Novel Action of Indoleamine 2,3-Dioxygenase Attenuating Acute Lung Allograft Injury
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《美国呼吸和危急护理医学》
Department of Pediatrics and Department of Pharmacology and Therapeutics, University of Florida
Medical Research Service, Department of Veteran Affairs Medical Center, Gainesville, Florida
ABSTRACT
Rationale: Lung allografts are prone to reperfusion injury and acute rejection, which, in addition to infiltrating lymphocytes, are accompanied by neutrophil infiltration and neutrophil-associated oxidative stress. Indoleamine 2,3-dioxygenase (IDO) is a unique cytosolic enzyme that possesses T-cell–suppressive and antioxidant properties.
Objectives: The purpose of this study was to determine if genetic up-regulation of IDO could ameliorate acute lung allograft injury.
Methods: Lung orthotopic transplants were performed using Lewis donors and Sprague-Dawley rat recipients (allografts) or the same strain (isografts). Plasmid-encoding human IDO was delivered to donor lungs in vivo using a nonviral gene-transfer vector, polyethylenimine. Transplanted lungs were evaluated at 6 d post-transplantation based on pulmonary function, histology, inflammatory responses, and their associated oxidative stress. Basic biology of the IDO-overexpressing lung cells was evaluated in vitro in response to external oxidant.
Measurements and Main Results: This gene delivery method led to uniform transgene expression in lung tissue distributed in airway, alveolar epithelial, and endothelial cells. IDO overexpression in lung allografts resulted in a significant protective effect with improvement in functional properties (peak airway pressure and oxygenation) and histologic appearance. Although IDO was able to block local T-cell responses, it failed to abrogate neutrophilic infiltration and the inflammation-associated oxidative stress. IDO-enhanced lung cells were resistance to oxidant-induced necrosis and apoptosis by limiting intracellular reactive oxygen species formation.
Conclusions: These results demonstrate that IDO prevents acute lung allograft injury through augmenting the local antioxidant defense system and inhibiting alloreactive T-cell responses.
Key Words: gene therapy lung transplantation oxidative stress T cells
The success of human lung transplantation, as shown by 1- and 5-yr survival rates, lags considerably behind those for other solid organ transplants (1, 2). Therapeutic strategies, such as the use of cyclosporine, have a relatively low therapeutic efficacy for lung transplant patients (3), suggesting that development of more specific and effective therapeutic intervention is desirable.
Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme catalyzing the oxidative cleavage of indole ring of L-tryptophan to N-formyl-kynurenine that decomposes spontaneously to formate and L-kynurenine; the latter compound can be metabolized further along the kynurenine pathway (4, 5). Increasing evidence indicates that, in addition to defense against pathogens, this enzyme exerts important immunomodulatory effects. IDO may suppress T-cell responses by depleting local L-tryptophan, an essential amino acid for T-cell proliferation and function, and by action of the tryptophan metabolites, such as kynurenine, which can have a profound inhibitory effect on T-cell viability (4). Munn and colleagues (6) demonstrated that IDO expression in mouse placenta down-regulated proliferation of alloreactive T cells and prevented rejection of the semiallogeneic fetuses. This observation led to the idea that increasing donor lung IDO may provide an efficient immunologic shield to prevent the destructive allograft-reactive response without requiring the systemic use of conventional immunosuppressive treatments (7). The lung allografts, unlike the semiallogeneic fetuses, can be attacked not only by T-cell–mediated rejection but also by ischemia– reperfusion injury and nonspecific inflammation, which are often accompanied by neutrophil infiltration in the post-transplant period (3, 8–13). There is convincing evidence that the nonspecific inflammatory process and its associated oxidative stress play crucial roles in the pathogenesis of acute lung allograft injury (8, 9, 11, 12, 14), indicating that the inhibitory effect of IDO on T cells alone may be insufficient to fully protect against acute lung allograft injury.
In this regard, it is potentially relevant that IDO uses superoxide anion radical (O2·) as a substrate and a cofactor in its catalytic process (15, 16). On consumption of O2·, the dioxygenase initiates the formation of tryptophan metabolites, including 3-hydroxyanthranilic acid and 3-hydroxykynurenine, which are potent radical scavengers (5). The capability of transferring a prooxidant (O2·) into antioxidants makes IDO a powerful modulator of oxidative stress (5, 17). Thus, it is interesting to determine if up-regulation of IDO in donor lung could protect against acute allograft injury through augmenting the local antioxidant defense system in addition to its inhibitory effect on T-cell responses. In this study, a nonviral gene transfer approach was used in which plasmid DNA was complexed to a linear polycationic polymer, polyethylenimine (PEI), and used for delivering human IDO (hIDO) gene to donor rat lung.
METHODS
Additional details on all methods are provided in the online supplement.
Construction of hIDO-expressing Plasmid, Cell Culture, and Transient Transfection
A plasmid encoding hIDO or an enhanced green fluorescent protein (GFP) was constructed and referred to as pCMV-hIDO and pCMV-GFP, respectively. The control plasmid containing no insert (empty vector) is referred to as pCMV-null. Transient transfection of cultured cells with pCMV-hIDO, pCMV-null, or pCMV-GFP was performed.
Intratracheal Gene Delivery to Rat Lungs
The DNA/PEI complex was prepared as described previously (18). For in vivo gene delivery, 0.2 ml of transfection solution containing 20 μg of plasmid DNA was delivered to rat lung via an intratracheal catheter.
