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FUNCTIONAL MODIFICATION OF DENDRITIC CELLS WITH RECOMBINANT ADENOVIRUS ENCODING INTERLEUKIN 10 FOR THE TREATMENT OF SEPSIS

Oberholzer, Andreas*; Oberholzer, Caroline*; Efron, Philip A*; Scumpia, Philip O*; Uchida, Takefumi*; Bahjat, Keith; Ungaro, Ricardo*; Tannahill, Cynthia L*; Murday, Michelle*; Bahjat, Frances R*; Tsai, Van; Hutchins, Beth; Moldawer, Lyle L*; LaFace, Drake; Clare-Salzler, Michael J

doi: 10.1097/01.shk.0000163208.29655.74
Basic Science Aspects
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Control of dendritic cell (DC) function is critical for strategies to modulate innate and acquired immune responses. We examined whether transduction of murine DCs with adenoviral vectors (Adv) expressing interleukin (IL)-10 could alter their phenotype and T cell stimulatory function. Murine bone marrow-derived DCs were transduced with AdV encoding human IL-10 or green fluorescent protein (GFP). Whereas transduction of immature DCs with AdV/GFP resulted in dose-dependent maturation, DCs transduced with Adv/IL-10 maintained an immature state with low major histocompatibility complex (MHC) class II, CD86, and IL-12 expression. The Adv/IL-10 transduced DCs were phenotypically unique, characterized by suppression of IL-12 expression, failure to stimulate Th1 or Th2 cytokine responses, and retained capacity to endocytose antigen. Importantly, Adv/IL-10-transduced DCs were biologically active in vivo, in that administration of these DCs into mice before a generalized peritonitis significantly improved survival. We conclude that Adv/IL-10 transduction of DCs provides an efficient means to modulate DC function. The capacity to modify DCs by adenoviral expression of IL-10 may provide a novel ex vivo or in vivo approach to mitigate acute and chronic inflammatory diseases like sepsis.

Departments of *Surgery and Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida; and Canji, Inc., San Diego, California

Received 11 Oct 2004; first review completed 26 Oct 2004; accepted in final form 9 Mar 2005

Address reprint requests to Lyle L. Moldawer, PhD, Department of Surgery, University of Florida College of Medicine, Room 6116, Shands Hospital, 1600 SW Archer Road, Gainesville, FL 32610. E-mail: moldawer@surgery.ufl.edu.

This work was supported in part by the National Institute of General Medical Sciences (grants R01 GM-63041 and R37 GM-40586 to L.L.M.), by the National Heart, Lung and Blood Institute (grant P30 HL-08912 to L.L.M.), and by the National Institute of Diabetes, Digestive and Kidney Disease and the National Institute of Allergy and Infectious Diseases (grant PO1 DK-14288 to M.C.S.). A.O. was supported in part by a grant from the Swiss National Science Foundation. P.E. was supported in part by a National Institute of General Medical Sciences training grant (grant T32 GM-09721-05) in burns and trauma research.

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INTRODUCTION

Immature myeloid dendritic cells (DCs) readily endocytose antigens and respond to various self and foreign antigens that subsequently modulate their development and function. Unless activated by environmental signals that promote maturation, immature DCs that migrate from tissue sites to draining lymph nodes are likely to be nonimmunogenic, as they express low levels of major histocompatibility complex (MHC) class II and costimulatory molecules, and present little peptide antigen (1). Because of their limited capacity to present antigen and to stimulate T cell responses, immature DCs may promote tolerance. In contrast, upon receiving an activating stimulus, immature DCs migrate to the draining lymph nodes and lose their capacity to take up antigen. As they proceed through the maturation process, DCs present antigen efficiently and readily activate T lymphocyte responses as they increase surface expression of MHC-peptide complexes, upregulate costimulatory molecules, and produce essential cytokines (2). Various stimuli are capable of triggering DC maturation, including proinflammatory cytokines, bacterial cell products, viral agents and CD40 ligand (3, 4). Furthermore, DC maturation and function are modulated by other factors from the microenvironment that influence the ability of these cells to stimulate specific lymphocyte responses (e.g., Th1 versus Th2).

