BACKGROUND
the skin is the largest organ of the body, accounting for about fifteen percent of our body weight, and covering the entire external surface.
while many believe its role is merely as an external covering, the functions of the skin are far more complex.
the skin consists of three main layers - the epidermis, the dermis, and the hypodermis, each with their own function.
it is the epidermis, which creates a barrier to and protects from pathogens of the outside world.
this highly specialised layer is mainly composed of keratinocytes, melanocytes and dendritic cells.
its accessibility and specific anatomical and biological properties make the skin a very interesting organ for in vivo and ex vivo gene therapy approaches.
in case of cutaneous gene therapy, gene delivery can be easily controlled and the skin surgically excised if any side effects occur <cit> .
keratinocytes, the predominant epidermal cell type, are responsible for establishing a physical barrier and guaranteeing the structural integrity of the epidermis <cit> .
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 <cit> .
in most gene therapy applications, a "normal" gene is inserted into the genome of an individuals cell or tissue to replace an "abnormal" disease-causing gene.
in addition, foreign, therapeutical active genes can be introduced in order to add any not normally in the body produced metabolite.
on this basis, gene therapy can be a promising tool for the treatment of a wide variety of inherited as well as acquired disease including genetically inherited skin disorders, tumours, metabolic disorders and infectious diseases  <cit> .
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 <cit> .
a carrier molecule called vector must be used to deliver the therapeutic gene to the target cells.
based on viral and non-viral vectors, different applications for gene delivery have been developed in the last decades <cit> .
for transient transduction of target cells, adenoviral vector systems possess the highest effectivity and have been used in  <dig> % of the official agency sources , recombinant dna advisory committee , etc.)
registered clinical trials of gene therapeutical applications for different indications, such as cancer, infectious or monogenic diseases worldwide in the last two decades <cit> .
adenoviridae are non-enveloped, double stranded , linear desoxyribonucleic acid  viruses with a genome of 35- <dig> kb and a particle size of 70- <dig> nm <cit> .
the adenoviral genome is well characterised 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 therapeutical active metabolite <cit> .
the process of adenoviral entry into the host is extremely efficient and has been intensively studied.
adenoviruses exhibit a wide host range in vitro and in vivo; this range was also seen in nondividing cells <cit> .
in addition, the well-defined and easily manipulated viral genome favours the development of adenoviral vectors for gene therapy applications <cit> .
this, together with information from the complete library of human dna opened up extensive opportunities for gene therapy in medical and surgical specialities <cit> .
the major disadvantage of adenoviral vectors is that they can effectively induce the adaptive and innate immune response immediately after infection, leading to an induction of proinflammatory cytokines and chemokines in mice, primates and humans <cit> .
activation of innate immunity is associated with a reduction in efficacy of gene delivery <cit> .
it may further cause significant inflammatory reactions leading to morbidity and mortality of the transduced host <cit> .
newer generations of helper-dependent, gutted adenoviral vectors or adeno-associated viruses, which are depleted of almost all viral coding sequences <cit> , cause diminished adaptive immune responses to these vectors and improve the duration of gene transfer <cit> .
however, acute toxicity and diminished vector persistence provoked by the innate immune response remain the most significant barriers to clinical application of this promising technology <cit> .
to improve safety, efficacy and duration of adenoviral gene transfer it is necessary to explore the mechanisms by which adenoviruses trigger the innate immune response.
the detection of microbial components by pattern recognition receptors  is one of the earliest defense mechanisms that is known to trigger innate immune responses against infections <cit> .
of the many classes of molecules detected by cells as pathogen associated molecular patterns , nucleic acids are potent and broadly recognised <cit> .
to sense nucleic acids the immune system employs several classes of receptors, including rna helicases that can respond to cytosolic ribonucleic acid  and dna under certain conditions /melanoma differentiation associated gene  <dig>  <cit> ), and toll-like receptors  that can recognise endosomal dsrna  <cit> , singlestranded rna  <cit> , and hypomethylated dna  <cit> .
activation of tlrs occurs on the cell membrane surface  or within endosomal compartments  and requires either a myeloid differentiation primary response gene  <dig>  or tir-domain-containing adapter-inducing interferon-β  adapter molecules <cit> .
these proteins facilitate activation of downstream signalling cascades, which lead to the activation of inflammatory transcription factors, including nuclear factor-kappa b , activator protein  <dig>  and interferon regulatory factors  <cit> .
the presence of viruses in cells is detected by tlr- <dig>  - <dig>  - <dig> and - <dig>  tlr- <dig> recognises double-stranded  ribonucleic acid  <cit> , tlr- <dig> and tlr- <dig> bind single-stranded rna <cit> , whereas tlr- <dig> senses dsdna which is also contained in adenoviruses <cit> .
since the discovery of tlr- <dig>  there has been a growing body of evidence that dna derived from microbial and host cells can be recognised via a tlr-9-independent mechanism.
dna recognition in these pathways is sequence independent and occurs in the cytoplasm of the cells <cit> .
different ligands, such as adenoviral, mammalian and vertebrate dna as well as dsdna have been characterised for tlr-independent recognition in antigen presenting cells  <cit> .
recently, a dna sensor and activator of innate immune responses has been identified and termed dna-dependent activator of ifn-regulatory factors  <cit> .
subsequent studies have shown the presence of an additional mechanism for dna-sensing and activation of the innate immune system, as well as a mechanism of negative regulation of cytosolic dna-mediated immune responses <cit> .
nacht-leucine-rich repeat-pyd containing protein  <dig>  also known as cryopyrin, and its adaptor protein apoptosis-associated speck-like protein containing a card , regulate secretion of interleukin -1β in response to an adenovirus infection <cit> .
inflammasome activation also occurs upon cytosolic exposure of dna, though in this case dna-sensing was shown to be dependent on asc and not nalp <dig> <cit> .
