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Acid Ceramidase Maintains the Chondrogenic Phenotype of Expanded Primary Chondrocytes and Improves the Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ∼2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Introduction
Cell-based therapy for cartilage repair has gained increasing popularity since the first reports of successful autologous chondrocyte implantation (ACI) over 10 years ago [1]. In ACI, primary chondrocytes are obtained from small biopsies of healthy articular cartilage, expanded, and then placed onto 3-D scaffolds for subsequent use in cartilage repair surgery (for review see [2]). Currently, ACI is used in ,10% of all cartilage repair procedures where the lesions are less than 2-4 cm 2 [3]. ACI also has been used in veterinary medicine to improve the outcome of cartilage repair surgery in large (equine) and small (dog) animals [4,5]. There have been many reports documenting the improved clinical effectiveness of ACI as compared to other cartilage repair procedures, and several large, multi-site clinical studies are currently underway [6]. However, an important limitation of this procedure is the requirement of two invasive surgeries, the first of which requires extraction of cells from healthy cartilage tissue, and the second to implant the cells that have been expanded ex vivo. Recent research has therefore focused on the use of alternative chondrocyte sources where the cells can be obtained less invasively (e.g., nasoseptal) [7], the generation of chondrocytes from adult stem cells (e.g., mesenchymal stem cells [MSC] from the bone marrow or adipose tissue), and/or the use of MSCs directly for transplantation [8][9][10].
A key factor in the development of any cell-based therapy is to find safe and effective methods to rapidly expand autologous cells in a manner that retains their phenotype and in vivo repair potential. For ACI, research has concentrated on defining the culture media and growth factors used for articular chondrocyte expansion, as well as the improved design and formulation of scaffolds used to adhere the cells and prepare them for surgical reimplantation. Currently, most culture medias used to expand primary articular chondrocytes contain serum supplemented with growth factors, including members of the transforming growth factor (TGF-beta1-3) and bone morphogenic families (BMPs), insulin growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and others (for review see [11]). Similarly, numerous transcription factors influence chondrogenesis, including Sox9, beta-catenin, Smads and others, resulting in optimal expression of chondrocytespecific markers. Sox9 in particular is required for pre-cartilage condensation and differentiation of chondroprogenitor cells into chondroblasts [12].
Several studies have demonstrated the importance of sphingolipid signaling on cartilage homeostasis. For example, an early report [13] showed that a synthetic ceramide derivative (C2 ceramide) stimulated the expression of matrix metalloprotease (MMP) 21, 3 and 13 in rabbit articular chondrocytes, and induced chondrocyte apoptosis. Gilbert et al [14] also showed that treatment of bovine articular chondrocytes with sphingomyelinase, an enzyme that produces ceramide by sphingomyelin hydrolysis, decreased expression of collagen II. Elevated ceramide also has been documented in patients with rheumatoid and osteoarthritis [15], and inhibition of sphingosine-1-phosphate (S1P) production in induced rodent models of arthritis has led to beneficial clinical results [16].
In addition, we have found that animals with genetic deficiencies of enzymes involved in glycosaminoglycan degradation (i.e., the mucopolysaccharidoses, MPS) have numerous abnormalities in sphingolipid metabolism in their connective tissues. For example, chondrocyte apoptosis and cartilage degradation in the MPS animals is associated with elevated ceramide, while synovial hyperplasia is associated with elevated S1P [17]. Elevated AC activity also can be detected in serum and synovial fluid from MPS animals, likely a response to the elevated ceramide (unpublished finding).
In the current study the use of rAC as a media supplement to improve the quality of expanded primary and MSC-derived chondrocytes for cartilage repair was evaluated. Rat, equine and Figure 1. Primary rat articular chondrocytes were isolated and grown in monolayer cultures for 48 h with or without rAC (rAC, 200 U/ml) added to the media. At the indicated times proteins and lipids were extracted from the cells, and A) AC activity, B) ceramide, C) sphingosine, and D) sphingosine-1-phosphate levels were determined. Note that the addition of rAC to the culture media (gray bars) led to markedly increased AC activity in the cells by 12 h that was sustained for at least 48 h. In addition, by 12 h ceramide levels were elevated compared to baseline, but significantly less so in cells treated with rAC (i.e., by 48 h cells treated with rAC had no detectable ceramide elevation). Finally, the levels of S1P and sphingosine were elevated in rAC-treated cells by 12 and 48 h, respectively. * = p,0.05. doi:10.1371/journal.pone.0062715.g001 human articular chondrocytes treated once with rAC in either monolayer or 3-D culture systems had an improved chondrogenic  phenotype after 2-3 week cell expansion, including enhanced  expression of Sox9, FGF2, collagen 2, aggrecan and other  chondrogenic markers, and decreased detection of collagen 10. In addition, rat, feline and equine bone marrow cells grown for only 1 week in the presence of rAC yielded ,2-fold more MSCs, and rAC treatment also led to enhanced chondrogenesis of MSCs. We propose that rAC may be an important supplement to include in chondrocyte and MSC growth media that improves the production and chondrogenic potential of cells used for cartilage repair.

Animals
Animals were raised under National Institutes of Health and USDA guidelines for the care and use of animals in research. All animal protocols were approved by the Mount Sinai Institutional Animal Care and Use Committee (protocol # 98-0108), and were performed in accordance with NIH guidelines. Normal rats were identified by genotyping from the MPS VI breeding colony [18] maintained at the Mount Sinai School of Medicine, while the larger animals (cats and horses) were maintained at the University of Pennsylvania School of Veterinary Medicine (Philadelphia and New Bolton Center, PA, USA, respectively). The animals were housed with ad libitum food and water. Euthanasia was performed on cats using 80 mg/kg of sodium pentobarbital (Veterinary Laboratories, Lenexa, KS, USA) in accordance with the American Veterinary Medical Association guidelines. Euthanasia of rats was performed using carbon dioxide inhalation. Equine cells were obtained during routine surgical procedures.

Primary Chondrocyte Isolation and Culture
Methods for rat articular chondrocyte isolation and culture have been published elsewhere [19]. Briefly, to establish rat primary chondrocyte cultures, femoral caps and meniscus were collected from 6 age and sex-matched adult animals and washed three times in 0.9% NaCl supplemented with 5% penicillin/streptomycin (v/ v) and fungizone 0.1% (v/v). The cartilage was digested with 0.5 mg/ml pronase prepared in 0.9% NaCl (v/v) +5% penicillin/ streptomycin (v/v) +0.1% fungizone for 2 h at 37uC, and with 1 mg/ml collagenase (Sigma Chemical Co.) prepared in DMEM containing 10% FBS (v/v), 1% penicillin/streptomycin (v/v) and 0.1% fungizone overnight at 37uC. The resulting media was then passed through a 40 mm cell strainer, and the cells were isolated by centrifugation (1200 rpm for 8 min). They were further washed twice in DMEM containing 10% FBS, and plated in 75 cm 2 flasks at a density of 7000 cells per cm 2 in DMEM containing 10% FBS (v/v), 1% penicillin/streptomycin (v/v) and fungizone 0.1%. The Figure 2. Rat articular chondrocytes were obtained from femurs and grown for 3 weeks in monolayer cultures using standard culture medium with or without recombinant human AC (rAC 200 U/ml). rAC was added once at the initiation of the cultures. At the end of the 3-week expansion period, the cells were harvested and analyzed. A) Total cell counts (left) revealed no differences in the presence of rAC. Western blotting for two apoptosis markers (Bax and PARP) similarly revealed no differences. B) The expanded chondrocytes were analyzed by western blots for several important chondrogenic markers, including collagens 1A2 and 2A1, aggrecan, Sox9, FGF2, and TGF-beta1. Note that in all cases these chondrocyte markers were elevated in the cells treated with rAC. In contrast to collagens 2A1 and 1A2, collagen 10 expression, a marker of hypertrophy, was lowered by rAC treatment. C) Horse articular chondrocytes were obtained surgically from femoral heads and frozen. The frozen cells were then recovered and grown in monolayer cultures for 3 weeks without rAC. At 3 weeks the cells were passaged and re-plated at a density of 1610 6 /cm 2 , and then grown for an additional 1 week with or without rAC. At the end of this 1-week growth period the cells were analyzed by western blots, revealing that the expression of two chondrogenic markers, aggrecan and Sox9, were highly elevated in the rAC-treated horse cells. Bax expression also was reduced in the rAC cells, suggesting a reduction in apoptosis by rAC treatment. All experiments have been repeated at least 36. Images are representative from individual experiments. doi:10.1371/journal.pone.0062715.g002 freshly plated cells were considered passage 0 (P0). When reaching subconfluency, cells were trypsinized and passaged (P1) for further expansion. For subsequent passages, the cells were grown for 7 days, trypsinized, and then re-seeded at the same cell density. In some experiments rAC was added to the media at P0 (200 U/ml final concentration). Unless otherwise noted rAC was added to the primary chondrocytes only once, and subsequent media changes did not include the enzyme.
To grow rat chondrocytes in 3-D cultures, 565 mm collagen sponges (CollaCote, Zimmer Dental Inc., Carlsbad, CA, USA) were used. The sponges were pre-wet in DMEM containing 10% FBS (v/v) and antibiotics, and the cells, which had been previously isolated as described above, were suspended in media and pipetted onto the sponge. To distribute the chondrocytes throughout the sponge it was compressed slightly to circulate the media and distribute the cells. The sponges were then placed in culture dishes and grown in DMEM containing 10% FBS and antibiotics with and without rAC. The media was changed every three days with fresh media containing rAC (200 U/ml).
To grow rat chondrocytes in fibrin gels, 7.5610 6 cells were seeded in 200 ml of TISSEELH (Baxter Healthcare, Deerfield, IL, USA) gel in 96 well plates and cultured in standard media (DMEM containing 10% FBS and antibiotics) with or without rAC (200 U/ml). Media was changed every 4 days with fresh rAC included each media change. Cells were maintained for 2 weeks prior to analysis.
To obtain equine articular chondrocytes, cartilage explant tissue was recovered from the articular surfaces of the stifle joints of geldings (age 3-12 years) by use of a 6-mm 2 biopsy punch. The punch was pushed into the cartilage perpendicular to the articular surface until contact was made with the surface of the bone. The punch was then rotated and removed, and the resultant explant was separated from the bone by use of a scalpel blade via dissection parallel to the articular surface. Cartilage explants were trimmed to a thickness of approximately 100 mm to eliminate any mineralized tissue that might have affected subsequent culture or analysis procedures. Chondrocytes were isolated from explant tissue as described elsewhere [20], and either frozen (P0) or used for cell expansion in DMEM/F12 media containing 50 mg of ascorbate-2-phosphate/ml (v/v), 1% (v/v) penicillin/streptomycin, 1% (v/v) glutamate and 0.1% (v/v) fungizone. Human articular cartilage was collected from patients undergoing total knee replacement surgery with written consent in accordance with the Institutional Review Board of the Mount Sinai Program for the Protection of Human Subjects (IRBapproved protocol #09-0248). Cartilage slices were collected into DMEM/F12 media without FBS (containing 1% (v/v) penicillin/ streptomycin, 1% (v/v) glutamate and 0.1% (v/v) fungizone), and then incubated with Pronase (1 mg/ml) in DMEM/F12 containing 10% FBS (v/v) for 30 min at 37uC in a shaker. Following centrifugation at 2006g, the cartilage was washed twice with PBS and then incubated with collagenase P (1 mg/ml) in DMEM/F12 containing 10% FBS (v/v) overnight at 37uC in a shaker. The cartilage was then filtered using a 40-70 mm filter, centrifuged and the supernatant was discarded. The cells were washed once with PBS, resuspended in DMEM/F12 media containing 10% FBS (v/ v), and plated at a density of 1610 5 /cm 2 . In some experiments rAC was added to the culture media immediately at the time of cell plating (200 U/ml final concentration).

