Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria

Steroids 76 (2011) 1204–1212

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Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria D. Delic´ a , H. Ellinger-Ziegelbauer b , H.W. Vohr b , M. Dkhil c,d , S. Al-Quraishy c , F. Wunderlich a,c,∗ a

Division of Molecular Parasitology and Centre for Biological and Medical Research, Heinrich-Heine-University, Universitaetsstr. 1, 40225 Duesseldorf, Germany Bayer Healthcare AG, Department of Molecular and Genetic Toxicology, 42096 Wuppertal, Germany Zoology Department, College of Science, King Saud University, 11451 Riyadh, Saudi Arabia d Department of Zoology and Entomology, Faculty of Science, Helwan University, Helwan, Egypt b c

a r t i c l e

i n f o

Article history: Received 7 March 2011 Received in revised form 20 May 2011 Accepted 25 May 2011 Available online 12 June 2011 Keywords: Testosterone Gene expression Liver Immunity Malaria

a b s t r a c t Blood-stage malaria of Plasmodium chabaudi is characterized by its responsiveness to testosterone (T): T suppresses development of protective immunity, whereas once acquired immunity is T-unresponsive. Here, we have analyzed the liver, a T target and lymphoid organ with anti-malaria activity, for its T-responsiveness of gene expression in immune mice. Using Affymetrix microarray technology, in combination with quantitative RT-PCR, we have identified (i) T-unresponsive expression of newly acquired mRNAs encoding diverse sequences of IgG- and IgM-antibodies, (ii) 24 genes whose expression has become T-unresponsive including those encoding the TH 2 response promoting EHMT2 and the erythrocyte membrane protein band 7.2 STOM, (iii) T-unresponsive expression of mRNAs for the cytokines IL-1␤, IL-6, TNF␣, and IFN␥, as well as iNOS, which are even not inducible by infection, and (iv) 35 genes retaining their T-responsiveness, which include those encoding the infection-inducible acute phase proteins SAA1, SAA2, and ORM2 as well as those of liver metabolism which encode the T-downregulated female-prevalent enzymes CYP2B9, CYP2B13, CYP3A41, CYP7A1, and SULT2A2 and the T-upregulated male-prevalent enzymes CYP2D9, CYP7B1, UGT2B1, HSD3B2, HSD3B5, respectively. The mRNA of the latter T-metabolizing enzyme is even 5-fold increased by T, suggesting a decrease in the effective T concentrations in the liver of immune mice. Collectively, our data suggest that the liver, which has acquired a selective T-unresponsiveness of gene expression, contributes to the acquired T-unresponsive, antibody-mediated protective immunity to blood-stage malaria of P. chabaudi. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Natural immunity to malaria is directed against the blood-stages of the infectious agent, parasitic protozoans of the genus Plasmodium [1]. This type of immunity can be acquired, though only very slowly, in malaria-endemic areas: it diminishes disease symptoms, but it cannot prevent parasitemia during malaria season [1]. A convenient model to investigate blood-stage malaria is Plasmodium chabaudi in the mouse. A major characteristic of P. chabaudi malaria is its responsiveness to testosterone (T). Indeed, T suppresses development of protective immunity thus causing a lethal

∗ Corresponding author at: Division of Molecular Parasitology and Centre for Biological and Medical Research, Heinrich-Heine-University, Universitaetsstr. 1, 40225 Duesseldorf, Germany. Tel.: +49 178 3524210; fax: +49 211 81 14827. ´ E-mail addresses: [email protected] (D. Delic), [email protected] (H. Ellinger-Ziegelbauer), [email protected] (H.W. Vohr), [email protected] (M. Dkhil), [email protected] (S. Al-Quraishy), [email protected] (F. Wunderlich). 0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2011.05.013

