murine-excisional-wound-healing-model-histological-morphometric-wound

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

University of Iowa

Regulation of epidermal proliferation and differentiation is critical for maintenance of cutaneous homeostasis. Interferon Regulatory Factor 6 (Irf6)-deficient mice die perinatally and exhibit ectopic proliferation and defective epidermal differentiation. We sought to determine whether these disruptions of epidermal function were cell autonomous, and used embryonic Irf6(-/-) keratinocytes to understand the specific role of Irf6 in keratinocyte proliferation and differentiation. In the absence of Irf6, keratinocytes exhibited a heterogeneous phenotype with the presence of large cells. Irf6(-/-) keratinocytes displayed increased colony-forming efficiency compared with wild-type cells, suggesting that Irf6 represses long-term proliferation. Irf6 was present at low levels in wild-type keratinocytes in culture, and upregulated after induction of differentiation in vitro, along with upregulation of markers of early differentiation. However, Irf6(-/-) keratinocytes did not express markers of terminal differentiation. Overexpression of Irf6 in wild-type keratinocytes was insufficient to induce expression of markers of differentiation under growing conditions. Together, these results indicated that Irf6 is necessary, but not sufficient, for keratinocyte differentiation. Finally, using a transgenic mouse expressing Lac-Z under the regulation of an enhancer element 9.7  kb upstream of the Irf6 start site, we demonstrated that this element contributes to the regulation of Irf6 in the epidermis and keratinocytes in culture.

Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation.

DNA variation in Interferon Regulatory Factor 6 (IRF6) contributes risk for orofacial clefting, including a common DNA variant rs642961. This DNA variant is located in a multi-species conserved sequence that is 9.7 kb upstream from the IRF6 transcriptional start site (MCS9.7). The MCS9.7 element was shown to possess enhancer activity that mimicked the expression of endogenous Irf6 at embryonic day 11.5 in transient transgenic embryos, and also contains a p63 binding site that transactivates IRF6 expression. To analyze whether the MCS9.7 enhancer is sufficient to drive IRF6 expression, we generated stable transgenic murine lines that carry a MCS9.7-lacZ transgene. We hypothesized that MCS9.7 was sufficient to recapitulate the endogenous expression of Irf6 at other time-points during embryonic development.

MCS9.7 enhancer activity is highly, but not completely, associated with expression of Irf6 and p63.

Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms-TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.

Transforming growth factor Beta 3 is required for excisional wound repair in vivo.

Preterm birth (PTB) is a complex disorder associated with significant neonatal mortality and morbidity and long-term adverse health consequences. Multiple lines of evidence suggest that genetic factors play an important role in its etiology. This study was designed to identify genetic variation associated with PTB in oxytocin pathway genes whose role in parturition is well known.

Sequence variants in oxytocin pathway genes and preterm birth: a candidate gene association study.

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production. 

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock.

Interferon Regulatory Factor 6 (IRF6) is critical for craniofacial development, epidermal differentiation, and tissue repair. IRF6 mutations cause Van der Woude Syndrome (VWS) and Popliteal Pterygium Syndrome. Individuals with VWS exhibit craniofacial anomalies, including cleft lip and palate and lip pits. Furthermore, they have an increased risk for wound-healing complications following surgical repair when compared with patients with nonsyndromic cleft lip and palate (NSCLP). However, nothing is known about the skin of these patients. The objective was to characterize the skin of patients with VWS. We hypothesize that IRF6 is required for proper skin homeostasis in humans.

Interferon Regulatory Factor 6 Controls Proliferation of Keratinocytes From Children With Van der Woude Syndrome.

Van der Woude syndrome (VWS) is the most common form of syndromic orofacial cleft caused predominantly by mutations in Interferon Regulatory Factor 6 (IRF6). We previously reported that individuals with VWS have increased risk of wound healing complications following cleft repair compared with individuals with nonsyndromic orofacial clefts (nonsyndromic cleft lip and palate-NSCLP). In vitro, absence of IRF6 leads to impaired keratinocyte migration and embryonic wound healing. However, there is currently no data on tissue repair in adult animals and cells with reduced levels of IRF6 like in VWS.

Article	Year
Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation. The Journal of investigative dermatology\| PubMed ID: 21918538
MCS9.7 enhancer activity is highly, but not completely, associated with expression of Irf6 and p63. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 22113860
Transforming growth factor Beta 3 is required for excisional wound repair in vivo. PloS one\| PubMed ID: 23110169
Sequence variants in oxytocin pathway genes and preterm birth: a candidate gene association study. BMC medical genetics\| PubMed ID: 23889750	2013
Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock. PloS one\| PubMed ID: 27035130
Interferon Regulatory Factor 6 Controls Proliferation of Keratinocytes From Children With Van der Woude Syndrome. The Cleft palate-craniofacial journal : official publication of the American Cleft Palate-Craniofacial Association\| PubMed ID: 27115562
Interferon regulatory factor 6 is required for proper wound healing in vivo. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 31724286

Lindsey Rhea

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