Zhaoxu Chen
| Zhaoxu Chen |
Zhaoxu Chen, Kang I Ko
Periodontics, University of Pennsylvania, School of Dental Medicine
Introduction
Prior study demonstrated that Prx1+ oral fibroblasts are essential for expedited wound healing and that they preferentially differentiate towards SCA1+ subpopulation. Differential gene expression analysis of SCA1+ fibroblasts revealed up-regulation of monocyte/neutrophil chemotactic genes such as Ccl2, Cxcl1 and Ccl7. Here, we aim to validate these findings at the protein level and hypothesize that SCA1+ oral fibroblasts are the major source of chemokines such as CCL2, CXCL1 and CCL7 in steady state and in wound healing.
Methods
CCL2-mCherry reporter mice were used to validate expression of Ccl2 in SCA1+ fibroblasts. Additionally, flow cytometry was carried out on unwounded and day 2 oral wound models to examine expression of CCL2 in SCA1+ oral fibroblasts (CCL2+PDGFRα+SCA1+) and non-fibroblasts (CCL2+PDGFRα-SCA1+) under homeostatic and wounded conditions. Wildtype B6 mice were used to analyze of CXCL1 and CCL7 expression under homeostatic conditions. CXCL1 expression was analyzed through immunofluorescence staining with CXCL1 antibody and quantified by measuring mean fluorescence intensity (MFI). CCL7 expression was examined via flow cytometry to determine the proportion of CCL7+ cells in fibroblasts and non-fibroblasts. N = 3-5 animals were used for analysis in each group. Statistical significance was determined at p<0.05 via student’s t-test or 2-way ANOVA.
Results
CCL2 -mCherry expression were shown to be localized in the submucosal layer of the palate, which corresponded to the anatomic niche of SCA1+ oral fibroblasts in the palatal gingiva. Flow cytometry analysis showed significantly more SCA1+ fibroblasts expressed CCL2 than SCA1- fibroblasts (p<0.001) in unwounded condition, which further increased upon wound induction. In contrast to CCL2, CXCL1 expression was highly upregulated in the epithelial layer of the gingiva (p<0.0001), with an average epithelial MFI of 49.91±2.71 compared to laminal propria MFI of 10.96±1.16. When CCL7 expressing gingival cells were examined by flow cytometry, 66.63±6.57% of CCL7+ cells were non-fibroblasts (p<0.01). However, majority of the CCL7+ fibroblast was SCA1+ (83.37±2.35%).
Conclusion
Our results demonstrate that SCA1+ oral fibroblasts are the major source of CCL2 but not CXCL1 or CCL7. The findings point to a potential role of SCA1+ oral fibroblasts during wound healing in modulation of monocyte and macrophage recruitment, as CCL2 is a potent chemokine for these cell types.