Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1ß, from these cells in AA-affected skin.

Increased expression of PD-L1 and PD-L2 in dermal fibroblasts from alopecia areata mice.

Alopecia areata (AA) is a common autoimmune disorder affecting millions of people worldwide, which manifests as a sudden, non-scarring hair loss. The expression of a pro-inflammatory cytokine, interferon-gamma (INF-γ), has been well established to be involved in the development of AA. As IFN-γ and other cytokines are also known to up-regulate programmed cell death ligand 1 and 2 (PD-L1 and PD-L2), which both negatively control immune responses, we asked whether or not a high number of infiltrated T cells, seen in AA lesions, can modulate the expression of PD-L1 and PD-L2 in skin cells. From a series of experiments, we showed that a significantly higher number of PD-L1 or PD-L2 positive cells affect the skin in AA mice, compared to the skin of non-AA mice. The number of PD-L1 positive cells was well correlated with the number of infiltrated T cells, especially CD8+ T cells. We also found that the expression of PD-L1 and PD-L2 was co-localized with type 1 pro-collagen, CD90 and vimentin, which are biomarkers for dermal fibroblasts. Further studies revealed that releasable factors from activated, but not inactivated, lymphocytes significantly increase the expressions of both PD-L1 and PD-L2 in cultured dermal fibroblasts. In conclusion, our findings suggest that the expression of PD-L1 and PD-L2 in dermal fibroblasts is up-regulated by activated T cells in AA-affected skin, and as such, these regulatory molecules may not exert a negative control of the immune activation seen in AA lesions.

Keratinocyte-Releasable Factors Stimulate the Expression of Granulocyte Colony-Stimulating Factor in Human Dermal Fibroblasts.

Interaction between keratinocytes and fibroblasts plays a critical role in maintaining skin integrity under both normal and pathological conditions. We have previously demonstrated that keratinocyte-releasable factors influence the expression of key extracellular matrix components, such as collagen and matrix metalloproteinases in dermal fibroblasts. In this study, we utilized DNA microarray analysis to examine the effects of keratinocyte-releasable factors on the expression of several cytokines in human dermal fibroblasts. The results revealed significantly higher granulocyte colony-stimulating factor (G-CSF) expression in fibroblasts co-cultured with keratinocytes relative to mono-cultured cells, which was verified by RT-PCR and western blot. G-CSF is an important hematopoietic factor also thought to play a beneficial role in wound healing through stimulating keratinocyte proliferation. To partially characterize the keratinocyte-releasable factors responsible for stimulating G-CSF production, keratinocyte-conditioned medium (KCM) was subjected to thermal treatment and ammonium sulfate precipitation before treating fibroblasts. The results showed that keratinocyte-releasable G-CSF-stimulating factors remain stable at 56°C and upon 50% ammonium sulfate precipitation. Knowing that keratinocytes release IL-1, which stimulates G-CSF expression in various immune cells, several experiments were conducted to ask whether this might also be the case for fibroblasts. The results showed that the addition of recombinant IL-1 markedly increased G-CSF expression in fibroblasts; however, IL-1 receptor antagonist only partially abrogated KCM-stimulated G-CSF expression, indicating the role of additional keratinocyte-releasable factors. These findings underline the importance of cross-talk between keratinocytes and fibroblasts, suggesting that communication between these cells in vivo modulates the production of cytokines required for cutaneous wound healing and maintenance. J. Cell. Biochem. 118: 308-317, 2017. © 2016 Wiley Periodicals, Inc.

Identification of a Hematopoietic Cell Dedifferentiation-Inducing Factor.

