by vitamin D3
Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium
(Dr. rer. nat.) im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin
von Dipl. Biochemiker Mykola Goncharenko geboren am 30.04.1978
in Kiew, Ukraine
Präsident der Humboldt-Universität zu Berlin Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Christian Limberg
Gutachter/innen: 1. Prof. Dr. Wolfgang Uckert 2. Prof. Dr. Claus Scheidereit 3. Prof. Dr. Martin Zenke Tag der mündlichen Prüfung: 07.04.2008
ACKNOWLEDGEMENTS
I would like to use this opportunity to express my deep gratitude to Prof. Martin Zenke, who over the last number of years has guided me in every possible way with his abundant knowledge and insightful ideas.
I appreciate the time invested by Prof. Wolfgang Lockau, Prof. Harald Saumweber, Prof.
Claus Scheidereit, Prof. Wolfgang Uckert and Prof. Martin Zenke for reading through the numerous pages of this doctoral thesis manuscript and evaluating my work.
I would like to thank all members of the Institute of Biomedical Technology -Cell Biology- of RWTH Aachen, especially Antonio, Piritta, David, Verena, Ivonne, Julia - thank you. I would like to mention Thomas Hieronymus for his everyday support and enlightening discussions, and Chris for making the lab go around. I deeply appreciated the help of Andrea who made my daily life bearable, by taking care of every small crisis.
Many thanks to the members of the old “Zenke lab” of the MDC in Berlin: Petra Haink, Signe Knespel, Christine Hacker and many others whom I have known only briefly. I would like to say a special thank you to Gitta Blendinger for teaching me tissue culture techniques and to Tatjana Gust for supervising my fist steps at the lab.
I cannot let this opportunity pass without extending my gratitude to Prof. Wolfgang Uckert for hosting me for a year at his lab at the MDC after the move of the “Zenke lab” to Aachen, as well as for his support and excellent judgement. Here I have to additionally thank Lisa, Irmgard, Uta, Heike, Boris, Lilian, Cordelia and Matthias for providing everyday help and support. Also special words of appreciation have to go to Prof. Thomas Blankenstein for his constructive advice, and to all members of his team for their input and helpful scientific opinions.
Additionally, I would like to thank Prof. Scheidereit and members of his group for some revealing discussions on NF-κB signalling. I would like to extend a special word of gratitude to Eva Kärgel for sharing the chromatin immunoprecipitation technique, for all joint experiments, and for providing the moral support when it was needed so much.
I must regret that the limits of space prevent me having the satisfaction of acknowledging all the generous assistance which I have received from many scientists during the years of my Ph.D.
At last I would like to thank the members of my family, both past and present, and my dear friends Thomas, Olga, Christian, Juri, Etienne, Adrian, Stefan, Matt and Anis for their invaluable help, support and care.
ZUSAMMENFASSUNG
Die Fähigkeit dendritischer Zellen (DC) Immunität zu erzeugen und Antigen-spezifische Toleranz zu induzieren macht DC zu idealen Zielen pharmakologischer Interventionen zur Beeinflussung von Immunreaktionen. NF-κB Faktoren sind eine Gruppe von Transkriptionsregulatoren, die für die Entwicklung und Funktion von DC bedeutend sind.
Trotz ihrer zentralen Bedeutung für die DC Biologie ist die Identität von NF-κB regulierten Genen in DC weitestgehend unbekannt. In der vorliegenden Studie wurde die Hemmung der NF-κB Aktivierung durch den IκBα Super Repressor (IκBα-SR) und die Analyse der Genexpression durch DNA Microarrays genutzt, um das Repertoire an NF-κB regulierten Genen in DC zu ermitteln. Mit diesem Ansatz wurden unter anderem Connexin-43 und Fascin als direkte NF-κB regulierte Gene identifiziert.
Die Beeinflussung des NF-κB Signalwegs wurde als möglicher Weg zur Modifizierung von Immunantworten vorgeschlagen. Es wird vermutet, dass verschiedene immunmoduloratorische Verbindungen wie z.B. Vitamin D3 (VD3) in NF-κB vermittelte Immunmechanismen eingreifen. Um die Effekte von VD3 in der Aktivierung von DC zu untersuchen, wurden DNA Microarray Analysen an DC von Mäusen mit mutiertem und wildtyp Vitamin D3 Rezeptor (VDR) durchgeführt und die Beteiligung der VDR vermittelten Repression von NF-κB regulierten Genen wie z.B. Connexin-43 untersucht. Die so identifizierten Gene stellen potentielle Ansatzpunkte für die Entwicklung von spezifischeren entzündungshemmenden Medikamenten für die klinische Anwendung dar.
ABSTRACT
The ability of dendritic cells (DC) to initiate immunity and induce antigen-specific tolerance makes DC ideal targets for pharmacological intervention into immune responses. NF-κB factors are a family of transcriptional regulators important for DC development and function.
However, the identity of NF-κB target genes that are central to DC biology is largely unknown. In the present study, inhibition of the NF-κB activation by the IκBα super repressor (IκBα-SR) and DNA microarray analysis were used to determine the repertoire of NF-κB responsive genes in DC. This approach identified, among others, connexin-43 and fascin as direct NF-κB regulated genes.
The targeting of the NF-κB signalling pathway has been suggested as a useful means to modify immune responses. A number of immunomodulatory compounds, such as vitamin D3 (VD3), are believed to affect NF-κB mediated immune mechanisms. Microarray analysis employing vitamin D3 receptor (VDR) mutant and wild-type mice was used to survey the effects of VD3 in DC. In this study, effects of VD3 on the activation of DC are evaluated, and involvement of VDR mediated repression of NF-κB regulated genes, such as connexin-43, is surveyed. Identified genes can be potentially useful targets for the development of more specific anti-inflammatory agents for clinical applications.
