scholarly journals Localization of IL-1α Responsive Dermal Populations in an Ex Vivo Human Model of Wound Healing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrej Sikoski ◽  
Krish Jayapranu ◽  
Hong-Ming Zhou ◽  
Yunglong Liu ◽  
Xiaoling Xuei ◽  
...  
Keyword(s):  
Wound Healing ◽  
Human Skin ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Critical Role ◽  
Mouse Skin ◽  
Human Model ◽  
Cell Populations ◽  
Skin Culture ◽  
Inflammatory Phase

While playing a critical role in skin wound healing, the inflammatory phase of this process is poorly understood. To gain a better understanding of the inflammatory phase of wound healing, we developed an ex vivo skin culture model of skin injury-induced inflammation. Previous work in our laboratory showed ex vivo culture of human skin induces an interleukin 1 alpha (IL-1α)-dependent response characterized by increased transcript and protein levels for the inflammatory cytokines/chemokines, IL-6, CXCL1, and CSF3. However, the cellular sources of these factors in ex vivo cultured human skin have not been determined. Prior work with ex vivo cultured mouse skin and single cell RNA sequencing suggested fibroblasts and endothelial cells were potential cellular sources for these inflammatory mediators. The current studies used spatial transcriptomics analysis of ex vivo cultured human skin to localize the IL-1α target cell populations/skin tissue regions that produce IL-6, CXCL1 and CSF3. The Visium Gene Expression Solution platform (10x Genomics Inc.) was used to generate spatial transcriptomics data from skin specimens preserved immediately after biopsy or after skin culture for 24 hours. Loupe Browser version 5.1.0 (10x Genomics Inc) was used for data analysis to identify and characterize cell populations/regions expressing IL6, CXCL1, and CSF3 and associated differentially expressed genes (including cell type-specific transcripts). Notably, these IL-1α-induced transcripts were localized to the parent dermis region cluster. Analysis of subclusters in the dermal region showed differential expression of these inflammatory transcripts in regions enriched with either or both fibroblast and endothelial cell specific-type markers. Potential novel markers of this inflammatory response, like SOD2, were identified and warrant future investigation. Subsequent studies in identifying the targets of IL-1α in skin inflammation is called for, as they may lead to better understanding of this processes in wound healing and better clinical outcomes.

scholarly journals scRNA-Seq Identifies IL-1 Responsive Cell Subsets in the Skin Injury-induced Inflammatory Response

2020 ◽  
Vol 3 ◽  
Author(s):  
Kayla Harpold ◽  
Hong-Ming Zhou ◽  
Radomir Slominski ◽  
Leroy Seymour ◽  
Maria Bell ◽  
...  
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Cell Populations ◽  
Skin Injury ◽  
Skin Wound Healing ◽  
Inflammatory Phase ◽  
Skin Biopsies

Inflammation is an integral aspect of skin wound healing; however, the mechanisms that regulate inflammatory cascades in this context are not well defined. To better understand how skin inflammation impacts wound healing, we developed an ex vivo skin culture system to model key aspects of the inflammatory phase of wound healing. In this model, a defined set of proinflammatory cytokines and chemokines, mirroring those produced in wounds in vivo, are produced when mouse or human skin biopsies are cultured ex vivo.  We refer to this pattern of cytokine and chemokine induction as the skin injury-induced inflammatory response. Previous studies in our laboratory demonstrated this response is initiated by the cytokine, interleukin 1 alpha (IL-1α). To understand the cellular sources and targets of IL-1α during the skin injury-induced inflammatory response, skin biopsies from mouse tail skin were cultured ex vivo for 8 hours followed by processing for single cell RNA sequencing (scRNAseq). Using bioinformatic software, R, and the package, Seurat, analysis of scRNAseq data from this experiment identified 22 distinct cell population clusters. While no populations exhibited significant expression of Il1a transcripts, multiple cell populations expressed Il1r1 transcripts, which encodes the ligand-specific subunit of the IL-1 receptor.  Notably, fibroblast, endothelial cell and stromal cell clusters were characterized by expression of Il1r1 and the skin injury-induced inflammatory response transcripts Il6, Cxcl1 and/or Csf3. Furthermore, Reactome Pathway Analysis suggested the Il-1 signaling axis was activated in these cell populations. This information provides a basis for future studies to understand how IL-1 signaling in fibroblasts, endothelial cells and stromal cells impacts wound healing in vivo, which could in turn lead to novel therapeutic approaches to clinically relevant outcomes.  

scholarly journals Bv8-Like Toxin from the Frog Venom of Amolops jingdongensis Promotes Wound Healing via the Interleukin-1 Signaling Pathway

Toxins ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 15 ◽  
Author(s):  
Jiajia Chang ◽  
Xiaoqin He ◽  
Jingmei Hu ◽  
Peter Muiruri Kamau ◽  
Ren Lai ◽  
...  
Keyword(s):  
Wound Healing ◽  
Signaling Pathway ◽  
Wide Spectrum ◽  
Interleukin 1 ◽  
Full Thickness ◽  
Mice Model ◽  
Small Peptides ◽  
Newborn Mice ◽  
Proliferative Effect ◽  
Inflammatory Phase

