Alternatively activated macrophages derived from THP-1 cells promote the fibrogenic activities of human dermal fibroblasts.

Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Fibroblasts maintain the physical integrity of connective tissue, participate in wound closure as well as produce and remodel extracellular matrix. Macrophages have a close relationship with fibroblasts by increasing the production of matrix metalloproteinase-1 (MMP-1) for faster wound closure and remodeling and myofibroblast differentiation from fibroblasts. In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP-1 cell line were used to co-culture with human dermal fibroblasts (HDF) for 48, 96 and 144 hours to investigate the effect of macrophages subsets on the fibrogenic activity of fibroblasts. The differentiation and polarization from THP-1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti-fibrotic and pro-fibrotic factors, MMP-1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co-cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha-smooth muscle actin expressed cells, expression of the pro-fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti-fibrotic genes and MMP-1 activity. These findings reinforce the pro-fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future.

Synergistic effect of vitamin D and low concentration of transforming growth factor beta 1, a potential role in dermal wound healing.

Dermal wound healing, in which transforming growth factor beta 1 (TGFß1) plays an important role, is a complex process. Previous studies suggest that vitamin D has a potential regulatory role in TGFß1 induced activation in bone formation, and there is cross-talk between their signaling pathways, but research on their effects in other types of wound healing is limited. The authors therefore wanted to explore the role of vitamin D and its interaction with low concentration of TGFß1 in dermal fibroblast-mediated wound healing through an in vitro study. Human dermal fibroblasts were treated with vitamin D, TGFß1, both, or vehicle, and then the wound healing functions of dermal fibroblasts were measured. To further explore possible mechanisms explaining the synergistic effect of vitamin D and TGFß1, targeted gene silencing of the vitamin D receptor was performed. Compared to either factor alone, treatment of fibroblasts with both vitamin D and low concentration of TGFß1 increased gene expression of TGFß1, connective tissue growth factor, and fibronectin 1, and enhanced fibroblast migration, myofibroblast formation, and collagen production. Vitamin D receptor gene silencing blocked this synergistic effect of vitamin D and TGFß1 on both collagen production and myofibroblast differentiation. Thus a synergistic effect of vitamin D and low TGFß1 concentration was found in dermal fibroblast-mediated wound healing in vitro. This study suggests that supplementation of vitamin D may be an important step to improve wound healing and regeneration in patients with a vitamin D deficiency.

Systemic depletion of macrophages in the subacute phase of wound healing reduces hypertrophic scar formation.

Hypertrophic scars are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Studies suggest that M2-like macrophages are pro-fibrotic and contribute to hypertrophic scar formation. A previous study from our lab showed that M2 macrophages were present in developing hypertrophic scar tissues in vivo at 3-4 weeks after wounding. In this study, the effect of systemic macrophage depletion on scar formation was explored at subacute phase of wound healing. Thirty-six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, and 13 postgrafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1, and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4, and 8 weeks postgrafting. The transplanted human skin from the control group developed contracted, elevated, and thickened scars while the grafted skin from the depletion group healed with significant less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis, and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks postgrafting as compared to the control group. These findings suggest that systemic macrophage depletion in subacute phase of wound healing reduces scar formation, which provides evidence for the pro-fibrotic role of macrophages in fibrosis of human skin as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.

The natural behavior of mononuclear phagocytes in HTS formation.

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.

Fibrocytes participate in the development of heterotopic ossification.

Heterotopic ossification (HO) is a complication of musculoskeletal injury characterized by the formation of mature bone in soft tissues. The etiology of HO is unknown. We investigated the role of bone marrow derived progenitor cells in HO pathophysiology. We isolated the cells from HO specimens by cell explantation. Using flow cytometry and immunofluorescence microscopy, we found that 35 to 65% of the HO cells exhibit a bone marrow derived fibrocyte profile consisting in spindle-shaped morphology associated with type 1 pro-collagen and LSP1 expression. When cultured in osteogenic differentiation medium, active machinery for bone mineralization (high gene expression of Anx2, TNAP, and Pit-1), and calcium/phosphate deposits were found. Interestingly, interferon-alpha 2b significantly reduced the proliferation rate and COL1 gene expression in HO cells. We have characterized a novel subset of bone marrow derived progenitor cells in the HO specimens. The findings from this research study will provide new insights into the development of HO in burn patients.