TABLE OF CONTENTS
Acknowledgements ...I ZUSAMMENFASSUNG ... II ABSTRACT ...III TABLE OF CONTENTS ...IV ABBREVIATIONS... VII
1. INTRODUCTION...1
1.1 DC biology ...1
1.1.1 Immunity versus tolerance ...1
1.1.2 DC as potent initiators of immune responses ...2
1.1.3 Functional maturation of DC...2
1.2 Molecular regulation of DC maturation and function ...4
1.3 NF-κB signalling pathway...5
1.4 Immune effects of vitamin D3 on DC ...9
1.4.1 Vitamin D3 and intracellular signalling ...11
1.5 Gene transfer into DC...14
1.5.1 Receptor-mediated gene delivery into DC with adenovirus as ligand ...14
1.5.2 Adenoviral gene-delivery vectors...14
1.5.3 CAR and CAR mice ...15
1.6 Aims and objectives ...16
2. MATERIALS AND METHODS ...17
2.1 Materials ...17
2.1.1 Appliances and computer software ...17
2.1.2 Chemicals, solutions and buffers...18
2.1.3 Kits ...20
2.1.4 Other materials ...20
2.1.5 Enzymes ...20
2.1.6 Antibodies...20
2.1.6.1 Surface marker expression analysis of DC...20
2.1.6.2 Western blotting ...21
2.1.6.3 Chromatin immunoprecipitation (ChIP)...21
2.1.6.4 Immunofluorescence ...22
2.1.7 Oligonucleotides for semi-quantitative RT-PCR analysis ...22
2.1.8 Oligonucleotides for real-time RT-PCR analysis...22
2.1.9 Oligonucleotides for PCR analysis of ChIP probes ...23
2.1.10 Oligonucleotides for EMSA probes ...23
2.1.11 Northern blot probes...24
2.1.12 Bacterial strains ...24
2.1.13 Plasmids...24
2.1.14 Mouse strains...24
2.1.15 Cell lines...25
2.1.16 Cell culture media and additives ...25
2.1.17 Growth factors for mouse bone marrow-derived DC...25
2.1.18 Hormones, cytokines and ligands for DC stimulation...26
2.2 Methods ...27
2.2.1 Production of recombinant adenoviral vectors...27
2.2.1.1 Generation of high titre adenovirus stocks...27
2.2.1.2 Titration of adenoviruses by limited dilution assay ...28
2.2.2 DC preparation and culture...28
2.2.2.1 Preparation of mouse bone marrow-derived DC (“one-step” protocol)...28
2.2.2.2 Preparation of mouse bone-marrow derived DC (“two-step” protocol)...28
2.2.3 Characterization of DC by flow cytometry ...29
2.2.4 Transduction protocols ...30
2.2.4.1 Transduction with Ad/PEI/DNA ...30
2.2.4.2 Adenoviral transduction ...30
2.2.4.3 Evaluation of transduction efficiency...30
2.2.5 Luciferase reporter assay...30
2.2.6 Western blotting ...31
2.2.7 Electrophoretic mobility shift assay (EMSA) ...32
2.2.8 Northern blotting ...32
2.2.9 RT-PCR ...33
2.2.9.1 Semi-quantitative RT-PCR...33
2.2.9.2 Real-time RT-PCR ...33
2.2.10 Chromatin Immunoprecipitation (ChIP) ...33
2.2.10.1 Optimisation of chromatin shearing ...34
2.2.11 Analysis of vitamin D3 receptor mutant mice...35
2.2.12 Immunofluorescence microscopy and image analysis ...35
2.2.13 Microarray preparation...36
2.2.14 Bioinformatics and data analysis...36
2.2.14.1 Quality control of microarray data ...37
2.2.14.2 Data normalisations ...37
2.2.14.3 Further analysis of data ...38
3. RESULTS...39
3.1 CAR BM progenitors differentiate into DC ...39
3.2 Transduction of CAR DC using Ad/PEI/DNA system and adenoviruses...39
3.2.1 Transduction of K562 cell line ...40
3.2.2 Transduction of Flt3+CD11b+ progenitors...41
3.2.3 Transduction of wild-type and CAR DC progenitors...43
3.2.4 Transduction of DC using Ad/PEI/DNA complexes is a CAR independent event...44
3.2.5 Optimisation of transduction of CAR DC with recombinant adenoviruses ...45
3.3 AdIκBα-SR suppresses NF-κB activity...47
3.4 IκBα-SR expression in DC ...47
3.5 Expression of IκBα-SR in DC blocks NF-κB dimer DNA binding ...48
3.6 Known NF-κB target genes are regulated by IκBα-SR ...49
3.7 Microarray analysis of IκBα-SR experiment ...51
3.7.1 Basic filtering ...51
3.7.2 Untreated and IκBα-SR expressing DC are similar ...53
3.7.3 Complex cumulative effects of adenovirus and TNFα...54
3.7.4 Filtering identifies genes positively and negatively regulated by NF-κB ...55
3.7.5 Analysis of NF-κB target genes ...57
3.7.6 Gene Ontology (GO) over-expression analysis...64
3.8 Verification of the NF-κB regulated genes by RT-PCR ...67
3.9 Chromatin Immunoprecipitation (ChIP) ...69
3.9.1 Kinetics of NF-κB and AP-1 signalling in DC...69
3.9.2 Effects of TNFα and LPS on NF-κB and AP-1 signalling ...70
3.9.3 Optimisation of chromatin shearing ...71
3.9.4 ChIP for NF-κB p65...72
3.10 Identification of genes regulated by vitamin D3 ...73
3.10.1 Basic filtering ...74
3.10.2 Condition tree clustering ...75
3.10.3 The impact of TNFα and suppression by vitamin D3...75
3.10.4 Gene tree clustering identifies clusters of TNFα and vitamin D3 responsive genes 77 3.10.5 Analysis of vitamin D3 responsive genes in DC...78
3.10.6 Verification of the selected genes by real-time RT-PCR ...85
3.10.7 Vitamin D3 affects NF-κB target genes ...87
3.10.8 Vitamin D3 hinders DC differentiation and activation ...91
4. DISCUSSION...94
4.1 Transduction of primary DC ...94
4.2 Inhibition of canonical NF-κB signalling blocks expression of target genes...95
4.3 Identification of NF-κB responsive genes by microarray analysis ...97
4.4 Analysis of the NF-κB target genes in DC...98
4.5 NF-κΒ specific regulation of identified genes ...99
4.6 Identification of genes regulated by vitamin D3 ...101
4.7 Vitamin D3 suppression of NF-κB responsive genes ...103
4.8 Vitamin D3 affects DC differentiation and activation ...103
5. CONCLUDING REMARKS ...105
SUPPLEMENTARY TABLES...106
REFERENCES ...107
APPENDIX ...118
Publications ...118
Curriculum vitae...119
Lebenslauf ...121
Ehrenwörtliche Erklärung...123
ABBREVIATIONS
Ab antibody Ad, Ad5 adenovirus serotype 5, human
AdGFP recombinant EGFP expressing adenovirus AdIκBα-SR recombinant IκBα-SR expressing adenovirus AdOVA recombinant OVA expressing adenovirus
ANK ankyrin repeat
APC antigen presenting cell
BSA bovine serum ovalbumin
CAR Coxsackie adenovirus receptor
CCL chemokine ligand
CCR chemokine receptor
CD cluster of differentiation
ChIP chromatin immunoprecipitation
CTL cytotoxic T lymphocyte
Cx43 connexin-43, Gja1
DC dendritic cell
DD death domain
Dex dexamethasone
dl1014 E4-deficient adenovirus
DMEM Dulbecco's Modified Eagles Medium
DNA desoxyribonucleid acid
dNTP desoxyribonucleic acid triphosphate
EDTA ethylenediaminetetraacetic acid
FACS fluorescence activated cell sorter (flow cytometry)
FCS fetal calf serum
FITC fluorescein isothiocyanate
Flt3L c-fms-like tyrosine kinase 3 ligand GFP green fluorescent protein
Gja1 gap-junction associated protein 1, Cx43
GM-CSF granulocyte macrophage-colony stimulating factor
HBS HEPES buffered saline
HRP horseradish peroxidase
hyperIL-6 IL-6/IL-6R fusion protein ICOS inducible T-cell co-stimulator
IFNα interferon alpha
IFNβ interferon beta
IGF-1 insulin-like growth factor 1 IKK IκB kinase
IL interleukin IκB inhibitor of kappa-B
IκBα inhibitor of kappa-B alpha
IκBα-SR IκBα super repressor
LPS lipopolysaccharide LSP1 lymphocyte specific protein 1
MDC macrophage derived chemokine, Ccl22
MFI mean fluorescence intensity
MHC major histocompatibility complex MIP macrophage inflammatory protein
MOI multiplicity of infection
NEMO non-essential NF-κB modulator NF-κB nuclear factor kappa-B
NIK NF-κB-inducing kinase
NK natural killer
NLS nuclear localisation signal
OVA chicken ovalbumin
OX40 tumour necrosis factor receptor superfamily, member 4, Tnfrsf4 PBS phosphate buffered saline
PCR polymerase chain reaction PE phycoerythrin PEI polyethylenimine
PI propidium iodide
RANK receptor activator of nuclear factor-κB; tumor necrosis factor receptor superfamily member 11a, Tnfrsf11a
RANTES regulated upon activation, normal T-cell expressed, and secreted; Ccl5
RGD arginine-glycine-aspartate sequence
RHD Rel homology domain
RLU relative light unit
RNA ribonucleic acid
RPM rotations per minute
RPMI Roswell Park Memorial Institute
RT reverse transcription reaction; reverse transcriptase RXR retinoid acid X-receptor
SCF stem cell factor, Kitl SDS sodium dodecyl sulfate
SDS-PAGE SDS polyacrylamide gel electrophoresis
SR super repressor
SV40 simian virus 40
TAD transcriptional activation domain
TCR T-cell receptor
TE Tris-EDTA Th T-helper Th1 T helper cell response type 1 Th2 T helper cell response type 2
TLR Toll-like receptor
TNFα tumor necrosis factor alpha
TNFR tumor necrosis factor alpha receptor
UV ultraviolet light
VD3 vitamin D3
VDR vitamin D3 receptor
VDRE vitamin D3 response element
1. INTRODUCTION
1.1 DC biology
The immune system of vertebrates is characterised by a capacity to respond to perturbations without destroying self-tissues. This function is implemented through the use of at least three different approaches. Firstly, it responds in a few hours to the infectious agents (innate immunity) by recognising molecular pathogen-associated patterns typical of microorganisms and absent in self-tissues. Secondly, it mounts a late response that discriminates between different pathogens, giving rise to memory (adaptive immunity). And, thirdly, it maintains tolerance against self-proteins (Granucci, et al., 2004).