It has long been realized that hematopoietic cells may have the capacity to trans-differentiate into non-lymphohematopoietic cells under specific conditions. However, the mechanisms and the factors for hematopoietic cell trans-differentiation remain unknown. In an in vitro culture system, we found that using a conditioned medium from proliferating fibroblasts can induce a subset of hematopoietic cells to become adherent fibroblast-like cells (FLCs). FLCs are not fibroblasts nor other mesenchymal stromal cells, based on their expression of type-1 collagen, and other stromal cell marker genes. To identify the active factors in the conditioned medium, we cultured fibroblasts in a serum-free medium and collected it for further purification. Using the fractions from filter devices of different molecular weight cut-offs, and ammonium sulfate precipitation collected from the medium, we found the active fraction is a protein. We then purified this fraction by using fast protein liquid chromatography (FPLC) and identified it by mass spectrometer as macrophage colony-stimulating factor (M-CSF). The mechanisms of M-CSF-inducing trans-differentiation of hematopoietic cells seem to involve a tyrosine kinase signalling pathway and its known receptor. The FLCs express a number of stem cell markers including SSEA-1 and -3, OCT3/4, NANOG, and SOX2. Spontaneous and induced differentiation experiments confirmed that FLCs can be further differentiated into cell types of three germ layers. These data indicate that hematopoietic cells can be induced by M-CSF to dedifferentiate to multipotent stem cells. This study also provides a simple method to generate multipotent stem cells for clinical applications.

Methotrexate modulates the expression of MMP-1 and type 1 collagen in dermal fibroblast.

Methotrexate (MTX), an anti-metabolite and anti-inflammatory drug, has been used to effectively manage and prevent keloids, but its mechanism(s) of action has not been elucidated. Our study sought to evaluate the effect of MTX on the production of key extra cellular matrix components, collagen, and matrix metalloproteinase-1 (MMP-1), produced by fibroblasts and involved in development of fibrosis. The proliferation and viability of cultured human dermal fibroblasts in response to different concentrations of MTX were determined using cell counting and MTT assay, respectively. Western blot analysis was used to determine the levels of both intracellular and secreted type 1 collagen and MMP-1. The results showed no significant changes in the proliferation of fibroblasts treated with 50 ng/ml of MTX as compared to that of control. Under the same experimental conditions, the level of secreted and intracellular type I collagen was markedly reduced and, conversely, the level of MMP-1 increased in treated neonatal, adult, and hypertrophic scar fibroblasts as compared with those of controls. The possible involvement of MTX-induced extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in MMP-1 production was also studied and the result showed an increase in phosphorylated ERK 1/2 in response to MTX treatment. In summary, the findings of this study revealed that MTX significantly reduced collagen production in different strains of fibroblasts derived from neonatal, adult, and hypertrophic scar tissues, while under the same experimental conditions, it increased the expression of MMP-1. As such, our findings validate and identify a potential mechanism through which MTX functions as an anti-fibrogenic factor in treating fibroproliferative disorders.

Effects of kynurenine on CD3+ and macrophages in wound healing.

As prolongation of the inflammation phase in a healing process frequently leads to wound impairment, here we queried whether kynurenine (Kyn) could modulate this phase of wound healing. To address this, a protein microarray, quantitative polymerase chain reaction (qPCR), flow cytometry for immune cells and immune cell proliferation in the presence and absence of Kyn were conducted and compared. The result of a protein microarray revealed that the expression of 12 pro-inflammatory cytokines and chemokines was modulated in Kyn-treated mouse splenocytes as compared with those of control. These findings were then evaluated by conducting a qPCR for the gene expression of these factors and showed a significant reduction in the gene expression of majority of these cytokines and chemokines (interleukin [IL]-2, IL-17, C-X-C motif chemokine ligand [CXCL] 10, CXCL1, C-C motif ligand [CCL] 12, CXCL9, CCL4, CXCL2, and CCL5) in response to Kyn treatment. To test the anti-inflammatory effect of Kyn in an animal model, dorsal surface wounds were generated in a mouse model and wounds received daily topical application of either nothing (control), dermal cream (second control), or Kyn cream using uninjured skin tissue as another control. The wounded tissues were harvested on days 3, 6, and 10 postwounding. As anticipated, the results of fluorescence-activated cell sorting analysis revealed that upon wounding, the number of total infiltrated CD3+ cells and macrophages (CD11b+) significantly increased on day 3, peaked on day 6, and reduced on day 10 post-wounding. Interestingly, as compared with those of uninjured and dermal cream alone-treated wounds, Kyn treatment significantly reduced the number of infiltrated CD3+ cells, but not CD11b+ cells, at different time intervals examined. These findings collectively suggest that Kyn, as a small molecule, can potentially be used to overcome the difficulties associated with persistency of inflammation in healing wounds.

