We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-C5ar1^fl/fl) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice. C5ar1^fl/fl.Lyz2Cre^+/− mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control C5ar1^fl/fl mice. Examination of C5aR1-expressing macrophage transcriptomes by gene set enrichment analysis demonstrated that these cells were enriched in pathways corresponding to the complement cascade, collagen formation, and the NABA matrisome, strongly pointing to their critical roles in tissue repair/scarring. Since C5aR1 was also detected in a small population of platelet-derived growth factor receptor-β⁺ GFP⁺ cells, we developed C5ar1^fl/fl.Foxd1Cre^+/− mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-β⁺ pericytes isolated from FA-treated C5ar1^fl/fl.Foxd1Cre^+/− mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-C5ar1^fl/fl mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis. NEW & NOTEWORTHY This study used novel green fluorescent protein C5a receptor 1 floxed mice and the model of folic acid-mediated kidney fibrosis to demonstrate the pathogenic role of increased expression of this complement receptor on macrophages.

Cutaneous Radiation Injuries (CRIs) and wounds within an area of radiation exposure (combined injury, CI) are a significant concern for nuclear accidents and radiation combat/terrorist events. CRIs and CI present unique clinical challenges, and effective countermeasures are urgently needed. Here we describe a murine model of CRI and CI in C57BL/6 mice using 16.9 Gy thoracic X-ray irradiation (5.3 Gy/min, 160 kV) ± experimental wound administered immediately. Wound repair and radiation-induced dermatitis were assessed after irradiation. Our previous studies showed that genistein (200 mg/kg, s.c.), administered 24 h prior to irradiation prevented radiation injuries in two murine models. We investigated the effects of genistein in the CI model. Macroscopic and histological analyses showed that radiation significantly delayed wound closure, although wounds did not significantly alter the progression of radiation dermatitis. Genistein improved the early rate of wound closure and significantly reduced dermatitis in mice. Histological analysis showed that genistein improved skin structure and reduced inflammation and fibrosis. Immunohistochemistry showed that genistein attenuated radiation-induced cyclin-dependent kinase inhibitor 1 (p21/waf1) and α-smooth muscle actin and preserved K15 positive skin adult stem cells. These findings suggest that genistein may be an effective prophylactic against CRIs and CI.

Bioengineered dermal–epidermal composites (DECs) have demonstrated promise initiating skin regeneration and hair follicle neogenesis after injury. DECs in our work comprise a collagen matrix embedded with human dermal papilla cells (HDPCs) overlaid with human keratinocytes. HDPCs, as three-dimensional spheroids, enhance hair follicle formation, working in tandem with keratinocytes. Herein, 3D printed stamped PDMS microwell arrays were used as a strategy for spatially patterning dermal papilla spheroids in the dermal components of the DEC. DECs were transferred to cell culture media for 5 days followed by air–liquid interface culture for 2 days. Spheroid diameter, cell viability, and qPCR gene expression analyses were conducted. DECs were surgically grafted on immunocompromised mice, and healing was followed for 10 weeks. HDPCs cultured in the microwell arrays formed patterned viable spheroids and successfully transferred to the collagen dermal matrix. RNA analysis using qPCR showed upregulation of key HDPC markers (VCAN and BMP6) in DC microwell patterned HDPC spheroids compared to monolayers. This work represents a novel 3D printing strategy optimizing designing patterned HDPC spheroids in the extracellular matrix to regenerate functional human skin instead of scar tissue after injury.