Prokineticins are highly conserved small peptides family expressed in all vertebrates, which contain a wide spectrum of functions. In this study, a prokineticin homolog (Bv8-AJ) isolated from the venom of frog Amolops jingdongensis was fully characterized. Bv8-AJ accelerated full-thickness wounds healing of mice model by promoting the initiation and the termination of inflammatory phase. Moreover, Bv8-AJ exerted strong proliferative effect on fibroblasts and keratinocytes isolated from newborn mice by activating interleukin (IL)-1 production. Our findings indicate that Bv8 is a potent wound healing regulator and may reveal the mechanism of rapid wound-healing in amphibian skins.

scholarly journals The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury

Endocrinology ◽  
2016 ◽  
Vol 157 (10) ◽  
pp. 4066-4075 ◽  
Author(s):  
Lige Song ◽  
Garyfallia Papaioannou ◽  
Hengguang Zhao ◽  
Hilary F. Luderer ◽  
Christine Miller ◽  
...  
Keyword(s):  
Vitamin D ◽  
Wound Healing ◽  
Vitamin D Receptor ◽  
Macrophage Polarization ◽  
Critical Role ◽  
Dermal Fibroblasts ◽  
Wild Type ◽  
Macrophage Response ◽  
Protein Levels ◽  
Inflammatory Phase

Ligand-dependent actions of the vitamin D receptor (VDR) play a pleiotropic role in the regulation of innate and adaptive immunity. The liganded VDR is required for recruitment of macrophages during the inflammatory phase of cutaneous wound healing. Although the number of macrophages in the granulation tissue 2 days after wounding is markedly reduced in VDR knockout (KO) compared with wild-type mice, VDR ablation does not alter macrophage polarization. Parabiosis studies demonstrate that circulatory chimerism with wild-type mice is unable to rescue the macrophage defect in the wounds of VDR KO mice and reveal that wound macrophages are of local origin, regardless of VDR status. Wound cytokine analyses demonstrated a decrease in macrophage colony-stimulating factor (M-CSF) protein levels in VDR KO mice. Consistent with this, induction of M-CSF gene expression by TGFβ and 1,25-dihydroxyvitamin D was impaired in dermal fibroblasts isolated from VDR KO mice. Because M-CSF is important for macrophage self-renewal, studies were performed to evaluate the response of tissue resident macrophages to this cytokine. A decrease in M-CSF induced proliferation and cyclin D1 expression was observed in peritoneal resident macrophages isolated from VDR KO mice, suggesting an intrinsic macrophage abnormality. Consistent with this, wound-healing assays in mice with macrophage-specific VDR ablation demonstrate that a normal wound microenvironment cannot compensate for the absence of the VDR in macrophages and thus confirm a critical role for the macrophage VDR in the inflammatory response to injury.

scholarly journals TFAM-deficient mouse skin fibroblasts: an ex vivo model of mitochondrial dysfunction

2021 ◽  
Author(s):  
Manuel J. Del Rey ◽  
Carolina Meroño ◽  
Cristina Municio ◽  
Alicia Usategui ◽  
María Mittelbrunn ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Ex Vivo ◽  
Stress Test ◽  
Critical Role ◽  
Substantial Reduction ◽  
Mouse Skin ◽  
Cell Types ◽  
Ex Vivo Model ◽  
Primary Mouse ◽  
Inflammatory Phenotype

Mitochondrial dysfunction in different cell types is associated to several pathological processes and potentially contributes to chronic inflammatory and ageing-related diseases. Mitochondrial Transcription Factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of the Tfamfl/fl UBC-Cre/ERT2+/+ mice, we sought to develop a cellular in vitro system to investigate the role of mitochondrial dysfunction in the stromal cell component. We describe an inducible model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblast (SK-FB) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction of Tfam and mtDNA-encoded mRNA expression in Cre(+) cultured for low (LP) and high passages (HP). Ultimately, Tfam knockout translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction of the mitochondrial respiratory chain (MRC) complexes that was exacerbated in HP SK-FB cultures. The assembly pattern showed that the respiratory complexes fail to reach the respirasome in 4-OHT Cre(+) SK-FB. Functionally, we determined the mitochondrial function and the glycolytic activity by mito-stress and glycolysis-stress test respectively. These analysis showed that mitochondrial dysfunction was developed after long-term 4-OHT treatment in HP Cre(+) SK-FB and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FB showed a senescent and pro-inflammatory phenotype. In conclusion, we have generated and validated the first ex vivo model of fibroblast mitochondrial dysfunction that results in a pro-inflammatory phenotype applicable to explore this process in other cell types in a variety of pathological conditions.

scholarly journals Ex vivo culture of mouse skin activates an interleukin 1 alpha‐dependent inflammatory response

