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Analysis of the function of human bactericidal, permeability-increasing protein (BPI) and of the expression of selected BPI-family members [Elektronische Ressource] / vorgelegt von Diana Aichele

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90 pages
Analysis of the function of human bactericidal/permeability-increasing protein (BPI) and of the expression of selected BPI-family members Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Diana Aichele aus Göppingen Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 07. August 2008 Vorsitzender der Promotionskommision: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Dr. André Gessner Zweitberichterstatter: Prof. Dr. Thomas Winkler Contents 1 Introduction..........................................................................................1 1.1 The role of Toll-like receptors (TLRs) in innate immunity............................1 1.1.1 Toll like receptor 4 (TLR4) activation and signalling ............................................... 1 1.2 Antimicrobial proteins as part of the innate immune response ..................2 1.3 The BPI-family proteins...................................................................................3 1.3.1 Expression and function of human BPI................................................................... 4 1.3.2 Exprenction of murine BPI 5 1.3.3 The PLUNC-family proteins, novel members of the BPI-family .........................
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Analysis of the function of human bactericidal/permeability-increasing protein (BPI) and of the expression of selected BPI-family members Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Diana Aichele aus Göppingen
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 07. August 2008 Vorsitzender der Promotionskommision: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Dr. André Gessner Zweitberichterstatter: Prof. Dr. Thomas Winkler
Contents1 Introduction..........................................................................................1
1.1 The role of Toll-like receptors (TLRs) in innate immunity ............................1
1.1.1 Toll like receptor 4 (TLR4) activation and signalling ...............................................1
1.2 Antimicrobial proteins as part of the innate immune response ..................2
1.3 The BPI-family proteins...................................................................................3
1.3.1 Expression and function of human BPI...................................................................4
1.3.2 Expression and function of murine BPI...................................................................51.3.3 The PLUNC-family proteins, novel members of the BPI-family ..............................6
1.4 Human BPI in cystic fibrosis ..........................................................................7
1.4.1 Cystic fibrosis and bacterial lung infections ............................................................7
1.4.2 The role of BPI in cystic fibrosis..............................................................................8
1.5. Aims of the study............................................................................................8
2 Material and Methods ..........................................................................9
2.1 Cell culture .......................................................................................................9
2.2Animals.............................................................................................................9
2.3 Isolation and cultivation of primary cells ....................................................10
2.3.1 Murine peritoneal exudate cells (PECs)................................................................10
2.3.2 Generation of murine bone marrow-derived dendritic cells (BM-DC) ...................10
2.3.3 Generation of murine bone marrow-derived macrophages (BM-Mφ) ...................11
2.3.4 Recovery of murine bronchoalveolar lavage (BAL) cells ......................................11
2.3.5 Extraction of human isolated granulocytes ...........................................................11
2.4 Samples from CF patients.............................................................................12
2.5 Bacteria...........................................................................................................12
2.5.1 Cultivation and treatment ofPseudomonas aeruginosa.......................................122.5.2 Generation of competent bacteria.........................................................................13
2.6 Bacterial killing by BPI ..................................................................................13
2.7 Stimulation of various cells ..........................................................................13
2.8 Isolation of RNA .............................................................................................14
2.9 Polymerase Chain Reaction (PCR)...............................................................15
2.9.1 Reverse Transcription PCR (RT-PCR) .................................................................15
2.9.2 Qualitative PCR ....................................................................................................15
2.9.3 Quantitative PCR ..................................................................................................16
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2.10 Cloning of the BPI promoter and BPI promoter fragments .....................18
2.10.1Vector..................................................................................................................18
2.10.2 Cloning of the putative full length BPI promoter ..................................................18
2.10.3 Cloning of BPI promoter fragments .....................................................................19
2.10.4 Purification of plasmid DNA ................................................................................20
2.11 Transient transfection.................................................................................20
2.12 Luciferase reporter assay...........................................................................20
2.13 ExperimentalP. aeruginosalung infection in mice..................................21
2.13.1 Intranasal lung infection ......................................................................................21
2.13.2 Preparation of lung tissue ...................................................................................21
2.13.3 BAL samples .......................................................................................................21
2.14 Enzyme-linked immunosorbent assay (ELISA) ........................................22
2.15 Measurement of iNOS (inducible nitric oxide synthase) activity ............22
2.16 Protein detection by immunoblots ............................................................23
2.16.1 Western Blot........................................................................................................23
2.16.2 Generation of an anti-mouse BPI antiserum .......................................................232.17 Flow Cytometry ...........................................................................................24
2.18 Statistics ......................................................................................................24
3 Results ................................................................................................25
  
3.1 The Role of BPI in cystic fibrosis.................................................................25
3.1.1 Expression of BPI in the lungs of cystic fibrosis patients ......................................25
3.1.2 BPI expression patterns of various cells types......................................................26
3.1.3 BPI and IL-8 in cell-free sputum samples of CF patients ......................................27
3.1.4 The mode of BPI-release from polymorphonuclear leukocytes ...........................28
3.1.5 BPI acts bactericidal and bacteriostatic against clinically
 relevant strains ofP. aeruginosa...........................................................................30
3.2 The expression of BPI and other BPI-family proteins in a mouse
 model ofP. aeruginosalung infection ........................................................333.2.1 Characterization of acuteP. aeruginosalung infection in a mouse model ...........33
3.2.2 Expression of BPI and PLUNC proteins in experimental
P. aeruginosalung infection..................................................................................35
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3.3 The expression of short and long PLUNC 1 mRNA in different cell types ...
