Gene Expression Signatures of Peripheral CD4 T Cells Clearly Discriminate Between Patients with Acute and Chronic Hepatitis B Infection title ( HEPATOLOGY.

Gene Expression Signatures ofPeripheral CD4 T Cells Clearly Discriminate Between Patients with Acute and Chronic Hepatitis B Infection

title

( HEPATOLOGY 2009;49:781-790.)

1.Background

2.Methods

3.Result

4.Disscussion

5.Abstract

background

Acute HBV 5% in adults

CHB

Asymptomatic CHBcirrhosis /carcinoma

A strong genetic component with modified gene expression seems to be a major driving force affecting the course of viral hepatitis.

background

T cell hyporesponsiveness/dysfunction

CD4+CD25+ regulatory Tcells (Tregs)persistent infection

limit excessive immunopathological damage

facilitate viral persistence

background

the frequency / function CD4+CD25+ regulatory Tcells

severe CHB infection mild CHB /early AVH-B infection≥

The molecular mechanisms of host response against HBV

is still unknown. ？

background

idea Tregs

CD4 T cells from peripheral blood of patients with AVH-B and CHB infection in comparison to HCs.

to gain insight on how the status of infection influences the overall expression pattern of CD4 T cells,

to further analyze their gene expression profiles using microarray technology.

to choose 350 CD4 T cell–specific genes, customized to investigate Treg associated genes.

● Patients and Samples.

● Isolation of CD4 T Cells from PBMCs and FlowCytometric Analysis.

● Microarray Analysis.

● Statistical Analysis.

Patients and Methods

Microarray Analysis

Microarray is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target .

Patients and Methods

Microarray Analysis

Patients and Methods

Patients and Methods

AVH-B: ALT>10ULN with no past history and clinical, biochemical, or radiological evidence, HBsAg +, HBsAb-,HBeAg+, anti-HBeAg–, HBcAg IgM+CHB: HBsAg + >6m, ALT>1.5ULN, and histological evidence of chronic hepatitisHCs: with no previous history or current evidence, HBsAb(+)>300 IU/mL

Patients and Methods

Exclusion criteria :

▲ regular alcohol consumption (40 g/day for the past 5 years), diabetes, severe systemic illness, pregnancy, hepatocellular carcinoma

▲ coinfection with human immunodeficiency virus or other hepatitis viruses

▲ immunosuppressive therapy for other associated illness.

● We analyzed peripheral blood from 19 AVH-B patients, 21 CHB patients, and 12 HCs to determine the percentage of CD4+ T cells and CD4+CD25+Foxp3 Tregs.

● Via flow cytometry

result

1. Frequency Analysis of Circulating CD4+CD25+ Foxp3 Tregs

·

result

result

The frequency of CD4+T cells ： similar (AVH-B, 29%; CHB, 34%; HC, 30%)

The frequency of CD4+CD25+Foxp3 Tregs ： different

(AVH-B, 7%; CHB, 3.96%; HC, 2.18%)

● CD4T cells were isolated from peripheral blood (92% to 96% purity) for RNA extraction.

● four (three for AVH-B) individual measures per donor pool

● A unique set of 350 genes to establish an expression

profile of CD4 cells.

● significant expression levels could be detected for 316 genes.

result

2. Functional Dissection of Expression Signatures in AVH-B and CHB Patients

result

2. Functional Dissection of Expression Signatures in AVH-B and CHB Patients

Fig: A clear separation of the three CD4poolsby the principal component analysisThe use of this statistical technique allowed us to determine the key variables in our multidimensional dataset.

● 118/316 differentially in (AVH-B and CHB)/HCs

● 100 genes were assigned to GO terms

using the Source annotation tool

result

GO(gene ontology) 是基因本体论联合会所建立的数据库，旨在建立一个适用于各个物种的，对基因和蛋白功能进行限定和描述的，并随着研究的不断深入而更新的语言词汇标准。 GO是多种生物学本体论语言中的一种，提供了三层结构的系统定义方式，

用于描述基因产物的功能。 通过 GoFigure 网站，可以提交 DNA 或者蛋白序列，搜查的结

果会清晰具体地注释目标序列在分子功能、生物学途径、细胞组件

三方面的功能。 http://www.geneontology.org/GO.doc.shtml

result

The majority of the genes were related to

cellular processes(24%), biological regulation (14%),

response to stimulus(13%), metabolic processes(12%),

developmental processes(11%), multicellular organismal processes (8%),and immune system processes (7%).

result

result

With respect to the microarray-based analysis

of the viral infection process,

we further dissected the GO term

“response to stimulus”(46 genes) (see Fig.)

and “immune system process” (24genes) (see Fig).

result

result

result

Cluster Mapping and Affected Signaling Modules

result

All genes displayed in the cluster map.The cluster map organized into six sectionsAccording to gene expression behavior .The average relative gene expression values were calculated from four (CHB patients and HCs) and three (AVH-B patients) individual measures, respectively.

