olympus software (version 2.2; Olympus, Japan) were used to create the pictures.

Figure 5

The CAWS-induced murine model of KD with reduced F4/80+ macrophages, CD11b+ monocytes and increased Ly-6G+ neutrophils in cardiac tissues. (A–C) Cardiac tissues were stained by (A) F4/80+ macrophages, (B) CD11b+ monocytes, and (C) Ly-6G+ neutrophils at 4 weeks after rhG-CSF administration in CAWS-induced mice. Bar=20 μm. * p<0.05; ** p<0.01; *** p<0.001; and **** p<0.0001. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) and Olympus software (version 2.2; Olympus, Japan) were used to create the pictures.

Figure 6

The CAWS-induced murine model of KD with decreased CD3+, CD8+ and CD8+CD45+ T lymphocyte subpopulations and increased CD45+ T lymphocyte subpopulation. (A, B) The levels of CD3+, CD8+, CD45+ and CD8+CD45+ T lymphocyte subpopulations were examined in serum of CAWS-induced mice at 4 weeks after rhG-CSF administration. * p<0.05; ** p<0.01; *** p<0.001. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) was used to create the pictures.

Figure 7

The CAWS-induced murine model of KD with decreased CD4+, Foxp3+, CD25+ and Foxp3+CD25+ T lymphocyte subpopulations. (A, B) The levels of CD4+, Foxp3+, CD25+ and Foxp3+CD25+ T lymphocyte subpopulations were examined in serum of CAWS-induced mice at 4 weeks after rhG-CSF administration. * p<0.05; ** p<0.01; *** p<0.001. GraphPad Prism software (version 8.0.1; Graph Pad; San Diego, CA, USA) was used to create the pictures.