Animal Model, Experimental Groups, and Lung Tissue Sampling
Specific, pathogen-free, male Lewis and Sprague-Dawley rats ( 300 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed and cared for by Animal Care Services at the University of Florida. Experimental protocols were approved by the Animal Care Committee of the University of Florida.
The orthotopic left lung transplants were performed using Lewis donors and Sprague-Dawley recipient (allografts) or the same strain (isografts) as described previously (19). Some rats receiving allografts were given IDO inhibitor 1-methyl-D-tryptophan (1-mT) in drinking water (5 mg/ml, pH 7) (20) immediately after surgery. Left (transplanted) lungs were harvested at 6 d post-transplantation in all animals.
Assessment of Pulmonary Functional and Morphologic Properties
Peak airway pressure and partial pressure of oxygen (PaO2) from the left (transplanted) lung was measured in vivo. Hematoxylin and eosin (H&E)–stained lung sections were used to determine the severity of acute cellular rejection (ACR) (8, 9).
IDO Assay, Myeloperoxidase Assay, and Western Blotting Analysis
IDO activity in cell lysate or tissue supernatant was determined as previously reported (21) with slight modifications. Human IDO protein levels were evaluated by Western blotting analysis using a primary mouse monoclonal antihuman IDO antibody (Upstate, Charlottesville, VA) at 1:1,000 dilutions. Lung myeloperoxidase (MPO) enzymatic activity was determined as described previously (8).
Immunofluorescence Staining
Lung tissue sections were immunofluorescent stained for CD3 (a marker for T cells), MPO (a marker for activated neutrophils), von Willebrand factor (a marker for endothelial cells), or surfactant protein A (SP-A; a marker for type II pneumocytes) using a corresponding primary antibody and an Alexa Texas red–labeled second antibody (Molecular Probes, Eugene, OR) (18).
Assessment of Intracellular Reactive Oxygen Species Level and Oxidant-induced Cellular Toxicity
The intracellular reactive oxygen species (ROS) level of lung cells in response to an external oxidant challenge (60 μM H2O2 for 30 min) was assessed using a membrane-permeable and nonfluorescent probe 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (Molecular Probes) (22, 23). Assessment of oxidant-induced (100 μM H2O2 for up to 8 h) necrosis and apoptosis in cultured cells was performed as described previously (24).
Statistical Analysis
Data are expressed as mean ± SEM, and statistical analyses were performed with the Prism statistical program (GraphPad, San Diego, CA). One-way analysis of variance with the Newman-Keuls test was used to evaluate differences between groups, and the ACR grade was compared by the nonparametric Mann-Whitney test. A p value less than 0.05 was considered significant.
RESULTS
Transgene Expression of hIDO In Vitro and In Vivo
Human 293 cells do not express IDO (20); therefore, we used this cell line to verify hIDO expression from the plasmid pCMV-hIDO. Western blotting analysis using a mouse monoclonal antihuman IDO antibody detected a band of approximately 42 kD from cells transfected with pCMV-hIDO. In contrast, no IDO protein was found in untransfected cells or in cells transfected with pCMV-null (Figure 1A). The hIDO protein expressed in pCMV-hIDO–transfected cells was functionally active because a relatively high level of IDO enzymatic activity was found (Figure 1B). On the basis of data obtained by counting GFP-positive cells using a fluorescence microscopy (18), the transfection efficiency was as high as approximately 80%.
To verify in vivo transgene expression and distribution, pCMV-GFP construct was complexed with the nonviral vector PEI and delivered intratracheally to rat lung. GFP was intensively expressed in most of the terminal airway epithelium at Days 1 and 3 and seemed to be reduced at Day 7 after gene administration (Figure 2A). Massive GFP-positive cells also appeared in the alveolar walls (Figure 2B) and endothelium of small blood vessels (Figure 2C). On the basis of the number of alveolar epithelial cells immunolabeled with SP-A or SP-A and GFP (18), a transfection rate of approximately 28% in type II pneumocytes was obtained at Days 1 and 3, whereas the transfection rate was reduced to approximately 16% at Day 7 (Figure 2B). Similarly, a higher number of endothelial cells seemed to be transfected at Days 1 and 3, whereas few endothelial cells expressed GFP at Day 7 (Figure 2C). There was no GFP expression found in lung tissue taken from the untreated animals or animals that received the pCMV-null/PEI complexes (data not shown).
On the basis of the above results, hIDO gene was delivered via the airway to donor lung using the PEI as a gene carrier 24 hours before transplantation. In addition, one group received the empty control vector pCMV-null; this group acted as a control group for vector delivery. Because the mouse monoclonal anti-human IDO antibody does not cross-react with rat IDO (Upstate), we were able to detect hIDO protein expression in rat lung tissue by Western analysis. Rat lung allografts from the pCMV-hIDO/PEI treatment group exhibited a high level of hIDO protein expression (Figure 3A). The functional activity of the hIDO protein was verified by IDO enzymatic assay, in which we found that the pCMV-hIDO/PEI–treated allografts had a significantly higher level of IDO activity (35.85 ± 2.63 nM kynurenine/mg protein/60 min) in comparison to untreated lung allografts (p < 0.01) or those treated with the empty vector pCMV-null/PEI (p < 0.01; Figure 3B). To assess the effect of IDO inhibitor 1-mT on IDO activity in vivo, we measured endogenous kynurenine content in lung tissue (25). The pCMV-hIDO/PEI significantly elevated kynurenine content in lung allografts (15.31 ± 1.12 nM/mg protein; p < 0.01 vs. all other groups). This effect was reversed by 1-mT treatment (Figure 3C).