Cells of the innate and adaptive immune systems reside in non-lymphoid and lymphoid tissues where they are maintained in the state of homeostasis. Disturbances, such as infection or injury, are recognised by various immune cell types, including dendritic cells (DC).
Amongst the cells of the immune system, dendritic cells are proficient in antigen capture. DC, being potent antigen-presenting cells (APC), play an important role in the immune system.
They initiate activation of the innate immunity by recruitment of NK and NKT cells, and also lead to subsequent priming of adaptive immune response (Steinman, 1991). In addition, by inducing thymic deletion of self-reactive T-cells and by contributing to the differentiation of regulatory T-cells, DC play a critical role in central tolerance and in maintenance of peripheral tolerance in the physiological steady state (Steinman, et al., 2003). There is also an accumulating evidence of DC being able to induce specific T-cell tolerance. Although the underlying mechanisms are not fully understood, the capacity to induce regulatory T-cells may be an important property of tolerogenic or regulatory DC. There is continuing effort by various research groups to manipulate DC in order to generate the cells with custom properties, either using specific cytokines, immunologic or pharmacologic reagents, or gene- modification approaches.
1.1.1 Immunity versus tolerance
Pathogens often invade peripheral tissues whereas naïve T-cells are confined in lymphoid organs. Thus, there is a special separation between the site of capture, and the site of presentation of antigens. The coordinated migration and maturation of DC is critical for T-cell priming, as mature DC express membrane co-stimulatory molecules and secrete cytokines that are required for optimal T-cell priming. Interaction of DC with T-cells involves a number of ligand/receptor interaction pairs, which together with communication through cytokines exert a polarising effect on the differentiation of T-helper cells.
Various mechanisms have been described by which DC may regulate the balance between Th1/Th2 responses in vivo. These include DC subset, the antigen dose, the recognition of
pathogen products by specific pathogen-recognition receptors (like Toll-like receptors), and the cytokines present in the microenvironment.
1.1.2 DC as potent initiators of immune responses
T-cells recognise antigens through interaction with APC. APC represent a heterogeneous family of cells able to (i) internalise extracellular antigens efficiently, (ii) process exogenous or endogenous antigens into 10-15 amino acid peptides, (iii) load antigen-derived peptides onto MHC molecules on the APC surface, and (iv) present peptide-MHC complexes to antigen-specific T-cells (Banchereau, et al., 2000; Bell, et al., 1999).
Based on their T-cell stimulatory activity, lineage-affiliation, tissue distribution and in vivo trafficking ability, APC may be further classified into “professional” and “non-professional”
APC. Professional APC, which include bone marrow-derived DC and macrophages, possess a unique ability to fully activate and induce clonal expansion of naïve and memory T-cells, and are therefore crucial for development of primary immune responses. Non-professional APC (B-cells, lymphocytes, monocytes, endothelial cells) are able to stimulate mainly memory T- cells, which require lower levels of co-stimulation compared to naïve cells (Morelli and Thomson, 2003).
As professional APC, DC exhibit the following properties. Firstly, they possess the ability to stimulate naïve CD4+ and CD8+ T-cells efficiently; secondly, they have the capacity of transporting antigen from peripheral tissues (where immature DC reside) to T-cell areas of secondary lymphoid organs (where naïve T-cell recirculate); and thirdly, they are capable to cross-present antigens, a phenomenon that requires ability to take up, process, and present antigenic peptides (derived from extracellular antigens) in the context MHC class I molecules to antigen-specific CD8+ T-cells (Banchereau, et al., 2000; Bell, et al., 1999).
1.1.3 Functional maturation of DC
DC exert their effects through the segregation in time of different functions starting from the arrival of the perturbation (Banchereau and Steinman, 1998). After recognition of an infectious agent, resting immature DC undergo a maturation process that leads to a strictly defined kinetic expression of cytokines and cell surface molecules important for the activation and control of innate and adaptive immunity.
In the normal steady state (absence of inflammation), DC reside as interstitial immature APC in most peripheral tissues, in particular in skin and mucosae that are sites of interface with the exterior (with the exception of the central cornea). Being highly phagocytic, DC continuously monitor the environment for the invading microorganisms. Immature DC internalise exogenous antigens efficiently and exhibit low naïve T-cell stimulatory ability (Banchereau, et al., 2000; Bell, et al., 1999).
During inflammation, maturation of peripheral tissue-resident DC is triggered by the synergistic action of different combinations of the following endogenous or exogenous mediators released locally within the DC micro-milieu:
(i) pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-1β (IL-1β); tumour necrosis factor-α (TNFα), interferon-α (IFNα), and cyclooxygenase metabolites (prostaglandin E2);
(ii) bacterial or viral components, lipopolysaccharide (LPS), unmethylated cytosine poly-guanine (CpG) motifs, double-stranded RNA; and
(iii) interaction with molecules of TNF receptor (TNFR) superfamily (CD40, RANK, TNFR) on the DC surface with their ligands during cognate T-cell-DC interaction (Banchereau, et al., 2000; Bell, et al., 1999).
Oligomerisation of TNFR family molecules (TNFR, CD40 and RANK) and ligation of Toll- like receptors (TLR) 2, 4, and 9 on the DC surface are two of the mechanisms that induce nuclear translocation of the specific transcription factor NF-κB subunits required for DC activation, T-helper driving capacity instruction, cell survival, and migration of immature DC (Kadowaki, et al., 2001; Liu, et al., 2002; Ouaaz, et al., 2002; Thoma-Uszynski, et al., 2000).