Granulomatous mastitis in a 50-year-old male: A case report and review of literature.

BACKGROUND: Granulomatous mastitis (GM) an inflammatory disease of the breast that usually affects women of childbearing age, occurs very rarely in males. CASE SUMMARY: We present a case study of a 50-year-old male patient with GM. The patient developed a breast lump following the cleaning of a previously embedded dirt-filled nipple. While an initial improvement was noted with antibiotic therapy, a recurrence occurred a year later, showing resistance to the previously effective antibiotics. Subsequently, the lesion was excised. The histopathological examination confirmed the diagnosis of GM. CONCLUSION: GM should be considered a possible diagnosis of male breast masses.

Assessing urban park equity in Chaoyang District, Beijing using online review data.

Urban parks are essential components of urban ecosystems, providing vital ecological resources for city residents. However, the rapid expansion of high-density urban areas has led to an unequal distribution of park resources, raising growing concerns about spatial equity. To address these challenges, we employed an improved Gaussian two-step floating catchment area (2SFCA) method, considering park quality variations and integrating sentiment scores from park reviews to calculate a comprehensive park accessibility index, accounting for both supply and demand dynamics among park users. The results demonstrate the significance of park management, as users prioritise convenience and cleanliness of public facilities. Recreational quality significantly influences park distribution equity, with areas near Beijing's initial greenbelt zone showing improved accessibility (IA). Nonetheless, our analysis exposes disparities in urban park resource allocation within the Chaoyang District, indicating relative inequity. Spatial supply and demand mismatches, especially in the northwest and southeast, are evident. To enhance park layout equity, we recommend strategies like identifying and repurposing underused spaces, establishing pocket parks and micro-green areas, and improving recreational facilities. It is crucial to address the needs of vulnerable groups such as older residents and children. These insights stress the importance of ensuring fair urban park access to enhance the well-being of all city residents.

Impact of ecological conservation policies on land use and carbon stock in megacities at different stages of development.

Urban expansion, especially the construction of megacities, increases carbon emissions and adversely affects the carbon storage of terrestrial ecosystems. However, scientific land-use management policies can increase carbon storage. This study takes two megacities at different stages of development, Beijing and Tianjin, as examples to explore the impact of different ecological conservation scenarios on both urban land use and carbon storage to provide recommendations for the construction planning of large cities with low-carbon development as the goal. Furthermore, we coupled the patch-generating land use simulation (PLUS) model with the integrated valuation of ecosystem services and tradeoffs (InVEST) model to simulate land use and carbon storage under a natural development scenario, a planned ecological protection scenario (PEPS), and a policy-based ecological restoration scenario (PERS). From 2000 to 2020, both cities had different degrees of construction land expansion and carbon loss, and Tianjin's dynamic degree of construction land was 0.94% higher than Beijing's, with a carbon loss 183,536.19 Mg higher than Beijing's; this trend of reducing carbon reserves will continue under the natural development scenario (NDS). Under the PEPS and PERS, the carbon stock of both cities increases, and the impact on Tianjin is greater, with an increase of 4.51% and 8.04%, respectively. Under PERS, the carbon stock increases the most, but the dynamic degree of construction land use is negative for both cities. Beijing's carbon stock is 0.40% lower than Tianjin's, which deviates slightly from the trend of urban economic development. Megacities in the rapid development stage can refer to Tianjin, strictly following the ecological protection land planning scope and vigorously implementing ecological restoration policies to effectively increase regional carbon stock. Megacities in the mature stage of development can refer to Beijing, and flexibly implement ecological restoration policies to increase regional carbon stock without affecting the city's economic development.

Clinicopathologic Features and Pathogens of Granulomatous Lobular Mastitis.