Effect of hIDO Expression on Allograft Functional and Morphologic Properties
The active hIDO protein expression was effective in attenuating acute lung allograft injury. First, treatment with pCMV-IDO/PEI significantly reduced peak airway pressure (25.2 ± 1.8 cm H2O; p < 0.01 vs. untreated allografts; Figure 4A) and increased PaO2 level (467 ± 57 mm Hg; p < 0.01 vs. untreated allografts; Figure 4B) in lung allografts. The protection provided by pCMV-IDO/PEI was abolished by 1-mT (Figures 4A and 4B), confirming that the improved pulmonary function was due to increased IDO activity. Consistent with these findings, a dramatic reduction in ACR with hIDO transgene expression was found based on histologic assessment (Figure 5). Representative microphotographs of H&E-stained lung tissue from each of the six groups are shown in Figures 5A–5F. An occasional lung isograft (Figure 5B) showed mild lymphocytic infiltration in perivascular locations, whereas the majority of lung isografts displayed no detectable pathologic lesions and were similar to normal lungs (Figure 5A), indicating that surgical trauma had little impact on graft morphology. In contrast, untreated (Figure 5C) and pCMV-null/PEI–treated (Figure 5D) lung allografts exhibited severe ACR along with areas of tissue necrosis and destruction of alveolar/interstitial structure. Treatment with pCMV-hIDO/PEI markedly reduced the infiltration of mononuclear cells around vessels and airway with a widespread preservation of the bronchus-alveolar architecture (Figure 5E), whereas pCMV-hIDO/PEI combined with 1-mT failed to improve the morphologic appearance and, similar to the untreated allografts, showed severe ACR (Figure 5F). The ACR grade in pCMV-hIDO/PEI–treated allografts was significantly lower (p < 0.05) than that from other allograft groups (Figure 5G).
Effect of hIDO Expression on Allograft Inflammatory Responses
We next investigated mechanism(s) for the observed beneficial effect provided by hIDO in lung allografts. First, we verified the inhibitory effect of IDO on local T-cell responses. Representative microphotographs of CD3-immunofluoresent stained lung sections of a normal left lung and an untreated lung allograft are shown in Figure 6A. A large number of CD3-positive cells were found in untreated allografts; this finding was not affected by pCMV-null/PEI or pCMV-hIDO/PEI in combination with 1-mT. However, treatment with pCMV-hIDO/PEI complexes strikingly reduced the number of CD3-positive cells to a level similar to that of normal lungs or isografts (Figure 6B), indicating that T-cell responses were almost completely abolished by the hIDO gene therapy. These observations are in contrast to the action of hIDO on neutrophils. Consistent with previous reports (8–11), intense MPO-positive cells were found in untreated lung allografts. Treatment with pCMV-hIDO/PEI partially reduced the number of MPO-positive cells (p < 0.01 versus untreated allografts); the number of MPO-positive cells in pCMV-hIDO/PEI–treated allografts was still significantly higher than that from normal lungs (p < 0.05) or isografts (p < 0.05; Figure 6C). The MPO immunofluorescent staining was mirrored by MPO enzymatic activity, in which we found that MPO activity was higher in pCMV-hIDO/PEI–treated allografts as compared with normal lungs (p < 0.05) or isografts (p < 0.05; Figure 6D).
Changes of Basic Biology of hIDO-expressing Lung Cells
The above findings that hIDO gene therapy failed to abolish neutrophil recruitment allow us to address the question on whether lung resident cells transfected with hIDO are more resistant to inflammation-associated oxidative stress. To clarify this issue, two sets of experiments were performed using hIDO-expressing lung cells. Human bronchial epithelial cells transfected with hIDO contained high levels of IDO protein and enzymatic activity, while there was no detectable IDO protein and enzymatic activity found in untransfected cells or cells transfected with pCMV-null, indicating successful hIDO transfection in these cells (Figure 7A).
In the first set of experiments, the bronchial epithelial cells were loaded with the redox-sensitive probe 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, followed by challenge with a relatively mild oxidant (60 μM of H2O2 for 30 min). Our preliminary data showed that H2O2 at this concentration for this duration did not affect cell viability. We observed that the baseline level of intracellular ROS was similar between all groups (data not shown). However, addition of H2O2 resulted in a near 100% increase in intracellular ROS in untransfected or pCMV-null transfected cells, whereas the increase of intracellular ROS was significantly attenuated ( 50%) in hIDO-transfected cells (p < 0.01). Moreover, the inhibitory effect of hIDO on ROS formation was abolished by 1-mT (1 mM) treatment (Figure 7B), indicating that IDO may serve as a homeostatic factor within intact cells when the cells face oxidant challenges. This notion allowed us to perform the second set of experiment aimed at exploring if overexpressed IDO protected the epithelial cells against oxidant-induced toxicity. For this, epithelial cells were exposed to an increased concentration of H2O2 (100 μM) for up to 8 h. The percentage of cell death in cells transfected with hIDO was significantly lower than untransfected or pCMV-null–transfected cells (p < 0.05), and the protection provided by hIDO was abrogated by 1-mT treatment (Figure 8A). A similar pattern regarding cell apoptosis was found. Scarce apoptag-positive cells can be seen before oxidant treatment in all groups (data not shown). However, after 8 h of exposure to H2O2 (100 μM), a large number of apoptag-positive cells were seen in untransfected or pCMV-null transfected cells. This is in sharp contrast to pCMV-hIDO transfected cells that showed reduced number of apoptag-positive cells (p < 0.01 vs. untransfected or pCMV-null transfected cells; Figure 8B). A separate study using alveolar type II pneumocytes gave equivalent results (data not shown).
