DKK3 driven Wnt pathway in radiation-induced inflammatory and fibrosing skin injury

Li, Li (2023): DKK3 driven Wnt pathway in radiation-induced inflammatory and fibrosing skin injury. Dissertation, LMU München: Fakultät für Biologie

Abstract

Radiation dermatitis is a common side effect of cancer radiotherapy. To some extent radiation dermatitis occurs in many patients, in particular if the lesion is close to the skin. The skin affections can range from mild and transient redness to severe inflammation, fibrosis, ulceration or necrosis, and may severely reduce the patients’ quality of life. The treatment of chronic radiation dermatitis and fibrosis is difficult, underscoring then unmet need for novel targets and treatment strategies. Previous studies have shown that the canonical Wnt signaling pathway plays an important role in driving fibrosis and represents a potential target for the treatment of radiationinduced dermatitis and fibrosis. The soluble factor Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) modulates canonical Wnt activity in a cell-specific manner. Our group’s previous study (non-radiation) in inflammatory renal fibrosis suggested that epithelial DKK3 may activate canonical Wnt signaling, and through this, play a profibrotic and immune-polarizing role in renal fibrosis. Moreover, our group’s pilot studies of irradiated skin suggested that a global knockout of DKK3 may partially protect mice from radiation-induced hyperplasia and skin fibrosis. It was thus hypothesized for this thesis that following radiation damage, DKK3 expression in concert with canonical Wnt activation are linked to a compensatory hyper reactive epidermal hyperplasia, inflammatory infiltration and fibrosis. To test this hypothesis, a model of radiation-induced skin hyperplasia and inflammatory fibrosis was developed based on previous experiments of the group investigating radiation-induced lung fibrosis. A single dose of 20Gy radiation delivered to C57BL/N wildtype mice was found to induce reproducible chronic radiation dermatitis. Four to eight weeks after radiation, significant hyperplasia, myeloid infiltration and fibrosis was seen in irradiated skin as compared to non-irradiated controls. To identify the relevant cellular source of DKK3, and to monitor the cellular activity of canonical Wnt signaling in irradiated mouse skin, a DKK3 promoter based, and canonical Wnt (TCF/LEF) dual reporter mouse (DKK3-LCh x TCF/LEF) was used. The activation of DKK3 expression and Wnt pathway activity was evaluated following a single dose of 20Gy. At 6 days and 14 days after radiation, increased DKK3 expression and canonical Wnt pathway activity was detected in basal keratinocytes of irradiated skin and in many instances was found to be coincident. To investigate the effect of targeted DKK3 depletion on radiation-induced dermatitis and inflammatory fibrosis in vivo, a series of DKK3 tissue-specific knockouts were evaluated following radiation injury. Both DKK3 global and DKK3 keratinocyte specific knockout mice were shown to be significantly protected from epithelial hyperplasia and fibrosis. Interestingly, both the DKK3 global knockout and DKK3 keratinocyte specific knockout mice showed even more myeloid infiltration at the radiation site than the already increased infiltration seen in WT mice. The results suggested that keratinocyte expression of DKK3 and accompanying activation of canonical Wnt pathway signaling may be linked to a tissue milieu conducive toward the establishment of a pro-fibrotic environment. By contrast, the absence of DKK3 expression by keratinocyte was linked to a non-fibrotic tissue environment. To evaluate the myeloid phenotypes seen in the presence or absence of DKK3, cells were isolated from the skin of both DKK3 global knockouts and DKK3 keratinocytespecific knockouts and compared to the cells present in WT mice skin at 4 weeks after radiation. FACS analysis of leukocyte and myeloid sub-group markers was employed to characterize the phenotype of the extracted cells. Both DKK3 global knockout and the keratinocyte-specific knockout mice exhibited a decreased CD206+ and CD163+ surface marker expression on macrophages that are known to represent M2-like profibrotic phenotypes in both human and mouse settings. To better characterize the potential effect of DKK3 on the keratinocyte secretome with accompanying effects on myeloid differentiation and polarization, a chemokine/growth factor array and RT-qPCR analysis was performed on human keratinocytes (N/TERT-1) after DKK3 modulation. The N/TERT-1 cell line was engineered using an in house developed vector platform using a doxycycline-inducible DKK3 over expression plasmid. DKK3 knockdown performed via siRNA transfection was found to increase expression of the M1-like phenotype activation factor of GM-CSF, and also increased expression of the proinflammatory factors CXCL8, CXCL10, and TNF-α, while over expression of DKK3 led to increased keratinocyte expression of the M2-like and profibrotic factor TGF-β1. The conditioned media from N/TERT-1 cells with DKK3 modulation was then used to mature human peripheral blood mononuclear cells (PBMC) from healthy donors. After seven days the resultant keratinocyte conditioned macrophages (KcMф) were collected for FACS analysis. DKK3 over-expression in the keratinocytes increased expression of the M2-like phenotype surface markers CD206 and CD163, while DKK3 knockdown decreased the expression of these markers in KcMф. By contrast, expression of MHCII, CD80 and CD86 in KcMф was increased in all settings with maturation of the monocytes, and was not significantly altered by modulation of the DKK3 expression status of the keratinocyte line. The N/TERT-1 cell line was further modified to include a synthetic TCF-based reporter gene to monitor activation of canonical Wnt pathway signaling. Using this system it was demonstrated that radiation increased both endogenous DKK3 expression and canonical Wnt activity. Radiation also induced ROS and chemical ROS stimulation was able to increase DKK3 expression and canonical Wnt signaling activity suggesting a second messenger path for the phenomena. Fibroblasts are important drivers of inflammatory fibrosis. Interestingly, DKK3 fibroblast-specific knockout mice showed increased radiation-induced hyperplasia and fibrosis over that seen in WT mice. In line with this in vivo data, it could be demonstrated in vitro, that DKK3 over-expression in a dermal fibroblast cell line effectively inhibited canonical Wnt activity. These results further indicated the pleotropic nature of the DKK3 protein and its tissue specific mode of action. The present study thus shows that radiation-induced ROS can lead to enhanced DKK3 expression with activation of canonical Wnt activity. This activation in basal keratinocytes caused radiation-induced epithelial hyperplasia and subsequent tissue fibrosis, while loss of DKK3 in keratinocytes protected mice partially against chronic radiation dermatitis. Interestingly, while radiation increased myeloid infiltration in all mice under study, the level of infiltrate in the DKK3 global and keratinocyte specific knockout animals was even enhanced over that seen in the WT mice. Mechanistically, the results suggest that the presence of DKK3 in keratinocytes with accompanying epithelial hyperplasia drives macrophage polarization more towards a M2-like profibrotic phenotype, while the absence of DKK3 leads to macrophages showing a more M1-like pro-inflammatory phenotype. Overall, the results suggest that DKK3 has an important role in radiation-induced dermatitis and fibrosis, that blocking DKK3 may attenuate the disease, and that DKK3 may serve as a potential therapeutic target for radiation-induced dermatitis and fibrosis.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 23. Juni 2023
• 1. Bericht­erstatter:in: Nelson, Peter