Background: Granulomatous lobular mastitis (GLM) is a rare, benign, and complex breast disease that can be easily misdiagnosed as breast cancer. The etiology of GLM is unclear, and optimal treatment has not been established. Methods: Medical records for 333 patients with GLM in recent 5 years at Longhua Hospital, Shanghai, China, were analyzed. Potential pathogens in 33 fresh tissue specimens were also analyzed using 16S rDNA sequencing technology, matrix-assisted laser desorption ionization time of flight mass spectrometry, and bacterial cultures. Results: The median age of patients was 32 years (range 22-47 years). Among 333 patients, 38.7% displayed elevated prolactin, while 23.7% displayed high interleukin-2. In the granulomatous lesion, CD3-positive T lymphocytes were significantly more than CD20-positive B lymphocytes around the vacuoles or microabscesses. Gram-positive organisms were observed in 82 cases, including in 22 cases from fresh tissue specimens. Thirty-three cases yielded associated pathogens and all displayed multiple pathogenic infections, as identified using 16S rDNA sequencing technology. Pathogenic infections were further identified as belonging to 16 main genera and 8 main pathogenic species. Conclusions: GLM displays distinct histological and clinical features similar to those that have been previously reported in the literature. Using 16S rDNA sequencing technology, all of our cases demonstrated multiple pathogenic infections, which provided more useful information for clinical treatment.

MAP kinase mediates silica-induced fibrotic nodule formation and collagen accumulation in fibroblasts.

It is well known that silica generates fibrosis around them in animals and human. However, the pathogenesis and mechanism of silica-induced fibrosis are still poorly understood. Here, we established a new strategy through which the effects of silica on fibrotic nodule formation, key extracellular matrix accumulation, and the mechanism involved were explored. To achieve this, human dermal fibroblasts were directly exposed to silica gel for various durations. Fibrotic nodule formation was evaluated by their microscopic appearance, type-1 procollagen, and fibronection expression in cell lysate and MMP-1 and-3 in conditioned media were analyzed by Western blotting. The results show an easily formation of nodule-like structures around silica gel in an in vitro-cultured system. The findings further revealed that silica gel stimulates collagen and fibronectin expression, while down-regulates matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) released in conditioned medium. To explore the mechanism involved, P38 and ERK1/2 Mitogen-Activated Protein Kinase (MAPK) signaling pathways were evaluated. Result showed that silica inhibits P38 and extracellular signal-regulated kinases (ERK1/2) MAP kinase phosphorylation. The addition of ERK1/2 inhibitor increases silica-stimulated type-1 collagen expression, reduces MMP-1 release and further enhances silica-induced nodule formation in dermal fibroblasts. These findings indicate that the inhibition of ERK1/2 MAPK signaling pathway may contribute to silica-caused fibrosis. In summary, our findings suggest that silica can directly cause fibrotic phenotype when fibroblasts contact with silica particles independent of any inflammation and other factors may exist in an in vivo condition.

Microarray-based identification of aminopeptidase N target genes in keratinocyte conditioned medium-stimulated dermal fibroblasts.

Of the many processes that affect the outcome of wound repair, epidermal-dermal interactions are essential to extracellular matrix (ECM) remodeling and in particular, soluble factors released by keratinocytes are known to have a direct impact on the production of ECM by dermal fibroblasts. Aminopeptidase N (APN) has recently been proposed as a cell-surface receptor for stratifin and is responsible for the stratifin-mediated matrix metalloproteinase-1 (MMP-1) upregulation in fibroblasts. The present study examines whether modulation of APN gene expression has any impact on the fibroblast ECM gene expression profile. The result reveals that in the presence of keratinocyte-derived soluble factors, transient knockdown of APN in dermal fibroblasts affects the expression of key ECM components such as fibronectin, tenascin-C, MMP-1, MMP-3, and MMP-12. The regulatory effects of APN on fibronectin and selective MMPs appear to be associated with receptor-mediated signal transduction independently of its peptidase activity. On the contrary, inhibition of the APN enzymatic activity by bestatin significantly reduces the tenascin-C expression and enhances the contraction of fibroblast-populated collagen gel, suggesting an activity-dependent regulation of fibroblast contractility by APN. The overall effects of APN on the expression of fibronectin, tenascin-C, and MMPs in fibroblasts propose an important role for APN in the regulation of keratinocyte-mediated ECM remodeling and fibroblast contractile activity.