DISCUSSION
IDO is a unique enzyme of tryptophan metabolism with multiple functions that suggest it may have particular therapeutic utility in lung transplantation. In the present study we found that nonviral vector-mediated gene delivery of hIDO led to a remarkable protection against acute lung allograft injury through inhibiting T-cell responses and augmenting the local antioxidant defense system.
There is convincing evidence that T cells, which mediate allograft rejection, are susceptible to inhibition by IDO (4). It has been reported that treatment with adenoviral vector containing IDO abrogated acute allograft rejection in a rat model of lung transplantation (7). However, this study provided no direct evidence showing that the protection was attributed to the inhibitory effect of IDO on local T-cell proliferation. In addition, a recent study revealed that adenoviral vectors are capable of inducing IDO in antigen-presenting cells (26), thereby complicating interpretation of the experimental results. The adenovirus- specific drawback was avoided by using the pharmacologic-grade gene carrier PEI in the present study, in which we found that control plasmid pCMV-null had no effect on IDO expression in vitro (Figure 1) and that treatment with pCMV-null/PEI complex was unable to induce lung IDO activity in vivo (Figure 3). In contrast, treatment with pCMV-hIDO/PEI complexes increased local IDO activity (Figure 3) and abolished T-cell responses in lung allografts (Figure 6). Further confirmation that the inhibitory effects on T cells was due to the high level of IDO activity was demonstrated by the fact that 1-mT, a competitive inhibitor of IDO activity (20), was able to almost completely reverse the inhibition of T-cell responses induced by the enzyme (Figures 3 and 6). Thus, the IDO-dependent inhibition on T-cell responses constituted a mechanism by which hIDO gene therapy conferred protection against the acute lung allograft injury. However, it is unclear whether the abolished T-cell responses seen in the pCMV-hIDO/PEI–treated allografts resulted from IDO-dependent inhibition on T-cell proliferation or T-cell infiltration to the lung allografts. Additional studies are required to clarify this issue.
On the basis of the previous reports that neutrophil infiltration, a marker of pulmonary inflammation, also occurs and contributes importantly to acute lung allograft injury (3, 8–13), we investigated the possible inhibitory effect of IDO on this type of cell. Similar to a recent study showing that IDO overexpression partially suppressed pulmonary neutrophil recruitment in an animal model of asthma (25), we observed that massive neutrophil infiltration occurring in lung allografts was incompletely prevented by pCMV-hIDO/PEI, leaving a substantial number of neutrophils in pCMV-hIDO/PEI–treated lung allografts (Figure 6). Neutrophils cause tissue damage mainly through generating ROS including O2· by a process known as respiratory burst (8, 27). A significantly higher level of MPO enzymatic activity, an indicator of the inflammation-associated oxidative stress (8, 28), was found in pCMV-hIDO/PEI–treated allografts compared with those from normal lungs or isografts (Figure 6). Our evidence supports the notion that IDO-dependent inflammatory cell inhibition is specific to certain cell types (e.g., T cells) (29) and suggests that the IDO-dependent block alone may not be sufficient to fully protect against acute allograft injury in light of the fact that infiltration of multiple inflammatory cell types occurred in lung allografts.
IDO is a cytosolic enzyme; therefore, its antioxidant activity occurs inside the cell. This biologic characteristic may constitute another way to provide protection against the acute lung allograft injury. The present study provided direct evidence that overexpressed IDO-limited intracellular ROS formation, hence protecting resident lung cells from oxidant-induced necrosis and apoptosis (Figures 7 and 8). These findings, together with the observation that pCMV-hIDO/PEI preferentially transfected pulmonary epithelial cells and vascular endothelial cells (Figure 2), provide a plausible mechanistic explanation as to why the widespread preservation of the bronchus-alveolar architecture (Figure 5) with a relative intact gas-exchange function (Figure 4) existed in the presence of substantial neutrophil infiltration and inflammation-associated oxidative stress (Figure 6) in pCMV-hIDO/PEI–treated allografts. Furthermore, it has been showed that endothelium is prone to oxidant damage because intracellular ROS of endothelial cells are inaccessible to circulating antioxidant enzymes, leading to an increased vascular endothelial permeability and leukocyte extravasation (30, 31). It is thus conceivable that hIDO protected pulmonary endothelial cells from transplantation-associated oxidative stress and consequently contributed to the reduced neutrophil recruitment seen in the pCMV-hIDO/PEI–treated allografts (Figure 6).
On the basis of the above considerations, we conclude that IDO mediates multiple mechanisms to prevent acute lung allograft injury. On one side, IDO overexpression in resident lung cells completely blocks neighboring T-cell responses. On the other side, the basic biology of IDO-overexpressing lung cells is altered, and these cells are endowed with an enhanced resistance to inflammation-associated, oxidant-induced injury. The mode of IDO action delineated here might have far-reaching implications for the pathogenesis of certain diseases that involve IDO induction. For example, it may provide an additional explanation as to why tumor cells, which constitutively express IDO, are resistant against immune attack (20).