outcome of otherwise self-healing malaria [2–5]. This immunosuppressive effect of T is not transient, but rather persists for a long time, even after withdrawal of T [6,7]. Remarkably, however, when once acquired, protective immunity to P. chabaudi malaria has become T unresponsive [8]. The liver is an important lymphoid organ [9,10]. Moreover, the liver is apparently a prominent effector site against malarial bloodstages [11–14]. For instance, lymphotoxin ␤ receptor-deficient mice are devoid of secondary lymphoid tissues and contain a defect spleen [15,16], which is currently thought as to be the major effector site against blood-stage malaria. Nevertheless, these mice are able to self-heal blood-stage infections with P. chabaudi and to develop long-lasting protective immune mechanisms [11]. In these defect LT␤−/− mice, however, T also suppresses development of protective immunity [11] just as in intact mice [2,5]. These data, together with the fact that the liver is a T-target site [17–19], support the view that the liver is involved in mediating the suppressive T effect [14]. Currently, however, it is not known as to whether the liver retains its T-responsiveness upon acquiring T-unresponsive

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protective immunity to P. chabaudi malaria. This prompted us to investigate the effect of T on the liver transcriptome in immune mice. 2. Experimental 2.1. Mice Mice of the inbred strain C57BL/6 were bred under specified pathogen-free conditions at the central animal facilities of our university at Duesseldorf. They were housed in plastic cages and received a standard diet (Wohrlin, Bad Salzuflen, Germany) and water ad libitum. Experiments were performed exclusively with female mice and were approved by the State authorities. 2.2. Infections P. chabaudi was passaged weekly in NMRI mice [11,20]. Female C57BL/6 mice were i.p. injected with 106 P. chabaudi-parasitized erythrocytes. Thin smears from tail blood were Giemsa-stained. Parasitemia and cell number were measured in a Neubauer chamber. 2.3. Mice immune to P. chabaudi Approximately 10–12 weeks old C57BL/6 mice were i.p. infected with 106 P. chabaudi-infected erythrocytes. Those mice, which survived the primary challenge for at least 42 days, were taken as immune mice for T-treatment. 2.4. Testosterone treatment Immune mice and naïve mice of the same age received subcutaneous injections of 100 ␮l sesame oil containing 0.9 mg T (Testosterone-Depot-50, Schering, Berlin, Germany) twice a week for 3 weeks [2,7]. Controls were treated only with the vehicle. 2.5. RNA-isolation Mice were killed by cervical dislocation, livers were aseptically removed, and liver pieces were rapidly frozen in liquid nitrogen and stored at −80 ◦ C until use. RNA was isolated using approximately 250 mg frozen liver homogenized in 5 ml Trizol (Peqlab biotechnology, Erlangen, Germany) as described recently [7]. RNA concentrations were determined at 260 nm and RNA-quality was examined by gel electrophoresis using agarose delivered from Biozym Scientific (Hessisch Oldendorf, Germany).

Fig. 1. Course of blood-stage infections with P. chabaudi in naïve and immune C57BL/6 mice. Mice were pre-treated with T or vehicle for 3 weeks before infecting with 106 P. chabaudi-parasitized erythrocytes. Values represent means ± SD (n = 8 mice). Only half of the SD bar is given for clarity reasons.

2.7. Data analysis The software ‘Expression Analyst’ (GeneDataAG, Basel, Switzerland) was applied for gene expression analysis. Only those probe sets were evaluated which revealed an expression intensity >20 in each sample and were more than 2-fold deregulated. These probe sets were then subjected to two-way ANOVA (p < 0.01). Genes were analyzed using the Database for Annotation, Visualization and Integrated Database (DAVID) [21]. Gene Cluster 3.0 [22–24] was used for principal component analysis. Data were log2 -transformed and normalized to the mean expression value for control mice. Hierachical clustering was done using uncentered correlation and average linkage mode. 2.8. Quantitative real-time PCR DNA-free RNA was used for synthesis of cDNA using the QuantiTectTM Reverse Transcription Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The QuantiTectTM SYBR® Green PCR kit (Qiagen) was applied for amplifications in the ABI Prism® 7500HT Sequence Detection System (AppliedBiosystems, Darmstadt, Germany) with gene-specific QuantiTectTM primers delivered by Qiagen (Hilden, Germany). PCR reactions were performed and evaluated as detailed recently [7].