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts.

During the early stage of wound healing process, blood clots can be served as a temporary extracellular matrix (ECM) to let skin cell migration and proliferation. The red blood cells are generally thought as inert bystanders in the early and inflammatory phase of wound healing. Here, we provide evidence that red blood cells (RBC) also play an important role in modulation of key ECM components such as type-I collagen, α-smooth muscle actin, fibronectin, and matrix metalloproteinases (MMPs). In this study, we used western blot analysis and showed a significant increase in the level of MMP-1, 2, 3. Furthermore, we found that RBC lysate significantly down-regulates type-I collagen and α-smooth muscle actin while up-regulates fibronectin expression in dermal fibroblasts. To further explore the mechanism by which RBC lysate modulates MMP-1 expression, the effect of inhibitors for three MAPK signaling pathways on RBC inducing MMP-1 expression by dermal fibroblasts were tested. The result showed that the inhibitor of ERK1/2 could abrogate the stimulatory effect of RBC lysate on MMP-1 expression in dermal fibroblasts. Consistently, RBC treatment results in an increase of ERK1/2 phosphorylation in dermal fibroblast. In conclusion, these findings suggest that RBC lysate can modulate the expression of MMPs and key ECM components which are important in healing process.

M-CSF-stimulated myeloid cells can convert into epithelial cells to participate in re-epithelialization and hair follicle regeneration during dermal wound healing.

Dermal wound healing is a complex process which requires the interaction of many cell types and mediators in a highly sophisticated temporal sequence. Myeloid cells which compose of a significant proportion of the inflammatory cells infiltrate to the to a wound site where they play important roles in clearance of damaged tissue and microorganisms. Myeloid cells have the capacity to be converted into fibroblast-like cells and endothelial cells during wound healing process. However, whether myeloid cells in wounds can convert into epithelial cells where they contribute to healing process is not clear. In this study, we performed double immunofluorescent staining with antibodies for hematopoietic cells and keratinocytes as well as cell tracing technique to investigate hematopoietic cell conversion. The result showed that during the healing process, some of the CD45-positive hematopoietic cells also expressed keratin 14, a marker for keratinocytes. Further, double immunofluorescent staining in dermal wounds, using CD11b and K14 antibodies indicated that CD11b-positive myeloid cells were the origin of newly generated epithelial cells. Through tracing injected labeled splenocyte-derived myeloid cells in skin, we confirmed that myeloid cells were able to convert into keratinocytes in repaired skin. Furthermore, our results from in vivo experiments provided new information on contribution of myeloid cells in hair follicle regeneration. In conclusion, this work highlights the myeloid cell contributions in wound repair and hair follicle regeneration through conversion of M-CSF-stimulated CD11b-positive myeloid cells into epithelial cells in a murine model.

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin.

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication.

Split Thickness Grafts Grow From Bottom Up in Large Skin Injuries.

Autologous split thickness skin graft is necessary for the survival of patients with large burns and skin defects. It is not clear how a thin split thickness skin graft becomes remarkably thicker within a few weeks following transplantation. Here, we hypothesized that growth of split thickness graft should be from bottom up probably through conversion of immune cells into collagen producing skin cells. We tested this hypothesis in a preclinical porcine model by grafting split thickness meshed skin (0.508 mm thickness, meshed at 3:1 ratio) on full thickness wounds in pigs. New tissue formation was evaluated on days 10 and 20 postoperation through histological analysis and co-staining for immune cell markers (CD45) and type I collagen. The findings revealed that a split thickness graft grew from bottom up and reached to almost the same level as uninjured skin within 60 days postoperation. The result of immune-staining identified a large number of cells, which co-expressed immune cell marker (CD45) and collagen on day 10 postoperation. Interestingly, as the number of these cells reduced on day 20, most of these cells became positive for collagen production. In another set of experiments, we tested whether immune cells can convert to collagen producing cells in vitro. The results showed that mouse adherent immune cells started to express type 1 procollagen and α-smooth muscle actin when cultured in the presence of fibroblast conditioned media. In conclusion, the early thickening of split thickness graft is likely happening through a major contribution of infiltrated immune cells that convert into mainly collagen producing fibroblasts in large skin injuries.