FOOTNOTES
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200509-1413OC on November 17, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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Medical Research Service, Department of Veteran Affairs Medical Center, Gainesville, Florida
ABSTRACT
Rationale: Lung allografts are prone to reperfusion injury and acute rejection, which, in addition to infiltrating lymphocytes, are accompanied by neutrophil infiltration and neutrophil-associated oxidative stress. Indoleamine 2,3-dioxygenase (IDO) is a unique cytosolic enzyme that possesses T-cell–suppressive and antioxidant properties.
Objectives: The purpose of this study was to determine if genetic up-regulation of IDO could ameliorate acute lung allograft injury.
Methods: Lung orthotopic transplants were performed using Lewis donors and Sprague-Dawley rat recipients (allografts) or the same strain (isografts). Plasmid-encoding human IDO was delivered to donor lungs in vivo using a nonviral gene-transfer vector, polyethylenimine. Transplanted lungs were evaluated at 6 d post-transplantation based on pulmonary function, histology, inflammatory responses, and their associated oxidative stress. Basic biology of the IDO-overexpressing lung cells was evaluated in vitro in response to external oxidant.
Measurements and Main Results: This gene delivery method led to uniform transgene expression in lung tissue distributed in airway, alveolar epithelial, and endothelial cells. IDO overexpression in lung allografts resulted in a significant protective effect with improvement in functional properties (peak airway pressure and oxygenation) and histologic appearance. Although IDO was able to block local T-cell responses, it failed to abrogate neutrophilic infiltration and the inflammation-associated oxidative stress. IDO-enhanced lung cells were resistance to oxidant-induced necrosis and apoptosis by limiting intracellular reactive oxygen species formation.
Conclusions: These results demonstrate that IDO prevents acute lung allograft injury through augmenting the local antioxidant defense system and inhibiting alloreactive T-cell responses.
Key Words: gene therapy lung transplantation oxidative stress T cells
The success of human lung transplantation, as shown by 1- and 5-yr survival rates, lags considerably behind those for other solid organ transplants (1, 2). Therapeutic strategies, such as the use of cyclosporine, have a relatively low therapeutic efficacy for lung transplant patients (3), suggesting that development of more specific and effective therapeutic intervention is desirable.
Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme catalyzing the oxidative cleavage of indole ring of L-tryptophan to N-formyl-kynurenine that decomposes spontaneously to formate and L-kynurenine; the latter compound can be metabolized further along the kynurenine pathway (4, 5). Increasing evidence indicates that, in addition to defense against pathogens, this enzyme exerts important immunomodulatory effects. IDO may suppress T-cell responses by depleting local L-tryptophan, an essential amino acid for T-cell proliferation and function, and by action of the tryptophan metabolites, such as kynurenine, which can have a profound inhibitory effect on T-cell viability (4). Munn and colleagues (6) demonstrated that IDO expression in mouse placenta down-regulated proliferation of alloreactive T cells and prevented rejection of the semiallogeneic fetuses. This observation led to the idea that increasing donor lung IDO may provide an efficient immunologic shield to prevent the destructive allograft-reactive response without requiring the systemic use of conventional immunosuppressive treatments (7). The lung allografts, unlike the semiallogeneic fetuses, can be attacked not only by T-cell–mediated rejection but also by ischemia– reperfusion injury and nonspecific inflammation, which are often accompanied by neutrophil infiltration in the post-transplant period (3, 8–13). There is convincing evidence that the nonspecific inflammatory process and its associated oxidative stress play crucial roles in the pathogenesis of acute lung allograft injury (8, 9, 11, 12, 14), indicating that the inhibitory effect of IDO on T cells alone may be insufficient to fully protect against acute lung allograft injury.
In this regard, it is potentially relevant that IDO uses superoxide anion radical (O2·) as a substrate and a cofactor in its catalytic process (15, 16). On consumption of O2·, the dioxygenase initiates the formation of tryptophan metabolites, including 3-hydroxyanthranilic acid and 3-hydroxykynurenine, which are potent radical scavengers (5). The capability of transferring a prooxidant (O2·) into antioxidants makes IDO a powerful modulator of oxidative stress (5, 17). Thus, it is interesting to determine if up-regulation of IDO in donor lung could protect against acute allograft injury through augmenting the local antioxidant defense system in addition to its inhibitory effect on T-cell responses. In this study, a nonviral gene transfer approach was used in which plasmid DNA was complexed to a linear polycationic polymer, polyethylenimine (PEI), and used for delivering human IDO (hIDO) gene to donor rat lung.
METHODS
Additional details on all methods are provided in the online supplement.
Construction of hIDO-expressing Plasmid, Cell Culture, and Transient Transfection
A plasmid encoding hIDO or an enhanced green fluorescent protein (GFP) was constructed and referred to as pCMV-hIDO and pCMV-GFP, respectively. The control plasmid containing no insert (empty vector) is referred to as pCMV-null. Transient transfection of cultured cells with pCMV-hIDO, pCMV-null, or pCMV-GFP was performed.
Intratracheal Gene Delivery to Rat Lungs
The DNA/PEI complex was prepared as described previously (18). For in vivo gene delivery, 0.2 ml of transfection solution containing 20 μg of plasmid DNA was delivered to rat lung via an intratracheal catheter.
