genomewide characterization of host-pathogen interactions by ...

More documents

Recommendations

Info

Maren Depke Discussion and Conclusions associated groups of genes, of which few were regulated at the early and several at the later time point of analysis. Examples are given with leukemia inhibitory factor LIF (induced at both time points) and its receptor (induced after 6.5 h), ligand CD274 (PD-L1, induced at both time points) and PD-L2 (induced after 6.5 h), several interferon regulated genes at both time points or additionally at the 6.5 h time point, and the histone genes, of which one was repressed at the 2.5 h time point and further 17 were added at the 6.5 h time point. Furthermore, indoleamine 2,3-dioxygenase 1 (IDO1) belonged to the genes with the highest induction factors at the 6.5 h time point. The gene codes for a key molecule in immunomodulation, microbial growth control, and pathogen immune escape (Puccetti 2007, Zelante et al. 2009). IDO is known to be induced by interferons (Carlin et al. 1989, Taylor MW/Feng 1991) e. g. during infection and inflammation, but also in conditions like stress, which result in a transient increase of proinflammatory cytokines (Kiank et al. 2010). The enzyme catalyzes the reaction of tryptophan to formylkynurenine. This is the first rate-limiting step of the so-called “kynurenine pathway”, which leads to NAD + biosynthesis or to the complete oxidative degradation of kynurenine (King/Thomas 2007, Xie et al. 2002). One of the main effects of induced IDO activity is the depletion of tryptophan, which is discussed to be a mechanism to inhibit microbial growth (Pfefferkorn 1984, Byrne et al. 1986, Müller et al. 2009). But this mechanism can be counteracted by bacteria, which might induce their tryptophan biosynthesis or which developed own counter-regulatory mechanisms. An example is Chlamydophila psittaci which owns adapted tryptophan biosynthesis genes that enable the bacterium to produce tryptophan from kynurenine taken from their host’s metabolism (Xie et al. 2002). During metabolism of tryptophan, several neuro- and immune-active substances are produced, of which some additionally exhibit toxic features and thus might act antimicrobially. The intermediates of tryptophan and kynurenine degradation, 3-hydroxykynurenine and α- picolinic acid, were reported to inhibit growth of S. aureus and other bacterial species in a murine transplantation model even in the presence of tryptophan (Saito et al. 2008, Narui et al. 2009). The most prominent characteristic of IDO is the induction of an anti-inflammatory bias after a proinflammatory stimulus. This effect aims to limit inflammation and the accompanying damage to the host tissue. It has to be considered that the experimental setting in this study only included one single cell type, the bronchial epithelial cell line S9. Thus, interpretation of an antiinflammatory effect assumes that bronchial epithelial cells might also in vivo induce IDO, e. g. during staphylococcal pneumonia. Tryptophan degradation intermediates were shown to inhibit proliferation of CD4 + and CD8 + T cells and of NK cells (Frumento et al. 2002). Other experiments supported a proposed mechanism of inhibition of T cell proliferation, which was triggered by the low concentration of tryptophan resulting from IDO activity and mediated by GCN2 kinase (EIF2AK4, eukaryotic translation initiation factor 2 alpha kinase 4), a sensor for uncharged tRNA (Munn et al. 2005). The contradiction that tryptophan depletion leads to an antimicrobial effect on the one hand and to an inhibition of T cell mediated immune response on the other hand was resolved by Müller and coworkers, who determined the concentration limit necessary for the two effects. They observed that bacterial inhibition (50 % inhibitory concentration of 1.9 µM) took already place at a higher tryptophan concentration whereas tryptophan had to be depleted even further (50 % inhibitory concentration of 0.1 µM) to achieve T cell inhibition (Müller et al. 2009). Besides the described aspects, in an in vivo infection situation dendritic cells (DC) will contribute to the immune response. Further IDO-mediated immune-modulation mechanisms involve DC mediated T cell tolerance (Popov/Schultze 2008). The induction of IDO in epithelial cells near 192
