In this thesis we used a functional genomic approach to study host-pathogen interactions [1]. We analyzed the interaction from the host point of view and in particular from the dendritic cells point of view. Dendritic cells (DCs) constitute a heterogeneous group of antigen-presenting leukocytes important in activation of both innate and adaptive immunity [2]. In the first part of this thesis we explored the possibility to use dendritic cell transcriptomes to generate biomarkers of inflammation that may be useful to test DC activation in vitro and in vivo. We considered DC transcriptomes upon stimulation with different microorganisms and molecules able to induce activation (Schistosoma mansoni eggs, Leishmania Mexicana promastigote, Listeria monocytogenes, LPS, CpG, polyIC, Pam3cys, zymosan) or inhibition (Schistosoma SLA, Leishmania Mexicana amastigote, dexamethasone). We applied a supervised classification method, random forest algorithm, in order to identify an inflammatory signature that can describe, at molecular level, the dendritic cells status in terms of inflammation. The informative genes were selected using a training set (77 samples) and then validated on a testing set (38 samples). The 54 predictive genes selected are able to distinguish very accurately between inflammatory and non inflammatory samples. Amongst these we found genes well known to be involved in the inflammatory process (Icam1, IL-6), as well as genes not tightly correlated with inflammation (Hdac5, Gadd45b). Surprisingly, some genes with unknown function (Txndc16, Isg15) were also selected. The diagnostic performance of the generated signature was assessed against an independent set of samples (D1 cells treated with IFNa, DEX, vitamin D, IL-10, Lactobacillus paracasei, Listeria monocytogenes, LPS, poly I:C, PAM3CYS, zymosan for 24h), by qPCR. Moreover, we validated the inflammatory signature in vivo, by testing the response in splenic DCs isolated from mice treated with LPS and dexamethasone. Most of the studied genes (80%), successfully characterized the activation state of splenic DCs, and differentiated the profile of these cells from that of DCs derived from untreated mice. The small number of genes in our signature allows to use simple, conventional assays, such as quantitative reverse transcriptase-polymerase chain reaction. The increasing availability of laboratory diagnosis by polymerase chain reaction has opened up new possibilities for genomic testing based on the use of genetic signatures, in routine clinical conditions. A second major outcome from whole gene expression studies is the discovery of the molecular pathways induced in complex systems such as host-bacteria interactions [3]. Therefore, in the second part of this thesis we analyzed dendritic cells-bacteria interactions and we focused on the identification of the specific molecular mechanisms induced in DCs upon infections. Among the 1500 genes modulated in DCs by bacteria, we have detected the up-regulation of the cytokine IL22. This interleukin has been shown to have both pro- and anti-inflammatory activity in different chronic inflammatory diseases, in mouse models of infection and in liver injury [4,5]. IL-22 was described to be produced mainly by CD4+ T cells, in particular by Th17, and by NK cells. Only few and controversial data are available on IL-22 production by DC. IL-22 does not seem to influence directly immune cells since the IL-22R1 chain of IL-22 heterodimeric receptor complex is present only in a range of non immune tissues (skin, liver, respiratory system and gastrointestinal tract) [6]. Therefore, in this study we focused on how IL-22 is produced and regulated in DCs. We first demonstrated that IL-22 is produced by DCs in different in vitro systems (D1 cells, BMDC, splenic DCs) and in vivo by using mouse models of systemic inflammation and infection induced by LPS and gram+ bacteria. We showed that IL-22 up-regulation by LPS reaches the maximum level within 2 h post stimulation. Instead IL-22 is induced by IL-23 in total splenocytes with a different kinetic suggesting the existence of different regulatory pathways in different cell types. Using cellular systems (BMDC and splenocytes) derived from wt or Myd88 ko mice, we established that Myd88 has a key role in IL-22 up-regulation induced by LPS, as well as, by IL-23. We identified a Myd88-dependent and IL-23-independent production of IL-22 in BMDC upon LPS stimulation. Moreover, we found an IL-23-dependent and Myd88-dependent up-regulation of IL-22 after induction with IL-23 in total splenocytes and in splenic DCs. Finally, we performed a preliminary experiment in order to unravel IL-22 role in the liver since we could measure IL-22 receptor expression in this organ. Microarray experiment on primary hepatocytes, stimulated for 24 h with recombinant IL-22, suggests a role for IL-22 in the liver as a cytokine that promotes innate immune activation in terms of induction of antimicrobial peptides and secretion of chemokines that recruit phagocytes. The cellular source of IL-22 in the liver is currently under investigation.

(2009). Gene expression profiling in host-pathogen interactions and identification of the molecular mechanisms involved in dendrictic cells activation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).