Given their central role in the innate and acquired immune responses, manipulation of DCs is a promising avenue for the development of cellular therapeutics for cancer, inflammatory diseases, autoimmunity, and other immune-mediated processes. Recent studies have demonstrated the feasibility of adenoviral gene transfer and expression to manipulate human and murine DCs (5-8). Adenoviral transduction of DCs with specific tumor genes resulted in increased immunogenicity and inhibition of tumor growth and metastases (9). In addition, adenovirus-induced gene transfer can be used to directly modulate DC function, and thus, T cell responses.

Interleukin 10 (IL-10) is a pleiotropic cytokine that regulates development and function of numerous cells essential for immunity. In addition to its effects on suppressing inflammatory and Th1 responses (10), IL-10 strongly inhibits DC maturation and IL-12 production (11, 12). Certain populations of DCs appear to produce IL-10, which may promote the generation of regulatory T cell populations (13). Importantly, localized and regulated IL-10 expression appears to be critical as the systemic presence of this cytokine has been associated with a poor outcome in a number of disease processes, including trauma and sepsis (14-16). Therefore, localized expression of IL-10 from DCs may provide a more direct therapeutic approach for diseases of exaggerated proinflammation or Th1-type responses, such as in autoimmune diseases, transplant rejection, acute bacterial infections, and sepsis.

The goal of the present study was to evaluate how adenoviral gene transfer of IL-10 modulates DC maturation and the capacity to stimulate specific T cell responses. Additionally, our goal was to examine whether modifying DC phenotype with adenoviral vectors (Adv)/IL-10 to promote localized expression of IL-10 could impact survival after an acute inflammatory challenge in vivo. The results in this report demonstrate that adenovirus provides an efficient vehicle for modulating immunity as it readily transduces DCs, resulting in stimulation of DC maturation in a dose-dependent fashion. Further modulation could be achieved after recombinant adenovirus-mediated transduction of DCs to express IL-10, which suppressed adenovirus-induced DC maturation. Whereas subsequent activation of Adv/IL-10 transduced DCs with LPS or CD40 agonists resulted in the typical upregulation of cell surface activation markers, expression of IL-10 transgene suppressed LPS induction of IL-12 expression, as well as the stimulation of Th1 and Th2 cytokine responses in a DC/lymphocyte coculture system. Finally, when Adv/IL-10-transduced DCs were readministered to mice, they improved survival in a model of generalized peritonitis.

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MATERIALS AND METHODS

Mice

Specific pathogen-free female C57BL/6 mice between 5 to 8 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained on standard rodent chow and water ad libitum. The studies were approved by the Institutional Animal Care and Use Committee at the University of Florida College of Medicine before initiation of these studies. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals.

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Generation of bone marrow-derived DCs

DCs derived from murine bone marrow were generated as previously described (17). Briefly, bone marrow cells harvested from the femur and tibia of C57BL/6 female mice were depleted of red blood cells by lysis with ammonium chloride. Thereafter, 106 cells were cultured on 24-well plates (Costar, Corning, NY) in RPMI 1640 (Cellgro, Herndon, VA) with 10% heat-inactivated fetal calf serum, 0.000375% 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO), and 1% penicillin-streptomycin-neomycin (Gibco, Grand Island, NY), pH 7.2 to 7.4, supplemented with 500 U/mL recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN) and 1,000 U/mL recombinant murine IL-4 (BD PharMingen, San Diego, CA), and were incubated in a 5% CO2 atmosphere at 37°C for a total of 4 to 5 days. The medium was replaced on day 2 with additional recombinant cytokines.

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Adenoviral transduction of bone marrow-derived DCs

Depending on the experiment, bone marrow-derived DCs were incubated on day 4 or 5 with varying quantities of the recombinant AdV for 2 h. The number of adenovirus particles varied from 106 to 1010 particles/mL for 106 DCs, yielding an approximate particle per cell transduction number ranging from 10° through 104. Thereafter, the plate was centrifuged (GPR centrifuge; Beckman, Fullerton, CA) at 1000 rpm for 5 min, and the media was completely replaced and supplemented with recombinant GM-CSF and IL-4. For experiments in which maturation of DCs was induced, 1 μg/mL LPS (Escherichia coli 0111:B4; Sigma Chemical Co.) or 5 μg/mL anti-CD40 antibody (no azide/low endotoxin levels, <0.01 ng/μg protein; BD PharMingen) was added to the DCs on day 4 (24 h before adenovirus transduction) or on day 5 (24 h after adenovirus transduction).