Maren Depke Discussion and Conclusions sites of infection might help to constrain the immune defense reactions to the actually infected cells and to protect the non-infected neighboring cells from damage by immune cells like cytotoxic T cells (King/Thomas 2007). This confinement of immune response evolutionarily developed in favor of the host. Nevertheless, in certain context it benefits pathogens, which might cause persistent infections since the immune system switched to an anti-inflammatory state (Zelante et al. 2009). In relation to IDO1, the increased expression of tryptophanyl-tRNA synthetase (WARS) was conspicuous, which was also confirmed on protein level. In contrast to bacterial tRNA synthetases, the eukaryotic or mammalian enzymes were studied to a lesser extent. The enzyme was identified and described to be IFN-γ induced by two groups, Fleckner and coworkers as well as Buwitt, Bange and colleagues almost 20 years ago (Fleckner et al. 1991, Buwitt et al. 1992, Bange et al. 1992) and separately, the inducibility by IFN-α and IFN-γ was demonstrated (Rubin et al. 1991). The protein domain structure (reviewed by Kisselev 1993) as well as the gene’s exonintron-organization and interferon response elements are long-known (Frolova et al. 1993). It was hypothesized that the induction of tryptophanyl-tRNA synthetase in parallel to the tryptophan catabolizing enzyme IDO aims to protect the host from the tryptophan starvation which is induced in response to proinflammatory stimuli. Tryptophan would be safe from degradation when complexed to tRNA and still available for host protein synthesis (Boasso et al. 2005, Murray MF 2003). Furthermore, the induction of tryptophanyl-tRNA synthetase by interferon was described to find its biological rationale in the enrichment of tryptophan in immune response related and equally interferon inducible proteins like MHC molecules (especially in the Ig-domains). Hence, induction of tryptophanyl-tRNA synthetase was suggested to reflect the heightened need of tryptophanyl-tRNA to sustain the ability to synthesize these proteins (Xue/Wong 1995). More recent literature data assign tRNA synthetases expanded functions (Yang XL et al. 2004). Proteolytic tryptophanyl-tRNA synthetase fragments (Favorova et al. 1989), splice variants (Tolstrup et al. 1995), and transcription starting from a newly identified IFN-γ sensitive promoter (Liu J et al. 2004) were reported. Smaller tryptophanyl-tRNA synthetase variants were shown to act anti-angiogenic (Otani et al. 2002, Wakasugi et al. 2002). In infected S9 cells 6.5 h after start of infection, expression changes of cytokine genes, GTPase/GTP binding proteins, genes involved in (anti)coagulation, anti-oxidant defense, and complement system, lysosomal proteins and adhesins were obvious. This time point also included repression of different branches of de novo lipogenesis like cholesterol, unsaturated fatty acid, and storage lipid biosynthesis. Fatty acid synthase FASN was repressed in transcriptome analysis and was equally reduced in abundance in proteome analysis. Contrarily, several genes which are involved in lipid messenger generation were induced. The hints for induced ceramide/sphingosine biosynthesis link the induction of lipid messenger generating genes to the observation of induced expression of genes associated with death receptor signaling and apoptosis. The transcriptome profiling of infected S9 cells revealed that in the death receptor signaling cascade, the receptor FAS and some initiating caspases were induced in parallel with the ligands TNFSF10 and TNFSF15 and signal transduction genes. But also a caspase inhibitor (CFLAR) and an anti-apoptotic gene (BAG1) were induced, and CASP9, link to the mitochondrial apoptosis pathway, was repressed. Among further pro-apoptotic genes, a set of five apolipoprotein L genes (APOL1, APOL2, APOL3, APOL4, APOL6) was detected with induced expression, of which one, APOL2, was also increased in protein abundance. The APOL genes were of special interest. The 193