Acid Ceramidase, Ceramide, Sphingosine and Sphingosine-1-phospate Measurements
Rat chondrocytes were grown in 12-well plates with or without rAC (200 U/ml), and AC activity, ceramide, sphingosine and sphingosine-1-phosphate levels were determined in cell lysates at 12, 24 and 48 h. Cells were harvested and total proteins were extracted using the Cell Lytic TM M Cell Lysis Reagent (Sigma, Saint Louis, MO, USA). Proteins were quantified (Bio-Rad, Hercules, CA, USA), and the lysates were subjected to AC enzymatic activity measurement as described previously [21]. Lipids also were extracted from the cell lysates, and ceramide, sphingosine and sphingosine-1-phosphate levels were quantified as already published [22,23].

Mesenchymal Stem Cell Isolation and Culture
Rat MSCs were obtained from the femoral and tibia cavities of adult rats. Femurs and tibias were isolated, the extremities were removed, and the bone marrow was flushed out using PBS. After 2 washings in PBS, bone marrow cells were counted and plated at a density of 5610 6 cells/cm 2 in Alpha MEM supplemented with Figure 5. Primary rat chondrocytes were seeded into 3-D collagen scaffolds and grown for 7 or 14 days with or without rAC (DMEM containing 10% serum). A) They were then analyzed for morphology and proteoglycan production by Safranin-O staining. Note that the cells grown with rAC were larger and maintained a round phenotype that stained positive with Safranin-O (arrows). Shown are representative images from experiments performed 36. B) Cell shrinkage was evaluated based on Safranin-O and H&E staining using the following equation: DNA unit size = p(fiber segment diameter/2) 2 6 (myofiber segment length)/myofiber nuclei]. * = p,0.05. C) Primary rat chondrocytes were grown for 2 weeks in biodegradable fibrin gels with or without rAC in the culture media. Alcian Blue staining, another marker of proteoglycan expression, indicated enhanced chondrogenesis (more intense blue color). Experiment was performed 26. doi:10.1371/journal.pone.0062715.g005 Bone marrow from cats was collected during post mortem procedures and shipped in transport medium (RPMI 1640, 1% (v/v) penicillin/streptomycin, For horses, bone marrow was collected aseptically from the sternum by use of an 11-gauge bone marrow biopsy needle and a 60 ml sterile Luer-tip syringe that contained 10 ml of sterile acid-citrate-dextrose solution. After aspiration of the bone marrow, the aspirate was briefly mixed with the acid-citratedextrose solution, and the syringe then was placed on ice and shipped overnight. Once received, cells were washed twice with PBS and plated at a density between 1-5610 6 cells/cm 2 (feline cells) and 3610 8 cells/cm 2 (equine cells) in Alpha MEM supplemented with 20% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 1% (v/v) glutamate and 0.1% (v/v) fungizone. The freshly plated bone marrow cells were treated with or without rAC at day 0 (200 U/ml final concentration). At day 3, medium was removed and replaced by fresh medium lacking rAC. Medium was subsequently changed twice a week, and cells were passaged when they reached subconfluency. In some experiments the cells were stained after one week of culture with crystal violet (0.5% [Sigma] w/v in methanol) to count the number of colonies. For these assays (CFU-F) cells were incubated 30 min at room temperature and rinsed 4 times with PBS, before a final washing in water. Only colonies with a diameter greater than 1 mm were counted.
In experiments using rat cells, flow cytometric analysis also was performed to assess the number of MSCs. For these analyses MSCs were collected, washed twice in PBS supplemented with 2% FBS and marked for CD90-FITC and CD45-PE (Cat# 11-0900-81 and Cat# 554878, respectively, BD Biosciences Pharmingen, San Diego, CA, USA) diluted in PBS+FBS 2% for 15 min on ice and in the dark. After subsequent washings, cells were analyzed in a LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometry analysis could not be performed on the cat and horse cells due to the lack of suitable antibody reagents.
To test the effect of rAC on the chondrogenic potential of BM-MSCs, bone marrow cells were amplified for 3 weeks in Alpha MEM supplemented with 20% (v/v) FBS, 1% (v/v) penicillin/ streptomycin, 1% (v/v) glutamate and 0.1% (v/v) fungizone. rAC was not included during this cell expansion phase. At the end of 3 weeks the cells were trypsinized, counted, and 5610 5 cells were differentiated in pellet cultures using conical culture tubes. Chondrogenic differentiation was performed in DMEM high glucose media containing 6.25 mg/ml insulin, 6.25 mg/ml transferin, 1.25 mg/ml bovine serum albumin, dexamethasone 100 nM, ascorbate-2-phosphate 50 mM, 5.33 mg/ml linoleic acid and 10 ng/ml TGF-beta1, as described previously [24]. To evaluate the effect of rAC on the chondrogenic differentiation, in some experiments rAC was added into the chondrogenic medium (200 U/ml final concentration). Medium was changed every 3 days, with or without rAC. Cells were maintained in a humidified incubator at 37uC under 5% CO 2 .

Recombinant AC Production and Purification
Human rAC was produced in Chinese hamster ovary (CHO) cells as described previously [25]. Briefly, rAC overexpressing CHO cells were grown to confluency in DMEM supplemented with 10% v/v FBS and 1% penicillin/streptomycin. The conditioned medium was collected, and rAC was concentrated by pressure filtration (cut off 30 kDa, Amicon, Billerica, MA, USA) and purified using a fast protein liquid chromatography system (Amersham Biosciences, Piscataway, NJ, USA). The amount of rAC was quantified by enzyme activity measurement and western blot analysis as previously described [21,26].

Processing of the Differentiated Mesenchymal Stem Cell Pellet Cultures
Once BM-MSCs were differentiated in the chondrogenic media with or without rAC, pellets were removed from the incubator, washed twice, and fixed in paraformaldehyde 4% for 15 min at room temperature. They were then dehydrated by successive incubations in 70% ethanol (30 min at room temperature), 95% ethanol (2630 min at room temperature) and 100% ethanol (2630 min at room temperature). Pellets were then cleared in two successive baths of xylene (2630 min at room temperature), and paraffin embedded and microtome processed to create 6 mm slices on polyprep slides (Sigma).
For histochemical staining, pellets were deparaffinized in xylene, hydrated with distilled water, and stained with either Alcian Blue 8GX 1% (w/v), pH 2.5 (Sigma) for 30 min at room temperature or Safranin-O (Sigma) 0.1% (w/v) for 5 min at room temperature. Slides were washed, dehydrated and cleared with xylene before covering with mounting medium.