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Construction of a recombinant adenovirus expressing human IL-10

A recombinant adenovirus expressing the human IL-10 cDNA transgene was constructed using standard homologous recombination methods, as previously described (18, 19). Briefly, the human IL-10 cDNA containing the full-length translated region (from pDSRG-IL10 plasmid, respectively, obtained from Kevin Moore at The DNAX Research Institute, Palo Alto, CA), was subcloned into the BamHI/XbaI cloning site of the pACN transfer plasmid (19, 20) just downstream from the human cytomegalovirus immediate early enhancer/promoter and the adenovirus type 2 tripartite leader sequence. Additionally, a recombinant adenovirus expressing green fluorescent protein (GFP) or containing an empty expression cassette was constructed for use as a control. All of the viral constructs were similar with the exception of the transgene, and the production and purification procedures were identical.

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Flow cytometric analysis of DCs

Twenty four hours after adenoviral transduction with or without stimulation by LPS or anti-CD40 antibodies, respectively, cells were washed twice with flow buffer (1% bovine serum albumin [BSA], 1 mM EDTA [Fisher Scientific, Atlanta, GA], and 0.1% sodium azide [NaN3; Sigma Chemical Co.] in Hanks' balanced salt solution (HBSS) without phenol red, calcium, and magnesium [Cellgro, Herndon, VA]), and were resuspended in 4% BSA flow buffer and blocked with CD16/CD32 Fc antibodies (BD PharMingen) followed by staining, as described below. DCs were identified using anti-CD11c and anti-CD8 antibodies, and DC maturation was determined based on the relative levels of CD86 and MHC Class II (Immunotech, Miami, FL) expression. Antibodies were directly conjugated with fluorescein isothiocyanate, PerCp, or R-phycoergthrin (PE) (BD PharMingen), or were indirectly conjugated to an antibiotinylated antibody labeled with allophycocyanin (APC) (Molecular Probes, Eugene, OR). Samples were analyzed on a FACSCalibur instrument with Lysis II Software (both Becton Dickinson Systems, San Jose, CA).

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DC/lymphocyte coculture

CD3+ lymphocytes from spleens of syngeneic mice were isolated with magnetic beads (StemCells, Vancouver, Canada). Thereafter, 2 × 106 CD3+ cells were incubated for 4 days with 4 × 105 DCs that had been previously transduced with 1010 particles/mL of Adv/empty or Adv/IL-10, and depending on the experiment, were additionally stimulated with LPS. We used a high ratio of DC-to-T cells (1:5) to achieve maximal effect of transduced DCs on T cell activation to maximize T cell responses. The appearance of Th1 (IL-2 and IFN-γ) and Th2 cytokines (IL-4) in the supernatants was determined by immunoassay.

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Measurement of endocytosis

DC uptake of rhodamine-green Dextran was evaluated as previously described (21). Very briefly, DCs were resuspended in flow tubes (Fisher Scientific) at a concentration of 5 × 105 cells in 100 μL of RPMI containing 25 mM HEPES (Cellgro) and were preincubated at 4°C (background) or 37°C for 15 min. Thereafter, 100 μg of Dextran labeled with rhodamine-green (10,000 D; Molecular Probes) per sample was added for 4 h. The uptake was stopped, and the remaining rhodamine-green Dextran was removed by washing the cells twice with cold flow buffer. Thereafter, the maturation state of the DCs was determined with CD86-PE and Class II-APC as described above. Viability was confirmed by 7-amino-actinomycin D (BD PharMingen). Endocytosis in living MHC class II and CD86 low-, as well as in MHC class II and CD86 high-expressing DCs was determined by evaluating the difference in rhodamine-green Dextran uptake at 37°C and 4°C cell incubation.

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Cytokine measurements

Human IL-10, as well as murine IL-2, IL-4, IL-12p40 and p70, and IFN-γ in cell supernatants were measured by specific enzyme-linked immunoabsorbant assay using commercially available reagents (Endogen, Inc., Woburn, MA for hIL-10, and PharMingen Inc., for IL-2, IL-4, IFN-γ, and both IL-12 subunits).

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Readministration of adenovirus-transduced DCs

DCs were transduced for 2 h with AdV containing an empty cassette or expressing GFP or human IL-10, as described above.