Animal Model, Experimental Groups, and Lung Tissue Sampling
Specific, pathogen-free, male Lewis and Sprague-Dawley rats ( 300 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed and cared for by Animal Care Services at the University of Florida. Experimental protocols were approved by the Animal Care Committee of the University of Florida.
The orthotopic left lung transplants were performed using Lewis donors and Sprague-Dawley recipient (allografts) or the same strain (isografts) as described previously (19). Some rats receiving allografts were given IDO inhibitor 1-methyl-D-tryptophan (1-mT) in drinking water (5 mg/ml, pH 7) (20) immediately after surgery. Left (transplanted) lungs were harvested at 6 d post-transplantation in all animals.
Assessment of Pulmonary Functional and Morphologic Properties
Peak airway pressure and partial pressure of oxygen (PaO2) from the left (transplanted) lung was measured in vivo. Hematoxylin and eosin (H&E)–stained lung sections were used to determine the severity of acute cellular rejection (ACR) (8, 9).
IDO Assay, Myeloperoxidase Assay, and Western Blotting Analysis
IDO activity in cell lysate or tissue supernatant was determined as previously reported (21) with slight modifications. Human IDO protein levels were evaluated by Western blotting analysis using a primary mouse monoclonal antihuman IDO antibody (Upstate, Charlottesville, VA) at 1:1,000 dilutions. Lung myeloperoxidase (MPO) enzymatic activity was determined as described previously (8).
Immunofluorescence Staining
Lung tissue sections were immunofluorescent stained for CD3 (a marker for T cells), MPO (a marker for activated neutrophils), von Willebrand factor (a marker for endothelial cells), or surfactant protein A (SP-A; a marker for type II pneumocytes) using a corresponding primary antibody and an Alexa Texas red–labeled second antibody (Molecular Probes, Eugene, OR) (18).
Assessment of Intracellular Reactive Oxygen Species Level and Oxidant-induced Cellular Toxicity
The intracellular reactive oxygen species (ROS) level of lung cells in response to an external oxidant challenge (60 μM H2O2 for 30 min) was assessed using a membrane-permeable and nonfluorescent probe 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (Molecular Probes) (22, 23). Assessment of oxidant-induced (100 μM H2O2 for up to 8 h) necrosis and apoptosis in cultured cells was performed as described previously (24).
Statistical Analysis
Data are expressed as mean ± SEM, and statistical analyses were performed with the Prism statistical program (GraphPad, San Diego, CA). One-way analysis of variance with the Newman-Keuls test was used to evaluate differences between groups, and the ACR grade was compared by the nonparametric Mann-Whitney test. A p value less than 0.05 was considered significant.
RESULTS
Transgene Expression of hIDO In Vitro and In Vivo
Human 293 cells do not express IDO (20); therefore, we used this cell line to verify hIDO expression from the plasmid pCMV-hIDO. Western blotting analysis using a mouse monoclonal antihuman IDO antibody detected a band of approximately 42 kD from cells transfected with pCMV-hIDO. In contrast, no IDO protein was found in untransfected cells or in cells transfected with pCMV-null (Figure 1A). The hIDO protein expressed in pCMV-hIDO–transfected cells was functionally active because a relatively high level of IDO enzymatic activity was found (Figure 1B). On the basis of data obtained by counting GFP-positive cells using a fluorescence microscopy (18), the transfection efficiency was as high as approximately 80%.
To verify in vivo transgene expression and distribution, pCMV-GFP construct was complexed with the nonviral vector PEI and delivered intratracheally to rat lung. GFP was intensively expressed in most of the terminal airway epithelium at Days 1 and 3 and seemed to be reduced at Day 7 after gene administration (Figure 2A). Massive GFP-positive cells also appeared in the alveolar walls (Figure 2B) and endothelium of small blood vessels (Figure 2C). On the basis of the number of alveolar epithelial cells immunolabeled with SP-A or SP-A and GFP (18), a transfection rate of approximately 28% in type II pneumocytes was obtained at Days 1 and 3, whereas the transfection rate was reduced to approximately 16% at Day 7 (Figure 2B). Similarly, a higher number of endothelial cells seemed to be transfected at Days 1 and 3, whereas few endothelial cells expressed GFP at Day 7 (Figure 2C). There was no GFP expression found in lung tissue taken from the untreated animals or animals that received the pCMV-null/PEI complexes (data not shown).
On the basis of the above results, hIDO gene was delivered via the airway to donor lung using the PEI as a gene carrier 24 hours before transplantation. In addition, one group received the empty control vector pCMV-null; this group acted as a control group for vector delivery. Because the mouse monoclonal anti-human IDO antibody does not cross-react with rat IDO (Upstate), we were able to detect hIDO protein expression in rat lung tissue by Western analysis. Rat lung allografts from the pCMV-hIDO/PEI treatment group exhibited a high level of hIDO protein expression (Figure 3A). The functional activity of the hIDO protein was verified by IDO enzymatic assay, in which we found that the pCMV-hIDO/PEI–treated allografts had a significantly higher level of IDO activity (35.85 ± 2.63 nM kynurenine/mg protein/60 min) in comparison to untreated lung allografts (p < 0.01) or those treated with the empty vector pCMV-null/PEI (p < 0.01; Figure 3B). To assess the effect of IDO inhibitor 1-mT on IDO activity in vivo, we measured endogenous kynurenine content in lung tissue (25). The pCMV-hIDO/PEI significantly elevated kynurenine content in lung allografts (15.31 ± 1.12 nM/mg protein; p < 0.01 vs. all other groups). This effect was reversed by 1-mT treatment (Figure 3C).