The therapeutic potential of a C-X-C chemokine receptor type 4 (CXCR-4) antagonist on hypertrophic scarring in vivo.

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell-derived factor 1/CXCR4 signaling is up-regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood-borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell-derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE-9908 on dermal fibrosis was determined in vivo using a human HTS-like nude mouse model, in which split-thickness human skin is transplanted into full-thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE-9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down-regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.

Impaired cutaneous wound healing in transforming growth factor-ß inducible early gene1 knockout mice.

Transforming growth factor-ß inducible early gene (TIEG) is induced by transforming growth factor-ß (TGF-ß) and acts as the primary response gene in the TGF-ß/Smad pathway. TGF-ß is a multifunctional growth factor that affects dermal wound healing; however, the mechanism of how TGF-ß affects wound healing is still not well understood because of the complexity of its function and signaling pathways. We hypothesize that TIEG may play a role in dermal wound healing, with involvement in wound closure, contraction, and reepithelialization. In this study, we have shown that TIEG1 knockout (TIEG1-/-) mice have a delay in wound closure related to an impairment in wound contraction, granulation tissue formation, collagen synthesis, and reepithelialization. We also found that Smad7 was increased in the wounds and appeared to play a role in this wound healing model in TIEG1-/- mice.

Temperature-sensitive polymer-conjugated IFN-gamma induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG).

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co-cultured with IDO-expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon-gamma (IFN-gamma) conjugated with a temperature-sensitive polymer to induce the expression of IDO mRNA and protein. SDS-PAGE showed successful conjugation of IFN-gamma with the temperature-sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer-conjugated IFN-gamma. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN-gamma-treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer-conjugated IFN-gamma was significantly longer than in those treated with free (non-conjugated) IFN-gamma (P < 0.001). IFN-gamma radiolabeling showed a prolonged retention of IFN-gamma within collagen gel in its polymer-conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN-gamma). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO-expressing FPCG treated with polymer-conjugated IFN-gamma were co-cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co-cultured with IFN-gamma-treated IDO-expressing fibroblasts, compared to those co-cultured with non-IDO-expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1-methyl-D-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature-sensitive polymer further prolongs the effect of IFN-gamma on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts.

Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions as a local immunosuppressive factor.

As a possible way of making a non-rejectable skin substitute, here, we ask the question of whether the expression of indoleamine 2,3-dioxygenase (IDO) selectively suppresses immune, but skin, cell proliferation. To address this question, a series of experiments in which adenovirus (Ad-IDO) infected IDO expressing dermal fibroblasts were co-cultured with different types of immune cells were carried out. The immune cells were then harvested and evaluated for propidium iodide (PI) positive cells by FACS analysis. TUNEL assay was also carried out to determine the apoptotic status of these cells. The results showed that the expression of IDO in dermal fibroblasts significantly induces apoptotic death of PBMC, CD4(+)-, CD8(+)- and B cell-riched primary lymphocytes, Jurkat cells, and THP-1 cells. IDO-mediated damage of immune cells was restored by an addition of tryptophan and IDO inhibitor. Using the same approaches, we also demonstrated that skin cells and endothelial cells are remarkably resistant to tryptophan-deficient environment. Furthermore, no significant difference in cell proliferation between Ad-GFP (control)- and Ad-IDO-GFP-infected either keratinocytes or fibroblasts, was found. The results of this study, therefore, suggest that the expression of IDO by dermal fibroblasts mediates immune cell damage and this may shed a new light toward developing a non-rejectable skin substitute in the future.