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

Alopecia 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.

Local expression of indoleamine 2,3-dioxygenase protects engraftment of xenogeneic skin substitute.

The expression of indoleamine 2,3-dioxygenase (IDO), which metabolizes tryptophan, an essential amino acid, into kynurenine, has been identified as having a key role in the prevention of the immune rejection of the semi-allogeneic fetus during pregnancy. We have previously demonstrated that IDO expressed in fibroblasts causes bystander CD4(+) T cell damage as well as THP-1 cell damage by apoptosis. As T cells are primarily responsible for graft rejection, here, we asked the question of whether engraftment of IDO-expressing xenogeneic fibroblasts populated in a collagen matrix can be immuno-protected in an animal model. The results show a significant reduction in the number of infiltrated CD3(+) T lymphocytes on days 14 and 28 post-transplantation in the wounds receiving IDO-expressing fibroblasts relative to controls. IDO-expressing human fibroblasts embedded in bovine collagen on wounds in a rat model accelerates wound healing by promoting neovascularization during the early stages and providing protection of the xenograft fibroblasts. Using a co-culture system, we further confirm that IDO can induce angiogenesis through the depletion of tryptophan. These findings suggest that IDO may have an application in promoting the engraftment of skin substitutes and other transplanted organs.

Accelerating skin wound healing by M-CSF through generating SSEA-1 and -3 stem cells in the injured sites.

Wound healing is a complicated process requiring the collaborative efforts of different cell lineages. Our recent studies have found that one subset of hematopoietic cells can be induced to dedifferentiate into multipotent stem cells by means of a proliferating fibroblast releasable factor, M-CSF. Understanding the importance of stem cells on skin wound healing, here we evaluate the biological significance of M-CSF on skin wound healing. In an in vivo mouse skin excisional wound model, we found that SSEA-positive stem cells were present in wounded but not normal skin. After isolating skin cells from either normal or wounded skin by collagenase digestion, and analyzing the SSEA-1 positive cells by flow cytometry, we found a significant increase in the number of SSEA-1 positive cells in wounded skin. Topical application of M-CSF in skin wounds accelerated healing remarkably, while application of M-CSF-neutralizing antibody slowed wound healing. Furthermore, injection of EGFP-labeled hematopoietic cell-derived stem cells generated from M-CSF treated splenocytes resulted in EGFP-labeled cells being enriched in the skin wound site and further differentiated into functional organ-specific cells. Together, these data demonstrated that M-CSF makes a significant contribution to the healing process by inducing hematopoietic cell dedifferentiation into stem cells.

Fibroblast Cell-Based Therapy for Experimental Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin producing ß cells of the pancreatic islets. Curbing autoimmunity at the initiation of T1D can result in recovery of residual ß cells and consequently remission of diabetes. Here we report a cell-based therapy for autoimmune diabetes in non-obese diabetic (NOD) mice using dermal fibroblasts. This was achieved by a single injection of fibroblasts, expressing the immunoregulatory molecule indoleamine 2,3 dioxygenase (IDO), into peritoneal cavity of NOD mice shortly after the onset of overt hyperglycemia. Mice were then monitored for reversal of hyperglycemia and changes in inflammatory/regulatory T cell profiles. Blood glucose levels dropped into the normal range in 82% of NOD mice after receiving IDO-expressing fibroblasts while all control mice remained diabetic. We found significantly reduced islet inflammation, increased regulatory T cells, and decreased T helper 17 cells and ß cell specific autoreactive CD8+ T cells following IDO cell therapy. We further showed that some of intraperitoneal injected fibroblasts migrated to local lymph nodes and expressed co-inhibitory molecules. These findings suggest that IDO fibroblasts therapy can reinstate self-tolerance and alleviate ß cell autoreactivity in NOD mice, resulting in remission of autoimmune diabetes.