Twenty-four hours later, the cells were subjected to density gradient centrifugation (Ficoll-Histopaque; Sigma Chemical Co.; d = 1.083) to eliminate dead cells. Thereafter, the interfacial cells were washed twice with sterile phosphate-buffered saline (PBS). In addition, some DCs transduced with the AdV expressing GFP were analyzed by flow cytometry to quantitate transduction efficiency. After anesthesia, 50 μL containing 3.2 × 105 DCs transduced with 1010 particles/mL of either Adv/empty or Adv/IL-10 was injected into the hind footpad of mice (n = 20 per study group). The dose and route of DC administration were based on earlier studies in the NOD model of type I diabetes with DCs obtained from draining lymph nodes of mice with acquired disease (22). Approximately, 1 to 8 × 104 infectious viral particles were associated with the administration of the transduced DCs (data not shown). Shortly thereafter (5 min), mice were challenged with a cecal ligation and puncture.

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In vivo tracking of readministered DCs

In a previous report, we demonstrated that labeled DCs could be recovered from the perifollicular regions of the popliteal lymph node after the footpad injection of fluorescently labeled DCs (22). Bone marrow-derived DCs (untreated or pretreated with LPS, Adv/empty, or Adv/IL-10) were labeled with carboxy-fluorescein diacetate succinimidyl ester (CFSE; Renovar, Madison, WI) using the manufacturer's instructions. Briefly, DCs were resuspended in PBS, and CFSE was added to a final concentration of 1 mM. The suspension was incubated for 10 min at 37°C. The labeled DCs were washed twice with PBS and were resuspended in normal saline for footpad injection.

After induction of anesthesia with pentobarbital, 106 DCs in 50 μL of PBS were injected into the hind footpads of B6 mice that underwent cecal ligation and puncture 24 h earlier. Twenty-four hours later, mice were sacrificed, and bilateral popliteal and inguinal lymph nodes were harvested. The lymph nodes were resuspended in 8 mL of HBSS, without Ca+3 and Mg+2 (Cellgro), and 400 U/mL collagenase D solution (Boehringer-Mannheim, Norwich, CT). Lymph nodes were placed in tissue culture dishes on ice and were dissected with two 30-gauge needles. The tissue fragments remaining in the dish were resuspended in 4 mL of HBSS with Ca+3 and Mg+2 and 400 U/mL collagenase D and placed in a 37°C water bath for 30 min. The solutions were pipetted vigorously, creating a single cell suspension, and the cells are washed twice in HBSS and centrifuged. CFSE, 7-AAD, and CD11c staining were determined by flow cytometry.

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Cecal ligation and puncture model of polymicrobial sepsis

For induction of a polymicrobial sepsis, mice were subjected to cecal ligation and puncture as previously described (16). In brief, a laparotomy was made and the cecum was isolated, ligated, and punctured through and through with a 22-gauge needle. Thereafter, mice were observed for 10 days to determine outcome.

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Presentation of data and statistics

Results are presented as the mean and SEM. Differences between experimental groups were considered significant at P < 0.05, as determined by a one-way analysis of variance or Student's t test. Post hoc analyses were performed with Tukey's multiple range test. Differences in survival were determined with Kaplan-Meier log-transformed survival analysis.

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RESULTS

Adenovirus transfects immature and mature DCs

Past reports vary as to the extent that DCs can be transduced with recombinant AdV (6, 9). Therefore, we sought to determine the transduction rate of immature (MHC class II and CD86 low-expressing) and mature (MHC class II and CD86 high-expressing) DCs. Unstimulated and LPS-matured bone marrow-derived DCs were readily transduced by a recombinant AdV encoding the reporter gene gfp (Adv/GFP) at doses ranging from 106 to 1010 particles/mL. The number of transduced DCs increased with the number of virus particles, with less than 10% of the cells transduced at particle concentrations less than 108 particles/mL (Fig. 1, A and B). At concentrations of 109 and 1010 particles/mL, transduction rates were approximately 30% and 70%, respectively. There was no significant difference in transduction rates between immature and LPS-stimulated DC cultures, or between the immature and mature subpopulations within these cultures (Fig. 1).