Gene expression profiling in host-pathogen interactions and identification of the molecular mechanisms involved in dendrictic cells activation

TORRI, ANNA
2009-11-26

Abstract

In this thesis we used a functional genomic approach to study host-pathogen interactions [1]. We analyzed the interaction from the host point of view and in particular from the dendritic cells point of view. Dendritic cells (DCs) constitute a heterogeneous group of antigen-presenting leukocytes important in activation of both innate and adaptive immunity [2]. In the first part of this thesis we explored the possibility to use dendritic cell transcriptomes to generate biomarkers of inflammation that may be useful to test DC activation in vitro and in vivo. We considered DC transcriptomes upon stimulation with different microorganisms and molecules able to induce activation (Schistosoma mansoni eggs, Leishmania Mexicana promastigote, Listeria monocytogenes, LPS, CpG, polyIC, Pam3cys, zymosan) or inhibition (Schistosoma SLA, Leishmania Mexicana amastigote, dexamethasone). We applied a supervised classification method, random forest algorithm, in order to identify an inflammatory signature that can describe, at molecular level, the dendritic cells status in terms of inflammation. The informative genes were selected using a training set (77 samples) and then validated on a testing set (38 samples). The 54 predictive genes selected are able to distinguish very accurately between inflammatory and non inflammatory samples. Amongst these we found genes well known to be involved in the inflammatory process (Icam1, IL-6), as well as genes not tightly correlated with inflammation (Hdac5, Gadd45b). Surprisingly, some genes with unknown function (Txndc16, Isg15) were also selected. The diagnostic performance of the generated signature was assessed against an independent set of samples (D1 cells treated with IFNa, DEX, vitamin D, IL-10, Lactobacillus paracasei, Listeria monocytogenes, LPS, poly I:C, PAM3CYS, zymosan for 24h), by qPCR. Moreover, we validated the inflammatory signature in vivo, by testing the response in splenic DCs isolated from mice treated with LPS and dexamethasone. Most of the studied genes (80%), successfully characterized the activation state of splenic DCs, and differentiated the profile of these cells from that of DCs derived from untreated mice. The small number of genes in our signature allows to use simple, conventional assays, such as quantitative reverse transcriptase-polymerase chain reaction. The increasing availability of laboratory diagnosis by polymerase chain reaction has opened up new possibilities for genomic testing based on the use of genetic signatures, in routine clinical conditions. A second major outcome from whole gene expression studies is the discovery of the molecular pathways induced in complex systems such as host-bacteria interactions [3]. Therefore, in the second part of this thesis we analyzed dendritic cells-bacteria interactions and we focused on the identification of the specific molecular mechanisms induced in DCs upon infections. Among the 1500 genes modulated in DCs by bacteria, we have detected the up-regulation of the cytokine IL22. This interleukin has been shown to have both pro- and anti-inflammatory activity in different chronic inflammatory diseases, in mouse models of infection and in liver injury [4,5]. IL-22 was described to be produced mainly by CD4+ T cells, in particular by Th17, and by NK cells. Only few and controversial data are available on IL-22 production by DC. IL-22 does not seem to influence directly immune cells since the IL-22R1 chain of IL-22 heterodimeric receptor complex is present only in a range of non immune tissues (skin, liver, respiratory system and gastrointestinal tract) [6]. Therefore, in this study we focused on how IL-22 is produced and regulated in DCs. We first demonstrated that IL-22 is produced by DCs in different in vitro systems (D1 cells, BMDC, splenic DCs) and in vivo by using mouse models of systemic inflammation and infection induced by LPS and gram+ bacteria. We showed that IL-22 up-regulation by LPS reaches the maximum level within 2 h post stimulation. Instead IL-22 is induced by IL-23 in total splenocytes with a different kinetic suggesting the existence of different regulatory pathways in different cell types. Using cellular systems (BMDC and splenocytes) derived from wt or Myd88 ko mice, we established that Myd88 has a key role in IL-22 up-regulation induced by LPS, as well as, by IL-23. We identified a Myd88-dependent and IL-23-independent production of IL-22 in BMDC upon LPS stimulation. Moreover, we found an IL-23-dependent and Myd88-dependent up-regulation of IL-22 after induction with IL-23 in total splenocytes and in splenic DCs. Finally, we performed a preliminary experiment in order to unravel IL-22 role in the liver since we could measure IL-22 receptor expression in this organ. Microarray experiment on primary hepatocytes, stimulated for 24 h with recombinant IL-22, suggests a role for IL-22 in the liver as a cytokine that promotes innate immune activation in terms of induction of antimicrobial peptides and secretion of chemokines that recruit phagocytes. The cellular source of IL-22 in the liver is currently under investigation.
FOTI, MARIA
dendritic cells, functional genomics, gram positive bacteria, inflammatory signature, interleukin-22
MED/04 - PATOLOGIA GENERALE
English
Scuola di Dottorato in Medicina Traslazionale e Molecolare
MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 45R
22
2008/2009
(2009). Gene expression profiling in host-pathogen interactions and identification of the molecular mechanisms involved in dendrictic cells activation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).
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