2.6. Hybridization of microarrays 2.9. Statistical analysis Quality of RNA was re-controlled with a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) on a RNA 6000 Nano chip. The One cycle kit (Affymetrix Inc., Santa Clara, USA) was then used to synthesize biotin-labeled cRNA from 5 ␮g total RNA according to the manufacturer’s protocol. Approximately 15 ␮g biotin-labeled cRNA was then hybridized to Affymetrix MOE430A Gene Chips® , and fluorescence of the hybridized cRNA was read with an Affymetrix 300 Scanner. The chips were quality-controlled with the software “Expressionist Refiner” (GeneDataAG, Basel, Switzerland). Each probe set is represented by 11 pairs of 25mer perfect match and mismatch oligonucleotides. The intensities of all 11 probe pairs per probe were condensed to one intensity value using the MAS 5.0 statistical algorithms implemented in the Expressionist software. For reasons of comparability, the microarrays were scaled after condensing to an average signal intensity of 100.

Significance was evaluated by two-way ANOVA with Dunnett and Bonferoni post-hoc-tests using the statistical package program SPSS version 17.0. 3. Results 3.1. T-unresponsiveness of acquired immunity Female mice of the inbred strain C57BL/6 are able to self-heal blood-stage infections with P. chabaudi malaria. When mice are challenged with 106 P. chabaudi-infected erythrocytes, approximately 87.5% of the mice survive the infections (Fig. 1). Peak parasitemia of approximately 43% occurs on day 8 p.i., which is followed by a smaller peak of approximately 12% on day 18 p.i. Testosterone (T) prevents self-healing and all mice succumb to

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as CYP2B9, CYP2B13, CYP3A41, CYP7A1, and SULT2A2, respectively. Their T-response is about the same in both immune and naïve mice. Only the male-prevalent HSD3B5 exhibits a T-induced 5-fold higher expression in immune than naïve mice (cf. Table 1 and Table S1). Moreover, the complement component C6 exhibits an approximately 2-fold higher expression in T-treated immune mice than in T-treated naïve mice (cf. Table 1 and Table S1). Such an upregulation of HSD3B5 and C6 can be also verified by quantitative RT-PCR (Fig. S3). 3.3. T-unresponsive genes in liver of immune mice

Fig. 2. Venn-diagram summarizing the numbers of T-affected genes in naïve mice (horizontal ellipses) and in immune mice (vertical ellipses). The overlap of the ellipses represents numbers of those genes deregulated by T in both groups of mice.