Fig. 1

Fig. 1

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Human IL-10 expression inhibits adenovirus-induced maturation of DCs

We next sought to determine whether transduction with the adenovirus itself would drive DC maturation, and whether this process could be influenced by expression of human IL-10. Note that human IL-10 has been reported to functionally interact with murine receptors (23). Untreated and LPS-stimulated bone marrow-derived DCs were transduced with a recombinant adenovirus containing an empty cassette (Adv/empty) or with the same vector engineered to express human IL-10 (Adv/IL-10). We observed a dose-dependent increase in the number of transduced cells that underwent maturation as assessed by the number of DCs with MHC class IIhi and CD86+ expression (Fig. 2A). Interestingly, transduction with the same concentration of Adv/IL-10 blocked the adenovirus-induced maturation. The percentage of mature DCs decreased from 50% to 80% to 20% to 30% when transduced with Adv/IL-10 at concentrations of 109 particles/mL or greater (Fig. 2A). LPS-stimulated controls showed a strong induction of DC maturation (Fig. 2C), and subsequent infection with Adv/empty only modestly enhanced further maturation. Importantly, DC maturation induced by LPS was not affected by subsequent transduction of DCs with Adv/IL-10 (Fig. 2C), indicating that IL-10 expression could not reverse maturation induced by LPS.

Fig. 2

Fig. 2

To assess the degree of transgene expression in DCs transduced by Adv/IL-10, we measured human IL-10 concentrations in the DC supernatants (Fig. 2, B and D). In unstimulated DCs as well as in LPS-stimulated DCs, there was a dose-dependent increase in human IL-10 secreted into the cell supernatants. IL-10 levels in the former group were approximately 2-fold increased (230,300 ± 1,400 pg/mL) compared with the LPS stimulated cells (131,800 ± 4,500 pg/mL) despite similar transduction rates (Fig. 1).

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DC function is modulated by IL-10 expression

IL-12p70 is produced by mature DCs (24), whereas p40, a component of the IL-12p70 heterodimer, is produced by immature and mature DCs (25). As anticipated, robust production of IL-12p40 (7000-10,000 pg/mL) was noted in the supernatants of LPS-treated DCs, and adenovirus transduction did little to increase this (Fig. 3B). In contrast, markedly lower levels of p70 were observed in unstimulated DCs, whereas there was a dose-dependent increase in IL-12p70 expression in DCs transduced with increasing quantities of adenovirus (Fig. 3A). On the other hand, DCs that were transduced with Adv/IL-10 showed a reduction in IL-12p70 release, with concentrations ranging from 47 to 108 pg/mL (Fig. 3A). However, transduction of DCs with Adv/IL-10 after maturation with LPS did not affect IL-12 p40 or p70 production, which is consistent with previous reports of an absence of IL-10 regulation of IL-12 production in mature DCs (Fig. 3, B and C) (26).

Fig. 3

Fig. 3

We next studied the effects of adenovirus transduction and IL-10 expression on the ability of DCs to activate CD3+ T lymphocyte responses. DC/CD3+ T cell cocultures were established in which DCs were transduced with 1010 particles/mL of Adv/empty or Adv/IL-10, washed, and then placed in culture with purified syngeneic CD3+ T cells. Production of human IL-10 and endogenous IL-12p40 were confirmed in this experiment. Human IL-10 concentrations were 126 ± 2 ng/mL, whereas IL-12p40 concentrations were 520 ± 150 pg/mL in cocultures containing DCs transduced with the Adv/IL-10 vector. In contrast, IL-12p40 concentrations were 1700 ± 350 pg/mL in the cocultures containing DCs transduced with the Adv/empty vector. Previous studies demonstrated that IL-12 produced by DCs promotes a Th1 cell response (e.g., IFN-γ production) (4, 26), whereas IL-10 is capable of suppressing Th1 responses in part through selective inhibition of the CD28 costimulatory pathway (27, 28). As IL-12 production was decreased after Adv/IL-10 transduction where considerable amounts of human IL-10 were secreted, it was anticipated that Th1 cytokine production by syngeneic T cells would also be decreased. Indeed, IFN-γ as well as IL-2 production was significantly reduced in the cocultures containing DCs transduced with Adv/IL-10 (Fig. 4, A and B). Furthermore, production of the Th2 cytokine, IL-4, by T-cells cocultured with Adv/IL-10-transduced DCs was reduced in comparison with T-cells cocultured with DCs transduced with Adv/empty control vector, although IL-4 levels were only slightly above the assay sensitivity (Fig. 4C).