As mature APC, DC exhibit decreased endocytosis of extracellular antigens. This phenomenon, regulated by actin cytoskeleton, has been associated with down-regulation of the activated form of small GTPase, Cdc42 (Garrett, et al., 2000).
Maturation confers DC the capacity to present in the lymphoid organs antigens they have encountered earlier in periphery. This stems from a unique regulation of antigen processing that leads to delayed presentation of antigenic MHC complexes. Thus, stimuli that lead to DC maturation increase the efficiency of antigen processing for both class I and class II pathways.
Half-life of peptide loaded major MHC molecules at the plasma membrane is also increased.
Thus, large number of long-lived peptide-loaded MHC class I and class II molecules persist at the cell surface and render DC capable of stimulating T-cells even after several days.
Additionally, DC up-regulate surface co-stimulatory molecules (CD80, CD86, OX40 ligand, and ICOS ligand) and the intracellular adhesion molecules - CD54 and CD58 - required for both physical interaction with T-cells and assembly of immunological synapse. They also increase expression of the chemokine receptor Ccr7 on their surface. As a result, mature DC stimulate naïve and memory T-cell-priming and home to T-cell areas of secondary lymphoid tissues in response to the Ccr7 ligands, CCL21 (secondary lymphoid tissue chemokine – SCL, or 6Ckine), and CCL19 (EBI-1 ligand chemokine – ECL) or macrophage-inflammatory protein (MIP)-3β (Banchereau, et al., 2000; Bell, et al., 1999).
Fully functional DC may be isolated from a variety of organs including the spleen, liver, kidney, heart, lymph nodes, and blood. Alternatively, DC may be generated from bone marrow progenitors by culturing freshly isolated bone marrow precursor cells in the presence of GM-CSF or Flt3L. A critical determinant of DC function is their level of maturation.
Immature DC are efficient at capturing antigen while they are relatively poor at presenting antigen to T cells. Conversely, mature DC do not capture antigen as well but potently
stimulate T cells. Phenotypically, immature DC are characterised by low surface expression of MHC class I and II proteins, integrins (CD54), and co-stimulatory molecules (CD40, CD80, and CD86). In contrast, mature DC possess high surface expression of these markers.
Maturation is a terminal and critical event in DC development. DC require maturation before exhibiting their full immunostimulatory and anti-tumour potential.
1.2 Molecular regulation of DC maturation and function
The interaction of DC with pathogens involves various classes of innate antigen receptors.
Stimulation of TLR family members (Medzhitov and Janeway, 2000; Pasare and Medzhitov, 2005) leads to DC maturation caused by signalling through the NF-κB pathway and/or stress- induced kinase activation (Rescigno, et al., 1998). DC also possess a palette of phagocytic receptors, such as scavenger receptors, that are important for bacterial internalisation (Rescigno, 2002).
Ten mammalian TLRs have been described (Akira, 2001; Medzhitov, et al., 1997). Various microbial components and products have been shown to activate different cellular responses as a consequence of signalling through TLR 2, 3, 4, 5 and 9 (Bendelac and Medzhitov, 2002).
Thus, TLR2 has been shown to be involved in detection of the products of gram-positive bacteria (peptidoglycans or lipoproteins) and yeast (Hirschfeld, et al., 1999; Underhill, et al., 1999; Yoshimura, et al., 1999). TLR4 is involved in initiation of response to gram-negative bacteria, reacting to bacterial lipopolysaccharide (LPS) (Poltorak, et al., 1998; Takeuchi, et al., 1999). The engagement of TLR4 induces nuclear translocation of the NF-κB transcription factor (Medzhitov, et al., 1997) through an adaptor protein MyD88 and serine/threonine kinase IRAK. LPS-induced DC maturation is mediated by NF-κΒ, and inhibition of NF-κB blocks the maturation process (Hofer, et al., 2001; Rescigno, 2002).
Interestingly, activation of DC by LPS also promotes survival of the cells in a growth-arrested state after deprivation of growth factors. This response has been shown to be dependent on mitogen-activated protein (MAP) kinases of the ERK family, but anti-apoptotic mediators have still not been described.
DC express a moderate level of Fc receptors, which are mostly not modulated during maturation. DC also express Mac-1 molecule (CD11b/CD18; αMβ2 integrin), which is the Cr3 complement receptor used for the phagocytosis of complement-coated bacteria. Similarly to the Fc receptors, the surface expression of Mac-1 molecule is not changed during DC activation. This is in contrast to monocytes and neutrophils which strongly up-regulate Mac-1 expression during differentiation and in the presence of inflammatory stimuli. DC are very efficient in the uptake and presentation of mannosylated proteins. Presence of mannose receptors can be explained by the fact that there is a high mannan content in the oligosaccharide cell envelope of both gram-positive and gram negative bacteria, making mannose receptor pathway important for bacterial internalisation. Interestingly, mannose receptor targeting has been used for gene delivery into primary DC (Diebold, et al., 2002).
1.3 NF-κB signalling pathway
Despite the well-known role of DC in regulating T lymphocyte activation, intracellular mechanisms directing DC function are largely undefined. It is known that DC function is regulated by their state of maturation (Banchereau, et al., 2000; Bell, et al., 1999). Maturation of DC can be induced by microbial stimuli, pro-inflammatory cytokines, as well as through interaction with T-cells that express CD40L (Banchereau, et al., 2000; Banchereau and Steinman, 1998; Caux, et al., 1994; Reis e Sousa, 2001). Interestingly, many inducers of DC maturation are also strong activators of NF-κB transcription factors (Baldwin, 1996; Ghosh, et al., 1998), indicating that these factors play an important role in DC maturation.
The family of NF-κB transcription factors co-ordinate a variety of cellular events that control such diverse biological processes as development, apoptosis and cell cycle progression, and play a crucial role in regulating inflammatory and immune response genes (Ghosh, et al., 1998; Hatada, et al., 2000; Hayden, et al., 2006). Additionally, pathological deregulation of the NF-κB has been determined to be the cause of many pathological conditions, including cancer (Perkins and Gilmore, 2006; Scheidereit, 2003).
NF-κB factors can both induce and repress gene expression by binding specific κB elements in the promoters and enhancers of various genes (Hayden and Ghosh, 2004). The NF-κB factors function in mammals as hetero- and homodimers of five proteins: p50/p105 [or NF- κB1], p52/p100 [or NF-κB2], p65/RelA, RelB, and c-Rel. Such NF-κB complexes are kept in the cytoplasm by the inhibitors of NF-κB, the IκBs. Phosphorylation of IκB by the IκB kinase (IKK) complex, leads to IκB degradation, resulting in NF-κB activation. Liberated NF-κB then becomes free to translocate to the nucleus and to exert its effects.
A characteristic feature of all NF-κB family members is the presence of an N-terminal Rel homology domain (RHD) of approximately 300 amino acids (Figure IA). RHD mediates DNA binding and dimerisation, and contains the nuclear-localisation domain. Rel subfamily members (RelA, RelB and c-Rel) contain unrelated C-terminal transcriptional activation domains (TADs) (Perkins, 2007). NF-κBs are maintained in the cytoplasm of the cell in inactive state by being associated with the inhibitory proteins of the IκB family. In mammalian cells, the family is represented predominantly by IκBα, IκBβ and IκBε (Figure IB). The IκB proteins contain characteristic ankyrin repeats. Ankyrin repeats (ANK) bind to NF-κB, thus sequestering NF-κB in the cytoplasm by masking the nuclear localisation signal (NLS), located at the RHD. For IκBα such masking is only partially effective and NF-κB- IκBα complexes may shuttle into the nucleus even in the absence of pathway signalling.