it remains unclear, however, as to whether the nalp3/asc inflammasome can directly detect cytosolic dna or if the response is triggered by another protein.
another negative regulator of the dna-mediated innate immune response, adenosine deaminase acting on rna  <dig> , has also been reported <cit> .
adar <dig> is an ifn inducible protein that possesses dna-binding domains similar to those found in dai.
several studies on  dna induced innate immune reaction have focused on antigen presenting cells  such as dendritic cells  or macrophages .
however, information about innate immune reactions and mechanisms in virus recognition in epithelial cells, such as keratinocytes, is very limited.
for optimization of cutaneous gene therapy, it is necessary to investigate the immune reaction of the predominant epidermal cell type after adenoviral challenge.
since keratinocytes represent the majority  of cells of the outermost epidermal layer <cit> , the aim of this study was to investigate the signal transduction of the innate immunity after adenoviral dna internalization in this specific cell type.
in addition, systemic effects of adenovirally induced immune reactions will be studied.
RESULTS
dna internalization induced innate immune reaction
in order to get a deeper insight into the molecular mechanisms of the innate immune system after  dna internalization into keratinocytes, we decided to use a gfp-encoding adenovirus for this study.
since this generation of adenoviral vectors exhibit a high immunogenicity, this vector seemed to be an interesting tool for the investigation of signal transduction moleculess involved in adenovirus detection.
the basal mrna levels of 18s ribosomal rna, type-i-interferons and cytokines  possessed a comparable profile in hacat cells hkc .
hkc possessed significant higher levels of il-1α  and il- <dig>  mrna compared to hacat cells.
in contrast, mrna level of il- <dig> was detected on a significant higher level in hacat cells .
in order to get any information whether cytoplasmatic localised adenoviral dna is capable to induce an innate immune reaction, human keratinocytes were transfected with dna.
adenoviral dna internalization led to a significantly increased  ifn-α expression  <dig> h post transfection in hacat cells.
the same cells demonstrated a significant increase in ifn-β expression  after stimulation for  <dig> h .
besides type-i-ifn expression, there was significant increase in expression of inflammatory cytokines, namely interleukin - <dig>  il- <dig>  il- <dig> and tnf-α recorded in hacat cells and hkc with a maximal induction  <dig> h  and  <dig> -  <dig> h  post transfection .
after detection of an adenoviral dna induced inflammatory response in human keratinocytes it was interesting to see whether dna from other species is also capable to activate an inflammatory reaction in these cells.
rt-pcr analysis showed an increased induction of ifn-α in both cell types after s. cerevisiae  and ad-dna treatment .
maximal induction of ifn-α after treatment with sc-dna was  <dig> -fold in hacat cells and 2-fold in hkc.
transfection of calf thymus  and plasmid  dna did not show any significant effect in either cell type.
the transfection with sc-dna led to a significant increase  of ifn-β in both cell types.
in hacat cells, a 2380-fold upregulation was detected  whereas hkc accounted for a 11-fold increase of transcription .
ad-dna stimulation of both cell types also induced an expression of ifn-β in keratinocytes .
no changes in ifn-β expression of both cell types were demonstrated post ct-dna stimulation and p-dna induced upregulation of ifn-β was only detected in the hacat cell line .
in order to get any information about the innate immune reaction after adenoviral transduction, hacat cells were transduced with different concentrations of adenoviral vectors  of the gfp-encoding adenoviral vector .
a concentration dependent induction of ifn-α and ifn-β was observed  <dig> h post transduction , 7-fold ,  <dig> -fold ; ifn-β: 2-fold ,  <dig> -fold ,  <dig> -fold .
an adenoviral transduction of human ex vivo full skin samples led to an induction of ifn-α ; ifn-β ; il-1α ; il- <dig> ; il- <dig>  and tnf-α  .
involvement of toll-like receptor family in dna recognition
toll-like receptors  are among the most important receptor of innate immunity.
for an investigation of the involvement of tlrs in adenoviral dna detection.
the expression levels of tlr- <dig>  - <dig> and - <dig> were determined after transfection of dna from different species.
there was no significant change in expression of tlr- <dig>  - <dig>  - <dig> and trif in both cell types .
an increased amount of myd <dig> mrna was detected in hacat cells stimulated with sc-dna , ad-dna  and p-dna .
an increased amount of myd <dig> mrna was detected in hkc cells stimulated with sc-dna  and ad-dna .
stimulation of hacat cells and hkc with ct-dna and p-dna stimulation of hkc did not show any significant differences.
a transfection of hacat cells with ad- and sc-dna led to an induction of dai.
transfection with ad-dna led to a  <dig> -fold  and  <dig> -fold  increase of dai mrna.
moreover, treatment of the cells with sc-dna resulted in a stronger induction of dai ,  <dig> -fold ).
dna recognition and signal transduction
to get a deeper indight into signal transduction mechanisms after adenoviral challenge, different signal transduction molecules .
the hacat cells and hkc demonstrated involvement of the same signal transduction pathways for ifn-α/β production , except for the expression of ifn-α in hkc, which did not show any significant regulation of ifn-α in this experiment.
figure  <dig> indicates the involvement of p <dig> mapk, pi3k, jnk and nfκb in triggering ifn-α/β production as the mrna expression ratio was significantly decreased to a maximum difference of 27% compared to non-inhibited positive controls.
samples treated with erk2/mapkk and jak/stat inhibitors showed significantly reduced ifn-α/β expression from  <dig> to 56% compared to the positive control .
ifn-α mrna levels of p <dig> mapk-inhibited samples were significantly different compared to the non-inhibited positive control  in hacat cells.
the lowest signals in ifn-β expression were obtained in pi3k inhibited samples  and 18% in hkc  compared to positive control and jnk blocked hkc .