infection, mainly during the crisis phase between day 8 and day 13 of infection, and peak parasitemia is raised to approximately 70%. Mice which have self-healed the infections have acquired protective immunity. This becomes evident upon homolog reinfections of those mice, which have survived primary infections, as survival of all mice without any visible symptoms of sickness and as a very low peak parasitemia of approximately 3.5% on day 8 p.i. (Fig. 1). T-pretreatment of immune mice for 3 weeks affects neither survival rate nor causes any significant rise in parasitemia (Fig. 1). 3.2. T-responsive gene expression in liver of immune mice In order to identify possible effects of T on hepatic gene expression in immune mice, we have compared 4 groups of mice, with 3 mice per group: (i) control naïve mice (C), (ii) naïve mice treated with T (T), (iii) control immune mice (Cimm ), and (iv) immune mice treated with T (Timm ), respectively. Individual livers are subjected to Affymetrix microarray analysis, with principal component analysis (Fig. S1) and expression profile clustering (Fig. S2). The Venn-diagram in Fig. 2 summarizes the numbers of T-upregulated and T-downregulated genes in immune vs. naïve mice identified by Affymetrix microarrays. In immune mice, the expression of 39 genes is affected by T, with 17 T-upregulated genes and 22 T-downregulated genes (vertical ellipses in Fig. 2). By contrast, T affects the expression of 156 genes in naïve mice with 51 Tupregulated and 105 T-downregulated genes (horizontal ellipses in Fig. 2). The overlap of the ellipses represents the same genes deregulated by T in both immune and naïve mice (Fig. 2). Tables 1 and 2 summarize the 17 T-upregulated and 22 Tdownregulated genes in the liver of immune mice. Among the 17 T-upregulated genes, there are the 4 genes Susd4, Lama3, BC024137, and Ugt2b1 the expression of which is T-responsive only in immune mice. The remaining 13 genes are also T-upregulated in naïve mice (Table S1), among which are the genes encoding the male-prevalent enzymes such as CYP2D9, CYP7B1, UGT2B1, HSD3B5, and HSD3B2, respectively. The 22 T-downregulated genes contain 9 genes which are only found in immune mice. The T-downregulated genes comprise also those, which code for the female-prevalent enzymes such

Table 3 summarizes those 24 genes, whose expression is T-responsive in naïve mice, but has become significantly Tunresponsive in immune mice. Among these genes are Ehmt2, Stom, and Hnf4a (Table 3). Moreover, we have wondered why our above microarray analyses have not revealed any changes in the expression of antibody-encoding genes. Indeed, protective immunity to P. chabaudi malaria is mediated by antibodies [8,25,26] and the liver as lymphoid organ contains intrahepatic B cells, but also circulating B cells [9,10]. In order to evaluate possible T-effects on antibodyencoding genes, we have first compared the ratio of Cimm to C. As expected, the liver of immune mice expresses a number of genes encoding diverse regions of IgG and IgM in immune mice (Table 4). Remarkably, however, none of these genes is significantly affected by pre-treatment with T, as deduced from the ratio Timm /Cimm (Table 4). 3.4. T-unresponsiveness of genes affected by P. chabaudi In order to explore possible T-effects on expression of P. chabaudi-responsive genes, immune mice (Cimm ) and T-treated immune mice (Timm ) were challenged with 106 P. chabaudi-infected erythrocytes for 1 day (Cinf , Tinf ), before individual livers of the three mice of the four groups were subjected to Affymetrix microarray analysis (Fig. S1). Two-way ANOVA analysis over the four groups Cimm , Cinf , Timm , and Tinf has surprisingly revealed that the P. chabaudi infections affected hepatic expression of only 4 genes in the control immune mice. Indeed, only the expression of the genes encoding the acute phase proteins SAA2, SAA1, and ORM2 are upregulated and that of TXNL1 is significantly downregulated. In T-pretreated immune mice, P. chabaudi infections upregulate the expression of Saa2, Saa1, Orm2, and Acnat2, and significantly downregulate the expression of Tenc1, Adamts5, Nt5e, Atoh8, and Lipg, respectively (Table 5). To our surprise, none of the genes encoding the cytokines IL-1␤, IL-6, TNF␣, and INF␥, as well as iNOS has been identified to be affected by infection in immune mice as analyzed by Affymetrix microarrays. This result can be confirmed using quantitative RT-PCR analysis (Fig. 3). Even on day 8 p.i., the expression of these genes is completely unresponsive to P. chabaudi infection in immune mice and is not changed by pre-treatment with T (Fig. 3). By contrast, hepatic mRNA expression of these genes in naïve mice is significantly induced by P. chabaudi infections, though pre-treatment with T does not significantly influence this infectioninduced upregulation in naïve mice (Fig. 3). 4. Discussion Mice which have acquired immunity to P. chabaudi malaria have concomitantly acquired an unresponsiveness to testosterone (T), evidenced as a T-induced lethal outcome of blood-stage malaria only in naïve mice, but not in immune mice (cf. Fig. 1). Here we demonstrate that the liver undergoes complex changes in its gene expression during acquiring T-unresponsive immunity.