Immunohistochemistry
For immunohistochemical analysis pellets were deparaffinized in xylene, hydrated and washed three times in PBS. If needed (i.e., for Sox9 immunostaining), cells were permeabilized in 0.2% (v/v) Triton X-100 made in PBS (pH 7.4) for 5 min at room temperature, post-fixed in paraformaldehyde, and washed in PBS several times. Slides were then blocked 2 h at room temperature in PBS supplemented with Tween 0.1% (v/v), and 10% (v/v) FBS. After blocking, slides were incubated with primary antibody diluted in PBS containing 0.1% Tween and 5% FBS overnight at 4uC. The following primary antibodies from Santa Cruz Biotechnology, Santa Cruz, CA, USA were used for immunostaining: Rabbit anti-Sox9 (H-90, sc-20095), rabbit anticollagen 2A1 (H-300, sc-28887), goat anti-collagen 10A1 (E-14, sc-323750), and goat anti-aggrecan (D-20, sc-16492). After exposure to the primary antibodies, slides were then incubated 1 h at room temperature with the corresponding secondary antibody conjugated with Cy3, diluted in PBS/Tween 0.1%/10% FBS. Finally, the slides were washed several times in PBS, and mounted with a DAPI containing medium (Vector Laboratories, Burlingame, CA, USA). Localization of the primary antibodies was visualized using the fluorescent Cy-3 second antibody and laser-scanning confocal microscopy (Zeiss LSM510).
For immunohistochemistry of the primary chondrocyte cultures, P3 cells were plated on chamber slides (5000 cells/chamber, lab-tek2 chamber slide, Thermo Fisher Scientific, USA) and grown until subconfluency. Cells were then washed twice in PBS, fixed in paraformaldehyde 4% for 15 min at room temperature, and immunostained as described above.  . Rat bone marrow cells were isolated and expanded for 3 weeks in standard culture media (without rAC) to prepare homogenous populations of MSCs. They were then placed into chondrocyte differentiation media (Stem Cell Technology) with or without TGF-beta1 and/or rAC. Pellet cultures were grown for 3 weeks to prepare chondrocytes, and then fixed and analyzed by Alcian Blue and Safranin-O staining, markers of chondrogenesis. A) Note the small and poorly formed pellets in the absence of TGF-beta1 and rAC (upper left). TGF-beta1 is a standard supplement used to induce the differentiation of bone marrow MSCs to chondrocytes. Addition of TGF-beta1 or rAC to the cultures independently had only a modest effect on the pellet size and staining (upper right and lower left). However, inclusion of both proteins in the culture media had a much more significant effect (lower right), both on the size of the pellets and staining intensity. Pellet size is an indicator of the number and/or size of chondrocytes, and staining intensity is a measure of proteoglycan deposition. Beside Alcian Blue and Safranin-O staining, pellets were subjected to immunostaining against B) Sox9, C) Aggrecan, and D) Collagen 2A1. Experiments have been performed with 3 independent rats. Representative images are shown from one experiment. Blue (DAPI) indicates nuclei, and red indicates Sox9, Aggrecan or Collagen 2A1, respectively.

RNA Isolation and Quantitative RT-PCR
For RNA isolation cells were trypsinized, washed twice in PBS, and resuspended in Trizol/chloroform (5/1 (v/v)) (Life Technologies, USA). After centrifugation, the aqueous upper phase was saved and purified through an affinity column (RNeasy Mini Kit, Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. RNA was resuspended in RNase-free water, quantified, and 1 mg of RNA was subjected to reverse transcription. For qPCR analysis of the human articular chondrocyte cultures, cDNA was synthesized with Superscript VILO (Invitrogen). SYBR green qPCR (Invitrogen Platinum Taq) was performed and gene expression was normalized to GAPDH (2-ddCt method).

Statistical Analysis
Where appropriate, statistical analysis was carried out using a standard student's t-test analysis, one-way analysis of variance (ANOVA) with the variable group, multivariate analyses of variance (MANOVAs) followed by post hoc Bonferroni adjustments. The results were considered significant at P,0.05. Statistics were performed using Sigma Stat 3.1 (Systat Software).

Changes in Sphingolipid Metabolism Following rAC Treatment of Primary Articular Chondrocytes
Primary rat articular chondrocytes were isolated and grown in monolayer cultures as described in the Materials and Methods using DMEM containing 10% FBS with or without rAC. For the baseline time point, cells were collected from 6 rats and pooled without culture. The AC activity, ceramide, sphingosine and sphingosine-1-phosphate (S1P) levels in these control cells were compared to cells grown for 12, 24 and 48 h with or without rAC. The data are summarized in Fig. 1.
As expected, cells grown in media supplemented with rAC exhibited markedly increased AC activity by 12 h, and this was sustained through 48 h. Ceramide levels also were increased compared to baseline by 12 h, but less in cells exposed to rAC. In fact, by 48 h the ceramide levels in the rAC-treated cells had returned to baseline, while in those without rAC treatment they remained significantly elevated. Surprisingly, despite the high ceramide levels we did not see elevation of sphingosine at 12 or 24 h. In contrast there was an early elevation of S1P, presumably due to the activity of sphingosine kinase. By 48 h significant elevations of sphingosine were detected in the rAC-treated cells. By 7 days AC, ceramide, sphingosine and S1P levels all had returned to baseline regardless of rAC treatment (data not shown).
Overall, these results demonstrated that rAC was taken up by rat chondrocytes and retained biological activity by hydrolyzing ceramide and producing sphingosine and S1P. The fact that AC levels returned to baseline by 7 days was consistent with its expected intracellular half-life of 48-72 h [27].

Effects of rAC Supplementation on the Phenotype of Expanded Articular Chondrocyte Monolayer Cultures
We next evaluated the effects of rAC supplementation on the phenotype of articular chondrocytes after cell expansion. For these experiments rat chondrocytes were grown for 3 weeks with and without rAC in DMEM containing 10% FBS. rAC was added once at the time of the initial cell plating (P0). All analyses were performed at the end of the 3-week expansion period (P3) unless otherwise mentioned.
No effect of rAC treatment on the total number of cells was observed ( Fig. 2A), consistent with the fact that the levels of two apoptotic markers, Bax and PARP, also were very similar with and without rAC treatment.
In contrast, there was a marked effect of rAC treatment on the cell quality after 3 weeks of expansion, as determined by the expression of various chondrocyte markers. As shown in Fig. 2B, western blot analysis revealed that the expression of collagen 2A1, collagen IA2, aggrecan, Sox9, TGF-beta1, and FGF2 were enhanced in cells treated with rAC. Similar effects were evident in cells grown using a different culture media, RPMI containing 10% FBS (data not shown). Expression of collagen 10, a marker of chondrocyte hypertrophy and de-differentiation [28], was reduced in these cultures.
To confirm that the effects of rAC were not rat-specific, we studied equine articular chondrocytes provided frozen at P0. The frozen cells were thawed and expanded for 3 weeks without rAC, and then re-plated with or without rAC in the culture media. They were grown for 1 additional week and then analyzed. As shown in Fig. 2C, the expression of two important chondrocyte markers, aggrecan and Sox9, were markedly enhanced in the rAC-treated equine cells, consistent with what was observed in the rat. The lack of other equine-specific antibodies precluded the analysis of additional chondrocyte markers in these cultures. Of interest, we also observed a reduction in the expression of the pro-apoptotic Bax protein in the rAC-treated equine cells, more than that seen in the rat cells. This could be due to the equine cells having been provided to us frozen, eliciting additional stress-related cell death during the culture period that was reduced by rAC.
We further evaluated rAC-treated and untreated rat chondrocytes by immunohistochemistry and confocal microscopy. These analyses confirmed that after 3 week expansion the number of cells expressing collagen 2A1 was increased ,40% following rAC treatment (Fig. 3A). In addition, confocal analysis revealed higher levels of Sox9 in the cells after exposure to rAC (Fig. 3B).
Osteoarthritis (OA) is a common, age-related disorder that results in cartilage degradation, and patients with OA frequently undergo surgical procedures to repair their defective joints. Articular chondrocytes from two aged OA patients was obtained Figure 8. Horse bone marrow cells were isolated and expanded for 3 weeks in standard culture media (without rAC) to prepare homogenous populations of MSCs. They were then placed into chondrocyte differentiation media containing TGF-beta1, but with or without rAC. A) Pellet cultures were grown for 3 weeks to prepare chondrocytes, and then fixed and analyzed by Alcian Blue (left panel) and Safranin-O (right panel) staining. Note the smaller, more diffuse pellets in the absence of rAC. Pellets were also submitted to immunostaining against B) Sox9, C) Aggrecan, or D) Collagen 2A1. Note the higher expression intensity of Sox9, Aggrecan and Collagen 2A1 in the pellets. E) Pellets were also subjected to an immunostaining against Collagen 10. Note the diminished expression of Collagen 10 in pellets exposed to rAC. DAPI (blue left panel) indicates nuclei, and the right panel (red) indicates Sox9, Aggrecan, Collagen 2A1, or Collagen 10, respectively. Merged images are to the right. Scale bars: 50 mm. * = p,0.05, ** = p,0.005, *** = p,0.001. doi:10.1371/journal.pone.0062715.g008 and expanded in monolayer culture for 3 weeks with or without rAC in the media (added once at the initial cell plating). As depicted in Fig. 4, qPCR analysis showed that cells from patient 1 exhibited significantly elevated expression of Sox9, collagen 2A1, aggrecan and TGF-beta mRNA in response to rAC treatment. In cells from patient 2, Sox9 expression was significantly elevated, and there was a trend towards elevated collagen 2A1 and aggrecan expression. As was observed with the rat and horse cells, the total number of chondrocytes obtained after the expansion period was similar, despite the differential gene expression pattern. It is again notable that no elevation of collagen 10 expression was observed in the rAC treated cells from either patient.
Overall, these results revealed that supplementing chondrocyte media with rAC once at the time of initial cell plating had a significant, positive effect on the chondrogenic phenotype after expansion.

