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 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 A Synthetic Curcuminoid Analog, (2E,6E)-2,6-bis(2-(trifluoromethyl)benzylidene)cyclohexanone, Ameliorates Impaired Wound Healing in Streptozotocin-Induced Diabetic Mice by Increasing miR-146a

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 920
Author(s):  
Jingjuan Huang ◽  
Jia Fu ◽  
Bing Liu ◽  
Rui Wang ◽  
Tianhui You
Keyword(s):  
Wound Healing ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Diabetic Wound ◽  
Diabetic Mice ◽  
Skin Wound Healing ◽  
Promising Alternative ◽  
Impaired Wound Healing ◽  
Diabetic Wound Healing

The impairment in diabetic wound healing represents a significant clinical problem, with no efficient targeted treatments for these wound disorders. Curcumin is well confirmed to improve diabetic wound healing, however, its low bioavailability and poor solubility severely limit its clinical application. This study aims to provide the pharmacological basis for the use of (2E,6E)-2,6-bis(2-(trifluoromethyl)benzylidene)cyclohexanone (C66). The results showed that topically applied C66 improved cutaneous wound healing in vivo. Further studies showed that C66 treatment increased the level of microRNA-146a (miR-146a) in the wounds in streptozotocin (STZ)-induced diabetic mice, downregulated the expression of interleukin-1 receptor-associated kinase 1 (IRAK1) and phosphorylated nuclear factor-κB (NF-κB) p65 subunit (p-p65) (both p < 0.05), and suppressed the mRNA expression of inflammation-related cytokines, tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and interleukin-6 (IL-6). The in vitro data obtained in human umbilical vein endothelial cells (HUVECs) showed that C66 could reverse high glucose (HG)-induced NF-κB activation due to upregulation of miR-146a expression, which matched the in vivo findings. In conclusion, the present study indicates that C66 exerts anti-inflammation activity and accelerates skin wound healing of diabetic mice, probably via increasing miR-146a and inhibiting the NF-κB-mediated inflammation pathway. Therefore, C66 may be a promising alternative for the treatment of diabetic wounds.

scholarly journals Modification of collagen-based sponges can induce an upshift of the early inflammatory response and a chronic inflammatory reaction led by M1 macrophages: an in vivo study

2020 ◽  
Vol 24 (10) ◽  
pp. 3485-3500 ◽  
Author(s):  
C. Herrera-Vizcaíno ◽  
S. Al-Maawi ◽  
R. Sader ◽  
C. J. Kirkpatrick ◽  
J. Choukroun ◽  
...  
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Inflammatory Reaction ◽  
Ex Vivo ◽  
Cell Interaction ◽  
Control Group ◽  
Ex Vivo Model ◽  
Inflammatory Proteins ◽  
Time Point

Abstract Background The present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group. Material and methods First, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group. Results At day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days. Conclusion Altering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial’s degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo. Clinical relevance A pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.

scholarly journals Immunohistochemical analysis of the inflammatory reaction within the implantation bed of a collagen hemostatic biomaterial

2021 ◽  
Author(s):  
◽  
Carlos Arturo Herrera Vizcaino
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Inflammatory Cytokines ◽  
Degradation Rate ◽  
Ex Vivo ◽  
The Body ◽  
Cellular Reaction ◽  
Intergroup Comparison ◽  
Pro Inflammatory Cytokines