investigation of adenovirus induced immune reaction in vivo
in vitro cell culture assays are not convenient for an investigation of systemic inflammatory effects after adenoviral challenge.
to get some information about the systemic influences on vector reapplication, in vivo studies have been performed.
in these experiments, immunocompetent  and t-cell deficient athymic  mice were twice transduced with the ad-gfp vector.
the transgene expression and inflammatory response was measured via life imaging and qrt-pcr.
an application of  <dig> iu ad-gfp into immunocompetent mice resulted in a gfp expression detectable for  <dig> days with a maximum at day  <dig> ; figure 4).
a second injection of the same vector dose  as well as first transduction of two new areas  on day  <dig> led to an increased gfp expression for  <dig> days with a maximum of  <dig>  ×  <dig> rlu  and  <dig> ×  <dig> rlu  after  <dig> h. an application of  <dig> iu adgfp into athymic mice resulted in a gfp expression of  <dig> ×  <dig> rlu two days post transduction.
the expression level was reduced to  <dig> ×  <dig> rlu five days post transduction and stayed on a relatively constant level until the end of the experiment on day  <dig> .
a second injection of the same vector dose into the same area did not show any changes in gfp expression.
a transduction of the pbs treated  areas on day  <dig> resulted in an induction of gfp synthesis converging a level of  <dig>  ×  <dig> rlu after  <dig> h. no significant changes were measured at the following time points until the end of the experiment at day  <dig>
in addition to transduction with  <dig> iu adgfp, two other virus concentrations of  <dig> iu and  <dig> iu were also tested for intradermal transduction of skh-1h/r mice.
the application of  <dig> iu ad-gfp did not show any detectable effects whereas the application  <dig> iu adgfp lead to a gfp expression over a period of  <dig> days with a maximum of  <dig> rlu on day  <dig> .
a reapplication of the same vector dose did not show any effect .
in contrast, the application of  <dig> iu ad-gfp resulted in a gfp expression for  <dig> days with maximum at day  <dig> .
reapplication of the same doses of ad-gfp on day  <dig> and  <dig> reduced gfp expression  and duration .
blood count analysis showed a constant increase in numbers of leukocytes, granulocytes, lymphocytes and monocytes after application of  <dig> iu ad-gfp; there were no significant differences after application of lower doses .
rt-pcr analysis of type-i-interferon expression after transduction of athymic and immunocompetent mice exhibited a lower expression of ifn-α ;  <dig> -fold, p =  <dig>  ) and ifn-β ;  <dig> -fold, p =  <dig>  ) in athymic compared to immunocompetent mice .
additionally, the expression of other transcripts encoded by the adenoviral vector, was determined in this study.
the viral early regions e <dig> and e <dig> are parts of the viral genome, possessing major regulatory functions.
while, there was a strong decrease in gfp mrna level in immunocompetent mice  <dig> h after second administration of the adenoviral vector, the viral early regions e <dig> and e <dig> showed an increasing expression for  <dig> h .
discussion
there are a number of studies that examine molecular mechanisms of innate immunity in mice, primates and humans <cit> .
however, these studies have a propensity to only address specialised antigen-presenting cells  such as dcs or mΦ <cit>  and rna virus-induced immune reactions of apc <cit> .
in addition, data on the role of keratinocytes in innate immunity, particularly towards dna internalization and dna virus infection is limited.
for optimization of a cutaneous gene delivery, it is critical to shed light on the molecular mechanisms a signalling pathways of the immunity of keratinocytes as they protect the body from the external environment.
to address these issues, we have investigated the molecular mechanisms and signal transduction molecules involved in innate immunity towards dna internalization into human keratinocytes.
this study was performed using a replication incompetent, gfp-encoding adenovirus in order to significantly induce an innate immune response for an investigation of signal transduction molecules involved in adenovirus recognition.
this hacat cell line was used as it is simple to cultivate and experiments may be carried out under highly standardised and reproducible conditions <cit> .
unlike other keratinocyte cell lines, the hacat cell acts like a primary keratinocyte under many experimental conditions <cit> .
the spontaneously immortalised human keratinocyte hacat cell line shows almost normal differentiation and keratinization in skin models <cit>  and was used throughout all experiments in comparison to primary keratinocytes.
to investigate differences between the hacat cell line and primary keratinocytes, this study examined the innate immune reaction after dna stimulation.
the results demonstrate the feasibility of both cell types for the investigation of innate immune responses.
cytokine expression in hacat displayed expected differences in comparison to hkc, but consistent with köllisch et al., results were broadly comparable in chronology of interferon and cytokine induction at both cell types <cit> .
any differences of induction intensity and kinetics between hacat cells and hkc were not unexpected due to altered growth conditions and the high number of passages of hacat cultures.
both cell types exhibit the same time-course with a primary induction of ifn-α , followed by an increased ifn-β expression  and cytokine expression .
transduction of ex vivo full skin samples with an adenoviral vector also led to an induction of inflammatory mediators.
this induction was measured on a comparable level to primary keratinocytes in vitro.
this issue confirms the validity of data obtained in in vitro experiments with hkc.
studies have described an activation of innate immunity independent of the species and sequence of internalised dna, only the presence of nucleic acid in the cytoplasm elicits an immune reaction.
the induction of type-i-interferon and cytokine synthesis during introduction of nucleic acids in the cytoplasm of apcs has been shown by different groups  <cit> .
limited data regarding immunogenicity of epidermal cells to different species of dna is available.
there is evidence for species independent induction of innate immunity when dna is localised in the cytoplasm of a cell <cit>  and immune induction via cytoplasmatic localised mammalian dna has been demonstrated especially by an organism's individual dna <cit> .
this study demonstrates a high induction of type-i-ifn by  dna internalization.