Table 1 T-upregulated genes in the liver of female C57BL/6 mice immune to P. chabaudi malaria. Gene no.

PMID

Functions

1420531 at

8319586, 8477648, 8647315

0.0044

1419349 a at

16547391, 4074718

Involved in steroid synthesis with both dehydrogenase and isomerase activity; male-specific Male-specific; regulate by growth hormone; sterol 16a-hydroxylase

16.23

0.0026

1449844 at

14.59

0.0003

1420379 at

16807376, 12399219

Gene symbol

Gene name

Timm /Cimm

p-Value

1

Hsd3b5

Hydroxysteroid dehydrogenase-5. Delta<5>-3-beta

248.63

0.0033

2

Cyp2d9

23.74

3

Slcolal

Cytochrome P450, family 2, subfamily d, polypeptide 9 Solute carrier organic anion transporter family, member lal Solute carrier organic anion transporter family, member lal

Slcolal

Affymetrix probe ID

C6

Complement component 6

9.46

0.0091

1449308 at

4007963

5 6

Susd4 Lama3

Sushi domain containing 4 Laminin. Alpha 3

5.45 4.87

0.0034 0.0003

1424221 at 1427512 a at

12079511

7

Hsd3b2

Hydroxysteroid dehydrogenase-2. Delta<5>-3-beta

3.28

0.0006

1425127 at

8647315

8 9

BC024137 Cyp7bl

cDNA sequence BC024137 Cytochrome P450, family 7, subfamily b, polypeptide 1

3.24 3.19

0.0071 0.0088

1427513 at 1421075 s at

9295351, 8530364, 11284740

10

Nox4

NADPH oxidase 4

2.67

0.0011

1451827 a at

11376945, 15155719

11

Tgfbli4

Transforming growth factor beta 1 induced transcript 4 Transforming growth factor beta 1 induced transcript 4 Aminolevulinic acid synthase 2, erythroid

2.62

0.0023

1454758 a at

19329776

2.56

0.0042

1425742 a at

2.55

0.0042

1451675 a at

10773455

SPRY domain-containing SOCS box 4 ATP-binding cassette, sub-family G (WHITE), member 2 Aldehyde oxidase 3

2.38 2.35

0.0029 0.0004

1451418 a at 1422906 at

19084913

2.31

0.0051

1418858 at

18757308

UDP glucuronosyltransferase 2 family, polypeptide Bl Choline phosphotransferase 1

2.04

0.0005

1424934 at

19131521

2.01

0.0006

1455901 at

12097155

Tgfbli4 12

Alas2

13 14

Spsb4 Abcg2

15

Aox3

16

Ugt2bl

17

Chptl

D. Deli´c et al. / Steroids 76 (2011) 1204–1212

4

Organic anion transporter with male predonderance in the liver; regulated by androgens and growth hormone secretion pattern Complement component produced in the liver; higher expression in males than in females Unknown Expression correlated with dedifferentiation of hepatocellular tumors Important enzyme for steroid synthesis with both dehydrogenease and isomerase activity; minor, male-preponderant isoform Unknown Oxysterol-7-a-hydroxylase involved in synthesis of 7-a-hydroxylated bile acids; male specific expression in the liver NAPDH oxidase generating ROS; Nox4 is most frequently expressed in tumor cells Contributes to the regulation of haematopoietic precurser cells

Catalyzes the first step in heme synthesis in erythroblasts and is induced by erythroblast Unknown Breast cancer resistance protein which mediates biliary elimination Maturation of secretory cells was associated with progressive increases in the expression of Fmo3, Ponl, Aox3, and Cyp2f2 between late embryonic and postnatal periods Male-predominant expression in the liver; inductive effects by testosterone Involved in phospholipid metabolism

Genes in grey indicate those which are also upregulated by T in naïve mice.