Effects of rAC Supplementation on 3-D Culture of Primary Articular Chondrocytes
To achieve effective implantation of expanded chondrocytes in patients, after the initial monolayer expansion period the cells used for ACI are generally seeded onto 3-D scaffolds for subsequent growth and transplantation. Therefore, the effects of rAC treatment on primary rat chondrocytes grown on collagen-coated scaffolds were evaluated (Fig. 5A). For this experiment, the cells were seeded directly onto the scaffolds bathed in DMEM containing 10% FBS with or without rAC. They were then grown for 7 or 14 days and analyzed. In order to obtain optimal penetration of the 3-D scaffolds with the enzyme, fresh rAC was added to the cells at each media change. As show in the representative images (Fig. 5A), cells grown in the presence of rAC were larger and maintained a rounder phenotype than those without rAC. Importantly, these cells also stained positive with Safranin-O, a commonly used marker of proteoglycan expression. Further quantification of cell shrinkage [29] revealed significantly less shrinkage in the rAC treated cells (Fig. 5B).
In addition to collagen-coated scaffolds, biodegradable fibrin gels also are frequently used for implantation of the cells into the damaged cartilage site. As shown in Fig. 5C, rAC treatment of rat chondrocytes grown in such gels for 2 weeks exhibited enhanced staining for Alcian Blue, an important marker of proteoglycan deposition.

Effects of rAC Supplementation on the Yield of Bone Marrow-derived Mesenchymal Stem Cells
As noted above, recent interest in cell-based cartilage repair has focused on the use of MSCs, which can be readily obtained from adult bone marrow, adipose tissue or other autologous sources, and may be induced in vitro or in vivo to form chondrocytes and other mesenchymal cell lineages. The effects of rAC on adult rat bone marrow-derived MSCs before and after their differentiation into chondrocytes were therefore evaluated. Addition of rAC to the culture media at the time of initial plating of the rat bone marrow cells led to a ,2-3 fold increase in the number of MSCs obtained at day 5, as judged by the number of colony forming fibroblast units (CFU-F) or by flow cytometry (Fig. 6A,B). To confirm that these findings were not rat-specific, the effect of rAC supplementation on bone marrow MSCs obtained from cats and horses also was evaluated (Fig. 6C,D). Due to the lack of suitable antibody reagents for flow cytometry, the number of MSCs in these species were quantified by CFU-F only, revealing increases similar to those observed with the rat cells.

Effects of rAC Supplementation on the Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells
The effects of adding rAC during the chondrogenic differentiation of MSCs also were evaluated. For these studies differentiation was carried out in high-density pellet cultures using standard media with or without TGF-beta1 (see Materials and Methods). Rat bone marrow cells were first grown for 3 weeks in the absence of rAC to obtain a population of adherent MSCs that was ,90% pure (CD90+/CD40-, not shown). They were then grown in high-density pellet cultures for an additional 3 weeks in chondrocyte differentiation media. As shown in Fig. 7A, rat pellets grown in the presence of rAC were larger (.2-fold) and stained more intensely for proteoglycans using Alcian Blue and Safranin-O. The effects of rAC were independent of TGF-beta1, and the two factors worked synergistically to yield optimal results. The rat pellets also were analyzed by immunohistochemistry and confocal microscopy for Sox9, aggrecan and collagen 2 expression. As illustrated by the images in Figs. 7B-D, the expression of these three chondrogenic markers were significantly elevated in cells supplemented with rAC. To confirm these findings, the same studies were performed using equine cells, and essentially the same results were obtained (Fig. 8A-D).
As described earlier for the chondrocyte expansion studies, rAC treatment led to either no change or a reduction in the levels of collagen 10, a marker of chondrocyte hypertrophy and dedifferentiation, in contrast to collagen 2, which was elevated. To examine this further the differentiated equine MSC-derived chondrocytes were stained for collagen 10, and the levels were found to be decreased in cells treated with rAC (Fig. 8E). Taken together, the elevated expression of collagen 2, aggrecan and Sox9 along with low collagen 10 expression suggests that the treated cells had a more chondrogenic phenotype conducive to cartilage repair.

Discussion
The dedifferentiation of chondrocytes during cell expansion remains one of the main barriers in ACI [30]. During the amplification process it takes only a few days for the cells to change their shape from rounded to fibroblastic and to start displaying abnormal features. For example, the cytoskeleton begins to express F-actin stress-fibers, important chondrocyte-specific extracellular matrix proteins and/or transcription factors are reduced (e.g., Col 2, proteoglycans, Sox9), hypertrophic makers (e.g., Col 10) may become elevated, and degradative proteases (e.g., MMPs, ADAM-TS) are activated. Moreover, although dedifferentiated cells can be re-differentiated when implanted in 3-D culture systems such as alginate, agarose or fibrin hydrogels, irreversible de-differentiation is usually observed in preparations of primary chondrocytes. Hence, during the expansion phase it is of primary interest to maintain primary chondrocytes as close as possible to their initial chondrogenic profile, and/or to improve their capacity to redifferentiate prior to re-implantation.
The current study shows that a single treatment of primary chondrocytes with rAC, although not improving the yield of cells after expansion, greatly improved their chondrogenic phenotype. In particular, the expression of collagen 2A1, aggrecan, FGF2 and Sox9 were markedly increased in rat cells at the end of the 3-week expansion period (Figs. 2 and 3). Similar findings were observed in equine and human cells (Figs. 2 and 4). When grown in 3-D culture systems, i.e. collagen sponges or fibrin gels, addition of rAC also improved the cell phenotype, as shown by H&E and Safranin-O staining (data not shown and Fig. 5). Cells were bigger and rounder, and the expression of proteoglycans was improved with rAC treatment (Fig. 5C).
It may be speculated that these changes were due to the shortterm effects of rAC treatment on sphingolipid metabolism (Fig. 1), presumably leading to transcriptional and post-translational modifications that resulted in these downstream effects. rAC activity was rapidly elevated in primary chondrocytes after addition of the enzyme to the culture media, lowering the levels of ceramide compared to controls, and resulting in the elevation of sphingosine by 48 h. Ceramide is a well characterized, stressresponse lipid that has many effects on cell survival and differentiation. Sphingosine, the product of AC activity, has similar biological effects, and both of these lipids have been shown to influence chondrogenesis. For example, ceramide may stimulate aggrecanse activity, leading to proteoglycan depletion in cells [13]. On the other hand, ABC29460, a selective inhibitor of sphingosine kinase 2, an enzyme that converts sphingosine to S1P, attenuated cartilage damage and reduced chondrocyte apoptosis in a rat model of osteoarthritis [16]. This suggests that sphingosine may have a protective effect on chondrocytes. The role of S1P itself also has been studied extensively in cultured chondrocytes and in animal models of OA with somewhat conflicting results; in some cases S1P has been shown to reduce aggrecan and proteoglycan expression [31], while in others it has a protective effect [32]. Clearly, sphingolipids have important effects on chondrocyte growth and differentiation, and manipulation of this pathway with rAC appears to be maintaining the cells in a more chondrogenic phenotype after expansion. Future research should focus on the early transcriptional and other changes occurring in chondrocytes treated with rAC to better understand the mechanism of these positive, downstream effects on the chondrocyte phenotype.
In addition to the use of primary chondrocytes for ACI, many investigators have turned to MSCs for cartilage repair. The main limitations in the use of MSCs for cell-based cartilage repair procedures are that they lose their ability to differentiate into chondrocytes very early (3 or 4 passages), shortening the time for amplification prior to differentiation, and proper ex vivo and in vivo protocols to differentiate MSCs into fully functional chondrocytes that do not undergo hypertrophy are still lacking. Numerous papers have been published describing the use of new agents for the differentiation of MSCs towards chondrocytes. Among the various molecules evaluated, proteins of the TGF-beta1 and bone morphogenic families (BMPs) are the most described. Another strategy relies on the engineering of 3-D scaffolds that release growth factors, anti-apoptotic or differentiation factors in which the MSCs can be seeded at high density to mimic the natural conditions of differentiation.
In this study a single addition of rAC into the culture media of rat bone marrow was shown to increase the yield of MSCs at 1 week (Fig. 6), as seen by CFU-F assay and flow cytometry analyses. Repeated treatment with rAC during the chondrogenic differentiation process also improved the quality of resulting MSC-derived chondrocytes, as seen by Alcian blue staining, Safranin-O staining, and immunostaining ( Figs. 7 and 8). Notably, the levels of Sox9, collagen 2A1 and aggrecan were more elevated in cells differentiated in the presence of rAC, but the level of collagen 10 was diminished, revealing a partial blockage of hypertrophy in cells differentiated in presence of rAC. Reduced collagen 10 also was observed in primary chondrocytes treated with rAC.
Thus, two positive influences of rAC treatment were observed with MSCs, although the mechanisms behind these observations might be different. For example, while the treatment of bone marrow cells with rAC at P0 may have been beneficial because the enzyme reduced the ceramide-related stress response induced by the extraction of the cells from their natural environment and plating in an artificial medium (thereby yielding more viable MSCs), the benefit of the treatment during the differentiation process may be due to other mechanisms such as better signaling through the TGF-beta pathway and/or effects of sphingolipid changes on chondrogenesis and cartilage homeostasis. In addition, reduction of the stress response during MSC chondrodifferentiation may be a factor, in particular since the differentiation medium is devoid of serum and hence a very potent stress inducer.
Overall, we show for the first time that the addition of rAC to culture media has a positive influence on the chondrogenic phenotype of expanded primary and MSC-derived chondrocytes, likely through alterations of the sphingolipid signaling pathway. These findings could have an important impact on cell-based cartilage repair by providing higher quality cells for transplantation, and/or by including the enzyme directly in 3-D scaffolds to improve chondrogensis in vivo. Future studies will focus on evaluating the effects of rAC treated cells in vivo, as well as exploring the mechanisms underlying these AC-induced changes more fully.


s2fieldsofstudy: Biology Engineering

text:
Jürgen Roth: Immunogold master

Roth pioneered immunogold EM on thin sections and studies protein glycosylation.