the results suggest that cytoplasmic localised dna triggers induction of type-i-interferon in hacat cells and hkc.
in contrast to shirota et al., we did not find any immune reaction after stimulation with dna from calf thymus  which does not support the hypothesis of immune induction through mammalian dna <cit> .
for apc, shirota et al.
showed an 800-fold induction  and a 200-fold induction  of ifn-β in consequence of ct-dna internalization <cit> .
in contrast to mammalian dna, fungal dna stimulation led to a significantly increased expression of type-i-ifn as well as adenoviral dna.
taken together, the results of the present study indicate an important role of keratinocytes in innate immunity.
keratinocytes may not recognise pathogens in the same manner as apcs, but the data proposes a major role of keratinocytes in innate immune defense against pathogens.
however, once induction of innate immunity by transfection of  dna was demonstrated, this study investigated signal transduction pathways involved in dna recognition.
the toll-like receptor family is the best known prr with tlr- <dig> being the unique dsdna detection receptor for this family <cit> .
downstream signalling in tlr pathways is dependent on the adapter molecules myd <dig> and trif <cit> .
studies investigating dna/rna recognition in different cell types propose tlr-dependent and -independent pathways for dna/rna recognition in apcs <cit> .
there is limited data regarding molecular mechanisms of pathogen recognition in keratinocytes, so the involvement of tlr- <dig> and the adapter molecules for tlr signalling  was investigated.
the results indicate tlr- <dig> and trif-independent recognition of dsdna in hacat cells and hkc.
interestingly, both cell types exhibit an upregulation of myd <dig> after dna internalization, which may denote involvement of other tlr receptors <cit> .
in addition to dna recognition via tlr- <dig>  there is also a possibility of rna transcript or another pathogen associated molecular patterns  induced tlr pathway induction.
therefore, this study investigated the induction of tlr- <dig>  and tlr- <dig> .
evidence supports a rna transcript and zymosan independent immune induction, since there were no changes in expression of these receptors detected.
in contrast, and induction of dna recognising receptor dai was observed in both cell types, indicating a tlr-independent dna recognition in keratinocytes.
besides involvement of tlr receptor pathways in dna recognition, this study examined participation of different effector molecules in cytokine induction.
inhibitor studies in hacat cell and hkc showed a 'two-step' inhibition in both cell types.
on the one hand, an interferon down-regulation of  <dig> to 54% compared to a non-inhibited positive control was detected in consequence of blocking erk2/mapkk- and jak/stat-regulated pathways.
on the other hand, inhibition of nfκb-, jnk-, p <dig> mapk- and pi3k-regulated pathways led to a maximal ifn down-regulation of 25% compared to non-inhibited positive control.
these results suggest a major participation of nfκb, jnk, p <dig> mapk and pi3k and also an involvement of erk2/mapkk and jak/stat in signal transduction after recognition of cytoplasmatic localised dna.
pi3k-mediated signal transduction plays a major role in ifn induction in keratinocytes.
in agreement with the present study, philpott et al.
described a pi3k-mediated tnf-α induction pathway <cit> .
a major involvement of jnk, p <dig> mapk and nfκb in ifn-induction was not unexpected as these factors play a crucial role in initialization of transcription which is mediated by different pathways like tlr-, pi3k- jak/stat and mapk-cascades <cit> .
secondary involvement of erk2/mapkk- and jak/stat-regulated pathways may be due to a feedback mechanism mediated by cytokine receptors in the cells.
the results suggest that induction of these pathways by cytokines may be primarily initiated by dna recognition involving pi3k-dependent signalling cascades.
the data for dose-dependence between vector dose and immune reaction for in vivo adenoviral gene delivery is supported by other studies  <cit> .
the results demonstrate that local and systemic immune reactions led to increased counts of leukocytes, lymphocytes, monocytes and granulocytes in mice.
the present study exhibits a comparable time course in gfp expression post reapplication of the identical vector dose into the same or non-transduced areas.
these results suggest not only a local but also a systemic reaction against adenovirus, leading to decreased intensity and duration of transgene expression.
hence, therapeutic reapplication of the vector to stabilise transgene expression may be hindered <cit> .
adenoviral in vivo transduction into athymic mice, which exhibit a deficient t-cell system and hence a reduced adaptive immune response, led to a stable transgene expression and reapplication of same vector doses into non-transduced areas showed an increase in transgene expression to the same level of areas of primary application.
this, taken together with a diminished expression of type-i-interferon and proinflammatory cytokines, implies potential for effective adenoviral gene delivery into immunosuppressed skin and a future direction of our study may be to investigate inhibition of factors like type-i-interferons in order to optimise gene therapy with adenoviruses.
interestingly, there was a decrease in gfp mrna level detected together with an increasing amount of viral region e <dig> and e <dig> mrna , suggesting a gene silencing mechanism to regulate transgene expression in the tissue.
since this mechanism could not be explained yet, subsequent experiments are needed to analyse mrna regulating effects.
the results of this study suggest an induction of the innate immunity triggered by cytoplasma localised dna which is mediated by pi3k-, p <dig> mapk-, jnk-, nfκb-, jak/stat- and erk1/2-dependent pathways.
after vector reapplication in vivo, immnocompetent mice possessed a decrease in duration and intensity in transgene expression.
a stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed, suggesting opportunities for a higher efficiency of cutaneous gene delivery in immunosuppressed skin.
it might be an interesting approach to influence in signal transduction cascades for an optimization of cutaneous gene delivery.
therefore, it is important to constitute any pathogen recognition receptors involved in recognition of adenoviral vectors.
since the present study did not observe any induction of tlrs  <dig>   <dig> and  <dig>  but an induction of dai, there is evidence for a tlr independent dna recognition pathway in human keratinocytes.
further investigation will be required to fully describe and understand the complex mechanism in signal transduction after adenoviral challenge in human epidermal cells.