1207

1208

Table 2 T-downregulated genes in the liver of female C57BL/6 mice immune to P. chabaudi malaria. Affymetrix probe ID

PMID

Functions

0.47

0.0001

1451681 at

10876094

CD36 antigen

0.47

0.0091

1450884 at

19471024

Zbpl

Z-DNA binding protein 1

0.46

0.0072

1429947 a at

4

Fmol

Flavin containing monooxygenase 1

0.45

0.0013

1417429 at

10564822, 17618271 10462538

Catalyze the dehydrogenation of retinols, including 9-cis retinol, and to exhibit 3a- and 17p-hydroxysteroid dehydrogenase activities CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior Upregulated in macrophages; cytosolic DNA sensor involved in type I interferon response Expression of Fmol is suppressed by overproduced nitric oxide

5

Cyp2a4/Cyp2a5

Cytochrome P450, family 2, subfamily a, polypeptide 4/5

0.41

0.0017

1422230 s at

10490589

6

Trim24

Tripartite motif protein 24

0.39

0.0018

1427259 at

19556538

Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP Trim24 ubiquitylates and negatively regulates p53 levels

7

Trim24 Prlr

Tripartite motif protein 24 Prolactin receptor

0.22 0.38

0.0038 0.0039

1427258 at 1451844 at

6329976

Treatment of female mice with testosterone reduces prlr levels

8

Prlr Prlr Rcan2

Prolactin receptor Prolactin receptor Regulator of calcineurin 2

0.22 0.21 0.31

0.0079 0.0057 0.0004

1450226 at 1421382 at 1421425 a at

16648267

Functions as calcineurin fascilitator

9

Abcd2

ATP-binding cassette, sub-family d (ALD), member 2

0.28

0.0001

1419748 at

10

Cyp2c38

Cytochrome P450, family 2, subfamily c, polypeptide 38

0.25

0.0001

1452501 at

8577752, 16249184 19358898

ABC transporter in the peroxisome membrane, LXR prevents expression by interfering with binding of SREBlc 91.8% sequence identity to Cyp2c39

11

CypJal

Cytochrome P450, family 7, subfamily a, polypeptide 1

0.24

0.0011

1438743 at

1036899

Gene symbol

Gene name

1

CRAD-L

Cis-retinol/3alphahydroxysterol short-chain dehydrogenase-like

2

Cd36

3

Timm /Cimm

Nt5e

5 nucleotidase. Ecto

0.21

0.0007

1422974 at

9306922

Cholesterol-7alpha-hydroxylase that is expressed in a female-specific manner Reduced activity by ischemia

12

Fmo2

Flavin containing monooxygenase 2

0.21

0.0003

1422904 at

16872995

Typically expressed at high levels in lung

13

Nt5e

5 nucleotidase. Ecto

0.21

0.0071

1422974 at

9306922

Reduced activity by ischemia

14 15

C730036D15Rik Cyp2c39

Acyl-coenzyme A amino acid N-acyltransferase 2 Cytochrome P450, family 2, subfamily c, polypeptide 39

0.17 0.14

0.0005 0.0013

1425150 at 1421363 at

16

Cypl7al

Cytochrome P450, family 17, subfamily a, polypeptide 1

0.11

0.0001

1417017 at

14623888, 9721182 15761033

17 18

AB056442 Cyp2b9

cDNA sequence AB056442 Cytochrome P450, family 2, subfamily b, polypeptide 9

0.04 0.04

0.0025 0.0008

1419751 x at 1419590 at

15381067



19

72% similar to bile acid-Coenzyme A: amino acid N-acyltransferase Acts as a retinoic acid 4-hydroxylase. Metabolizes arachidonic acid to 14, 15-ds-epoxyeicosatrienoic Exhibits 17alpha-hydroxylase/17,20-lyase activity, a crucial enzyme for the synthesis of all steroid hormenes, and squalene monooxygenase (epoxidase) activity involved in cholesterol biosynthesis Organic anion transporter 6 Female specific expression depends on growth hormone