G lycosylation, the synthesis of branched sugar chains on proteins, is a posttranslational modifi cation commonly found on membrane and secretory proteins. Glycosylation is important for proper protein folding, and the composition of the sugar chains can also be used to track a glycoprotein's maturation.
When Jürgen Roth started working on glycoproteins, researchers had only recently begun probing the mechanisms and machinery of glycosylation using biochemical approaches. Roth applied his expertise in electron microscopy (EM) to develop methods for visualizing carbohydrates (1) and protein glycosylation. Using electrondense colloidal gold particles covered with protein A to detect antibodies bound to thin sections by electron microscopy (2), Roth's seminal studies have shown where (3,4) and why (5) protein glycosylation takes place. Yet there's still more work he wants to do, as we learned when we spoke with him recently at home on holiday in Switzerland.

I understand you grew up in East Germany…
Yes, that's right. Actually, I was born a year and a half before World War II ended, before the state of Thuringia was liberated by the American army and then left to the Russians. I grew up in what was initially the Russian military zone, which later became the German Democratic Republic.

What was it like growing up there?
Those initial postwar yearsand many years afterwards-were diffi cult for everybody. It was a hard life. We were not poor, but we were struggling. But my memories are of a happy early childhood. My parents had to earn a living, so I was not really supervised all day. I grew up outdoors. I think for a young kid there's nothing nicer than to be in the forest or a fi eld with friends.
Of course, when I was young, I did not understand the political situation in East Germany. Sometimes people-including an uncle of mine-would disappear for years and then come back and not speak about what happened to them. The eyeopener for me as a young boy was June 17, 1953: the fi rst-ever uprising of the people against the Communist regime. I vividly remember the state of emergency. I was much older when they started to build the Wall in Berlin.

You fi rst trained as a medical doctor…
Yes. I wished primarily to study biology, but there was simply no hope because of the very limited number of places. So I studied medicine because I hoped this would later give me a chance to do experiments like ones I had done in secondary school, such as dissecting an earthworm to look at the nervous system. It was very diffi cult to get into medical school; I was rejected from the university on my fi rst try. But the following year I was admitted to study medicine at the Friedrich Schiller University in Jena.
After six years studying medicine, I became a resident in the Institute of Pathology at the University. I had purposefully chosen pathology because it is related on one side to clinical medicine and on the other side to experimental work. I was really lucky to join a research team lead by Professor Helmut W. Meyer, who was a microbiologist and electron microscopist. That's how I entered electron microscopy.

What sorts of techniques were you using?
Meyer's lab studied proteins and lipids in biological membranes using freeze-fracture EM. But I was interested in the sweet aspect of membranes, the carbohydrates at the cell surface, which are called the glycocalyx. At that time, there were some histochemical techniques for carbohydrates, but they were rather nonspecifi c. I stumbled onto some early work on plant lectins, proteins with carbohydrate-binding properties that had a much higher specifi city. I obtained some concanavalin A from Wilhelm Bernhard in Villejuif, France, and later purifi ed other lectins myself. Then we used peroxidase or ferritin to label the lectins. This gave us probes to detect specifi c oligosaccharide structures by electron microscopy.

How did you get out from behind the Berlin Wall?
In the early 1970s I had started to use particles of collodial gold as a highly electron-dense marker to label lectins. In this, I was following on from the work of Marc Horisberger in Switzerland, who was using lectin-gold complexes in scanning electron microscopy to study microbes. My publications on this caught the attention of Lelio Orci from the University of Geneva, who invited me to his institute to teach this technique to his lab. At that time, I was living in East Germany, and I was a nobody. Ordinarily, there would have been no hope to follow this invitation, but the director of our Institute of Pathology, Professor Franz Bolck, was very supportive of me and the other PhDs. He also had a good contact with the Minister of Education. In 1976, I received a prestigious East German science prize, and, as he presented the award to me, the Minister of Education said, "Your travel to Geneva will be okay." So, I traveled to Orci's lab, and together we quickly generated a publication using ferritin-labeled lectins. He invited me for another visit, this time for a year. After that visit, I just stayed on in Geneva.

What did you work on there?
Before I came to Geneva, I used lectin-gold complexes to study the distribution and dynamics of glycoproteins on the cell surface. But this did not tell me anything I was really interested in. I wanted to know more about glycosylation as it occurs in the ER and the Golgi apparatus. I tried to get a more direct view of this using lectin-gold complexes to visualize carbohydrates and antibodies to detect glycosylation enzymes in the ER and Golgi on thin sections. Later on, the low-temperature resins developed in the lab of the late Eduard Kellenberger became very important for my work.
I had done some quite unsuccessful pilot experiments with antibodies and protein A-gold while I was still in Jena, but, on my second trip to Geneva, I met Moïse Bendayan, a postdoc in the lab. He had made a number of antibodies against exocrine pancreatic enzymes, so we joined forces. We used his antibodies, and I made a protein A-gold complex to specifi cally detect the antibodies bound to Epon thin sections. It worked! After incubating thin sections with antibodies against amylase and with protein A-gold, we saw gold particle labeling over the ER, the Golgi apparatus, and zymogen granules. This experiment was followed by many studies of other secretory, membrane, and cytosolic proteins. We also extended the stereology methods originally developed by Ewald Weibel, counting gold particles in our samples to achieve quantitative immunogold EM.

Had anyone done something similar?
Others had used gold-labeled reagents for cell-surface labeling. But we were the fi rst to succeed at using protein A-gold on ultra-thin sections of tissues to visualize antibodies bound to antigenic sites exposed on the surface of the thin sections. This technique became the gold standard in immunoelectron microscopy.


extfieldsofstudy: Biology Medicine

text:
Innate immune response after adenoviral gene delivery into skin is mediated by AIM2, NALP3, DAI and mda5

Methods for human skin gene therapy requires efficient and stable introduction of genes into skin cells. Transient cutaneous gene therapy is an attractive approach in the treatment of skin diseases. The ‘Achilles heel’ of adenoviral gene therapy is its immunogenicity and many aspects of adenovirus induced cutaneous immune reaction still remain unanswered, particularly the role of keratinocytes. Therefore, human keratinocytes were transfected with adenoviral DNA and cytokine expression was analyzed. Moreover, adenoviral transduction of full-skin was performed ex vivo and in vivo. We observed cytokine induction after cytoplasmatic internalization of adenoviral DNA into epidermal cells. Inhibition of AIM2, NALP3, DAI or mda5 downregulated the cytokine response. Transduction of immunocompetent mice led to a detectable transgene expression for 12 days. Re-application of the vector led to a decrease in intensity and duration of transgene expression limited to 4 days and an increased cytokine expression. In contrast, immunodeficient mice showed a reduced expression of cytokines after DNA internalization. AIM2, NALP3, DAI and mda5 are essential in the induction of an innate immune response towards adenoviral DNA. This immune reaction leads to a decrease in transduction efficiency of the vector after re-application and modulation of these receptor systems stabilizes transgene expression.