Current research on medical biomaterials have shown that the physical and chemical characteristics of biomaterials determine the body inflammatory cellular reaction after their implantation. The aim of this study was to evaluate the individual effects of the physical characteristics over the initial biomaterial-cellular interaction and the inflammatory cellular reaction. For this purpose, an equine-derived collagen hemostatic sponge (E-CHS) was modified by pressing and evaluated using ex vivo, in vitro and in vivo methods. The E-CHS was pressed by applying constant pressure (6.47± 0.85 N) for 2 min using a sterile stainless-steel cylinder and cut in segments of 1cm2. Subsequently, E-CHS and the pressed equine-derived collagen hemostatic sponge (P-E-CHS) were studied as two independent biomaterials and compared to a control group (CG). A blood concentrate containing inflammatory cells known as platelet rich fibrin (PRF) was used to mimic the initial biomaterial-cell interaction and to measure the absorption coefficient of the biomaterials to liquid PRF (iPAC). Additionally, the biomaterials were cultivated together with PRF for 3 and 6 days to measure the induction of pro-inflammatory cytokines (TNF-α and IL-8). The results were obtained through enzyme-linked immunosorbent assay (ELISA) and histological methods. PRF cultivated without biomaterials served as the CG. Additionally, the biomaterials were evaluated in vivo using a subcutaneous model in Wistar rats and compared to sham operated animals (CG) representing physiologic wound healing. After 3, 15 and 30 days, the explanted samples were evaluated using histochemical and immunohistochemical (IHC) staining using the following markers: CD68 (pan macrophages), CCR7 (pro-inflammatory macrophages, M1), CD206 (pro-wound healing macrophages, M2) and α-Smooth Muscle Actin (α-SMA; vessel identification). After the mixture of liquid PRF with both biomaterials for 15 minutes, the ex vivo results showed that E-CHS was penetrated by cells, whereas P-E-CHS was cell-occlusive. Additionally, P-E-CHS induced a higher release of pro-inflammatory cytokines compared to liquid PRF alone (CG) and E-CHS after 3 days (P< 0.05). Although the biomaterial was pressed, the difference of the iPAC value did not show statistical differences. In vivo, the CG induced at day 3 a higher inflammatory response compared to the experimental groups (EG) (P< 0.05). The intergroup comparison showed that P-E-CHS induced a higher presence of macrophages (CD68+/CC7+) compared to E-CHS at day 3 (P< 0.05). Only CD68+/CCR7+ mononuclear cells (MNCs) were observed without multinucleated giant cells (MNGCs). After 15 days, the presence of macrophages (CD68+ P<0.01 /CCR7+ P<0.001 /CD206+ P<0.05) reduced considerably in the CG. On the contrary, the inflammatory response increased in the EGs (CD68+/CCR7+). The intergroup comparison showed that this increment was statistically significant when comparing E-CHS and P-E-CHS to the CG at day 15 (P<0.01 and P< 0.05 respectively). At this time point, a reduced number of MNGCs were observed in the EGs. In the CG no MNGCs were observed. Furthermore, E-CHS showed a faster degradation rate and was fully invaded by cells and vessels formed in its interior region. On the other hand, P-E-CHS remained occlusive to cell penetration and vessels were formed only in the periphery. After 30 days, the cellular reaction shifted to a higher number of M2 macrophages (CD260+) in all groups and a reduced presence of CD68+ and CCR7+ MNCs. Both biomaterials degraded and only small fragments were found in the implantation bed surrounded by MNGCs (CCR7+). These results are of high clinical relevance and show that changes in biomaterial properties have a significant impact on their interaction with the body. They also serve as insight into the possibility to develop versatile biomaterials with different applications. For example, E-CHs can be applied to support hemostasis in a bleeding alveolar socket and P-E-CHs by being cell occlusive and having a delayed degradation rate can be applied for guided bone and tissue regeneration.

Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production

2020 ◽  
Vol 21 ◽  
Author(s):  
Hana M. Hammad ◽  
Amer Imraish ◽  
Maysa Al-Hussaini ◽  
Malek Zihlif ◽  
Amani A. Harb ◽  
...  
Keyword(s):  
Wound Healing ◽  
Rural Communities ◽  
Ex Vivo ◽  
Ethanol Extract ◽  
Aortic Ring ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Achillea Fragrantissima

Objective: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. Methods: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using rat aortic ring assay and in vivo using rat excision wound model. Results: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulates the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and was found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline-treated group. This effect was comparable to that induced by MEBO, the positive control. Conclusion: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

scholarly journals Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

2019 ◽  
Vol 20 (15) ◽  
pp. 3679 ◽  
Author(s):  
Lin Chen ◽  
Alyne Simões ◽  
Zujian Chen ◽  
Yan Zhao ◽  
Xinming Wu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Oral Mucosa ◽  
Wound Closure ◽  
Expression Patterns ◽  
Skin Wound ◽  
Skin Wound Healing ◽  
Specific Expression ◽  
Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

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.

