Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.     

Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.     

Expression of a releasable form of annexin II by human keratinocytes

Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining.     

Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts

Success of transplantation of pancreatic islets which is a promising way for restoring efficient insulin regulation in type 1 diabetes depends on lifelong use of immunosuppressive drugs. To eliminate the use of systemic immunosuppressive drugs for islet transplantation, we examined the potential use of a local immunosuppressive factor, indoleamine 2,3-dioxygenase (IDO). Thus, the aim of this study was to determine whether local expression of IDO in bystander syngeneic fibroblasts could prevent islet allogeneic immune response in vitro. C57BL/6 (B6) mouse fibroblasts were induced to express IDO by either IFN-gamma treatment or transduction with an adenoviral vector and were co-cultured with B6 mouse lymphocytes and BALB/c mouse pancreatic islets in the presence or absence of an IDO inhibitor. Proliferation of lymphocytes were then assessed using [(3)H]-thymidine incorporation assay. IDO-expression by co-cultured syngeneic fibroblasts resulted in a five-fold decrease in lymphocyte proliferation rate upon stimulation of lymphocytes by allogeneic mouse pancreatic islets (21.9% +/- 5.3 and 22.1% +/- 4.9 in the preparations with IFN-gamma treated and genetically modified IDO-expressing fibroblasts, respectively vs. 100% in control groups, P < 0.01). Allogeneic response was restored when IDO inhibitor was added to the culture indicating that suppression was due to IDO. In conclusion, this study shows that local expression of IDO by syngeneic bystander fibroblasts can suppress in vitro proliferation of lymphocytes in response to stimulation with allogeneic pancreatic islets. This local immunosuppressive function of IDO may be employed for development of a novel alternative strategy for preventing allogeneic islet graft rejection.     

The role of stratifin in fibroblast–keratinocyte interaction

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a “stemness condition” of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.     

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. J. Cell. Physiol. 225: 196–205, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression

Airway remodelling in asthma involves various mediators modulating the production/breakdown of collagen by lung fibroblasts. Matrix metalloproteinase-1 (MMP-1) plays an important role in collagen breakdown. We recently showed that epithelial cell-derived extracellular form of 14-3-3σ is an important inducer of MMP-1 expression in skin fibroblasts. Thus, we hypothesized that 14-3-3 proteins are important regulators of MMP-1 expression in the respiratory airway. We examined the presence of extracellular 14-3-3 proteins in conditioned media obtained from primary lung epithelial cells, A549 and HS24 cells, and their effect on MMP-1 expression by lung fibroblasts (IMR-90). In addition, we evaluated IMR-90 response to 14-3-3 proteins in the presence of transforming growth factor-β(1) (TGF-β(1)), a cytokine known to decrease MMP-1 expression by fibroblasts. Extracellular 14-3-3α/β, but not -σ, is released by the human-derived lung epithelial cell lines, A549 and HS24. Unlike dermal fibroblasts, IMR-90 cells do not produce MMP-1 in response to 14-3-3σ. Conversely, MMP-1 production was induced following treatment of IMR-90 with recombinant or lung epithelial cell-derived 14-3-3α/β. These findings were also confirmed using primary human bronchial epithelial cells and lung fibroblasts obtained from non-asthmatic patients. The MMP-1-inducing effect of 14-3-3α/β on IMR-90 was not inhibited by TGF-β(1). Lung epithelial cell-derived 14-3-3α/β has a potent MMP-1-inducing effect on airway fibroblasts. Modulation of MMP-1 by 14-3-3α/β, may be important in the alteration of collagenase production associated with airway remodelling in obstructive lung diseases. Our data indicate that 14-3-3 proteins may be potential targets for future therapeutic strategies aimed at modulating tissue remodelling in asthma.     

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

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.