Fig. 4

Fig. 4

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Pre-exposure of DCs to adenovirus-expressing IL-10 does not influence subsequent phenotypic maturation to LPS/anti-CD40, but alters their function

As a potential therapeutic use of ex vivo-manipulated DCs is being considered for modulation of inflammatory diseases, we wished to determine whether DCs transduced with Adv/IL-10 are resistant to further maturation with factors they may be exposed to in vivo, such as LPS or CD40L. After ex vivo transduction of DCs with 1010 particles/mL Adv/empty or Adv/IL = 10 (Fig. 5), cells were stimulated with LPS or anti-CD40 antibody. Stimulation with anti-CD40 and LPS induced maturation (MHC class IIhi and CD86+ expression) in over 80% of the cells (Fig. 5A). There was no difference in the percentage of mature cells between LPS and anti-CD40 stimulus, nor were there any differences in the proportion of mature cells with regard to whether cells were transduced with Adv/empty or Adv/IL-10. Therefore, unlike previous reports in which exogenous IL-10 added to DC cultures blocked the maturation responses to LPS (26), production of this cytokine by DCs transduced with adenovirus could not prevent subsequent maturation when these cells were exposed to activating stimuli.

Fig. 5

Fig. 5

Importantly, these DCs first transduced with Adv/empty or Adv/ IL-10 and then activated with LPS or anti-CD40 did not behave functionally like mature DCs. The DCs first transduced with adenovirus-expressing IL-10 and then activated with LPS or anti-CD40 showed marked differences in cytokine expression profiles. For example, IL-12p70 secretion by the mature DCs exposed to LPS or anti-CD40 was suppressed by previous human IL-10 expression, although slightly increased compared with baseline (Fig. 5B). Of interest, there appeared to be a general inhibitory effect of adenovirus transduction on IL-12p70 production when stimulated with LPS, which was not seen with a CD40 stimulus. The lack of IL-12p70 secretion is consistent with suppression of DC stimulatory function essential for activation of T cell responses.

We further sought to determine whether other characteristics of immature DCs were maintained, as previous reports demonstrated that recombinant adenovirus-matured DCs retain the capacity for efficient antigen uptake by endocytosis (29, 30). Therefore, we examined the endocytic capacity of DCs transduced with Adv/IL-10 and then exposed to strong maturation stimuli (i.e., LPS). Specifically, we examined Dextran uptake mediated by the mannose receptor typically expressed on immature but not mature DCs (21). DCs were cultured at 37°C or 4°C (background), and the Dextran-rhodamine green uptake was determined after 4 h of incubation. As shown in Figure 6, unstimulated immature DCs (MHC IIlo CD86lo) continued to endocytose, whereas unstimulated mature DCs (MHC IIhi CD86hi) had low rates of endocytosis. Upon transduction with either of the adenoviral vectors, mature and immature DCs retain their endocytic capacity, in contrast to noninfected LPS-matured DCs (Fig. 6).

Fig. 6

Fig. 6

To evaluate the function of these unique DCs on T cell activation, DC/CD3+ T cell cocultures were conducted. DCs were first incubated with 1010 particles/mL Adv/IL-10 or Adv/empty control vectors, and thereafter were stimulated with LPS for 24 h and were then extensively washed. After 4 days of coculture of DCs with syngeneic splenocytes, the T cell cytokines IL-2, IL-4, and IFN-γ were determined in the cell supernatants (Fig. 7). IL-12p70 was also measured in the cell supernatants as a parameter for DC maturation. Interestingly, we observed suppression of Th1 and Th2 cytokine responses in cocultures stimulated by Adv/IL-10-transduced DCs that were subsequently activated with LPS. Similar results were also seen using CD40 ligand-activated DCs (data not shown). As anticipated, IL-12p70 concentrations were also reduced. These data suggest that DCs transduced with an AdV-expressing IL-10 and then matured with LPS or CD40 do not stimulate either type of Th response. Thus, recombinant AdV-mediated autocrine expression of IL-10 in recombinant adenovirus-stimulated and LPS-activated DCs could suppress the capacity to stimulate T cell cytokine responses.

