Yunyuan Li

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-γ showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-γ-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-γ-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-γ alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-γ. Coculture of eosinophils with an IFN-γ-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

Kynurenine increases matrix metalloproteinase-1 and -3 expression in cultured dermal fibroblasts and improves scarring in vivo.

We previously demonstrated that the formation of hypertrophic scarring on the wounds of a rabbit ear fibrotic model was significantly reduced by grafting a bilayer skin substitute expressing indoleamine 2,3-dioxygenase (IDO). Here, we hypothesize that the improved healing quality is due to extracellular matrix modulatory effect of IDO-mediated tryptophan metabolites. To test this hypothesis, a series of in vitro and in vivo experiments were conducted and the findings revealed a significant increase in the expression of matrix metalloproteinase 1 (MMP-1) in fibroblasts either transduced with human IDO gene or cultured with conditioned media obtained from IDO-expressing cells. Consistent with this finding, kynurenine (Kyn) treatment markedly increased the levels of MMP-1 and MMP-3 expression through activation of the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase)-ERK1/2 MAPK signaling pathway. On the other hand, Kyn significantly suppressed the expression of type I collagen in fibroblasts as compared with that of control. To test the anti-fibrogenic effect of Kyn in an in vivo model, rabbit ear fibrotic wounds were topically treated with cream containing 50 μg Kyn per l00 μl of cream per wound. The result showed a marked improvement in scar formation relative to the controls. These findings collectively suggest that Kyn can potentially be used as an anti-fibrogenic agent for treating hypertrophic scarring.

Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2, 3-dioxygenase expression of interferon-γ-treated skin cells in a co-culture system

Indoleamine 2,3‐dioxygenase (IDO) is an intracellular tryptophan‐oxidizing enzyme possessing various immunosuppressive characteristics. In this study, we report the possible use of this enzyme in an allogenic skin substitute to suppress the proliferation of immune cells. Human fetal skin fibroblasts and keratinocytes were treated with the cytokine interferon‐γ to induce expression of IDO mRNA and protein. IDO enzyme activity was evaluated by measurement of kynurenine levels in the interferon‐γ–treated and –untreated cells. Results of Northern analysis showed a dose‐dependent response in expression of IDO mRNA to the various concentrations of interferon‐γ used. Northern blot analysis also showed a time‐dependent expression of IDO in response to different durations of interferon‐γ treatment. The level of kynurenine measured, as the bioactivity of IDO enzyme, was significantly higher in the interferon‐γ–treated fibroblasts and keratinocytes compared to those of controls (p < 0.001). To illustrate the immunosuppressive effects of IDO on immune cell proliferation, IDO‐expressing fibroblasts were cocultured with human peripheral blood mononuclear cells for a period of 5 days. Results of 3H‐thymidine incorporation assays showed a significant reduction in proliferation of the mononuclear cells cocultured with IDO‐expressing skin cells compared to monocytes cocultured with control (non‐IDO‐expressing) skin cells (p < 0.001). Furthermore, addition of the IDO‐inhibitor (1‐methyl‐D‐tryptophan) significantly reversed the immunosuppressive effects of IDO on monocyte proliferation (p < 0.001). In conclusion, suppression of peripheral blood mononuclear cell proliferation due to interferon‐γ–induced IDO‐expression in allogenic human skin cells might shed new light on developing a nonrejectable allogenic skin substitute. (WOUND REP REG 2003;11:337–345)

Temperature-sensitive polymer-conjugated IFN-γ 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‐γ) conjugated with a temperature‐sensitive polymer to induce the expression of IDO mRNA and protein. SDS–PAGE showed successful conjugation of IFN‐γ 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‐γ. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN‐γ‐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‐γ was significantly longer than in those treated with free (non‐conjugated) IFN‐γ (P < 0.001). IFN‐γ radiolabeling showed a prolonged retention of IFN‐γ 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‐γ). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO‐expressing FPCG treated with polymer‐conjugated IFN‐γ 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‐γ‐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‐γ on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts. J. Cell. Physiol. 201: 146–154, 2004.

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

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

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 σ 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 Dulbeccos modified Eagles 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 (Mol Cell Biochem 266: 167–174, 2004)

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. J. Cell. Biochem. 113: 1061–1068, 2012.

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. J. Cell. Physiol. 226: 3114–3120, 2011.

14-3-3 sigma isoform interacts with the cytoplasmic domain of the transmembrane BP180 in keratinocytes.

The protein bullous pemphigoid antigen‐2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin α6, integrin β4, or bullous pemphigoid antigen‐1 (BPAG1/BP230) is critical for this attachment in skin. The protein 14‐3‐3 σ, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14‐3‐3 protein family naturally found in all eukaryotic cells. Here, we have used a 14‐3‐3σ GST pull‐down screening assay and showed that sigma (σ) isoform of the 14‐3‐3 protein family interacts with the cytoplasmic N‐terminal domain of BP180. Analysis of a series of truncated or deleted 14‐3‐3σ revealed that only intact 14‐3‐3σ molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14‐3‐3 σ is essential for this interaction. Further, a BP180 co‐immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14‐3‐3 σ interacts with cytoplasmic domain, but not ecto‐domain of the BP180. In conclusion, the finding of this study provides evidence that 14‐3‐3σ isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored. J. Cell. Physiol. 212:675–681, 2007.

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.