Additionally, a nuclear export sequence present in the IκBα allows its fast deportation from the nucleus into the cytoplasm (Hayden and Ghosh, 2004).
p50/NF-κB1 and p52/NF-κB2 are distinctive members of the NF-κB proteins, as they are synthesised as longer p105 and p100 precursor proteins. p105 and p100 are characterised by containing the RHD in their N-terminus, and a sequence of ankyrin repeats (a feature of the IκB family), followed by the death domain (DD) at the C-terminal end of the protein.
Proteolytic processing of these precursor proteins leads to the generation of the p50 and p52 forms. Both p50 and p52 homodimers can interact with BCL-3 IκB family member. BCL-3 is localised in the nucleus of the cell and functions as a transcriptional co-activator (Hayden and Ghosh, 2004; Perkins, 2007).
NF-κB factors possess a particular dimerisation preference. p50/p65 combination represents an archetypical dimer. But p65 can also dimerise with p52 and c-Rel. RelB usually forms homodimers, or RelB/p65 or RelB/c-Rel heterodimers. Similarly to the NF-κB members, IκB proteins also possess an interaction partner preference. IκBα and IκBβ typically interact with p65/p50, p65/p52, p65/p65, c-Rel/p50, c-Rel/p52 and c-Rel/c-Rel dimers, but they are not engaged in the complexes containing RelB. IκBε joins into complexes containing p65 or c- Rel.
NF-κB pathway embraces several convergent cascades (Figure II). In response to particular stimuli, such as pro-inflammatory cytokines (e.g. TNFα, IL-1), engagement of T-cell receptor (TCR) or stimulation by certain TLR ligands (e.g. LPS), activation of the NF-κB canonical signalling takes place, followed by the induction of responsive genes. This occurs as a result of nuclear translocation of NF-κB, which is a consequence of inducible phosphorylation of IκB proteins on specific residues by the IKK (IκB kinase) complex (Karin and Ben-Neriah, 2000). Phosphorylation of IκB permits its recognition by the ubiquitination complex, and after subsequent polyubiquitination, IκBs are degraded by the 26S proteosome. Thus, IκBα, IκBβ and IκBε are completely degraded. The kinase phosphorylating the IκBs, IKK, is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit NEMO/IKKγ.
Majority of signalling pathways that activate NF-κB converge on IKK (Scheidereit, 2006).
Depending on the nature of stimulation, NF-κB can be activated by the non-canonical pathway. Both canonical and non-canonical pathways act in co-operation, with the canonical pathway excreting a licensing role over the non-canonical one. Thus, the canonical pathway, activated by the stimuli such as TNFα or LPS, has the ability to activate the components of the non-canonical pathway, e.g. CD40, LTβR (Perkins, 2007; Scheidereit, 2006). Ligation of those receptors activates NF-κB-inducing (NIK) kinase, which then causes processing and partial degradation of p100, and subsequent formation of a p52-containg dimer. p52-RelB dimers, which are often activated following non-canonical signalling, have a higher affinity for distinct κB elements and hence might regulate a distinct set of NF-κB target genes. It is thought that distinct NF-κB dimers activate particular groups of genes in a time dependent manner, and that binding by multiple members increases RNA polymerase recruitment and subsequent gene expression (Saccani, et al., 2003; Schreiber, et al., 2006).
Moreover, canonical pathway activation is also possible as a result of genotoxic stimuli, such as ionising radiation, and can also be induced by some chemotherapeutic drugs, causing NEMO-dependent IKKβ activation. Other stimuli, such as hypoxia, hydrogen peroxide stimulation, UV light, etc. might activate NF-κB in atypical, IKK-independent manner (Perkins, 2007).
(A) The NF-κB family
RelA(p65)
RelB c-Rel p105(p50) p100(p52)
RHD RHD RHD
RHD RHD LZ
TA2 TA1 TAD
TA1 TA2 ANK
ANK
DD DD
IκBα
Bcl-3
(B) The IκB family
IκBβ IκBε
PEST PEST ANK
(C) The IKK family
NEMO(IKKγ)
IKKα IKKβ
Kinase domain
CC2 LZ
CC1 ZF
LZ HLH NBD
Kinase domain LZ HLH NBD
(A) The NF-κB family
RelA(p65)
RelB c-Rel p105(p50) p100(p52)
RHD RHD RHD
RHD RHD LZ
TA2 TA1 TAD
TA1 TA2 ANK
ANK
DD DD
IκBα
Bcl-3
(B) The IκB family
IκBβ IκBε
PEST PEST ANK
(C) The IKK family
NEMO(IKKγ)
IKKα IKKβ
Kinase domain
CC2 LZ
CC1 ZF
LZ HLH NBD
Kinase domain LZ HLH NBD
Figure I. The mammalian members of the NF-κB, IκB and IKK families. (A). There are five NF-κB family members found in mammals: RelA/p65, RelB, c-Rel, p50/p105 [or NF- κB1] and p52/p100 [or NF-κB2]. Members are characterised by the N-terminal Rel-homology domain (RHD) that mediates DNA binding and dimerisation, and possesses the nuclear- localisation signal. The Rel subfamily (comprised of RelA, RelB and c-Rel) contain C- terminal transcription activation domains (TADs). TA1 and TA2 are subdomains of TAD.
(B). The IκB family consists of IκBα, IκBβ, IκBε and BCL-3. Like p105 and p100, IκB proteins possess ankyrin repeat motifs (ANK) in their C-termini. (C). Major subunits of the IκB kinase (IKK) complex: catalytic subunits IKKα and IKKβ and the regulatory subunit, NF-κB essential modifier, NEMO/IKKγ.
Principal structural motifs of each protein are depicted. CC - coiled coil; DD - region with homology to death domain; HLH - helix-loop-helix; LZ - RelB transactivation domain, containing a putative leucine-zipper-like motif; NBD - NEMO-binding domain; PEST - domain rich in proline (P), glutamate (E), serine (S) and threonine (T); ZF - zinc finder domain. Adapted from (Perkins, 2007).