16150593

64% identical and 79% similar to Slc22a9, anorganic anion transporter in transepithelial transport of steroid sulfates

20

BC014805 BC014805 Cyp3a41

cDNA sequence BC014805 cDNA sequence BC014805 Cytochrome P450, family 3, subfamily a. polypeptide 41

0.03 0.03 0.01

0.0002 0.0005 0.0004

1425752 at 1425751 at 1419704 at

10775455

Female-specific isoenzyme

21

Cyp2bl3

Cytochrome P450, family 2, subfamily b, polypeptide 13

0.01

0.0071

1449479 at

15155787

Female-specific expression

22

Sult2a2

Sulfotransferase family 2A. Dehydroepiandrosterone (DHEA)-preferring. member 2

<0.01

0.0026

1419528 at

8570624, 9566751, 16864508

Female specific expression in the liver; involved in control of the amounts of active androgens in cells; protects against the toxic effects of lithocholic acid

Genes in grey indicate those which are also downregulated by T in naïve mice.

D. Deli´c et al. / Steroids 76 (2011) 1204–1212

p-Value

Gene no.

Table 3 T-responsive genes in the liver of naive mice becoming T-unresponsive in mice immune to P. chabaudi malaria. Gene no.

Gene symbol

Gene name

T/C

p-Value

Timm /Cimm

p-Value

Affymetrix probe ID

PMID

Functions

1

Uspl2

Ubiquitin specific protease 12

0.08

0.0006

0.95

0.72

1425805 a at

20147737

2

Ehmt2

0.24

0.0087

1.01

0.99

1426888 at

17898715

3

Ndrgl/Ndrl

Euchromatic histone lysine N-methyltransferase 2 N-myc downstream regulated gene 1/N-myc downstream regulated-like

WDR20 regulates activity of the USP12/UAF1 deubiquitinating enzyme complex Arginine methylation on chromatin

0.35

0.0031

1.12

0.57

1450977 s at

17916902

4

Rnfl4

Ring finger protein 14

0.28

0.0011

0.84

0.04

1431030 a at

10085091

5

Sellh

0.34

0.0014

1.03

0.73

1453559 a at

6

Slc9a8

0.34

0.0032

1.01

0.96

1426274 at

12845533

7 8 9

Zfml Stom Hnf4a

Sell (suppressor of lin-12) 1 homolog (C. elegans) Solute carrier family 9 (sodium/hydrogen exchanger), member 8 Zinc finger, matrin-like Stomatin Hepatic nuclear factor 4, alpha

0.47 0.43 0.41

0.0061 0.0088 0.0022

1.22 1.07 0.98

0.14 0.03 0.87

1417792 at 1419098 at 1450447 at

10090952 19689247

0.34

0.0039

0.80

0.16

1448538 a at

0.41 0.43 0.48

0.0048 0.0094 0.0014

0.95 0.99 1.11

0.45 0.91 0.45

1443696 s at 1425461 at 1422772 at

20042707

HABP2 negatively regulates vascular integrity

17113876

0.44

0.0012

0.97

0.85

1451623 at

19619308

Targeted disruption of the gene causes embryonic lethality and defective angiogenesis in mice Regulator of embryonetic stem cell pluripotency and self-renewal Heparan sulfate proteoglycan mediates the clearance of both hepatic and intestinally derived triglyceride-rich lipoproteins Plays a role in nucleotide excision repair, cell cycle regulation and the ubiquitination pathways Bone marrow-derived mast cells and eosinophils from Spred-1−/− mice were more sensitive to IL-3 Presumably involved in cation transport (based on domain architecture) Regulates HCC cell growth Key factor modulating actin polymerization in migrating cells Involved in G1/S phase transition and expression peaks in G1 phase Required for normal bile duct development through prevention of excess cholangiocyte proliferation Important for NIC cell effector function Involved in liver cell proliferation, as well as to apoptotic liver cell death, and represents an important regulator of hepatic regeneration