Cold Plasma Treatment Promotes Full-thickness Healing of Skin Wounds in Murine Models

2021 ◽  
pp. 153473462110021
Author(s):  
Joon M. Jung ◽  
Hae K. Yoon ◽  
Chang J. Jung ◽  
Soo Y. Jo ◽  
Sang G. Hwang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Plasma Treatment ◽  
Cold Plasma ◽  
Smooth Muscle Actin ◽  
Treated Group ◽  
Control Group ◽  
Alpha Smooth Muscle Actin ◽  
Skin Wound Healing

Cold plasma can be beneficial for promoting skin wound healing and has a high potential of being effectively used in treating various wounds. Our aim was to verify the effect of cold plasma in accelerating wound healing and investigate its underlying mechanism in vitro and in vivo. For the in vivo experiments, 2 full-thickness dermal wounds were created in each mouse (n = 30). While one wound was exposed to 2 daily plasma treatments for 3 min, the other wound served as a control. The wounds were evaluated by imaging and histological analyses at 4, 7, and 11 days post the wound infliction process. Immunohistochemical studies were also performed at the same time points. In vitro proliferation and scratch assay using HaCaT keratinocytes and fibroblasts were performed. The expression levels of wound healing–related genes were analyzed by real-time polymerase chain reaction and western blot analysis. On day 7, the wound healing rates were 53.94% and 63.58% for the control group and the plasma-treated group, respectively. On day 11, these rates were 76.05% and 93.44% for the control and plasma-treated groups, respectively, and the difference between them was significant ( P = .039). Histological analysis demonstrated that plasma treatment promotes the formation of epidermal keratin and granular layers. Immunohistochemical studies also revealed that collagen 1, collagen 3, and alpha-smooth muscle actin appeared more abundantly in the plasma-treated group than in the control group. In vitro, the proliferation of keratinocytes was promoted by plasma exposure. Scratch assay showed that fibroblast exposure to plasma increased their migration. The expression levels of collagen 1, collagen 3, and alpha-smooth muscle actin were elevated upon plasma treatment. In conclusion, cold plasma can accelerate skin wound healing and is well tolerated.

In Vivo and ex Vivo Approaches to Studying the Biomechanical Properties of Healing Wounds in Rat Skin

2013 ◽  
Vol 135 (10) ◽  
Author(s):  
Clare Y. L. Chao ◽  
Gabriel Y. F. Ng ◽  
Kwok-Kuen Cheung ◽  
Yong-Ping Zheng ◽  
Li-Ke Wang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Ex Vivo ◽  
Normal Skin ◽  
Biomechanical Properties ◽  
Skin Wound ◽  
Sprague Dawley ◽  
Rat Skin ◽  
Mechanical Stiffness ◽  
Air Jet

An evaluation of wound mechanics is crucial in reflecting the wound healing status. The present study examined the biomechanical properties of healing rat skin wounds in vivo and ex vivo. Thirty male Sprague-Dawley rats, each with a 6 mm full-thickness circular punch biopsied wound at both posterior hind limbs were used. The mechanical stiffness at both the central and margins of the wound was measured repeatedly in five rats over the same wound sites to monitor the longitudinal changes over time of before wounding, and on days 0, 3, 7, 10, 14, and 21 after wounding in vivo by using an optical coherence tomography-based air-jet indentation system. Five rats were euthanized at each time point, and the biomechanical properties of the wound tissues were assessed ex vivo using a tensiometer. At the central wound bed region, the stiffness measured by the air-jet system increased significantly from day 0 (17.2%), peaked at day 7 (208.3%), and then decreased progressively until day 21 (40.2%) as compared with baseline prewounding status. The biomechanical parameters of the skin wound samples measured by the tensiometer showed a marked reduction upon wounding, then increased with time (all p < 0.05). On day 21, the ultimate tensile strength of the skin wound tissue approached 50% of the normal skin; while the stiffness of tissue recovered at a faster rate, reaching 97% of its prewounded state. Our results suggested that it took less time for healing wound tissues to recover their stiffness than their maximal strength in rat skin. The stiffness of wound tissues measured by air-jet could be an indicator for monitoring wound healing and contraction.

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