Roles of cutaneous cell-cell communication in wound healing outcome: An emphasis on keratinocyte-fibroblast crosstalk.

Tissue repair is a very complex event and involves a continuously orchestrated sequence of signals and responses from platelets, fibroblasts, epithelial, endothelial and immune cells. The details of interaction between these signals, which are mainly growth factors and cytokines, have been widely discussed. However, it is still not clear how activated cells at wound sites lessen their activities after epithelialization is completed. Termination of the wound healing process requires a fine balance between extracellular matrix (ECM) deposition and degradation. Maintaining this balance requires highly accurate epithelial-mesenchymal communication and correct information exchange between keratinocytes and fibroblasts. As it has been reported in the literature, a disruption in epithelialization during the process of wound healing increases the frequency of developing chronic wounds or fibrotic conditions, as seen in a variety of clinical cases. Conversely, the potential stop signal for wound healing should have a regulatory role on both ECM synthesis and degradation to reach a successful wound healing outcome. This review briefly describes the potential roles of growth factors and cytokines in controlling the early phase of wound healing and predominantly explores the role of releasable factors from epithelial-mesenchymal interaction in controlling during and the late stage of the healing process. Emphasis will be given on the crosstalk between keratinocytes and fibroblasts in ECM modulation and the healing outcome following a brief discussion of the wound healing initiation mechanism. In particular, we will review the termination of acute dermal wound healing, which frequently leads to the development of hypertrophic scarring.

Studying the in vivo application of a liquid dermal scaffold in promoting wound healing in a mouse model.

Lack of matrix deposition is one of the main factors that complicates the healing process of wounds. The aim of this study was to test the efficacy and safety of a liquid dermal scaffold, referred to as MeshFill (MF) that can fill the complex network of tunnels and cavities which are usually found in chronic wounds and hence improve the healing process. We evaluated in vitro and in vivo properties of a novel liquid dermal scaffold in a delayed murine full-thickness wound model. We also compared this scaffold with two commercially available granular collagen-based products (GCBP). Liquid dermal scaffold accelerated wound closure significantly compared with no-treated control and collagen-based injectable composites in a delayed splinted wound model. When we compared cellular composition and count between MF, no treatment and GCBP at the histology level, it was found that MF was the most analogous and consistent with the normal anatomy of the skin. These findings were matched with the clinical outcome observation. The flowable in situ forming scaffold is liquid at cold temperature and gels after application to the wound site. Therefore, it would conform to the topography of the wound when liquid and provides adequate tensile strength when solidified. This patient-ready gelling dermal scaffold also contains the nutritional ingredients and therefore supports cell growth. Applying an injectable liquid scaffold that can fill wound gaps and generate a matrix to promote keratinocytes and fibroblasts migration, can result in improvement of the healing process of complex wounds.

Intraperitoneal injection of IDO-expressing dermal fibroblasts improves the allograft survival.

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme with tolerogenic effects on different immune cells. Our group has previously shown that co-transplantation of IDO-expressing fibroblasts with donor tissues can delay immune rejection by inducing local immunosuppression. In this study, we have employed a systemic approach to improve allograft survival without using any immunosuppressive medication. To achieve this, 10 million lentiviral transduced IDO-expressing donor derived fibroblasts were injected into the peritoneal cavity of allograft recipients. We showed that IDO-fibroblast therapy increases the survival of both islets and skin allografts and decreases the infiltration of immune cells in subcutaneous transplanted skins. Indirect pathway of allo-reactive T cell activation was suppressed more than the direct pathway. Injected IDO-fibroblasts were found in peritoneal cavity and mesenteric lymph nodes of the recipient mice. In conclusion, IDO-expressing fibroblast therapy proved to be a novel approach in improving the allogeneic graft survival.

Kynurenine Modulates MMP-1 and Type-I Collagen Expression Via Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts.

Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti-fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up-regulates cytochrome-P450 (CYP1A-1) expression, the AHR target gene. A specific AHR antagonist, 6,2',4'-trimethoxyflavone, inhibited the Kyn-dependent modulation of CYP1A-1, MMP-1, and type-I collagen expression. Establishing the anti-fibrogenic effect of Kyn and its mechanism of action, we then developed nano-fibrous Kyn slow-releasing dressings and examined their anti-fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated-dressings significantly improved the dermal fibrosis indicated by MMP-1 induction, alpha-smooth muscle actin and type-I collagen suppression, and reduced tissue cellularity, T-cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow-releasing anti-fibrogenic dressing. J. Cell. Physiol. 231: 2749-2760, 2016. © 2016 Wiley Periodicals, Inc.

IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1ß and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1ß levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 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.

Tolerogenic effect of mouse fibroblasts on dendritic cells.

There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). To clarify this issue, in this study, we have evaluated different features of fibroblast-primed DCs including their ability to express co-inhibitory and co-stimulatory molecules, pro-inflammatory and anti-inflammatory cytokines and their ability to induce T-cell proliferation. We also examined migratory capacity of DCs to lymphatic tissues and present fibroblast-derived antigens after encountering fibroblasts. The results of our in vitro study showed that both co-inhibitory (programmed death ligand 1 and ligand 2 and B7H4) and co-stimulatory (CD86) molecules were up-regulated when DCs were co-cultured with fibroblasts. In an animal model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased both total DC count and expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4(+) and CD8(+) T cells. Even activation of fibroblast- primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4(+) T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and indoleamine 2, 3 dioxygenase. Fibroblast-primed DCs had a significantly reduced interleukin-12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin). In conclusion, after priming with fibroblasts, DCs gain tolerogenic features. This finding suggests the potential role of fibroblasts in the maintenance of immune tolerance.

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.

Three-dimensional scaffolds reduce islet amyloid formation and enhance survival and function of cultured human islets.

Islet transplantation provides a promising approach for treatment of type 1 diabetes mellitus. Amyloid formation and loss of extracellular matrix are two nonimmune factors contributing to death of isolated human islets. We tested the effects of two types of three-dimensional scaffolds, collagen matrix (CM) and fibroblast-populated collagen matrix (FPCM), on amyloid formation, viability, and function of isolated islets. Islets from cadaveric donors were cultured in FPCM, CM, or two-dimensional plate (2D) for 7 days. After 7 days, compared with the 2D culture condition, CM and FPCM markedly reduced amyloid formation of cultured islets and decreased apoptotic ß-cell rate by ∼75%. IL-1ß and Fas levels were also reduced in scaffold-embedded islets. Furthermore, ß/α cell ratios were increased by ∼18% and ∼36% in CM- and FPCM-embedded islets, respectively. Insulin content and insulin response to elevated glucose were also enhanced by both three-dimensional scaffolds. Moreover, culture in CM and FPCM (but not 2D) preserved insulin, GLUT-2, and PDX-1 mRNA expression. FPCM-embedded islets had significantly higher insulin response and lower amyloid formation than CM-embedded islets. These findings suggest that three-dimensional scaffolds reduce amyloid formation and improve viability and function of human islets in vitro, and that CM and fibroblasts have additive effects in enhancing islet function and reducing amyloid formation. Using this strategy is likely to improve outcome in human islet transplantation.

Antibacterial Thermosensitive Silver-Hydrogel Nanocomposite Improves Wound Healing.

Bacterial infection and poor cell recruitment are among the main factors that prolong wound healing. To address this, a strategy is required that can prevent infection while promoting tissue repair. Here, we have created a silver nanoparticle-based hydrogel composite that is antibacterial and provides nutrients for cell growth, while filling cavities of various geometries in wounds that are difficult to reach with other dressings. Silver nanoparticles (AgNPs) were synthesized by chemical reduction and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and inductively coupled plasma-mass spectroscopy (ICP-MS). Using varying concentrations of AgNPs (200, 400, and 600 ppm), several collagen-based silver-hydrogel nanocomposite candidates were generated. The impact of these candidates on wound healing was assessed in a rat splinted wound model, while their ability to prevent wound infection from a contaminated surface was assessed using a rat subcutaneous infection model. Biocompatibility was assessed using the standard MTT assay and in vivo histological analyses. Synthesized AgNPs were spherical and stable, and while hydrogel alone did not have any antibacterial effect, AgNP-hydrogel composites showed significant antibacterial activity both in vitro and in vivo. Wound healing was found to be accelerated with AgNP-hydrogel composite treatment, and no negative effects were observed compared to the control group. The formulations were non-cytotoxic and did not differ significantly in hematological and biochemical factors from the control group in the in vivo study. By presenting promising antibacterial and wound healing activities, silver-hydrogel nanocomposite offers a safe therapeutic option that can be used as a functional scaffold for an acceleration of wound healing.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts.