Introduction
The skin is the biggest and most important organ in protecting the body from a hostile environment. The epidermis, its outside layer, is mainly composed of keratinocytes, which guard the body against physical, chemical, or biological damage by establishing a protective layer (Bouwstra et al. 2003). Its accessibility makes the skin an easily approachable target for the treatment of both local and systemic diseases via gene therapy (Kim et al. 2000). Gene therapy is a promising tool for the treatment of a wide variety of inherited as well as acquired disease including genetically inherited skin disorders, tumors, metabolic disorders and infectious diseases (Mulligan 1993). Specific anatomical and biological properties make the skin a very interesting organ for in vivo and ex vivo gene therapy approaches. Gene delivery can be easily controlled and the skin surgically excised if any side effects occur (Christensen et al. 2001). Keratinocytes are responsible for establishing a physical barrier and guaranteeing the structural integrity of the epidermis (Bouwstra et al. 2003). As the epidermis is known to produce a variety of cytokines and growth factors, keratinocytes may also be engineered as bioreactors to secrete gene products, which have local or systemic effects (Tomic-Canic et al. 1998). Its accessibility suggests that different methods for gene delivery can be pursued, depending on the desired application. The approach used to deliver DNA into the skin will have an influence not only on the efficiency of DNA delivery, but also on the level and duration of transgene expression (Worgall et al. 1997). For transient transduction of target cells, adenoviral vector systems possess the highest efficacy and have been used in 23.3% of the registered clinical trials worldwide in the last two decades (JGT 2012).
Adenoviridae are non-enveloped, double stranded (ds), linear desoxyribonucleic acid (DNA) viruses with a genome of 35-40 kb and a particle size of 70-100 nm (Rux & Burnett 2004). The adenoviral genome is well characterized and comparatively easy to manipulate. Most adenoviruses cause mild diseases in immunocompetent human adults and by deletion of crucial regions of the viral genome the vectors can be rendered replication defective, which increases their predictability and reduces unwanted side effects. Moreover, deleted regions of the viral genome can easily be replaced by foreign genomic material encoding the therapeutically active metabolite (Tatsis & Ertl 2004). The process of adenoviral entry into the host is extremely efficient and has been intensively studied (Douglas 2007). Adenoviruses exhibit a wide host range in vitro and in vivo; this range was also seen in nondividing cells (Zhang 1999). In addition, the welldefined and easily manipulated viral genome favors the development of adenoviral vectors for gene therapy applications. A major disadvantage of adenoviral vectors is that viral DNA can effectively elicit the innate and adaptive immune response immediately after infection, leading to the secretion of pro-inflammatory cytokines in mice, primates and humans (Raper et al. 2003;Schnell et al. 2001;Zhang et al. 2001). Activation of innate immunity is associated with a reduction in efficacy of gene transfer (Worgall et al. 1997) but also in profound damage to healthy tissue and significant morbidity in transduced hosts (Raper et al. 2003;Schnell et al. 2001). Several studies focused on the immune reaction elicited through cytoplasmatic localized adenoviral DNA. This led to the development of newer generations of adenoviral vector systems that were depleted of a number of viral genes in order to reduce the immune reaction. Helper-dependent adenoviral vectors lack almost all viral coding sequences and lead to diminished adaptive immune responses and improved duration of gene transfer (Muruve 2004). However, acute toxicity and reduced vector persistence provoked by the innate immune response remain the most significant barriers to an effective clinical application of this promising technology (Brunetti-Pierri et al. 2004).
Several studies on adenoviral DNA induced innate immune reaction have focused on antigen presenting cells (APCs) such as dendritic cells (DCs) or macrophages (MΦ) (Nociari et al. 2007;Zhu et al. 2007) and RNA virus-induced immune reactions of APC (Lopez et al. 2006). In addition to adenoviral DNA, activation of innate immunity has also been described for vertebrate and mammalian DNA, and synthetic oligonucleotides (Nociari et al. 2007;Zhu et al. 2007). Moreover, a sequence independent mechanism for cytoplasmatic DNA recognition and immune activation has been specified (Suzuki et al. 1999). The detection of microbial components by pattern recognition receptors (PRRs) is one of the earliest defense mechanisms to trigger innate immune responses against infections (Janeway & Medzhitov 2002). Of the many classes of molecules detected by cells as pathogen associated molecular patterns (PAMPs), nucleic acids are potent and broadly recognized (Isaacs et al. 1963). In order to sense nucleic acids the immune system employs several classes of receptors. The family of Toll-like receptors (TLRs) in this context is the best described group of PRRs. TLRs can recognize endosomal doublestranded (ds)RNA (TLR-3) (Alexopoulou et al. 2001), singlestranded (ss)RNA (TLR-7/-8) (Diebold et al. 2004), or hypomethylated DNA (TLR-9) (Hemmi et al. 2000). Activation of nucleic acid sensing TLRs occurs within endosomal compartments (TLR-3, -7, -8 and −9) and requires either a myeloid differentiation primary response gene 88 (MyD88) or TIRdomain-containing adapter-inducing interferon-β (TRIF) adapter molecules. These proteins facilitate activation of downstream signaling cascades, which lead to the activation of inflammatory transcription factors, including nuclear factor-kappa B (NFκB), activator protein 1 (AP-1) and interferon regulatory factors (IRF) 3 and 7. An activation of downstream cascades leads to a release of inflammatory cytokines which play an inportant role in direct or indirect viral clearing mechanisms. A direct mechanism is associated with a recruitment of inflammatory cells, activation of adaptive immune system and complement system whereas an indirect mechanism is represented by a further induction of inflammatory signalling, for example an activation of JAK/STAT-signalling. The JAK/STAT-signal cascade is activated mainly through cytokine receptors on the cell surface and thereby plays during an infection in the communication between cells play a central role. An activation of this signaling cascade leads to further induction of cytokine synthesis in the cell (Kanehisa 2012).
Since the discovery of TLR-9, there has been a growing body of evidence that DNA derived from microbial and host cells can be recognized via a TLR-9-independent mechanism. DNA recognition in these pathways is sequence independent and occurs in the cytoplasm of the cells (Suzuki et al. 1999). Different ligands, such as adenoviral, mammalian and vertebrate DNA as well as dsDNA have been characterized for TLR-independent recognition in APCs (Martin & Elkon 2006;Nociari et al. 2007;Zhu et al. 2007). In addition to TLRs, the RIG-like receptor (RLR) mediated signal transduction has also been discussed for DNA-recognition wherein a corresponding DNA-sensing receptor in this signaling cascade has not been identified yet (Nociari et al. 2007). RLR-dependent signaling is induced by recognition of cytosolic ribonucleic acid (RNA) via retinoic acidinducible gene I (RIG-I) and melanoma differentiation associated gene 5 (mda5)) and requires an adapter molecule IPS-1 (mitochondrial antiviral signaling protein 1). Downstream signaling results in an NFκB-, AP-1-and IRF-3/7-dependent induction of cytokine expression (Yoneyama & Fujita 2007). In 2009, a DNA sensor and activator of innate immune responses has been identified and termed DNA-dependent activator of IFN-regulatory factors (DAI and also known as DLM-1 and ZBP1) (Takaoka et al. 2007). The activation of DAI leads to a RIP1/3-dependent activation of NFκB. Hence, an activation of IRF-3 and IRF-7 has also been reported (Kanehisa 2012). Subsequent studies have shown the presence of an additional mechanism(s) for DNAsensing and activation of the innate immune system. NACHT-leucine-rich repeat-PYD containing protein 3 (NALP3) also known as cryopyrin, and its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), regulate secretion of interleukin (IL)-1β in response to an adenovirus infection. Inflammasome activation also occurs upon cytosolic exposure of DNA, though in different other reports DNA-sensing was shown to be dependent on ASC and not NALP3 (Muruve et al. 2008). In particular a group of proteins of the HIN-200 (hematopoietic interferon-inducible nuclear proteins with a 200-amino-acid repeat) protein family, which exhibit a DNA-binding domain along with a CARD-domain, became more and more important in the last years. For this protein family, an induction of innate immunity dependent of AIM2 (absent in melanoma 2) and IFI16 (gamma-interferon inducible protein 16) via ASC has been described (Roberts et al. 2009;Unterholzner et al. 2010).
However, data on the role of epithelial cells in innate immunity, particularly in response to DNA internalization and DNA virus infection is limited. Also, there is a lack of information about constitutive expression of inflammatory factors in keratinocytes, as well as data about the induction of inflammatory factors after adenoviral DNA internalization still are missing. On the way to improve safety, efficacy and duration of cutaneous adenoviral gene therapy it is necessary to get a basic knowledge on the reaction of epidermal cells in response to adenoviral gene delivery. This study was performed in order to depict the important role of human epidermal cells in innate immunity towards adenoviral vector systems. This will help to fine tune various therapeutic intervention strategies. Therefore, this study observed the mechanisms of innate immune reaction of cultured keratinocytes in vitro, the immune response to adenoviral gene delivery into human skin samples ex vivo by using a human full skin culture system (Steinstraesser et al. 2009) and in vivo, using a murine transduction model.

Keratinocyte cell culture
Fresh human skin was obtained after abdominoplasty surgery (informed consent was given by the patient) and washed in PBS (PAA Laboratories, Coelbe, Germany). The skin was placed in a sterile petri dish and the hypodermis was excised. The skin was disinfected with Lavasept (Braun AG, Melsungen, Germany) for 5 min and washed with PBS, the tissue was sliced into pieces of 1 cm 2 . Skin pieces were transferred into a new petri dish with the epidermal side up and the skin was completely immersed with freshly prepared 0.2% dispasesolution (4.7 U/ml, Gibco, Paisley, United Kingdom [UK]) and incubated overnight at 4°C. The epidermis was peeled off and placed in Trypsin/EDTA-solution (0.05%/0.02%, Gibco, Paisley, UK) and reduced to pieces as small as possible. The pieces were incubated at 37°C for 20 min in a gently shaking (180 rpm) waterbath (GFL Burgwedel, Germany). The cell suspension was vortexed and the trypsin digestion was stopped by adding fetal bovine serum (FBS, HyClone, Logan, USA). The suspension was filtered through a 100 μm cell strainer (Becton Dickinson Heidelberg, Germany) and centrifuged at 400 × g, 20°C for 5 min. The cells were resuspended in a 5 ml keratinocyte medium ( Louis, USA), 20 ng/ml hEGF (Sigma, St. Louis, USA) and counted by CASY-1 (Schärfe-System, Reutlingen, Germany). Cells were seeded at a density of 75,000 cells/cm 2 into collagen type I (Becton Dickinson Falcon, Heidelberg, Germany) precoated culture flasks. All different cell types including HaCaT (kindly provided by Prof. Fusenig, University of Heidelberg) cell lines were cultured at 37°C in humidified atmosphere of 5% CO 2 . HaCaT cells were cultured in DMEM containing 10% FBS (Hyclone, Logan, USA) and 1% Penicillin/Streptomycin. Medium was changed every second day.

Human full skin culture
Fresh, sterile human skin explants were obtained in the operating room of the University Hospital Bergmannsheil, Bochum, Germany from six adult healthy patients (six different donors; age range: 19-43 years) undergoing abdominoplasty surgery. The study was approved by the local ethics committee (registration number: 2501; institutional review board of the Faculty of Medicine, Ruhr-University Bochum), and all of the patients gave written informed consent.
Skin explants were cultured as described by Steinstraesser et al. (Steinstraesser et al. 2010). For transduction, 10 10 infectious units (IU) Ad-GFP (green fluorescent protein) in 50 μl PBS were intradermally injected (n = 6 samples from two different donors per group). 3 to 96 h post transduction, tissue biopsy specimens were harvested for total RNA isolation. Transgene expression was localized via Kodak Imaging Station 4000MM and Kodak MI software.
DNA purification, RNA isolation and Reverse Transcription were performed as described by Steinstraesser et al. (Steinstraesser et al. 2011).