CONCLUSIONS
in summary, the results of this study suggest an tlr-independent induction of the innate immunity triggered by cytoplasm localised dna which is mediated by pi3k-, p <dig> mapk-, jnk-, nfκb-, jak/stat- and erk1/2-dependent pathways in human keratinocytes.
this activation of innate immunity leads to a decrease in intensity and duration of adenovirally induced transgene expression.
in contrast, a stable transgene expression and a reduced inflammatory response have been observed in immunodeficient mice.
these results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

 the skin is the largest organ of the body, accounting for about fifteen percent of our body weight, and covering the entire external surface. while many believe its role is merely as an external covering, the functions of the skin are far more complex. the skin consists of three main layers - the epidermis, the dermis, and the hypodermis, each with their own function. it is the epidermis, which creates a barrier to and protects from pathogens of the outside world. this highly specialised layer is mainly composed of keratinocytes, melanocytes and dendritic cells. its accessibility and specific anatomical and biological properties make the skin a very interesting organ for in vivo and ex vivo gene therapy approaches. in case of cutaneous gene therapy, gene delivery can be easily controlled and the skin surgically excised if any side effects occur. keratinocytes, the predominant epidermal cell type, are responsible for establishing a physical barrier and guaranteeing the structural integrity of the epidermis. 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. in most gene therapy applications, a "normal" gene is inserted into the genome of an individuals cell or tissue to replace an "abnormal" disease-causing gene. in addition, foreign, therapeutical active genes can be introduced in order to add any not normally in the body produced metabolite. on this basis, gene therapy can be a promising tool for the treatment of a wide variety of inherited as well as acquired disease including genetically inherited skin disorders, tumours, metabolic disorders and infectious diseases. 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. a carrier molecule called vector must be used to deliver the therapeutic gene to the target cells. based on viral and non-viral vectors, different applications for gene delivery have been developed in the last decades. for transient transduction of target cells, adenoviral vector systems possess the highest effectivity and have been used in % of the official agency sources, recombinant dna advisory committee, etc.) registered clinical trials of gene therapeutical applications for different indications, such as cancer, infectious or monogenic diseases worldwide in the last two decades. adenoviridae are non-enveloped, double stranded, linear desoxyribonucleic acid viruses with a genome of 35- kb and a particle size of 70- nm. the adenoviral genome is well characterised 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 therapeutical active metabolite. the process of adenoviral entry into the host is extremely efficient and has been intensively studied. adenoviruses exhibit a wide host range in vitro and in vivo; this range was also seen in nondividing cells. in addition, the well-defined and easily manipulated viral genome favours the development of adenoviral vectors for gene therapy applications. this, together with information from the complete library of human dna opened up extensive opportunities for gene therapy in medical and surgical specialities. the major disadvantage of adenoviral vectors is that they can effectively induce the adaptive and innate immune response immediately after infection, leading to an induction of proinflammatory cytokines and chemokines in mice, primates and humans. activation of innate immunity is associated with a reduction in efficacy of gene delivery. it may further cause significant inflammatory reactions leading to morbidity and mortality of the transduced host. newer generations of helper-dependent, gutted adenoviral vectors or adeno-associated viruses, which are depleted of almost all viral coding sequences, cause diminished adaptive immune responses to these vectors and improve the duration of gene transfer. however, acute toxicity and diminished vector persistence provoked by the innate immune response remain the most significant barriers to clinical application of this promising technology. to improve safety, efficacy and duration of adenoviral gene transfer it is necessary to explore the mechanisms by which adenoviruses trigger the innate immune response. the detection of microbial components by pattern recognition receptors is one of the earliest defense mechanisms that is known to trigger innate immune responses against infections. of the many classes of molecules detected by cells as pathogen associated molecular patterns, nucleic acids are potent and broadly recognised. to sense nucleic acids the immune system employs several classes of receptors, including rna helicases that can respond to cytosolic ribonucleic acid and dna under certain conditions /melanoma differentiation associated gene ), and toll-like receptors that can recognise endosomal dsrna, singlestranded rna, and hypomethylated dna. activation of tlrs occurs on the cell membrane surface or within endosomal compartments and requires either a myeloid differentiation primary response gene or tir-domain-containing adapter-inducing interferon-β adapter molecules. these proteins facilitate activation of downstream signalling cascades, which lead to the activation of inflammatory transcription factors, including nuclear factor-kappa b, activator protein and interferon regulatory factors. the presence of viruses in cells is detected by tlr- - - and - tlr- recognises double-stranded ribonucleic acid, tlr- and tlr- bind single-stranded rna, whereas tlr- senses dsdna which is also contained in adenoviruses. since the discovery of tlr- there has been a growing body of evidence that dna derived from microbial and host cells can be recognised via a tlr-9-independent mechanism. dna recognition in these pathways is sequence independent and occurs in the cytoplasm of the cells. different ligands, such as adenoviral, mammalian and vertebrate dna as well as dsdna have been characterised for tlr-independent recognition in antigen presenting cells. recently, a dna sensor and activator of innate immune responses has been identified and termed dna-dependent activator of ifn-regulatory factors. subsequent studies have shown the presence of an additional mechanism for dna-sensing and activation of the innate immune system, as well as a mechanism of negative regulation of cytosolic dna-mediated immune responses. nacht-leucine-rich repeat-pyd containing protein also known as cryopyrin, and its adaptor protein apoptosis-associated speck-like protein containing a card, regulate secretion of interleukin -1β in response to an adenovirus infection. inflammasome activation also occurs upon cytosolic exposure of dna, though in this case dna-sensing was shown to be dependent on asc and not nalp. it remains unclear, however, as