Cluster Mapping and Affected Signaling Modules

result

So: Different outcome of HBV (CHB/ AVH-B)/ HC

s)

different cluster mapping

result

Cluster Mapping and Affected Signaling Modules

result

All genes displayed in the cluster map were linked to signaling maps stored in public databases by using MAPP Findersoftware. T cell receptor (TCR) signaling, cell adhesion,mitogen-activated protein kinase kinase (MAPKK) signaling,cell cycle/cell proliferation, apoptosis, cytokines,and chemokines were identified as the most prominentlyaffected modules (see Fig. ).

result

Fig:Signaling module depicting gene sets activated in CD4 T cells from AVH-B patients

result

TIAM AVH>CVH

IL-9, IL-4, IL1R2, IFNA1

PECAM1

result

resultQuantitative RT-PCR

Fig: Quantitative RT-PCR analysis of selected genes

The frequency of CD4+CD25+Foxp3 Tregs ： different

(AVH-B, 7%; CHB, 3.96%; HC, 2.18%)

Tregs AVH-B> CHB> HC

discussion

discussion

the number of Tregs during the convalescence phase of AVH-B infection increases before the normal level is restored after resolutionof the infection

Discussion1:the molecular events are largely unknown.

discussion

This dynamic modulation of the antiviral immune response via self-limited action of suppressorTregs might support the healing process at the site of infection. The activity of Tregs might prevent the extended tissue damage resulting from an unchecked proinflammatory response.

On the other hand, a spatiotemporal dysregulation of Treg activity—that is, uncontrolled establishment of suppressor activity at the site of infection— could promote viral persistence andprogression to chronicity.

discussion

Microarray technology has a tremendous potential forcomplementing classic biological and clinical parameters;therefore, we used a human Treg chip16 to dissect specificgene networks affected by the action of Tregs on CD4T cells, which may contribute to the pathogenic stages ofacute and chronic HBV infection.

Disscussion 2:

an extremely large prospective patient cohort will be needed

T cell receptor (TCR) signaling, cell adhesion,

mitogen-activated protein kinase kinase (MAPKK) signaling,cell cycle/cell proliferation, apoptosis, cytokines,and chemokines

discussion

Disscussion3:

the relationship between the related genes with HBV

The information generated in this study suggests

that the in-depth investigation of the changes in major gene programs during the follow-up of AVH-B and CHB patients would allow us to elucidate the molecular pathways underlying the decision-making processes.

discussion

Abstract

CD4 T and regulatory T cells (Tregs) seem to play a key role in persistence of hepatitis B virus (HBV) infection.

However, the molecular events by which Tregs exert their modulatory activity are largely unknown.

Abstract

The transcriptional profiles of CD4 T cells of healthy controls (HCs) and patients affected by acute hepatitis B (AVH-B) or chronic hepatitis B (CHB) infection were established using a custom expression array consisting of 350 genes relevant for CD4 T cell and Treg function.

These studies were complemented by real-time reversetranscription polymerase chain reaction.

Abstract基因表达谱

Peripheral blood mononuclear cells (PBMCs) were

also analyzed for the presence of Tregs,

which Were more abundant in the acute stage of the

disease (7%) than in HCs and CHB infection

(HCs versus AVH-B, P0.003; AVH-B versusCHB, P 0.04).

180 genes (34%) intrinsically differentiate

HBV-infected patients from HCs.

Abstract

Using gene ontology, we identified T cell receptor signaling and clusterization, mitogen-activated protein kinase kinase signaling, cell adhesion, cytokines and inflammatory responses, cell cycle/cell proliferation, and apoptosis as the most prominentaffected modules.

A higher expression of CCR1, CCR3, CCR4, CCR5, and CCR8 was seen in AVH-B than in CHB-infected patients and HCs.

Annotation of the interconnected functional network of genes provided a unique representation of global immune activation during acute infection. Almost all genes were down-regulated in patients with CHB infection.

Abstract

Conclusion: The fingerprints enable clear discrimination b

etween patients suffering from AVH-B or CHB infection.

The observed profiles suggest accumulation of effector Tcells with a potential role in necro-inflammation during the acute stage.

Subsequent downregulated effector functions support the hypothesis of suppressed CD4 effector T cells favoring viral persistence in the chronic infection stage.

Abstract

Tips:

★ 调节性 T 细胞是热点研究问题 （分子水平）

★ 生物信息学技术 的应用

★ 展望