Transfection
Cells were grown in 6-well plates until 90-100% confluency. DNA transfection complexes were prepared according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany). Briefly, ΔE1-Ad5-CMV-GFP DNA was mixed in ratio of 2:5 with the Fugene® HD transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) in PCRgrade water (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at room temperature and then added to cells. If not mentioned otherwise, all transfection experiments were performed in triplicate for each group.
For siRNA transfection, keratinocytes were sown in a density of 15.000 cells/cm 2 (HaCaT) or 35.000 cells/cm 2 (HKC) in 12-well cell culture plates. After 24 hours the medium was changed and 400 μl serum-reduced medium. The siRNA transfection complex was generated by combining siRNA (Eurofins MWG Operon, Ebersberg, Germany) and X-tremeGENE® siRNA Transfection Reagent (Roche, Mannheim, Germany) in Opti-MEM (PAA Laboratories, Coelbe, Germany) according to manufacturer's instructions (ratio of gene X-tremeGENE®: siRNA = 1: 0.2). Before DNA transfection, the cells were cultivated for another 48 hours with siRNA-transfection complexes. Specific siRNA sequences are listed in Table 1.

Real-time PCR
Relative Quantification of mRNA was performed in a two-step real-time RT-PCR procedure using the fluorescent dye SYBR Green I (Light Cycler FastStart DNA Master SYBR Green I, Roche, Mannheim, Germany) and a Light Cycler 480 (Roche, Mannheim, Germany). The first step consisted of an RT reaction as described above, the second step of PCR amplification with specific primers listed in Table 2. These primer pairs were validated to generate a single PCR-product. The PCR reactions were performed with 2 μl of cDNA, 0.5 μM of sense and antisense primers, 3 mM MgCl 2 and 2 μl of FastStart SYBR Green reaction mix in a total volume of 20 μl. The cycling conditions were as follows: 95°C for 10 min at a ramp speed of 20°C/sec, 40 cycles (if not differently described) consisting of 94°C for 15 sec at a ramp speed of 20°C/sec, A primer specific annealing temperature (Table 1) for 10 sec at a ramp speed of 20°C/sec, 72°C for 10 sec at a ramp speed of 20°C/sec, followed by a melting point analysis: 95°C for 0 sec at a ramp speed of 20°C/sec, 65°C for 15 sec at a ramp speed of 20°C/sec, 95°C for 0 sec at a ramp speed of 0.1°C/sec, and finally a cooling phase: 40°C for 30 sec at a ramp speed of 20°C/sec. mRNA concentrations were corrected to 18S rRNA in each sample and were normalized to an untreated control (x-fold expression).

Animal studies
The research protocol described below conformed to all regulations related to animal use and other German federal statues. It was performed in compliance with the 'Guide for the Care and Use of Laboratory Animals' associated with the German Animal Welfare Act. The animals were housed at an ambient temperature of 20 ± 2°C and on a 12: 12 h light/dark cycle. Both food and water were available ad libitum. Athymic mice (Foxn1 nu ) were obtained from Harlan Winkelmann (Borchen, Germany) and immunocompetent mice (SKH-1 h/r ) were obtained from Charles River (Wilmington, Sulzfeld, Germany). In Table 1 List of oligonucleotides used for siRNA mediated gene silencing the first experiment, immunocompetent mice were randomized into three groups with n = 3 mice. Each animal was intradermally transduced with 10 8 -10 10 IU Ad-GFP in 50 μl PBS at two discrete areas on the back. Transgene expression was localized and quantified every second day via Kodak Imaging Station 4000 MM and Kodak MI software. 14 and 28 days after the first injection a second and third virus application was administered into the same areas and into a non-treated area.
In the second experiment, SKH-1 h/r and Foxn1 nu mice were each divided into six groups with n = 3 mice. Four distinct areas at the back of each mouse were marked. On day 0, 10 10 IU Ad-GFP in 50 μl PBS or PBS alone were injected intradermally into two areas per mouse followed by a second injection of 10 10 IU Ad-GFP into all four areas on day 14. 1, 6, 24, 48, 72 and 120 h after the second injection, one group was euthanized by intraperitoneal injection of 0.5 ml T61 (MSD Animal Health GmbH, Luzern, Switzerland), the transduced skin areas were excised and snap frozen in liquid nitrogen for RNA isolation.

Statistical analysis
Differences were analyzed for statistical significance with the student's t-test. Error bars represent standard errors of the mean (SEM). RT-PCR analysis was displayed as expression ratio of treated and untreated (vehicle control) cells (x-fold expression).

Applicability of HaCaT cells and primary human keratinocytes (HKC)
Initially baseline expression of potential key molecules of the innate immune reaction after adenoviral transduction have been analyzed in primary human keratinocytes (HKC) and a keratinozyte cell line (HaCaT) via qRT-PCR (n = 6 samples).

Comparative analysis of type-I-interferon expression
Type-I-interferons play a key role in the induction of immune responses to viral infection und are induced immediately after detection of viral components in the host cell. In addition to its direct antiviral function, these cytokines play an important role in the induction of cytokine expression in surrounding tissue (Kawai & Akira 2006). In summary, showed up with the exception of IFN-β expression in HaCaT cells, a higher induction of the expression of type-I-IFN after transduction with 1.1 × 10 10 IU of Ad-GFP transfection compared with an equivalent amount of DNA. Taking account of the transduction and transfection efficiency the data demonstrates the immunogenic potential of adenoviral DNA in keratinocytes ( Figure 1D).

Evaluation of cytokine expression after adenoviral DNA internalization
In order to get a deeper insight into cytokine expression after adenoviral DNA internalization into keratinocytes, an enlarged number of samples was analyzed. For this purpose the data from tests on different days with cells of different passages (HaCaT) and different patients (HKC) were examined at different time-points (type-I-IFN: n = 18 samples; cytokines: n = 6 samples).
The data ( Figure 1E) showed a 1.7-fold increased expression of IFN-α in HaCaT cells (p = 0.0836) and 1.5fold in HKC (p = 0.3329). For the expression of IFN-β mRNA a 26.1-fold, significant increase in HaCaT cells (p = 0.0029) could be detected. Transfection of primary keratinocytes, however, resulted in a 11.4-fold increase (p = 0.0439). The study showed an induction of cytokine gene expression of IL-1α and IL-6 in HaCaT cells by a factor of 10.3 (IL-1α; p = 0.0972) and 22.1 (IL-6; p = 0.0074) respectively. In contrast, the induction of IL-1α (2.6-fold; p = 0.0233) and IL-6 (2.1-fold; p = 0.0566) in HKC was comparatively lower. In addition to the induction of interferons and interleukins showed the transfection of keratinocytes also a significant increase of IL-8-encoding mRNA. In HaCaT cells, a 11.9-fold (p = 0.0038) induction has been detected whereas HKC possessed a 6.9-fold induction (p = 0.0312). The expression of TNFα showed mean values of 4.9-fold induction in HaCaT cells (p = 0.0478) and 1.6-fold increase in HKC (p = 0.5123).
Since there is a lack of systemic influences by using the human ex vivo full skin transduction for an investigation of adenovirus induced systemic immune reactions, the aim of our in vivo study was gaining a deeper insight into systemic influences towards adenovirus induced inflammatory response in a murine skin transduction model.

Adenovirally induced immune reaction in an in vivo murine transduction model
In our in vivo study, an application of 10 10 IU Ad-GFP resulted in strong GFP expression with a peak at day 2 and remained detectable for 12 days ( Figure 2C). Reapplication of the same dose of Ad-GFP on days 14 and 28 led to a reduced in intensity and duration (5 days) of GFP fluorescence.

DNA recognition and signal transduction
An induction of innate immunity requires different key molecules for inflammatory signal transduction, including MAPK-dependent signaling pathways (Erk2, MAPKK, p38 MAPK), JNK, JAK/STAT-cascades and the transcription factor NFκB. These factors represent key molecules of the toll-like receptor signaling pathway, but they can also interact with members of the RIG-like receptor family or the DNA-binding receptor DAI (Akira & Hoshino 2003;Kanehisa 2012;Yoneyama & Fujita 2007). In contrast to p38 MAPK, JNK and NFκB, an activation of Erk2 has not been described for the RIG-like receptor and DAI pathway (Kanehisa 2012).
The following section describes the data of a further stimulation of keratinocytes using specific inhibitors. This showed a predominantly significant reduction of cytokine expression after inhibition of NFκB, JNK, p38 MAPK and JAK/STAT in HaCaT cells and HKC ( Figure 3). In contrast, inhibition of Erk2 did not show any significant reduction of cytokine expression. This data also represents an indication for induction of cytokine expression by RIG-like receptors.

Expression of nucleic acid sensing receptors after adenoviral DNA delivery
For an examination of the involvement of TLRs, RLRs and DAI, an analysis of the expression profiles of these receptors and specific adapter molecules using qRT-PCR has been performed (Figure 4). In HaCaT cells, highest induction of mRNA expression after transfection was measured for mda5 (52.08-fold; 12 h post trasfection (p = 0.0267)), DAI (23.86-fold; 15 h post transfection (p = 0.1349)) and AIM2 (36.81; 12 h post transfection (p = 0.0421)) whereas HKC showed a significant increase in the NALP3 mRNA (3.29-fold, p = 0.0243) only 3 h post transfection.
The data of the strong induction of AIM2 expression in HaCaT cells suggests a key role of this receptor in eliciting an anti-adenoviral immune reaction. Furthermore, the expression of NALP3 in HKC was one of pronounced increase in this cell type and may therefore constitute a key role in the detection of adenoviral DNA. Additionally, an involvement of IFI16 could not be excluded. Due to the comparably lower regulation of IFI16 mRNA expression, further analysis focused on the factors AIM2, NALP3, mda5 and DAI.