to whether the nalp3/asc inflammasome can directly detect cytosolic dna or if the response is triggered by another protein. another negative regulator of the dna-mediated innate immune response, adenosine deaminase acting on rna, has also been reported. adar is an ifn inducible protein that possesses dna-binding domains similar to those found in dai. several studies on dna induced innate immune reaction have focused on antigen presenting cells such as dendritic cells or macrophages. however, information about innate immune reactions and mechanisms in virus recognition in epithelial cells, such as keratinocytes, is very limited. for optimization of cutaneous gene therapy, it is necessary to investigate the immune reaction of the predominant epidermal cell type after adenoviral challenge. since keratinocytes represent the majority of cells of the outermost epidermal layer, the aim of this study was to investigate the signal transduction of the innate immunity after adenoviral dna internalization in this specific cell type. in addition, systemic effects of adenovirally induced immune reactions will be studied.  dna internalization induced innate immune reaction in order to get a deeper insight into the molecular mechanisms of the innate immune system after dna internalization into keratinocytes, we decided to use a gfp-encoding adenovirus for this study. since this generation of adenoviral vectors exhibit a high immunogenicity, this vector seemed to be an interesting tool for the investigation of signal transduction moleculess involved in adenovirus detection. the basal mrna levels of 18s ribosomal rna, type-i-interferons and cytokines possessed a comparable profile in hacat cells hkc. hkc possessed significant higher levels of il-1α and il- mrna compared to hacat cells. in contrast, mrna level of il- was detected on a significant higher level in hacat cells. in order to get any information whether cytoplasmatic localised adenoviral dna is capable to induce an innate immune reaction, human keratinocytes were transfected with dna. adenoviral dna internalization led to a significantly increased ifn-α expression h post transfection in hacat cells. the same cells demonstrated a significant increase in ifn-β expression after stimulation for h. besides type-i-ifn expression, there was significant increase in expression of inflammatory cytokines, namely interleukin - il- il- and tnf-α recorded in hacat cells and hkc with a maximal induction h and - h post transfection. after detection of an adenoviral dna induced inflammatory response in human keratinocytes it was interesting to see whether dna from other species is also capable to activate an inflammatory reaction in these cells. rt-pcr analysis showed an increased induction of ifn-α in both cell types after s. cerevisiae and ad-dna treatment. maximal induction of ifn-α after treatment with sc-dna was -fold in hacat cells and 2-fold in hkc. transfection of calf thymus and plasmid dna did not show any significant effect in either cell type. the transfection with sc-dna led to a significant increase of ifn-β in both cell types. in hacat cells, a 2380-fold upregulation was detected whereas hkc accounted for a 11-fold increase of transcription. ad-dna stimulation of both cell types also induced an expression of ifn-β in keratinocytes. no changes in ifn-β expression of both cell types were demonstrated post ct-dna stimulation and p-dna induced upregulation of ifn-β was only detected in the hacat cell line. in order to get any information about the innate immune reaction after adenoviral transduction, hacat cells were transduced with different concentrations of adenoviral vectors of the gfp-encoding adenoviral vector. a concentration dependent induction of ifn-α and ifn-β was observed h post transduction, 7-fold, -fold; ifn-β: 2-fold, -fold, -fold. an adenoviral transduction of human ex vivo full skin samples led to an induction of ifn-α; ifn-β; il-1α; il-; il- and tnf-α. involvement of toll-like receptor family in dna recognition toll-like receptors are among the most important receptor of innate immunity. for an investigation of the involvement of tlrs in adenoviral dna detection. the expression levels of tlr- - and - were determined after transfection of dna from different species. there was no significant change in expression of tlr- - - and trif in both cell types. an increased amount of myd mrna was detected in hacat cells stimulated with sc-dna, ad-dna and p-dna. an increased amount of myd mrna was detected in hkc cells stimulated with sc-dna and ad-dna. stimulation of hacat cells and hkc with ct-dna and p-dna stimulation of hkc did not show any significant differences. a transfection of hacat cells with ad- and sc-dna led to an induction of dai. transfection with ad-dna led to a -fold and -fold increase of dai mrna. moreover, treatment of the cells with sc-dna resulted in a stronger induction of dai, -fold ). dna recognition and signal transduction to get a deeper indight into signal transduction mechanisms after adenoviral challenge, different signal transduction molecules. the hacat cells and hkc demonstrated involvement of the same signal transduction pathways for ifn-α/β production, except for the expression of ifn-α in hkc, which did not show any significant regulation of ifn-α in this experiment. figure indicates the involvement of p mapk, pi3k, jnk and nfκb in triggering ifn-α/β production as the mrna expression ratio was significantly decreased to a maximum difference of 27% compared to non-inhibited positive controls. samples treated with erk2/mapkk and jak/stat inhibitors showed significantly reduced ifn-α/β expression from to 56% compared to the positive control. ifn-α mrna levels of p mapk-inhibited samples were significantly different compared to the non-inhibited positive control in hacat cells. the lowest signals in ifn-β expression were obtained in pi3k inhibited samples and 18% in hkc compared to positive control and jnk blocked hkc. investigation of adenovirus induced immune reaction in vivo in vitro cell culture assays are not convenient for an investigation of systemic inflammatory effects after adenoviral challenge. to get some information about the systemic influences on vector reapplication, in vivo studies have been performed. in these experiments, immunocompetent and t-cell deficient athymic mice were twice transduced with the ad-gfp vector. the transgene expression and inflammatory response was measured via life imaging and qrt-pcr. an application of iu ad-gfp into immunocompetent mice resulted in a gfp expression detectable for days with a maximum at day; figure 4). a second injection of the same vector dose as well as first transduction of two new areas on day led to an increased gfp expression for days with a maximum of × rlu and × rlu after h. an application of iu adgfp into athymic mice resulted in a gfp expression of × rlu two days post transduction. the expression level was reduced to × rlu five days post transduction and stayed on a relatively constant level until the end of the experiment on day. a second injection of the same vector dose into the same area did not show any changes in gfp expression. a transduction