Chronic wounds are a major source of morbidity in the United States, affecting an estimated 5.7 million people per year at a cost of 20 billion dollars annually. Underlying factors, such as diabetes, poor perfusion due to vascular disease, or infection, complicates many of these wounds. The normal wound healing process proceeds through three stages, which are precisely choreographed. Appropriate early inflammation is required to sterilize and debride the wound, followed by an orderly transition to repair processes. Inappropriate inflammation can result in pathologic wound healing. It is well recognized that the commensal organisms present in the gut play an important role in mediating gastrointestinal immunity and inflammatory responses through their interaction with innate pathogen recognition receptors, such as TLRs. The skin is also continuously exposed to potential pathogens and possesses unique microflora. Recently, the common cutaneous commensal, Staphylococcus epidermidis, was shown to help establish an inflammatory homeostasis by reducing inflammation in the skin under normal conditions. This is accomplished through activation of TLR-2 by the bacteria's unique lipotechoic acid, which leads to suppression of a NF-kB mediated inflammatory pathway activated by TLR3. Recent research has also shown that TLR3 recognizes cutaneous injury in addition to its traditional dsRNA ligand, and is important for inducing inflammation and in wound healing. Inflammation is essential early in the wound healing process, but detrimental later on. Therefore, the timing and degree of inflammation must be tightly regulated. In light of these interactions, and the presence of much greater amounts of commensal bacteria in chronic wounds, I evaluated the role of TLRs and S. epidermidis LTA in modulating the wound healing process. TLR3 KO mice demonstrated altered gene expression following wounding, but unlike other reports, we observed delayed healing only on days 2-4. Exposure to LTA significantly delayed the wound healing respons.

Purpose: As established by the AccreditationCouncil for Graduate Medical Education (ACGME),dermatology residents in the United States mustparticipate in continuity clinic. This requirementmay be achieved through multiple means, allowingfor program variation. To better assess continuityclinic’s role in resident learning, more data onthis component of graduate medical education isneeded. Methods: An anonymous online survey wasdistributed via the American Board of Dermatologylistserv to all U.S. dermatology residents. Continuityclinic organization, setting, frequency, and patientand preceptor characteristics were assessed; residentsatisfaction and learning were compared. Results:Of 231 responses, 7.8% reported continuity clinicdaily, 77.1% weekly, 9.1% every other week, 3.0%monthly, 0.4% once every several months, and 2.2%only during certain blocks. Of the clinics reported,80.1% were “resident-run with attending” and 11.3%were attending-run. The rest were “resident-run withno attending” (0.9%), both resident and attendingrun(3.0%), or “other” (4.8%). Trainees in resident-runclinics (with attendings) reported greater continuityof care than those in attending-run clinics (p<0.001).Residents reported better teaching with attendingpresence during patient encounters than whenattendings were present only if concerns were raised(p<0.01).

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39^-/- mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R^-/- mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair.

The complex molecular microenvironment of the wound bed regulates the duration and degree of inflammation in the wound repair process, while its dysregulation leads to impaired healing. Understanding factors controlling this response provides therapeutic targets for inflammatory disease. Esophageal cancer-related gene 4 (ECRG4) is a candidate chemokine that is highly expressed on leukocytes. We used ECRG4 knockout (KO) mice to establish that the absence of ECRG4 leads to defective neutrophil recruitment with a delay in wound healing. An in vitro human promyelocyte model identified an ECRG4-mediated suppression of the hyaluronic acid receptor, CD44, a key receptor mediating inflammation resolution. In ECRG4 KO mouse leukocytes, there was an increase in CD44 expression, consistent with a model in which ECRG4 negatively regulates CD44 levels. Therefore, we propose a previously unidentified mechanism in which ECRG4 regulates early neutrophil recruitment and subsequent CD44-mediated resolution of inflammation.

Rapid neutrophil recruitment is critical for controlling infection, with dysfunctional neutrophil responses in diseases like diabetes associated with greater morbidity and mortality. We have shown that the leukocyte protein ECRG4 enhances early neutrophil recruitment to cutaneous wounds and hypothesized that ECRG4 regulates the early host response to infection. Using a cutaneous infection model, we found that ECRG4 KO mice had decreased early neutrophil recruitment with persistent larger lesions, increased bacterial proliferation and systemic dissemination. Although previous work identified ECRG4 as a negative regulator of CD44 on neutrophils, the mechanism regulating neutrophil recruitment remained unknown. We demonstrated that pro-inflammatory responses were intact in ECRG4 KO mice, but found decreased neutrophil mobilization from bone marrow and decreased migration to chemokines. ECRG4 KO mouse neutrophils demonstrated an increase in adhesion molecules that regulate recruitment, including enhanced induction of integrin CD11b and increased L-selectin and CD44 on bone marrow neutrophils. Analysis of gene expression in leukocytes from diabetic patients found decreased ECRG4 expression with similar increased L-selectin and CD44. We propose a previously unrecognized mechanism governing neutrophil recruitment, whereby ECRG4 mediates neutrophil surface adhesion molecules that determine both recruitment and outside-in signaling that modulates neutrophil response to pro-inflammatory stimuli.

Chronic metabolic diseases such as diabetes are characterized by delayed wound healing and a dysregulation of the inflammatory phase of wound repair. Our study focuses on changes in the payload of extracellular vesicles (EVs) communicating between immune cells and stromal cells in the wound bed, which regulate the rate of wound closure. Adoptive transfer of EVs from genetically defined mouse models are used here to demonstrate a functional and molecular basis for differences in the pro-reparative biological activity of diabetic (db/db) vs. wildtype EVs in wound healing. We identify several members of the Serpin family of serine protease inhibitors that are absent in db/db EVs, then we overexpress Serpin A1, F2 and G1 in EVs to evaluate their effect on wound healing in db/db mice. Serpins have an important role in regulating levels of elastase, plasmin and complement factors that coordinate immune cell signaling in full thickness wounds in a diabetic model. Here, we establish a novel therapeutic approach by engineering the payload of EVs based on proteomic analysis. Serpin-loaded EVs were used to rescue the Serpin deficiency identified by proteomics and promote wound healing in db/db mice, as well as evaluated how EVs affected extracellular matrix remodeling and the resolution of tissue injury. Therefore, we propose that the identification of EV payloads that are downregulated in diabetic wounds can be systematically analyzed for their functional activity and potential as a therapeutic, based on whether their re-expression in engineered EVs restores normal kinetics of tissue repair in chronic wounds.

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.