Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway

Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model

YesAlopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss.Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945)

Evaluating the Biocompatibility of an Injectable Wound Matrix in a Murine Model

(1) Background: Developing a high-quality, injectable biomaterial that is labor-saving, cost-efficient, and patient-ready is highly desirable. Our research group has previously developed a collagen-based injectable scaffold for the treatment of a variety of wounds including wounds with deep and irregular beds. Here, we investigated the biocompatibility of our liquid scaffold in mice and compared the results to a commercially available injectable granular collagen-based product. (2) Methods: Scaffolds were applied in sub-dermal pockets on the dorsum of mice. To examine the interaction between the scaffolds and the host tissue, samples were harvested after 1 and 2 weeks and stained for collagen content using Masson’s Trichrome staining. Immunofluorescence staining and quantification were performed to assess the type and number of cells infiltrating each scaffold. (3) Results: Histological evaluation after 1 and 2 weeks demonstrated early and efficient integration of our liquid scaffold with no evident adverse foreign body reaction. This rapid incorporation was accompanied by significant cellular infiltration of stromal and immune cells into the scaffold when compared to the commercial product (p < 0.01) and the control group (p < 0.05). Contrarily, the commercial scaffold induced a foreign body reaction as it was surrounded by a capsule-like, dense cellular layer during the 2-week period, resulting in delayed integration and hampered cellular infiltration. (4) Conclusion: Results obtained from this study demonstrate the potential use of our liquid scaffold as an advanced injectable wound matrix for the management of skin wounds with complex geometries.Medicine, Faculty ofNon UBCSurgery, Department ofReviewedFacultyResearcherOthe

An Evaluation of the Treatment of Full-Thickness Wounds Using Adipose Micro-Fragments within a Liquid Dermal Scaffold

In full-thickness wounds, inflammation, lack of matrix deposition, and paucity of progenitor cells delay healing. As commercially available solid (sheet) scaffolds are unable to conform to wounds of varying shapes and sizes, we previously generated a nutritious, injectable, liquid skin substitute that can conform to wound topography. In combination with adipose micro-fragments as a viable source of progenitor cells, a composite, in situ forming skin substitute was tested for the treatment of silicon ring splinted full-thickness wounds in rats. The in vitro survivability and migratory capacity of adipocytes derived from rat micro-fragmented fat cultured in our scaffold was examined with a Live/Dead assay, showing viability and migration after 7 and 14 days. In vivo, the efficacy of our scaffold alone (LDS) or with adipose micro-fragments (LDS+A) was compared to a standard dressing protocol (NT). LDS and LDS+A showed ameliorated wound healing, including complete epithelialization and less immune cell infiltration, compared to the NT control. Our findings demonstrate that a 3D liquid skin scaffold is a rich environment for adipocyte viability and migration, and that the addition of adipose micro-fragments to this scaffold can be used as a rich source of cells for treating full-thickness wounds

A Pilot Trial Assessing the Feasibility and Efficacy of a Novel Powder for Rapid Wound Healing

It is well-understood that wound care poses a significant burden on the healthcare system and patient well-being. As such, it is imperative to develop efficient methods that facilitate tissue repair. Our group previously developed a nutritional gel scaffold, proven to accelerate wound repair. Due to its gel-like properties, this scaffold requires a time-consuming reconstitution, and is optimized for cavernous wounds. This pilot study examined the feasibility of a powdered form of this scaffold to accelerate healing of full-thickness wounds, thus broadening the range of applications, while providing a practical product. Splinted full-thickness wounds were generated on the backs of 6 mice, and treated with either powder, the original gel scaffold, or no treatment. Feasibility and efficacy of the powder was assessed through comparison of clinical wound measurements and histological assessments. There was a significant effect of treatment on rate of epithelialization [H(3) = 8.346, p = 0.0024] and on days to epithelial closure [H(3) = 8.482, p = 0.0061]. Post hoc analysis revealed that while requiring no reconstitution and simple to apply, the powder was sufficient to accelerate epithelialization compared to untreated wounds (p < 0.05). Furthermore, our results suggest that application of this powder did not alter certain processes associated with healing progress, such as epidermal thickness and collagen deposition. As such, this powder may provide a novel alternative to our previously developed gel scaffold by accelerating epithelialization, while providing a practical product. Future studies necessitate further evaluation of healing measures with a larger sample size.Medicine, Faculty ofAlumniMedicine, Department ofSurgery, Department ofReviewedFacult

Anti-Scarring Properties of Different Tryptophan Derivatives

<div><p>Hypertrophic scars are associated with prolonged extracellular matrix (ECM) production, aberrant ECM degradation and high tissue cellularity. Routinely used antifibrotic strategies aim to reduce ECM deposition and enhance matrix remodeling. Our previous study investigating the antifibrotic effects of indoleamine2, 3 dioxygenase (IDO) led to the identification of kynurenine (Kyn) as an antiscarring agent. A topical antifibrogenic therapy using Kyn is very attractive; however, it is well established that Kyn passes the blood brain barrier (BBB) which causes complications including excitatory neuronal death. Here we investigated the antiscarring properties of kynurenic acid (KynA), a downstream end product of Kyn that is unlikely to pass the BBB, as an effective and safe replacement for Kyn. Our results indicated that while not having any adverse effect on dermal cell viability, KynA significantly increases the expression of matrix metalloproteinases (MMP1 and MMP3) and suppresses the production of type-I collagen and fibronectin by fibroblasts. Topical application of cream containing KynA in fibrotic rabbit ear significantly decreased scar elevation index (1.13±0.13 vs. 1.61±0.12) and tissue cellularity (221.38±21.7 vs. 314.56±8.66 cells/hpf) in KynA treated wounds compared to controls. KynA treated wounds exhibited lower levels of collagen deposition which is accompanied with a significant decrease in type-I collagen and fibronectin expression, as well as an increase in MMP1 expression compared to untreated wounds or wounds treated with cream only. The results of this study provided evidence for the first time that KynA is promising candidate antifibrogenic agent to improve healing outcome in patients at risk of hypertrophic scarring.</p></div

Stimulatory effect of kynurenines on MMP1 expression.

<p><b>A</b>: Dermal fibroblasts were treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. Following 24 hours of treatment cells were collected, and MMP1 expression was determined by Q-PCR after RNA extraction and cDNA synthesis. <b>B</b>: Evaluation of MMP1 expression at the protein level by Western blotting after 48 hours of treatment. <b>C</b>: The Mean±SEM ratio of MMP1 to β-actin density at the protein level. β-actin and GAPDH were used as loading controls for western blotting and Q-PCR, respectively.</p

Inhibition of type-I collagen and fibronectin expression in dermal fibroblasts by kynurenines.

<p>Type-I Collagen and fibronectin expression at the mRNA and protein level in cultured fibroblasts treated with increasing concentrations (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. <b>A & B</b>: Relative type-I collagen and fibronectin mRNA expression in treated fibroblasts, respectively. GAPDH was used as the reference gene. <b>C</b>: Evaluation of type-I collagen and fibronectin expression at the protein level using Western blotting. <b>D & E</b>: The Mean±SEM ratio of type-I collagen and fibronectin density to β-actin at the protein level, respectively. β-actin was used as protein loading control.</p