2019 ◽  
Vol 29 (1) ◽  
pp. 102-106 ◽  
Author(s):  
Hong‐Ming Zhou ◽  
Radomir M. Slominski ◽  
Leroy J. Seymour ◽  
Maria C. Bell ◽  
Priya Dave ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Mouse Skin ◽  
Ex Vivo Culture

scholarly journals Effect of Hypoxia on Gene Expression in Cell Populations Involved in Wound Healing

2019 ◽  
Vol 2019 ◽  
pp. 1-20 ◽  
Author(s):  
Sarah D’Alessandro ◽  
Andrea Magnavacca ◽  
Federica Perego ◽  
Marco Fumagalli ◽  
Enrico Sangiovanni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Endothelial Cells ◽  
Current Knowledge ◽  
Expression Profiles ◽  
Healing Process ◽  
Critical Role ◽  
Dermal Fibroblasts ◽  
Cell Populations ◽  
Wound Healing Process

Wound healing is a complex process regulated by multiple signals and consisting of several phases known as haemostasis, inflammation, proliferation, and remodelling. Keratinocytes, endothelial cells, macrophages, and fibroblasts are the major cell populations involved in wound healing process. Hypoxia plays a critical role in this process since cells sense and respond to hypoxic conditions by changing gene expression. This study assessed the in vitro expression of 77 genes involved in angiogenesis, metabolism, cell growth, proliferation and apoptosis in human keratinocytes (HaCaT), microvascular endothelial cells (HMEC-1), differentiated macrophages (THP-1), and dermal fibroblasts (HDF). Results indicated that the gene expression profiles induced by hypoxia were cell-type specific. In HMEC-1 and differentiated THP-1, most of the genes modulated by hypoxia encode proteins involved in angiogenesis or belonging to cytokines and growth factors. In HaCaT and HDF, hypoxia mainly affected the expression of genes encoding proteins involved in cell metabolism. This work can help to enlarge the current knowledge about the mechanisms through which a hypoxic environment influences wound healing processes at the molecular level.

scholarly journals Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model

2014 ◽  
Vol 190 (1) ◽  
pp. 358-366 ◽  
Author(s):  
Swathi Balaji ◽  
Chad M. Moles ◽  
Sukanta S. Bhattacharya ◽  
Maria LeSaint ◽  
Yashu Dhamija ◽  
...  
Keyword(s):  
Wound Healing ◽  
Organ Culture ◽  
Human Skin ◽  
Ex Vivo ◽  
Interleukin 10 ◽  
Dermal Wound Healing ◽  
Culture Model ◽  
Dermal Wound

scholarly journals Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes

2020 ◽  
Author(s):  
Md. Mehedi Hassan ◽  
Shirina Sharmin ◽  
Hyeon-Jin Kim ◽  
Seong-Tshool Hong
Keyword(s):  
Wound Healing ◽  
Ex Vivo ◽  
Mouse Skin ◽  
Bleeding Risk ◽  
Fibrinolytic Enzymes ◽  
New Class ◽  
Hemorrhagic Complications ◽  
Shock Proteins ◽  
Thrombotic Diseases

Rationale: Current thrombolytic agents activate plasminogen to plasmin which triggers fibrinolysis to dissolve thrombi. Since plasmin is a nonspecific proteolytic enzyme, all of the current plasmin-dependent thrombolytics lead to serious hemorrhagic complications, demanding for a new class of fibrinolytic enzymes independent from plasmin activation and undesirable side effects. We speculated that the mammalian version of bacterial heat shock proteins could selectively degrade intravascular thrombi, a typical example of a highly aggregated protein mixture. Objective: The objective of this study is to identify enzymes that can dissolve intravascular thrombi specifically without affecting fibrinogen and fibronectin so that the wound healing processes remains uninterrupted and tissues are not damaged. In this study, high-temperature requirement A (HtrA) proteins were tested for its specific proteolytic activity on intravascular thrombi independently from plasmin activation. Methods and Results: HtrA1 and HtrA2/Omi proteins, collectively called as HtrAs, lysed ex vivo blood thrombi by degrading fibrin polymers. The thrombolysis by HtrAs was plasmin-independent and specific to vascular thrombi without causing the systemic activation of plasminogen and preventing non-specific proteolysis of other proteins including fibrinogen and fibronectin. As expected, HtrAs did not disturb clotting and wound healing of excised wounds from mouse skin. It was further confirmed in a tail bleeding and a rebleeding assay that HtrAs allowed normal clotting and maintenance of clot stability in wounds, unlike other thrombolytics. Most importantly, HtrAs completely dissolved blood thrombi in tail thrombosis mice, and the i.v. injection of HtrAs to mice with pulmonary embolism completely dissolved intravascular thrombi and thus rescued thromboembolism. Conclusions: Here, we identified HtrA1 and HtrA2/Omi as plasmin-independent and highly specific thrombolytics which can dissolve intravascular thrombi specifically without bleeding risk. This work is the first report of a plasmin-independent thrombolytic pathway, providing HtrA1 and HtrA2/Omi as ideal therapeutic candidates for various thrombotic diseases without hemorrhagic complications.

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