Fig. 7

Fig. 7

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Adv/IL-10 transduced DCs mitigate the in vivo response to sepsis

Our laboratory has previously demonstrated that DCs migrate to the draining lymph node after injection into the footpad, and potently modulate immune responses (22). In the present report, we quantitated the migration of the administered DCs into the draining lymph node by labeling the cells with the fluorescent dye, CFSE. When approximately 106 bone marrow-derived DCs transduced were injected into the footpad, we could demonstrate that approximately 18% to 32% of the CD11c+ cells in the draining popliteal and inguinal lymph nodes were CFSE positive (Fig. 8). Expectedly, myeloid DCs stimulated with adenovirus or LPS migrated more efficiently to the draining lymph nodes than unstimulated DCs, which were generally more immature phenotypically.

Fig. 8

Fig. 8

We subsequently examined the effect of DCs transduced with Adv/IL-10 on the outcome of an acute polymicrobial sepsis. DCs were transduced with 1010 particles/mL Adv/IL-10 or Adv/empty or were incubated with buffer alone. These cells were washed repeatedly and were then injected into the hind footpad (3.2 × 105 DCs/recipient) of recipient mice that immediately underwent cecal ligation and puncture. Animals receiving DCs transduced with Adv/IL-10 had significantly improved survival compared with animals receiving DCs transduced with Adv/empty or just buffer (Fig. 9). The production of human IL-10 was apparently restricted to the lymph nodes because human IL-10 was not recovered from the plasma of animals with sepsis pretreated with Adv/IL-10 transduced DCs (data not shown). The in vivo results from the polymicrobial sepsis model provided a link between the results demonstrating the capacity of Adv/IL-10 transduction of DCs to suppress LPS-activated DC stimulatory function and the capacity to mitigate host responses to sepsis. Interestingly, both models require transduction with Adv/IL-10 before polymicrobial infection for efficacious suppression of the respective in vitro and in vivo responses.

Fig. 9

Fig. 9

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DISCUSSION

The present study explores the capacity of Adv/IL-10 to modulate DC maturation and cytokine expression profiles in response to inflammatory stimuli. First, we have demonstrated that adenoviral-induced expression of human IL-10 prevents adenovirus-induced maturation of DCs. Additionally, we have demonstrated that transduction of DCs with Adv/IL-10 can modify subsequent cytokine responses induced by LPS or anti-CD40. These Adv/IL-10-transduced DCs are poor stimulators of a DC/syngeneic T cell coculture reaction, lacking the capacity to stimulate Th1 and Th2 type cytokine responses. Finally, we have shown that adoptive transfer of Adv/IL-10-transduced DCs into animals before induction of an acute, lethal, inflammatory challenge improves outcome.

Our results also demonstrate that transduction of DCs with the AdV increased the maturation status of the cells, as determined by high MHC class II and CD86 expression. Endotoxin contamination of the AdV preparation is unlikely to account for the observed activation of DC, as the levels in the vector stocks were less than 0.06 EU/mL before dilution. Other studies have also noted that transduction of adenovirus induces maturation of DCs with upregulation of surface molecules such as CD54, CD86, and MHC class II via an NF-κB-dependent pathway (8, 29, 31). Our results extend these findings by demonstrating that this effect can be blocked by transduction with 1010 particles/mL an AdV-expressing IL-10. Exogenous IL-10 administration has previously been shown to decrease expression of MHC class II and costimulatory molecules such as CD58 and CD86 on antigen-presenting cells (26, 32). This study further demonstrates that autocrine expression of IL-10 in transduced DCs can limit DC maturation in response to the AdV itself. Moreover, the effects of this cytokine are dependent upon the maturation state of the DC, as Adv/IL-10 transduction does not inhibit DC stimulatory function (e.g., IL-12p70 production) of cells that have already been induced to mature with LPS or anti-CD40. These results are consistent with the lack of previously described effects of exogenous recombinant IL-10 on mature DCs (33). An alternative explanation is that autocrine production of IL-10 may be more efficient at blocking adenoviral-induced maturation than maturation induced by TLR4 signaling.