The JCR:LA-cp rat: a novel model for impaired wound healing.

JCR:LA-cp/cp obese rats and their lean controls were evaluated as a type 2 diabetic wound healing model and the healing quality was characterized. This model of insulin resistance has been used extensively to study atherosclerosis but has not previously been used to study wound healing. Six circular excisional wounds were made on the dorsum of each rat and followed to day 21. Tracings of the wounds were made and used to assess the rate of wound closure. Planimetry showed a significantly diminished contraction of wounds in obese rats, but no significant difference in reepithelialization was observed. Collagen content was determined from the hydroxyproline content in wounded and unwounded skin. There were significantly lower levels of hydroxyproline in the wounds of obese compared to lean animals at day 21. Histology showed adipose tissue in place of dermal tissue in the JCR:LA-cp/cp rat in both unwounded tissue and in the wound at day 21. Active transforming growth factor-beta 1 (TGF-beta 1) was measured in the serum using the plasminogen activator inhibitor-1/luciferase assay and serum total TGF-beta was measured using an enzyme-linked immunosorbent assay. Active TGF-beta was significantly higher in the serum of obese animals compared with lean animals, while total TGF-beta 1 was not significantly different between the groups. Both active and total TGF-beta was measured in tissue sections using the plasminogen activator inhibitor-1/luciferase assay. There was no significant difference in active TGF-beta between genotypes, while obese rats had significantly higher levels of total TGF-beta at day 21. These results indicate a deficiency in wound healing in obese animals characterized by decreased wound contraction, decreased collagen production, and changes in histology. The JCR:LA-cp rat develops insulin resistance, atherosclerosis and early type 2 diabetes and may be a good model for impairment of wound healing in humans with metabolic syndrome.

Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG).

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment.

Development, characterization, and wound healing of the keratin 14 promoted transforming growth factor-beta1 transgenic mouse.

Transforming growth factor-beta1 is a fibrogenic cytokine that is important in the development of fibroproliferative disorders of the skin after injury. To investigate the role of transforming growth factor-beta1 produced by keratinocytes during wound healing, a plasmid with the human transforming growth factor-beta1 gene coupled with the keratin 14 promoter (pG3Z: K14-TGF-beta1) was constructed. The construct was tested successfully in vitro before being used to generate transgenic animals, which were subsequently bred into homozygous and heterozygous lines. Genotype screening of founders and progeny was performed by Southern blotting and targeting of the transgene to the epidermis by the keratin 14 promoter was shown by reverse transcription polymerase chain reaction. The major phenotypic change observed in the transgenic animals was "scruffiness" of the fur attributed to transgene expression in the skin, seen primarily in the homozygous line. A significant reduction in the rate of reepithelialization of full-thickness excisional wounds of dorsal skin was seen in homozygous animals compared with normal litter-mate controls at day 7 (p < 0.05, Fisher's Exact test) and day 9 (p < 0.01) postwounding. Wounds in heterozygous animals also healed more slowly at day 9 (p < 0.01). Northern analysis of mRNA extracted from the wounds showed increased human transforming growth factor-beta1 message levels in homozygous and heterozygous animals, maximal at day 5. Significant increases in transforming growth factor-beta1 activity in healing wounds measured using the plasminogen activator inhibitor-1/luciferase assay were found in the transgenic strains at day 9 postinjury as compared with the normal litter-mate control mice (p < 0.001, ANOVA). Type I procollagen mRNA expression was higher in the homozygous and heterozygous animals, with the highest levels reached at day 9. By day 5 postwounding, biopsies of both homozygous and heterozygous tissues were significantly higher in collagen as compared with wounds in control animals (p < 0.05, ANOVA). Based on these data, the K14-TGF-beta1 transgenic mouse shows that excessive latent transforming growth factor-beta1 produced in the epidermal layer of the skin delays reepithelialization in excisional wounds but subsequently the cells of the epidermis stimulate dermal fibroblasts leading to fibrosis through a paracrine mechanism.