of the pbs treated areas on day resulted in an induction of gfp synthesis converging a level of × rlu after h. no significant changes were measured at the following time points until the end of the experiment at day in addition to transduction with iu adgfp, two other virus concentrations of iu and iu were also tested for intradermal transduction of skh-1h/r mice. the application of iu ad-gfp did not show any detectable effects whereas the application iu adgfp lead to a gfp expression over a period of days with a maximum of rlu on day. a reapplication of the same vector dose did not show any effect. in contrast, the application of iu ad-gfp resulted in a gfp expression for days with maximum at day. reapplication of the same doses of ad-gfp on day and reduced gfp expression and duration. blood count analysis showed a constant increase in numbers of leukocytes, granulocytes, lymphocytes and monocytes after application of iu ad-gfp; there were no significant differences after application of lower doses. rt-pcr analysis of type-i-interferon expression after transduction of athymic and immunocompetent mice exhibited a lower expression of ifn-α; -fold, p = ) and ifn-β; -fold, p = ) in athymic compared to immunocompetent mice. additionally, the expression of other transcripts encoded by the adenoviral vector, was determined in this study. the viral early regions e and e are parts of the viral genome, possessing major regulatory functions. while, there was a strong decrease in gfp mrna level in immunocompetent mice h after second administration of the adenoviral vector, the viral early regions e and e showed an increasing expression for h. discussion there are a number of studies that examine molecular mechanisms of innate immunity in mice, primates and humans. however, these studies have a propensity to only address specialised antigen-presenting cells such as dcs or mΦ and rna virus-induced immune reactions of apc. in addition, data on the role of keratinocytes in innate immunity, particularly towards dna internalization and dna virus infection is limited. for optimization of a cutaneous gene delivery, it is critical to shed light on the molecular mechanisms a signalling pathways of the immunity of keratinocytes as they protect the body from the external environment. to address these issues, we have investigated the molecular mechanisms and signal transduction molecules involved in innate immunity towards dna internalization into human keratinocytes. this study was performed using a replication incompetent, gfp-encoding adenovirus in order to significantly induce an innate immune response for an investigation of signal transduction molecules involved in adenovirus recognition. this hacat cell line was used as it is simple to cultivate and experiments may be carried out under highly standardised and reproducible conditions. unlike other keratinocyte cell lines, the hacat cell acts like a primary keratinocyte under many experimental conditions. the spontaneously immortalised human keratinocyte hacat cell line shows almost normal differentiation and keratinization in skin models and was used throughout all experiments in comparison to primary keratinocytes. to investigate differences between the hacat cell line and primary keratinocytes, this study examined the innate immune reaction after dna stimulation. the results demonstrate the feasibility of both cell types for the investigation of innate immune responses. cytokine expression in hacat displayed expected differences in comparison to hkc, but consistent with köllisch et al., results were broadly comparable in chronology of interferon and cytokine induction at both cell types. any differences of induction intensity and kinetics between hacat cells and hkc were not unexpected due to altered growth conditions and the high number of passages of hacat cultures. both cell types exhibit the same time-course with a primary induction of ifn-α, followed by an increased ifn-β expression and cytokine expression. transduction of ex vivo full skin samples with an adenoviral vector also led to an induction of inflammatory mediators. this induction was measured on a comparable level to primary keratinocytes in vitro. this issue confirms the validity of data obtained in in vitro experiments with hkc. studies have described an activation of innate immunity independent of the species and sequence of internalised dna, only the presence of nucleic acid in the cytoplasm elicits an immune reaction. the induction of type-i-interferon and cytokine synthesis during introduction of nucleic acids in the cytoplasm of apcs has been shown by different groups. limited data regarding immunogenicity of epidermal cells to different species of dna is available. there is evidence for species independent induction of innate immunity when dna is localised in the cytoplasm of a cell and immune induction via cytoplasmatic localised mammalian dna has been demonstrated especially by an organism's individual dna. this study demonstrates a high induction of type-i-ifn by dna internalization. the results suggest that cytoplasmic localised dna triggers induction of type-i-interferon in hacat cells and hkc. in contrast to shirota et al., we did not find any immune reaction after stimulation with dna from calf thymus which does not support the hypothesis of immune induction through mammalian dna. for apc, shirota et al. showed an 800-fold induction and a 200-fold induction of ifn-β in consequence of ct-dna internalization. in contrast to mammalian dna, fungal dna stimulation led to a significantly increased expression of type-i-ifn as well as adenoviral dna. taken together, the results of the present study indicate an important role of keratinocytes in innate immunity. keratinocytes may not recognise pathogens in the same manner as apcs, but the data proposes a major role of keratinocytes in innate immune defense against pathogens. however, once induction of innate immunity by transfection of dna was demonstrated, this study investigated signal transduction pathways involved in dna recognition. the toll-like receptor family is the best known prr with tlr- being the unique dsdna detection receptor for this family. downstream signalling in tlr pathways is dependent on the adapter molecules myd and trif. studies investigating dna/rna recognition in different cell types propose tlr-dependent and -independent pathways for dna/rna recognition in apcs. there is limited data regarding molecular mechanisms of pathogen recognition in keratinocytes, so the involvement of tlr- and the adapter molecules for tlr signalling was investigated. the results indicate tlr- and trif-independent recognition of dsdna in hacat cells and hkc. interestingly, both cell types exhibit an upregulation of myd after dna internalization, which may denote involvement of other tlr receptors. in addition to dna recognition via tlr- there is also a possibility of rna transcript or another pathogen associated molecular patterns induced tlr pathway induction. therefore, this study investigated the induction of tlr- and tlr-. evidence supports a rna transcript and zymosan independent immune induction, since there were no changes in expression