Previous studies have shown that IL-10 (12) and recombinant AdV (30) have the capacity to induce retention of phenotypes generally associated with immature DCs, such as sustaining the capacity of these cells to macropinocytose and endocytose particles. In these studies, we extend those results by demonstrating that adenovirus transduction sustained the capacity of DCs for uptake of Dextran particles after a strong maturation stimulus (i.e., LPS-activation; see Fig. 6). These data further support the hypothesis that recombinant AdV can modify the LPS-stimulated developmental programs of murine DCs. Additionally, these results suggest that adenoviral transduction and expression of human IL-10 can induce a phenotypically unique regulatory DC population characterized by increased surface expression of costimulatory molecules associated with more mature DC populations, but reduced IL-12 production and a retained capacity for endocytosis consistent with less mature phenotypes.

The capacity of Adv/IL-10-transduced DC to suppress T cell immunity in vitro provided the opportunity to test the hypothesis that suppression of T cell stimulatory function may be of importance for modulating disease processes associated with an exaggerated immune response, such those occurring in acute infections. We have demonstrated that the footpad injection of DCs results in their migration to the draining lymph node and beyond (Fig. 8). Mice with sepsis pretreated with ex vivo transduced DCs expressing IL-10 demonstrated a significant reduction in mortality. In two previous studies, we observed that footpad or direct thymic injection of the same adenoviral recombinant expressing human IL-10 also improved survival in this model of cecal ligation and puncture (15, 16). In both cases, DCs were the primary in vivo target for the adenoviral recombinants. Moreover, administration of Adv/IL-10 into the footpad induced suppression of DC maturation in the draining lymph node (15, 16). The current studies extend these findings by demonstrating that comparable improvements in survival can be obtained when the DC populations are transduced ex vivo and readministered to the animal, thus providing a more direct association between DC transduction and improved outcome. Although we cannot rule out the possibility that some free Adv/IL-10 vector, remaining after the washes, could be partly responsible for the improved outcome, it nonetheless underscores the capacity of targeted IL-10 expression in DC to mitigate the effects of sepsis.

Together, these studies highlight a central role that DC subpopulations may play to regulate T cell responses in septic hosts and to alter clinical outcome. It is interesting that pretreatment of DCs with adenoviral recombinants expressing IL-10 before LPS stimulation was required to modify their response to this potent activation stimulus in in vitro and in vivo models. This effect on DCs was very similar to animal studies where anticytokine therapies had to be administered before to the induction of sepsis (34), indicating that prophylactic treatment of sepsis is more effective than treatments applied subsequent to sepsis induction.

Although mortality from polymicrobial sepsis is complex and multifactorial, recent studies have suggested that increased activation induced cell death of CD4+ T-helper cell populations may contribute to this process (35). One possible explanation for the capacity of adv/IL-10 transduced DCs to protect septic hosts may be that suppression of antigen presentation to T cells may inhibit deletion of CD4+ T cells through activation-induced cell death (apoptosis). An alternative explanation is that the interaction between IL-10-expressing DCs and resting T cells results in a T cell population phenotypically distinct from Th1 or Th2 cells capable of regulating the inflammatory process. This may explain our findings in which incubation of DCs expressing IL-10 with splenocytes resulted in a secreted cytokine profile consistent with a decrease in Th1 and Th2 responses. Jonuleit et al. (36) have shown that repeated stimulation of T cells with immature DCs induces a T cell response different from mature DCs, and consistent with the generation of a regulatory, Tr1-like cell population. Our experiments suggest that the presence of this unique DC population, induced by IL-10-expressing adenovirus transduction, may be essential in this model. Recent studies support this suggestion as pulmonary DCs with high costimulatory molecule and MHC expression that produce IL-10, similar to our Adv/IL-10 transduced population, induce regulatory T cell responses (13). These T cells may be responsible for maintaining peripheral tolerance during the healthy state, and may play a critical role in modulating the magnitude of the inflammatory response to the microbial infection. The generation of these regulatory cells has been proposed as a potential mechanism for the treatment of experimental colitis (37) and as a means to directly modulate the inflammatory response (38).

In conclusion, these studies provide important information regarding the effects of AdV on DC biology that have important implications for host-vector interactions. In addition, they also demonstrate that the expression of human IL-10 by DCs can alter their phenotype and function such that they down-modulate the capacity to stimulate Th1 and Th2 type responses.

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Keywords:

Gene therapy; sepsis; interleukin 12; innate immunity; acquired immunity

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