Effect of hIDO Expression on Allograft Functional and Morphologic Properties
The active hIDO protein expression was effective in attenuating acute lung allograft injury. First, treatment with pCMV-IDO/PEI significantly reduced peak airway pressure (25.2 ± 1.8 cm H2O; p < 0.01 vs. untreated allografts; Figure 4A) and increased PaO2 level (467 ± 57 mm Hg; p < 0.01 vs. untreated allografts; Figure 4B) in lung allografts. The protection provided by pCMV-IDO/PEI was abolished by 1-mT (Figures 4A and 4B), confirming that the improved pulmonary function was due to increased IDO activity. Consistent with these findings, a dramatic reduction in ACR with hIDO transgene expression was found based on histologic assessment (Figure 5). Representative microphotographs of H&E-stained lung tissue from each of the six groups are shown in Figures 5A–5F. An occasional lung isograft (Figure 5B) showed mild lymphocytic infiltration in perivascular locations, whereas the majority of lung isografts displayed no detectable pathologic lesions and were similar to normal lungs (Figure 5A), indicating that surgical trauma had little impact on graft morphology. In contrast, untreated (Figure 5C) and pCMV-null/PEI–treated (Figure 5D) lung allografts exhibited severe ACR along with areas of tissue necrosis and destruction of alveolar/interstitial structure. Treatment with pCMV-hIDO/PEI markedly reduced the infiltration of mononuclear cells around vessels and airway with a widespread preservation of the bronchus-alveolar architecture (Figure 5E), whereas pCMV-hIDO/PEI combined with 1-mT failed to improve the morphologic appearance and, similar to the untreated allografts, showed severe ACR (Figure 5F). The ACR grade in pCMV-hIDO/PEI–treated allografts was significantly lower (p < 0.05) than that from other allograft groups (Figure 5G).
Effect of hIDO Expression on Allograft Inflammatory Responses
We next investigated mechanism(s) for the observed beneficial effect provided by hIDO in lung allografts. First, we verified the inhibitory effect of IDO on local T-cell responses. Representative microphotographs of CD3-immunofluoresent stained lung sections of a normal left lung and an untreated lung allograft are shown in Figure 6A. A large number of CD3-positive cells were found in untreated allografts; this finding was not affected by pCMV-null/PEI or pCMV-hIDO/PEI in combination with 1-mT. However, treatment with pCMV-hIDO/PEI complexes strikingly reduced the number of CD3-positive cells to a level similar to that of normal lungs or isografts (Figure 6B), indicating that T-cell responses were almost completely abolished by the hIDO gene therapy. These observations are in contrast to the action of hIDO on neutrophils. Consistent with previous reports (8–11), intense MPO-positive cells were found in untreated lung allografts. Treatment with pCMV-hIDO/PEI partially reduced the number of MPO-positive cells (p < 0.01 versus untreated allografts); the number of MPO-positive cells in pCMV-hIDO/PEI–treated allografts was still significantly higher than that from normal lungs (p < 0.05) or isografts (p < 0.05; Figure 6C). The MPO immunofluorescent staining was mirrored by MPO enzymatic activity, in which we found that MPO activity was higher in pCMV-hIDO/PEI–treated allografts as compared with normal lungs (p < 0.05) or isografts (p < 0.05; Figure 6D).
Changes of Basic Biology of hIDO-expressing Lung Cells
The above findings that hIDO gene therapy failed to abolish neutrophil recruitment allow us to address the question on whether lung resident cells transfected with hIDO are more resistant to inflammation-associated oxidative stress. To clarify this issue, two sets of experiments were performed using hIDO-expressing lung cells. Human bronchial epithelial cells transfected with hIDO contained high levels of IDO protein and enzymatic activity, while there was no detectable IDO protein and enzymatic activity found in untransfected cells or cells transfected with pCMV-null, indicating successful hIDO transfection in these cells (Figure 7A).
In the first set of experiments, the bronchial epithelial cells were loaded with the redox-sensitive probe 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, followed by challenge with a relatively mild oxidant (60 μM of H2O2 for 30 min). Our preliminary data showed that H2O2 at this concentration for this duration did not affect cell viability. We observed that the baseline level of intracellular ROS was similar between all groups (data not shown). However, addition of H2O2 resulted in a near 100% increase in intracellular ROS in untransfected or pCMV-null transfected cells, whereas the increase of intracellular ROS was significantly attenuated ( 50%) in hIDO-transfected cells (p < 0.01). Moreover, the inhibitory effect of hIDO on ROS formation was abolished by 1-mT (1 mM) treatment (Figure 7B), indicating that IDO may serve as a homeostatic factor within intact cells when the cells face oxidant challenges. This notion allowed us to perform the second set of experiment aimed at exploring if overexpressed IDO protected the epithelial cells against oxidant-induced toxicity. For this, epithelial cells were exposed to an increased concentration of H2O2 (100 μM) for up to 8 h. The percentage of cell death in cells transfected with hIDO was significantly lower than untransfected or pCMV-null–transfected cells (p < 0.05), and the protection provided by hIDO was abrogated by 1-mT treatment (Figure 8A). A similar pattern regarding cell apoptosis was found. Scarce apoptag-positive cells can be seen before oxidant treatment in all groups (data not shown). However, after 8 h of exposure to H2O2 (100 μM), a large number of apoptag-positive cells were seen in untransfected or pCMV-null transfected cells. This is in sharp contrast to pCMV-hIDO transfected cells that showed reduced number of apoptag-positive cells (p < 0.01 vs. untransfected or pCMV-null transfected cells; Figure 8B). A separate study using alveolar type II pneumocytes gave equivalent results (data not shown).