37
3.4 Molecular regulation of murine BPI..............................................................38
3.4.1 Activity and characteristics of the murine BPI promoter .......................................39
3.4.2 LPS-mediated transcriptional control of murine BPI .............................................41
3.4.2.1 Previous data ...............................................................................................................41
3.4.2.2 BPI mRNA and protein are induced via the TRIF-dependent
 signalling pathway of TLR 4 .........................................................................................42
3.4.2.3 BPI protein induction can be partially blocked by the soluble TNFR II ........................45
3.4.2.4 BPI protein induction is partially blocked in DCs from TNFR I-deficient mice .............46
3.4.2.5 BPI protein induction is partially blocked in DCs from LTα-deficient mice....................47
4 Discussion..........................................................................................50
4.1 Expression of BPI in the lungs of cystic fibrosis patients .........................50
4.2 Secretion of BPI from human PMNs.............................................................50
4.3 Antimicrobial capacity of human BPI againstP. aeruginosa.....................524.4 Expression of BPI in a lung infection mouse model...................................53
4.5 Expression of PLUNC proteins and possible immunological implications
...............................................................................................................................55
4.6 Molecular regulation of murine BPI..............................................................56
4.7 LPS-mediated transcriptional control of murine BPI..................................58
5 Summary ............................................................................................61
6 Zusammenfassung ............................................................................637 Bibliography.................................................................................... ...65
8 Abbreviations.....................................................................................79Danksagung ..........................................................................................81
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1 Introduction  
1.1 The role of Toll-like receptors (TLRs) in innate immunity  The innate immune system is characterized by the ability to recognize a broad spectrum of pathogens with only a limited number of different receptors. These so-called pattern recognition receptors (PRRs) are located on leukocytes and epithelial cells and are part of the first-line defence against invading microorganisms. PRRs recognize conserved structures on pathogens (pathogen-associated molecular patterns; PAMPs) that can be clearly distinguished from eukaryotic structures (1, 2). One family of PRRs that is well characterized are the toll-like receptors (TLRs) (3). The family of mammalian TLRs is homologue of the drosophila Toll 1 receptor, which was first identified as an important component in the embryonic development (4), but later was found to be crucial for the recognition and elimination of fungal infections (5).This shows that TLRs are highly conserved throughout evolution (6). So far, 10 human and 12 murine TLRs are known, recognizing a diverse spectrum of pathogenic compounds including nucleic acids, proteins and lipids. The recognition of such PAMPs by TLRs results in the initiation of an immune response to eliminate the invading pathogens (3, 7). This is partially accomplished by the induction and release of pro-inflammatory cytokines and chemokines following TLR activation (2, 8). Another key feature of this recognition is the initiation and regulation of the adaptive immune response (9, 10). This highlights the importance of the initial pathogen recognition by innate immune receptors for a successful immune response.