of these receptors detected. in contrast, and induction of dna recognising receptor dai was observed in both cell types, indicating a tlr-independent dna recognition in keratinocytes. besides involvement of tlr receptor pathways in dna recognition, this study examined participation of different effector molecules in cytokine induction. inhibitor studies in hacat cell and hkc showed a'two-step' inhibition in both cell types. on the one hand, an interferon down-regulation of to 54% compared to a non-inhibited positive control was detected in consequence of blocking erk2/mapkk- and jak/stat-regulated pathways. on the other hand, inhibition of nfκb-, jnk-, p mapk- and pi3k-regulated pathways led to a maximal ifn down-regulation of 25% compared to non-inhibited positive control. these results suggest a major participation of nfκb, jnk, p mapk and pi3k and also an involvement of erk2/mapkk and jak/stat in signal transduction after recognition of cytoplasmatic localised dna. pi3k-mediated signal transduction plays a major role in ifn induction in keratinocytes. in agreement with the present study, philpott et al. described a pi3k-mediated tnf-α induction pathway. a major involvement of jnk, p mapk and nfκb in ifn-induction was not unexpected as these factors play a crucial role in initialization of transcription which is mediated by different pathways like tlr-, pi3k- jak/stat and mapk-cascades. secondary involvement of erk2/mapkk- and jak/stat-regulated pathways may be due to a feedback mechanism mediated by cytokine receptors in the cells. the results suggest that induction of these pathways by cytokines may be primarily initiated by dna recognition involving pi3k-dependent signalling cascades. the data for dose-dependence between vector dose and immune reaction for in vivo adenoviral gene delivery is supported by other studies. the results demonstrate that local and systemic immune reactions led to increased counts of leukocytes, lymphocytes, monocytes and granulocytes in mice. the present study exhibits a comparable time course in gfp expression post reapplication of the identical vector dose into the same or non-transduced areas. these results suggest not only a local but also a systemic reaction against adenovirus, leading to decreased intensity and duration of transgene expression. hence, therapeutic reapplication of the vector to stabilise transgene expression may be hindered. adenoviral in vivo transduction into athymic mice, which exhibit a deficient t-cell system and hence a reduced adaptive immune response, led to a stable transgene expression and reapplication of same vector doses into non-transduced areas showed an increase in transgene expression to the same level of areas of primary application. this, taken together with a diminished expression of type-i-interferon and proinflammatory cytokines, implies potential for effective adenoviral gene delivery into immunosuppressed skin and a future direction of our study may be to investigate inhibition of factors like type-i-interferons in order to optimise gene therapy with adenoviruses. interestingly, there was a decrease in gfp mrna level detected together with an increasing amount of viral region e and e mrna, suggesting a gene silencing mechanism to regulate transgene expression in the tissue. since this mechanism could not be explained yet, subsequent experiments are needed to analyse mrna regulating effects. the results of this study suggest an induction of the innate immunity triggered by cytoplasma localised dna which is mediated by pi3k-, p mapk-, jnk-, nfκb-, jak/stat- and erk1/2-dependent pathways. after vector reapplication in vivo, immnocompetent mice possessed a decrease in duration and intensity in transgene expression. a stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed, suggesting opportunities for a higher efficiency of cutaneous gene delivery in immunosuppressed skin. it might be an interesting approach to influence in signal transduction cascades for an optimization of cutaneous gene delivery. therefore, it is important to constitute any pathogen recognition receptors involved in recognition of adenoviral vectors. since the present study did not observe any induction of tlrs and but an induction of dai, there is evidence for a tlr independent dna recognition pathway in human keratinocytes. further investigation will be required to fully describe and understand the complex mechanism in signal transduction after adenoviral challenge in human epidermal cells.  in summary, the results of this study suggest an tlr-independent induction of the innate immunity triggered by cytoplasm localised dna which is mediated by pi3k-, p mapk-, jnk-, nfκb-, jak/stat- and erk1/2-dependent pathways in human keratinocytes. this activation of innate immunity leads to a decrease in intensity and duration of adenovirally induced transgene expression. in contrast, a stable transgene expression and a reduced inflammatory response have been observed in immunodeficient mice. these results provide potential for an effective adenoviral gene delivery into immunosupressed skin. 
there is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. the aim of this study was to investigate the signal transduction of the innate immunity after adenoviral dna internalization in keratinocytes. a stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. for optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. the results suggest an induction of the innate immunity triggered by cytoplasm localised dna which is mediated by pi3k-, p mapk-, jnk-, nfkappab-, jak/stat- and erk1/2-dependent pathways. the results show an acute induction of type-i-interferon after in vitro transfection. these results provide potential for an effective adenoviral gene delivery into immunosupressed skin. in vivo, immunocompetent and athymic mice were twice transduced with an ad-vector. in contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications.
 adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. however, these vectors can induce immune responses that may severely affect the ability of vector re-application. there is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. for optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. the aim of this study was to investigate the signal transduction of the innate immunity after adenoviral dna internalization in keratinocytes. methods in vitro, keratinocytes were transfected with dna, in the presence and absence of inhibitors for signalling molecules. in vivo, immunocompetent and athymic mice were twice transduced with an ad-vector.  the results show an acute induction of type-i-interferon after in vitro transfection. inhibition of pi3k, p mapk, jnk and nfkappab resulted in a decreased expression of type-i-interferon. in contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo.  the results suggest an induction of the innate immunity triggered by cytoplasm localised dna which is mediated by pi3k-, p mapk-, jnk-, nfkappab-, jak/stat- and erk1/2-dependent pathways. a stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. these results provide potential for an effective adenoviral gene delivery into immunosupressed skin. 