DISCUSSION
IDO is a unique enzyme of tryptophan metabolism with multiple functions that suggest it may have particular therapeutic utility in lung transplantation. In the present study we found that nonviral vector-mediated gene delivery of hIDO led to a remarkable protection against acute lung allograft injury through inhibiting T-cell responses and augmenting the local antioxidant defense system.
There is convincing evidence that T cells, which mediate allograft rejection, are susceptible to inhibition by IDO (4). It has been reported that treatment with adenoviral vector containing IDO abrogated acute allograft rejection in a rat model of lung transplantation (7). However, this study provided no direct evidence showing that the protection was attributed to the inhibitory effect of IDO on local T-cell proliferation. In addition, a recent study revealed that adenoviral vectors are capable of inducing IDO in antigen-presenting cells (26), thereby complicating interpretation of the experimental results. The adenovirus- specific drawback was avoided by using the pharmacologic-grade gene carrier PEI in the present study, in which we found that control plasmid pCMV-null had no effect on IDO expression in vitro (Figure 1) and that treatment with pCMV-null/PEI complex was unable to induce lung IDO activity in vivo (Figure 3). In contrast, treatment with pCMV-hIDO/PEI complexes increased local IDO activity (Figure 3) and abolished T-cell responses in lung allografts (Figure 6). Further confirmation that the inhibitory effects on T cells was due to the high level of IDO activity was demonstrated by the fact that 1-mT, a competitive inhibitor of IDO activity (20), was able to almost completely reverse the inhibition of T-cell responses induced by the enzyme (Figures 3 and 6). Thus, the IDO-dependent inhibition on T-cell responses constituted a mechanism by which hIDO gene therapy conferred protection against the acute lung allograft injury. However, it is unclear whether the abolished T-cell responses seen in the pCMV-hIDO/PEI–treated allografts resulted from IDO-dependent inhibition on T-cell proliferation or T-cell infiltration to the lung allografts. Additional studies are required to clarify this issue.
On the basis of the previous reports that neutrophil infiltration, a marker of pulmonary inflammation, also occurs and contributes importantly to acute lung allograft injury (3, 8–13), we investigated the possible inhibitory effect of IDO on this type of cell. Similar to a recent study showing that IDO overexpression partially suppressed pulmonary neutrophil recruitment in an animal model of asthma (25), we observed that massive neutrophil infiltration occurring in lung allografts was incompletely prevented by pCMV-hIDO/PEI, leaving a substantial number of neutrophils in pCMV-hIDO/PEI–treated lung allografts (Figure 6). Neutrophils cause tissue damage mainly through generating ROS including O2· by a process known as respiratory burst (8, 27). A significantly higher level of MPO enzymatic activity, an indicator of the inflammation-associated oxidative stress (8, 28), was found in pCMV-hIDO/PEI–treated allografts compared with those from normal lungs or isografts (Figure 6). Our evidence supports the notion that IDO-dependent inflammatory cell inhibition is specific to certain cell types (e.g., T cells) (29) and suggests that the IDO-dependent block alone may not be sufficient to fully protect against acute allograft injury in light of the fact that infiltration of multiple inflammatory cell types occurred in lung allografts.
IDO is a cytosolic enzyme; therefore, its antioxidant activity occurs inside the cell. This biologic characteristic may constitute another way to provide protection against the acute lung allograft injury. The present study provided direct evidence that overexpressed IDO-limited intracellular ROS formation, hence protecting resident lung cells from oxidant-induced necrosis and apoptosis (Figures 7 and 8). These findings, together with the observation that pCMV-hIDO/PEI preferentially transfected pulmonary epithelial cells and vascular endothelial cells (Figure 2), provide a plausible mechanistic explanation as to why the widespread preservation of the bronchus-alveolar architecture (Figure 5) with a relative intact gas-exchange function (Figure 4) existed in the presence of substantial neutrophil infiltration and inflammation-associated oxidative stress (Figure 6) in pCMV-hIDO/PEI–treated allografts. Furthermore, it has been showed that endothelium is prone to oxidant damage because intracellular ROS of endothelial cells are inaccessible to circulating antioxidant enzymes, leading to an increased vascular endothelial permeability and leukocyte extravasation (30, 31). It is thus conceivable that hIDO protected pulmonary endothelial cells from transplantation-associated oxidative stress and consequently contributed to the reduced neutrophil recruitment seen in the pCMV-hIDO/PEI–treated allografts (Figure 6).
On the basis of the above considerations, we conclude that IDO mediates multiple mechanisms to prevent acute lung allograft injury. On one side, IDO overexpression in resident lung cells completely blocks neighboring T-cell responses. On the other side, the basic biology of IDO-overexpressing lung cells is altered, and these cells are endowed with an enhanced resistance to inflammation-associated, oxidant-induced injury. The mode of IDO action delineated here might have far-reaching implications for the pathogenesis of certain diseases that involve IDO induction. For example, it may provide an additional explanation as to why tumor cells, which constitutively express IDO, are resistant against immune attack (20).
FOOTNOTES
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200509-1413OC on November 17, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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