1.1.1 Toll like receptor 4 (TLR4) activation and signalling
TLR4 is the best characterized of all TLRs and recognizes lipopolysaccharide (LPS) / endotoxin of the cell wall of gram-negative bacteria. This recognition was first identified in mice that have a natural defect in the TLR4 gene (C3H/HeJ and C57BL/10ScCr) and are therefore resistant to endotoxic shock (11). TLR4 builds a receptor complex together with CD14 and MD-2. All three proteins are essential for the recognition of LPS. CD14 efficiently binds LPS, but as a glycosyl-phosphatidyl-inositol (GPI)-anchored protein it does not have an intracellular signalling domain (12). Soluble CD14 has in principle the same function as cell-anchored CD14 and
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Introduction
determines the LPS responsiveness of CD14 negative cells. Due to the fact that CD14 is a GPI-anchored protein, CD14-bound LPS has to be transferred to the TLR4/MD-2 receptor complex to initiate a signalling cascade. MD-2 mediates the localisation of TLR4 on the cell surface and the overall cellular distribution (13). However, very recent work shows that MD-2 might have a more prominent role in the recognition of LPS itself. Crystal structures of TLR4/MD-2 with the LPS antagonist Eritoran revealed that Eritoran binds to MD-2 and not TLR4 (14), which is in line with earlier observations that LPS has a similar affinity for MD-2 as for the TLR4/MD-2 complex. This indicates that the results observed for Eritoran might also hold true for LPS (15). TLRs are type I transmembrane proteins with a cytoplasmic domain that is homologous to the one of the type I interleukin-1 receptor (IL-1R) and therefore was termed toll-interleukin1-receptor homology (TIR)-domain (16). The intracellular signal transduction is further mediated by cytoplasmic adaptor molecules, which also comprise a TIR domain and form a homophilic interaction with the TIR domain of the TLR. So far, five intracellular adaptor proteins for TLRs are known. These proteins are MyD88 (myeloid differentiation primary response gene 88), TRIF (TIR-domain-containing adapter inducing interferon-β), TIRAP (TIR-domain containing adaptor protein, also called MAL), TRAM (TRIF-related adaptor molecule) and SARM (sterile α-and armadillo-motif-containing protein) (17).
TLR 4 signals via two different intracellular pathways, one employing the adaptor molecules TIRAP and MyD88, the other one using TRAM and TRIF. The MyD88 dependent pathway mainly leads to the activation of the transcription factor nuclear factorκB (NFκB) as well as mitogen activated protein (MAP)-kinases resulting in the induction of proinflammatory cytokines. The TRIF-dependent pathway was shown to induce type I interferons through the activation of the interferon regulatory factor 3 (IRF3), but also leads to the activation of NFκB and MAP-kinases (18).
1.2 Antimicrobial proteins as part of the innate immune response  To combat infections, the innate immune system provides an arsenal of different effector mechanism. One important function is the direct killing of pathogens, among others, accomplished by antimicrobial peptides and proteins (APPs). In concert with acute phase proteins, the complement system and the oxidative burst, APPs
2
Introduction
contribute largely to the elimination of infections in mammals (19). However, representing an ancient immune mechanism conserved throughout evolution, APPs are also expressed in plants (20), insects (6) and frogs (21). The expression of APPs in mammals is most prominent in neutrophil granulocytes, which are rapidly recruited to the site of infection. Moreover, antimicrobial proteins and peptides are expressed in mucosal epithelial cells that line the inner organ surfaces. High concentrations of APPs are present in tear fluid, saliva and nasal fluid.
The function of APPs is mostly based on their cationic charge to interact with pathogenic structures, which leads to the disintegration of the pathogenic cell wall or membrane (e.g. lysozyme, defensins, bactericidal/permeability-increasing protein [BPI]) (reviewed in (22)). Another important feature is the binding and neutralization of LPS, which is released from gram-negative bacteria and can otherwise lead to endotoxic shock or sepsis (23). One protein that combines direct antimicrobial activity and LPS neutralizing capacity is BPI (24). -
1.3 The BPI-family proteins  Bactericidal/permeability-increasing protein (BPI) is eponymous for a whole family of proteins that, besides BPI, includes the lipid binding proteins CETP (cholesterol ester transfer protein) and PLTP (phospholipid transfer protein) as well as the LPS binding protein (LBP) (25). All four proteins show a similar genetic organisation and are expected to show a BPI-like fold (see Figure 1) (26). So far, no immunological functions were shown for CETP or PLTP.
LBP is an acute phase protein which is mainly produced in the liver and is known to bind to the lipid A moiety of LPS (27). LPS bound to LBP is then delivered to its receptor CD14 and subsequently to the TLR4/MD-2 complex on innate immune cells, thereby triggering an antimicrobial response via TLR4 signalling (11, 12, 28).
LBP and BPI share a sequence homology of only 45% (29). Nevertheless, the two proteins reveal a highly conserved genomic organisation (30) and therefore a very similar structure (see Figure 1) (31). This results in similar functional properties, as both proteins bind to the lipid A moiety of LPS (32). Interestingly the fate of LPS bound to either LBP or BPI is quite contrary. LPS molecules bound to BPI are lost for immune recognition and therefore neutralized (32, 33).
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