NEMO IKKα IKKβ
NEMO ATM
Ub
NEMO ATM
Ub NEMO
ATM SUMO NEMO Atypical pathway (genotoxic stress, IKK dependent)
Canonical pathway (TNFα, IL-1, LPS)
P P p50IκBαRelA (Ser36)
(Ser32)
Uniquitination and proteosomal degradation of IκB Activation of
IKK complex
p50 RelA
Kinases, acetylases, phosphotases RelA
RelA P
p50IκBαRelA RelA
Degradation or
dissociation of IκB Degradation of IκB (Tyr42)
Tyr kinase Hypoxia, H2O2
Atypical pathway (IKK independent)
P P p50IκBαRelA
RelA CK2
UV, HER2/Neu
RelBRHD RelB TAD p52 Uniquitination and proteosomal degradation of p100
RelB RelB
P P
(Ser866) (Ser870) p100
ANK p52
IKKα IKKβ NIK Non-canonical pathway (LPS, CD40 and lymphotoxin Receptors. LMP1)
Cytoplasm Nucleus
p50 RelA RelA
Transcriptional activation
P P
Ac Ac Co-activator
p50 RelA RelA
Transcriptional repression
P P Co-repressor
P
p50 RelA RelA
Promoter targeting and sensitivity
P
TF
Distinct κB elements RelBRHD RelB TAD p52
bZIP bZIP
HMG-I
Zn-finger TFs NEMO
IKKα IKKβ
NEMO ATM
Ub
NEMO ATM
Ub NEMO
ATM SUMO NEMO Atypical pathway (genotoxic stress, IKK dependent)
Canonical pathway (TNFα, IL-1, LPS)
P P p50IκBαRelA (Ser36)
(Ser32)
Uniquitination and proteosomal degradation of IκB Activation of
IKK complex
p50 RelA
Kinases, acetylases, phosphotases RelA
RelA P
p50IκBαRelA RelA
Degradation or
dissociation of IκB Degradation of IκB (Tyr42)
Tyr kinase Hypoxia, H2O2
Atypical pathway (IKK independent)
P P p50IκBαRelA
RelA CK2
UV, HER2/Neu
RelBRHD RelB TAD p52 Uniquitination and proteosomal degradation of p100
RelB RelB
P P
(Ser866) (Ser870) p100
ANK p52
IKKα IKKβ NIK Non-canonical pathway (LPS, CD40 and lymphotoxin Receptors. LMP1)
Cytoplasm Nucleus
p50 RelA RelA
Transcriptional activation
P P
Ac Ac Co-activator
p50 RelA RelA
Transcriptional repression
P P Co-repressor
P
p50 RelA RelA
Promoter targeting and sensitivity
P
TF
Distinct κB elements RelBRHD RelB TAD p52
bZIP bZIP
HMG-I
Zn-finger TFs
Figure II. Pathways leading to the activation of the NF-κB. The canonical pathway is induced by the TNFα, IL-1 and many other stimuli, and is dependent on activation of IKKβ.
This activation results in phosphorylation of IκBα, leading to its ubiquitylation and subsequent degradation. Release of the NF-κB complex allows its translocation to the nucleus. Atypical IKK-dependent activation of NF-κB can occur following genotoxic stress. Here NEMO localises to the nucleus, where it is sumoylated and then ubiquitylated, in a process dependent on the ataxia telangiectasia mutated (ATM) checkpoint kinase. NEMO relocates back to the cytoplasm together with ATM, where activation of IKKβ occurs.
Atypical IKK-independent pathways of NF-κB activation include casein kinase-II (CK2) and tyrosine-kinase-dependent pathways. The non-canonical pathway results in the activation of IKKα by the NF-κB-inducing kinase (NIK), followed by phosphorylation of p100 by IKKα. This results in proteosome-dependent processing of p100 to p52, which can lead to the activation of p52-RelB heterodimers that target distinct κB elements.
Ac - acetylation; bZIP - leucine-sipper-containing transcription factor; HMG-I - high- mobility-group protein-I; LMP1 - latent membrane protein-1; RHD - Rel-homolgy domain;
TAD - transcription activation domain; TF - transcription factor; UV - ultraviolet light; Zn- finger TF - zinc-finger-containing transcription factor. Adapted from (Perkins, 2007).
Phosphorylation of translocated NF-κB by nuclear kinases, and its modification by acetylases and phosphatases, leads to transcriptional activation and repression of target genes, well as more complex promoter-specific effects. Additionally, interactions with other transcription factors can target NF-κB complexes to specific promoters, resulting in the selective activation of gene expression following cellular exposure to distinct stimuli.
Knockouts for all five members of the NF-κB family have been produced in mice (Beg, et al., 1995; Burkly, et al., 1995; Caamano, et al., 1998; Doi, et al., 1997; Franzoso, et al., 1998;
Kontgen, et al., 1995; Sha, et al., 1995; Weih, et al., 1995). These studies have identified key roles for NF-κB proteins in regulation of innate immunity, lymphocyte function, and regulation of cell survival (Alcamo, et al., 2001; Beg, et al., 1995; Grumont, et al., 1999;
Kontgen, et al., 1995; Ouaaz, et al., 1999; Sha, et al., 1995; Zheng, et al., 2001). Studies of RelB-/- mice have indicated a specific requirement for this protein in development of CD11c+CD8α- but not CD11c+CD8α+ DC (Burkly, et al., 1995; Weih, et al., 1995; Wu, et al., 1998). Quaaz et al. have later analysed the function of other NF-κB subunits in DC (Quaaz, et al., 2002). They have shown that κB site binding complexes in DC consist in large part of p50, RelA, and c-Rel subunits. In experiments on knockout mice, it has been shown that the absence of individual subunits does not effect DC survival, maturation, or T-cell stimulatory capacity. However, profound defects in DC function were found in the combined absence of p50+RelA or p50+cRel, respectively. There appears to be a key function of NF-κB complexes comprising of p50, RelA, and c-Rel in regulation of DC development, survival, and IL-12 production.
A major question in the NF-κB signalling remains the mechanism by which different NF-κB activatory stimuli lead to the activation of specific response genes, and what is the specific function exerted by each of the members of the NF-κB family. Part of this specificity seems to lie in the cross-talk between the NF-κB and other signalling pathways (Natoli and De Santa, 2006).
1.4 Immune effects of vitamin D3 on DC
DC can induce not only the state of immunity but also tolerance, both in the thymus and at the periphery (Steinman, et al., 2003), depending on the nature of the antigen processed and the stage of maturation. DC with an immature phenotype have been described to be tolerogenic in nature and to induce T-cells with suppressive properties (Jonuleit, et al., 2002). Thus, DC uptake, process and present antigens, but the outcome of these processes appears to be strongly dependent on the adjacent innate immune signals. Self-antigens acquired from the apoptotic cells, processed and further presented by the DC tend to generate regulatory T-cells and induce self-tolerance. In contrast, antigens taken up by the DC in the context of the activated innate response strongly stimulate antigen-specific T- and B-cells. T-cell responses are further skewed due to the secretion of immunomodulatory cytokines, such as IL-10 or IL- 12. Therefore, the context at which an antigen has been acquired is a crucial factor in determining the outcome of an immune reaction. For this reason development of
pharmacological agents that modify DC function in a way that would induce tolerogenic DC, which would subsequently lead to the development of regulatory T-cells, would be of great importance in the treatment of both autoimmune diseases and graft rejection.
Significant evidence has accumulated that the dihydroxyvitamin D3 (VD3), which has been previously known only as calcium and phosphorus metabolism regulator, can affect the function of the immune system. Several studies have shown that VD3 can affect various cells of the immune system, and that its immunomodulatory (often suppressive) effects can be exploited therapeutically for the potential treatment of certain immune disorders.
At first vitamin D3 receptor (VDR) was found to be expressed on the cells of human thymus and peripheral blood leukocytes. Then VDR was identified in human and murine monocytes, and was described to be induced in lymphocytes upon their activation. Studies investigating VD3 deficiency have pointed towards immune defects in patients and animal models. Thus, defects in inflammatory responses in macrophages have been published, and impaired delayed type hypersensitivity responses as a consequence of lack of VD3 have been described (Yang, et al., 1993).