Page 1 and 2:
G E N O M E W I D E C H A R A C T E
Page 3 and 4:
Maren Depke C O N T E N T S GENOMEW
Page 5 and 6:
Maren Depke Z U S A M M E N F A S S
Page 7 and 8:
Maren Depke Zusammenfassung der Dis
Page 9 and 10:
Maren Depke S U M M A R Y O F D I S
Page 11 and 12:
Maren Depke Summary of Dissertation
Page 13 and 14:
Maren Depke I N T R O D U C T I O N
Page 15 and 16:
Maren Depke Introduction Besides op
Page 17 and 18:
Maren Depke Introduction Extracellu
Page 19 and 20:
Maren Depke Introduction with heigh
Page 21 and 22:
Maren Depke Introduction 2005). SaP
Page 23 and 24:
Maren Depke Introduction contains a
Page 25 and 26:
Maren Depke Introduction via fibron
Page 27 and 28:
Maren Depke Introduction cathelicid
Page 29 and 30:
Maren Depke Introduction shock are
Page 31 and 32:
Maren Depke Introduction STUDIES OF
Page 33 and 34:
Maren Depke Introduction are effect
Page 35 and 36:
Maren Depke Introduction cell stimu
Page 37 and 38:
Maren Depke Introduction channel CF
Page 39 and 40:
Maren Depke M A T E R I A L A N D M
Page 41:
Maren Depke Material and Methods Li
Page 44 and 45:
Maren Depke Material and Methods Ki
Page 47 and 48:
Maren Depke Material and Methods GE
Page 49:
Maren Depke Material and Methods Ge
Page 52 and 53:
Maren Depke Material and Methods Ho
Page 54 and 55:
Maren Depke Material and Methods Ho
Page 56 and 57:
Maren Depke Material and Methods Pa
Page 58 and 59:
Page 60 and 61:
Page 63 and 64:
corticosterone [pg/ml plasma] corti
Page 65 and 66:
Maren Depke Results Liver Gene Expr
Page 67 and 68:
lood glucose levels [nmol/l] resist
Page 69 and 70:
LBP [ng/ml] CPR [ng/ml] Maren Depke
Page 71 and 72:
Maren Depke Results Liver Gene Expr
Page 73 and 74:
liver weight [g] Maren Depke Result
Page 75 and 76:
infection rate cfu / 10 mg tissue c
Page 77 and 78:
Maren Depke Results Kidney Gene Exp
Page 79 and 80:
Table R.2.1: Genes displaying stati
Page 81 and 82:
Maren Depke BR1 sign DsigB vs wt BR
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Maren Depke Results GENE EXPRESSION
Page 93 and 94:
Maren Depke Results Gene Expression
Page 95 and 96:
log2(ratio C57BL/6 / BALB/c) at IFN
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109:
Page 112 and 113:
Maren Depke Results Host Cell Gene
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Page 132 and 133:
Maren Depke Results Pathogen Gene E
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
S. aureus RN1HG sample conditions a
Page 140 and 141:
Page 142 and 143: Maren Depke Results Pathogen Gene E
Page 148 and 149: mean normalized intensity mean norm
Page 158 and 159: mean normalized intensity Maren Dep
Page 168 and 169: log 2 (ratio) log 2 (ratio) log 2 (
Page 171 and 172: Maren Depke D I S C U S S I O N A N
Page 173 and 174: Maren Depke Discussion and Conclusi
Page 191: Maren Depke Discussion and Conclusi
Page 205: Maren Depke Discussion and Conclusi
Page 208 and 209: Maren Depke References Athanasopoul
Page 210 and 211: Maren Depke References Byrne GI, Le
Page 212 and 213: Maren Depke References Demling R. T
Page 214 and 215: Maren Depke References Foss GS, Pry
Page 216 and 217: Maren Depke References Hagopian K,
Page 218 and 219: Maren Depke References Jin T, Bokar
Page 220 and 221: Maren Depke References Kwan T, Liu
Page 222 and 223: Maren Depke References Luster AD, U
Page 224 and 225: Maren Depke References Namiki S, Na
Page 226 and 227: Maren Depke References Puccetti P.
Page 228 and 229: Maren Depke References Siewert E, B
Page 230 and 231: Maren Depke References van den Akke
Page 232 and 233: Maren Depke References Yang XL, Sch
Page 235: Maren Depke A F F I D A V I T / E R
Page 238 and 239: Maren Depke Acknowledgments Kidney
show all

genomewide characterization of host-pathogen interactions by ...

Create successful ePaper yourself

Delete template?

Save as template?