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.

Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia.

Due to the poor prognosis and limited therapeutic options for adult patients with acute lymphoblastic leukemia (ALL), development of novel therapies is much needed to prolong patient survival and increase the efficacy of their treatment. Malignant T cells need high levels of nutrients to maintain their proliferation rate. Borrelidin, a small molecule nitrile-containing macrolide, is an inhibitor of bacterial and eukaryal threonyl-tRNA synthetase. Borrelidin-mediated inhibition of aminoacyl-tRNA synthesis, leads to an induction in the levels of uncharged tRNA, nutritional stress and ultimately inhibition of protein synthesis. The aim of the present study was to investigate whether borrelidin treatment inhibits the proliferation of malignant ALL cell lines, Jurkat and CEM cells, and study the mechanism by which this drug acts. Our results show that borrelidin was able to potently inhibit the proliferation of ALL cell lines with a half maximal inhibitory concentration of 50 ng/ml. Borrelidin showed a greater inhibitory effect on ALL cell lines compared to primary fibroblasts. Flow cytometry and western blot analysis indicated that borrelidin was able to increase the level of apoptosis and cause G(1) arrest in ALL cell lines. Activation of the general control nonderepressible-2 (GCN2) kinase stress responsive pathway and induction of CHOP protein was significantly higher in ALL cell lines treated with borrelidin. These findings collectively suggest for the first time that borrelidin targets ALL cell lines by inducing apoptosis and mediating G(1) arrest and that borrelidin treatment in ALL cell lines is correlated with activation of the GCN2 kinase pathway.

Establishing a cGMP pancreatic islet processing facility: the first experience in Iran.

It has been predicted that one of the greatest increase in prevalence of diabetes will happen in the Middle East bear in the next decades. The aim of standard therapeutic strategies for diabetes is better control of complications. In contrast, some new strategies like cell and gene therapy have aimed to cure the disease. In recent years, significant progress has occurred in beta-cell replacement therapies with a progressive improvement of short-term and long term outcomes. In year 2005, considering the impact of the disease in Iran and the promising results of the Edmonton protocol, the funding for establishing a current Good Manufacturing Practice (cGMP) islet processing facility by Endocrinology and Metabolism Research Center was approved by Tehran University of Medical Sciences. Several islet isolations were performed following establishment of cGMP facility and recruitment of all required equipments for process validation and experimental purpose. Finally the first successful clinical islet isolation and transplantation was performed in September 2010. In spite of a high cost of the procedure it is considered beneficial and may prevent long term complications and the costs associated with secondary cares. In this article we will briefly describe our experience in setting up a cGMP islet processing facility which can provide valuable information for regional countries interested to establish similar facilities.

Inflammatory Immune Responses Trigger Rejection of Allogeneic Fibroblasts Transplanted into Mouse Skin.

Fibroblasts, or their homolog stromal cells, are present in most tissues and play an essential role in tissue homeostasis and regeneration. As a result, fibroblast-based strategies have been widely employed in tissue engineering. However, while considered to have immunosuppressive properties, the survival and functionality of allogeneic fibroblasts after transplantation remain controversial. Here, we evaluated innate and adaptive immune responses against allogeneic fibroblasts following intradermal injection into different immune-deficient mouse strains. While allogeneic fibroblasts were rejected 1 week after transplantation in immunocompetent mice, rejection did not occur in immunodeficient γ chain-deficient NOD-SCID (NSG) mice. T-cell- and B-cell-deficient RAG1 knockout mice showed greater loss of fibroblasts by day 5 after transplantation compared with NSG mice (P ≤ 0.05) but prolonged persistence compared with wild-type recipient (P ≤ 0.005). Loss of fibroblasts correlated with the expression of proinflammatory chemokine genes and infiltration of myeloid cells in the transplantation site. Depletion of macrophages and neutrophils delayed rejection, revealing the role of innate immune cells in an early elimination of fibroblasts that is followed by T-cell-mediated rejection in the second week. These findings indicate that the application of allogeneic fibroblasts in tissue engineering products requires further improvements to overcome cell rejection by innate and adaptive immune cells.

Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal.

Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.

Incorporation of Silver Nanoparticles in Hydrogel Matrices for Controlling Wound Infection.

For centuries, silver has been recognized for its antibacterial properties. With the development of nanotechnology, silver nanoparticles (AgNPs) have garnered significant attention for their diverse uses in antimicrobial gel formulations, dressings for wound healing, orthopedic applications, medical catheters and instruments, implants, and contact lens coatings. A major focus has been determining AgNPs' physical, chemical, and biological characteristics and their potential to be incorporated in biocomposite materials, particularly hydrogel scaffolds, for burn and wound healing. Though AgNPs have been rigorously explored and extensively utilized in medical and nonmedical applications, important research is still needed to elucidate their antibacterial activity when incorporated in wound-healing scaffolds. In this review, we provide an up-to-date, 10-yr (2010-2019), comprehensive literature review on advancements in the understanding of AgNP characteristics, including the particles' preparation and mechanisms of activity, and we explore various hydrogel scaffolds for delivering AgNPs.

In Situ Forming Nutritional and Temperature Sensitive Scaffold Improves the Esthetic Outcomes of Meshed Split-Thickness Skin Grafts in a Porcine Model.

Objective: Full-thickness burn wounds require immediate coverage, and the primary clinical approaches comprise of skin allografts and autografts. The use of allografts is often temporary due to the antigenicity of allografts. In contrast, the availability of skin autografts may be limited in large burn injuries. In such cases, skin autografts can be expanded through the use of a skin mesher, creating meshed split-thickness skin grafts (MSTSGs). MSTSGs have revolutionized the treatment of large full-thickness burn injuries since the 1960s. However, contractures and poor esthetic outcomes remain a problem. We previously formulated and prepared an in situ forming skin substitute, called MeshFill (MF), which can conform to complex shapes and contours of wounds. The objective of this study was to assess the esthetic and wound healing outcomes in full-thickness wounds treated with a combination of MF and MSTSG in a porcine model. Approach: Either MSTSGs or MSTSG+MF was applied to full-thickness excisional wounds in Yorkshire pigs. Wound healing outcomes were assessed using histology, immunohistochemistry, and wound surface area analysis from day 10 to 60. Clinical evaluation of wounds were utilized to assess esthetic outcomes. Results: The results demonstrated that the combination of MSTSGs and MF improved wound healing and esthetic outcomes. Innovation: Effects of MSTSGs and reconstitutable liquid MF in a full-thickness porcine model were investigated for the first time. Conclusion: MF provides promise as a combination therapeutic regimen to improve wound healing and esthetic outcomes.

High expression of IMPACT protein promotes resistance to indoleamine 2,3-dioxygenase-induced cell death.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.

Highly efficient stable expression of indoleamine 2,3 dioxygenase gene in primary fibroblasts.

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

Mouse pancreatic islets are resistant to indoleamine 2,3 dioxygenase-induced general control nonderepressible-2 kinase stress pathway and maintain normal viability and function.

Islet transplantation is a promising treatment for diabetes. However, it faces several challenges including requirement of systemic immunosuppression. Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. Local expression of IDO in bystander fibroblasts suppresses islet allogeneic immune response in vitro. The aim of the present study was to investigate the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblast-populated collagen scaffold. Mouse islets were embedded within collagen matrix populated with IDO adenovector-transduced or control fibroblasts. Proliferation, insulin content, glucose responsiveness, and activation of general control nonderepressible-2 kinase stress-responsive pathway were then measured in IDO-exposed islets. In vivo viabilities of composite islet grafts were also tested in a syngeneic diabetic animal model. No reduction in islet cells proliferation was detected in both IDO-expressing and control composites compared to the baseline rates. Islet functional studies showed normal insulin content and secretion in both preparations. In contrast to lymphocytes, general control nonderepressible-2 kinase pathway was not activated in islets cocultured with IDO-expressing fibroblasts. When transplanted to diabetic mice, syngeneic IDO-expressing composite islet grafts were functional up to 100 days tested. These findings collectively confirm normal viability and functionality of islets cocultured with IDO-expressing cells and indicate the feasibility of development of a functional nonrejectable islet graft.