However, there was a controversy in the literature describing the effects of VD3. A number of researchers have pointed towards VD3-induced differentiation of monocytes and monocyte- derived cell lines towards a macrophage-like phenotype. Interestingly, monocytes are able to synthesise 25-hydroxyvitamin D3 by 1α-hydrolase activity, which was shown to be induced by certain activating stimuli (IFN-γ and LPS) or by viral infection. This suggests a paracrine role of dihydroxyvitamin D3 in promoting macrophage-dependent inflammation. Other research projects focusing on antigen presentation have revealed a different picture.
Treatment of monocytes with VD3 resulted in reduced MHC class II expression, the amount of co-stimulation, and consequently impaired T-cell stimulatory capacity.
Identification of DC and characterisation of co-stimulatory molecules on APC have provided a further insight into the function of VD3 (Griffin, et al., 2003). A number of reports published recently have shown that binding of VD3 to its receptor, VDR, activates pathways inhibiting DC maturation. The expression of MHC class II, co-stimulatory mediators CD80, CD86, CD40, and surface antigens CD1a and CD83 were all reduced upon VD3 treatment.
Additional data indicate that VD3 inhibits secretion of IL-12 and may promote IL-10 production. Those profound changes in DC surface molecules lead inevitably to reduced T- cell activation, proliferation and cytokine production. Interestingly, vitamin D3 treated DC have been used to endorse tolerance in female mice to male skin grafts. Nonetheless, more specific effects on various DC sub-types remain largely unknown.
Like DC, other cells of the immune system are also reactive to the presence of VD3. Ability of VD3 to exhibit inhibitory effects on T-cells has been demonstrated. The significance of those effects in vivo on the background of the inhibition of antigen-presenting function of DC and macrophages is not clear. Published data describing the effects of dihydroxyvitamin D on
B-cells and NK-cells are even more controversial than the current T-cell data. Much additional research will be required to draw more solid conclusions.
1.4.1 Vitamin D3 and intracellular signalling
Activation of DC, as well as activation of other lymphocytes, is regulated by a number of simultaneous and sequential events involving various signalling pathways. Signalling cascades operational in DC and other cells of the immune system are similar to those functional in other cell types and systems of the organism. But there appears to be additional components and cross-talk mediators specific to certain cell types. Moreover, the target genes regulated as a result of activation of those pathways have been postulated to be different in diverse cell types. VDR is known to regulate transcription of a number of target genes by binding to the so-called “vitamin D response elements” (VDRE), usually in the form of a heterodimeric complex with other binding partners, such as retinoid acid X-receptor (RXR) (Carlberg and Polly, 1998; Jones, et al., 1998). But VDR is also thought to exert its immunomodulatory properties through direct or indirect influence on signalling cascades. The pathways shown to be important for lymphocyte activation are summarised in Table I.
NF-κB pathway has been shown to be particularly important for the DC development and function. There also appear to be differences in the activation of particular NF-κB dimers and their effects on gene regulation. VD3 alone or in combination with other immunomodulatory agents, such as glucocorticoid dexamethasone, have been shown to regulate intracellular levels of the most common NF-κB proteins in DC - c-Rel and Rel-B. Moreover, inhibition of NF-κB dimer binding to the promoter upon VD3 treatment has been reported for some genes, e.g. IL-12p40. Such regulation of those transcription factor activity is most likely to be responsible for the down-regulation of MHC and IL-12 production, and hence T-cell stimulatory activity. Additionally, interference with recruitment of additional transcription factors or transcriptional machinery units might be involved. The current concepts describing the cross-talk between NF-κB and VDR signalling pathways are depicted in the cartoon below (Figure III).
A number of agents have been described to effect DC maturation process. Indeed, human DC matured in the presence of IL-10, have been shown to induce anergic T-cells which inhibited T-cell proliferation and reduced IL-12 and IFNγ production by the T-cells (De Smedt, et al., 1997; Enk, et al., 1993; Steinbrink, et al., 1997). Amongst other substances being able to inhibit DC maturation and immunostimulatory capacity are VD3 its active form - dihydroxyvitamin D3 (Piemonti, et al., 2000), glucocorticoids (Piemonti, et al., 1999) and vascular endothelial growth factor (VEGF) (Oyama, et al., 1998).
VD3 has been published to exert anti-inflammatory effects. VD3 is known to inhibit T-cell proliferation and cytokine secretion (Bhalla, et al., 1984; Rigby, et al., 1987), and similarly to the glucocorticoids has been shown to effect T-cell activation by inhibitory function on DC
Signalling pathway
Cell types identified
Mechanisms of pathway modulation
Target genes References
Nuclear factor kappa B (NF-κB)
T-cells Dendritic cells Dendritic cells
Inhibition of total intracellular levels of multiple NF-κB proteins Indirect inhibition of NF- κB heterodimer binding to promoter site
P105/p50 c-Rel Rel-B IL-12p40 (IL-12p35)
Yu et al., 1995;
Xing et al., 2002
D'Ambrossio et al., 1998
Nuclear factor of activated T-cells (NFAT)
Activated T-cells
Direct DNA binding of VDR/RXR and interface with NFATp/AP-1 complex assembly
IL-2 (GM-CSF, IL-4, TNFα)
Alroy et al., 1995;
Takeuchi et al., 1998
Phosphatidylinosito l-3-kinase (PI3-K)
Promonocytic cell lines Monocytes
Activation of PI3-K activity by formation of VD3-dependent VDR/
PI3-K complex
CD14, CD11b Hmama et al., 1999;
Mitogen activated protein kinase (MAPK) pathways
Promonocytic cell lines T-cell hybridoma
Activation of erk1/2 kinase Indirect inhibition of c-myc-mediated transcriptional activity
CD14, CD11b Fas ligand
Marcinkowska, 2001 Cippitelli et al. 2002
Table I. Regulation of signalling pathways by vitamin D3 in the cells of immune system.
Adapted from Griffin et al. (Griffin, et al., 2003). References in the table: (Alroy, et al., 1995;
Cippitelli, et al., 2002; D'Ambrosio, et al., 1998; Hmama, et al., 1999; Marcinkowska, 2001;
Takeuchi, et al., 1998; Xing, et al., 2002; Yu, et al., 1995).
activation (Penna and Adorini, 2000). Interestingly, the glucocorticoid dexamethasone and VD3 have been shown to be synergistic in induction of DC-mediated tolerance (Pedersen, et al., 2004). This has been hypothesized to be linked to the inhibition of transcription factor NF- κB and increased IL-10 secretion.
VD3 has been shown to inhibit CD86 up-regulation upon treatment of DC with maturation stimuli such as TNFα and IFNγ (Clavreul, et al., 1998), as well as repressed expression of CD40, CD58, CD80 and CD86 during LPS-induced DC maturation. VD3, similarly to dexamethasone, can inhibit the ability of DC to initiate Th1 response by shifting from IL-12 to IL-10 production upon LPS treatment (Penna and Adorini, 2000). At least some of these effects of VD3, such as inhibitory effect on IL-12 production, have been associated with the inhibition of the NF-κB (D'Ambrosio, et al., 1998).
PP
p50IκBαRelA RelA
p50 RelA RelA
VDR RXR
p50
Inflammatory protein TNFαLPS
IKK complex
AAAAAA
- +
Anti- inflammatory
protein
?
?