Recognition of adenoviral DNA in keratinocytes
For a more detailed statement about the involvement of specific receptors on the adenoviral-induced immune response, the expression of receptors AIM2, NALP3, mda5 and DAI was inhibited in a further experiment using siRNA over a period of 48 hours. Hence, the cells were transfected with adenoviral DNA (5 μg/ml medium), and the impact on interferon expression was determined by qRT-PCR.
For an accurate interpretation, the results of type-I-IFN expression in different samples were normalized to the corresponding siRNA efficiency (Table 3). The data generated here will give a closer insight into the role of individual receptors in the induction of immune response. The ratio of siRNA efficiency and type-I-IFN expression correlates with the importance of each receptor in the induction of the immune system, meaning a higher ratio indicates a greater participation in eliciting an innate immune response.
For this, AIM2 plays an important role in the induction of IFN-β. In contrast, NALP3, however causes a stronger induction of IFN-α. DAI plays a major role in the induction of both IFN-α and IFN-β whereas the RNA-binding receptor mda5 also plays a role in type-I-IFN induction, which, however, is lower when compared to AIM2, NALP3 and DAI.

Vector efficacy after inhibition of potential DNAreceptors
In order to investigate the effects of siRNA -mediated gene silencing on efficacy of transgene expression, HaCaT cells were incubated with previously mentioned siRNAs over a period of 48 hours and further transduced with 1 × 10 8 IU of Ad5-GFP. After another 48 hours, the reporter fluorescence was quantified using a kodak life-imaging station.
Qualitative data analysis of the transduced keratinocytes ( Figure 6A) possessed a stronger transgene fluorescence in cells treated with siRNA targeting AIM2, NALP3, mda5 and DAI. Quantitative analysis of GFP fluorescence ( Figure 6B) illustrated a 2.24-fold, significantly increased after inhibition of NALP3 (p = 0.0018). Pre-incubation with anti-AIM2-siRNA and anti-mda5 siRNA also leads to a significant increase in reporter fluorescence by a factor of 1.77 (AIM2, p = 0.0186) and 1.59 (mda5, p = 0.0244). A 1.34-fold amplified signal intensity was found after using anti-DAI-siRNA (p = 0.0902).
Hence, the data generated in this experiment indicates a primary involvement of the receptors AIM2, NALP3 and mda5 but also DAI in the induction of the (See figure on previous page.) Figure 3 Inhibition of signaling cascades. Cytokine expression after transfection of HaCaT cells and HKC with 5 μg/ml of adenoviral DNA with a follow-up of 15 h (HaCaT) or 6 h (HKC). One hour before transfection, inhibitors of indicated signaling molecules were added to the cell culture medium (10 mM final concentration). Data is indicated as percentage expression of cytokines in relation to a non-inhibited positive control (* = p <0.05, ** = p <0.005). innate immune response after adenoviral gene transfer keratinocytes.

Discussion
There are several studies that examine adenovirally induced innate immune reactions in mice, primates and humans (Raper et al. 2003;Schnell et al. 2001;Zhang et al. 2001). However, these studies have a propensity to only address specialized APCs such as DCs or MΦ (Nociari et al. 2007;Zhu et al. 2007) and RNA virusinduced immune reactions of APC (Diebold et al. 2004). In addition, data on the role of keratinocytes in innate immunity, particularly towards DNA internalization and DNA virus infection is limited. For optimization of adenoviral gene delivery into skin it is critical to shed light on the molecular mechanisms of immunity of epidermal cells. To address these issues, we have investigated the role of keratinocytes in innate immunity in response to DNA internalization.
This study used the HaCaT cell line as it is simple to cultivate and experiments were carried out under highly standardized and reproducible conditions (Boukamp et al. 1988). The data of constitutive cytokine expression in HaCaT cells displayed expected differences in comparison to HKC. However, these relate primarily to differences in expression levels of these factors, while all measured factors were expressed in both cell types. Consistent with Köllisch et al., HKC and HaCaT cells also revealed generally comparable data for induction of inflammatory factors after transfection of adenoviral DNA (Köllisch et al. 2005). In comparing both cell types, a difference in induction intensity and time response of these inflammatory factors has been detected in a previous study and could be confirmed by our experiments. However, HaCaT cells possessed a higher cytokine induction than HKC whereas HKC exhibited a faster reaction towards adenoviral DNA and might be caused by different factors like genetic abberations described for HaCaT cells and due to comparing highly proliferative HaCaT cell line and predominantly terminal differentiated HKCs (Steinstraesser et al. 2011).
In order to determine the differences in transfection and transduction efficacy of keratinocytes, cells were stimulated isolated adenoviral DNA or an equal dose of adenoviral vectors. The transduction efficacy of adenoviral vectors was 83% for HaCaT and 92% for HKC respectively, while transfection of keratinocytes with an equivalent dose of adenoviral DNA possessed a significant lower efficacy (HaCaT: 15%; HKC: 9%). In regard to transfection and transduction efficacy, transfection revealed a type-I-IFN expression on a comparative level as measured after transduction of keratinocytes and points out an important role of adenoviral DNA in the induction of innate immune response in keratinocytes.
As discussed above, recognition of cytoplasmatic localized viral DNA triggers the antiviral innate immune  response. Studies have described an activation of innate immunity independent of the species and sequence of the internalized DNA; the induction of type-I-interferon and cytokine synthesis during introduction of nucleic acids into the cytoplasm of APCs has been shown by different groups (Ishii et al. 2001;Yasuda et al. 2005). Data regarding immunogenicity of cytoplasmatic localized DNA in epidermal cells is limited. For this purpose, this study demonstrates a high induction of IFN-α/β, proinflammatory cytokines and chemokines by adenoviral DNA internalization in vitro, ex vivo and in vivo (Steinstraesser et al. 2011). Obtained in vivo data suggests immunization against adenovirus, which led to decreased intensity and duration of transgene expression after reapplication of the identical vector dose into the same or non-transduced areas in immuncompetent mice. Therefore, therapeutic reapplication of the vector into keratinocytes to stabilize transgene expression may be unefficient (Nociari et al. 2007;Zhu et al. 2007). Moreover, athymic mice showed a diminished type-I-IFN and cytokine expression ( Figure 2D) and a stable GFP level starting from day 5 (Steinstraesser et al. 2011), suggesting the possibility to optimize adenoviral gene delivery by modulating the inflammatory responses of the skin. An early decrease in GFP level detected in immunocompetent and athymic mice from day 2 to 5 denotes a T-cell independent mechanism for a reduction of GFP level (Steinstraesser et al. 2011) and was dedicated to an induction of innate immunity (Muruve 2004). Moreover, data from athymic mice and in vitro stimulation of keratinocytes suggests an important role of keratinocytes in eliciting an innate immune reaction after adenoviral challenge.
In previous years, many studies regarding the molecular mechanisms in DNA recognition were evaluated for the stimulation of different cell types with various species of DNA. For example, several groups have shown a major role of TLRs, the DNA -binding protein DAI as well as special forms of the inflammasome (AIM2, NALP3, IFI16) in macrophages, dendritic cells and fibroblasts (Goubau et al. 2010;Nociari et al. 2007;Takaoka et al. 2007;Unterholzner et al. 2010;Zhu et al. 2007).
However, our current understanding of the signal transduction after recognition of adenoviral DNA in keratinocytes is still rather limited. For the establishment of cutaneous adenoviral gene delivery, information about immune reactions towards adenoviral DNA is of high importance.
The data from this study depicted a special role of the JAK/STAT signaling pathway in induction of type-I-IFN. Since JAK/STAT plays a major role in signal transduction of type-I-IFN-and cytokinereceptors, the involvement of the JAK/STAT signaling pathway in antiviral immunity was not unexpected.
Additionally, NFκB, JNK and p38 MAPK but not Erk2 were also characterized as important factors in adenovirally induced signal transduction. The proteins NFκB, p38 MAPK and JNK play an important role not only in the TLR mediated signal transduction but also in RLRdependent (RIG-I and mda5) signaling (Kanehisa 2012). Since Erk2/MAPKK are not addressed by the TLR pathway, the lack of participation of Erk2/MAPKK in activation of type-I-IFN expression suggests an RLR-dependent pathway for adenoviral DNA recognition (Kanehisa 2012).
In analyzing potentially in adenovirus recognition involved receptor systems, a significant induction of mda5 was observed. An activation of RLRs is associated with an involvement of IRFs, NFκB, p38 MAPK and JNK, confirming the previous results of the inhibitor study. Since a signal transduction occurs via RLRs, which do not address Erk2/MAPKK, thus would explain the results of the type-I-IFN expression after inhibition of Erk2/MAPKK.
Additionally, there was also an induction of mRNA expression of NALP3-and AIM2 inflammasomes. Hence, a significant induction of inflammasome-activated caspase-1 and IL-1β, which is processed by caspase-1, was shown for both cell types. This fact suggests an involvement of inflammasomes in the adenovirally induced immune response.
In addition to the previously detected increased expressions of AIM2, NALP3 and mda5, the analysis of the DAI expression possessed a significant increase in HaCaT cells. This also indicates an involvement of this protein out of the adenovirally induced immune response and may also explain the NFκB dependence seen in the inhibitor study.
Since an analysis of keratinocytes derived from expression profiles only permits a tendency of the molecular Figure 6 Transgene expression after siRNA-mediated gene expression of AIM2, NALP3, mda5 and DAI. Quantitative (A) and qualitative (B) representation of the GFP expression in HaCaT cells 48 h after siRNA -mediated inhibition of AIM2, NALP3, mda5 and DAI and subsequent transduction with 1 × 10 8 IU Ad5-GFP or PBS (control) (* = p < 0.05; ** = p < 0.005).