+ Y
+ X
Y X
anti-inflammatory protein gene (?) Z
inflammatory protein gene Y X
-
Z
squelching (?) Vitamin D3 1α,25(OH)2D3
NEMO IKKα IKKβ
(Ser36) (Ser32) Uniquitination
and proteosomal degradation of IκB
RXR
RXR VDR VDR
VDR Phosphorylation
of IκB
RelA RelA
Cytoplasm Nucleus
p50
Co-factors
RXR PP
p50IκBαRelA RelA
p50 RelA RelA
RXR VDR RXR VDR
p50
Inflammatory protein TNFαLPS
IKK complex
AAAAAA AAAAAA
- +
Anti- inflammatory
protein
?
?
+ Y
+ X
Y X
anti-inflammatory protein gene (?) Z
inflammatory protein gene Y X
-
Z
squelching (?) Vitamin D3 1α,25(OH)2D3
NEMO IKKα IKKβ
NEMO IKKα IKKβ
(Ser36) (Ser32) Uniquitination
and proteosomal degradation of IκB
RXR
RXR VDR VDR
VDR Phosphorylation
of IκB
RelA RelA
Cytoplasm Nucleus
p50
Co-factors
RXR
Figure III. Cross-talk between NF-κB and VDR pathways. Multiple mechanisms might contribute to the ability of VDR to repress NF-κB pathway, and thereby exerting its anti- inflammatory effects. Different mechanisms may regulate distinct subsets of genes. Direct repression of transcription of pro-inflammatory genes by VDR through interaction with the NF-κB-containing transcription complex is possible. RXR and other co-factors (X, Y and Z) may or may not be necessary. Squelching is the mechanism by which a limited co-factor (Z), essential for transcription, is competitively withheld from the NF-κB-containing complex.
Induction of anti-inflammatory genes may directly influence RNA stability or translational efficiency of pro-inflammatory proteins.
VDR - vitamin D receptor; RXR - retinoid acid X-receptor; X, Y - co-factors; Z - other co- factors/regulators; (+) positive regulation; (-) negative regulation; (?) unknown/undefined mechanisms.
1.5 Gene transfer into DC
1.5.1 Receptor-mediated gene delivery into DC with adenovirus as ligand Targeted gene delivery capitalises on the presence of specific cell surface receptors for uptake of nucleic acids into cells. In this way receptor-binding ligands are coupled to polycationic compounds such as polylysine or polyethylenimine (PEI) that condense and bind nucleic acids. Among the polycationic compounds PEI was described to be especially useful. It possesses a high binding and condensing activity of nucleic acids, as well as a high pH buffering capacity that protects nucleic acids from degradation and enhances the exit from the endosomal compartment (Oh, et al., 2002). PEI has been shown to be effective for gene delivery into a variety of cell types even without the addition of cell binding ligands or endosomolytic agents (Abdallah, et al., 1996; Boussif, et al., 1995). To combine this high intrinsic transfection efficacy of PEI with the concept of receptor mediated gene transfer, Ad particles were included in PEI/DNA transfection complexes and were demonstrated to efficiently transduce a variety of cell types including DC (Baker and Cotten, 1997; Baker, et al., 1997; Diebold, et al., 1999; Gust, et al., 2004). PEI binds to virus particles through charged interactions with negative domains on the viral hexon. Thus, the generated Ad/PEI/DNA complexes contain plasmid DNA that is condensed and bound to the outside of the adenovirus carrier via PEI, and deliver DNA into cells via the adenovirus infection route, e.g., binding to αv integrins on the cell surface (Meier and Greber, 2003).
The Ad/PEI transfection method offers several advantages over recombinant adenovirus vectors. (1) Transgene expression does not rely on a transcriptionally active viral genome and, thus genetically and chemically inactivated adenovirus can be employed (Diebold, et al., 1999) thereby avoiding interference of adenovirus with DC function. (2) The same adenovirus component can be used to deliver different nucleic acids thus abolishing the need to prepare different adenoviral vectors for each individual cDNA to be transfected. (3) Different plasmids can be transduced simultaneously. (4) DNA as well as RNA can be delivered by Ad/PEI transfection complexes (Gust, et al., 2004).
1.5.2 Adenoviral gene-delivery vectors
Viral gene transfer is a powerful means to manipulate the properties of DC. A variety of vectors have been used for gene transfer into DC including adenovirus, adeno-associated virus, retrovirus, and lentivirus. Adenoviruses (Ad) have also been extensively tailored for use as vectors in vaccination and gene therapy. Ad are non-enveloped DNA viruses that cause respiratory and gastrointestinal infections as well as infections of the heart. The defective vectors in present use, such as AdOVA and AddIκBα-SR (see Materials and Methods) employed as gene delivery agents in this study, are commonly derived from adenovirus serotypes 2 and 5.
The ability of animal viruses to infect host cells is dependent on the presence of appropriate cellular receptor. In general, viruses of different families do not compete for binding to a common receptor. However, Ad serotypes 2 and 5 and the group B coxsackieviruses are human pathogens that share a common receptor - coxsackie-adenovirus receptor (CAR), although they belong to divergent virus families (Bergelson, et al., 1997). CAR is a 46-kDa transmembrane protein that contains two extracellular immunoglobulin-like domains and therefore belongs to this superfamily (Tomko, et al., 1997). CAR was shown to function as an adhesion molecule and to possess a tumour inhibitory activity. The expression of CAR varies, not only at different developmental stages and tissues, but also between species. In mice, although CAR is abundantly expressed in epithelial cells during embryogenesis, its expression in adults is restricted to fewer cell types.
Ad attachment to cells is mediated by fibres projecting from the 5-fold vertices of an icosahedral adenoviral capsid. It is the terminal portion of the fibre, known as a “knob”, that is responsible for receptor binding. Ad entry is also facilitated by the interaction between the penton base protein and integrins on the cell surface. Once fibre-mediated virus attachment to CAR has occurred, internalisation occurs through the interaction of an arginine-glycine- aspartate (RGD) sequence on the Ad penton base with αVβ3 or αVβ5 integrins on the cell surface (Bergelson, 1999; Hidaka, et al., 1999). In contrast to CAR, expression of αV- integrins is more homogenous, suggesting that limiting expression of CAR influences susceptibility to Ad-infection more than that of αV-integrins. Because CAR expression greatly enhances gene transfer, manipulation of the receptor expression may be useful in achieving efficient adenovirus-mediated transduction both in vivo and in vitro.
1.5.3 CAR and CAR mice
Frequently primary lymphocytes are resistant to most currently available gene transfer methods, including Ad vectors. The low expression level of CAR is the main reason why Ad transduction of lymphocytes is inefficient (Wan, et al., 2000). CAR is not present on all cell types, thus restricting such gene delivery systems. DC are relatively resistant to Ad-mediated gene delivery and high viral titres (multiplicity of infection (MOI) >100) are required to achieve a somehow significant gene transfer because mature DC do not express CAR.
According to the published data, approximately 80% of wild-type mature DC can be transduced but with MOIs of 100 or higher. To bypass this problem a transgenic mouse has been generated that expresses a truncated form of human CAR ubiquitously (Tallone, et al., 2001). These mice allow efficient in vitro transductions at low MOIs into lymphoid, myeloid, and endothelial cells. In vivo use of Ad-vectors resulted in gene delivery into macrophages, lymphocytes, and endothelial cells.