D4Wsu53e

11 12 13

Habp2 Fbxwll Clgaltl

14

MrpllS

DNA segment, Chr 4, Wayne State University 53, expressed Hyaluronic acid binding protein 2 F-box and WD-40 domain protein 11 Core 1 UDP-galactoseiN-acetylgalactosaminealpha-Rbeta 1.3-galactosyl transferase Mitochondrial ribosomal protein L15

15

Sdcl

Syndecan 1

0.33

0.0003

0.71

0.03

1448158 at

16

Rad23a

RAD23a homolog (S. cerevisiae)

0.46

0.0059

0.97

0.81

1453623 a at

11788722

17

Spredl

Sprouty protein with EVH-1 domain 1, related sequence

0.44

0.0048

0.90

0.64

1423161 s at

19597303

18

Chacll

Cation transport regulator-like

2.48

0.0009

1.23

0.48

1451382 at

19 20

Laspl Sorbsl

LIM and SH3 protein 1 Sorbin and SH3 domain containing 1

2.43 2.06

0.0043 0.0024

1.19 1.00

0.11 0.98

1455470 x at 1436737 a at

19155088 16483316

21

Plk3

Polo-like kinase 3 (Drosophila)

3.21

0.0052

1.40

0.33

1434496 at

17264206

22

Foxal

Forkheadbox Al

2.07

0.0006

0.87

0.49

1418496 at

20347503

23 24

Foxql Egfr

Forkheadboxql Epidermal growth factor receptor

3.80 5.77

0.0003 0.0087

0.74 0.84

0.39 0.49

1422735 at 1432647 at

15719160 19558330

Unknown Erythrocyte membrane protein 7.2b A direct transactivator of numerous xenobiotic-metabolizing cytochrome P450 (CYP) genes Unknown

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Ubiquitously expressed in tissues in response to cellular stress signals; involved in cellular differentiation, proliferation and growth arrest, neoplasia, tumor progression and metastasis, heavy metal response, the hypoxia response and DNA damage response Ubiquitin E3-ligase; might function as coactivator for AR-mediated transactivation in prostate cancer Involved in Notch signaling pathway, functions as a tumor suppressor Na+/H+-antiporter/exchanger

1209

1210

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Table 4 T-unresponsive expression of immunoglobulins in the liver of C57BL/6 mice immune to P. chabaudi malaria. Gene symbol

Gene name

Affymetrix probe ID

Cimm /C

p-Value

Timm /Cimm

p-Value

Igh-la Ighg Ighg Igh-4 Igk-V21 Igk-V8 Igk-V28 Igk-V28 Igk-V28 Igk-V32 Igl-Vl Igj IgM Igh-6 Igh-6

Immunoglobulin heavy chain la (serum IgG2a) Immunoglobulin heavy chain (gamma polypeptide) Immunoglobulin heavy chain (gamma polypeptide) Immunoglobulin heavy chain 4 (serum IgGl) Immunoglobulin kappa chain variable 21 (V21)-12 Immunoglobulin kappa chain variable 8 (V8) Immunoglobulin kappa chain variable 28 (V28) Immunoglobulin kappa chain variable 28 (V28) Immunoglobulin kappa chain variable 28 (V28) Immunoglobulin kappa chain variable 32 (V32) Immunoglobulin lambda chain, variable 1 Immunoglobulin joining chain Immunoglobulin heavy chain (V7183 family) Immunoglobulin heavy chain 6 (heavy chain of IgM) Immunoglobulin heavy chain 6 (heavy chain of IgM)

1425385 a at 1424631 a at 1426174 s at 1427756 x at 1425738 at 1452463 x at 1452417 x at 1427455 x at 1427660 x at 1427837 at 142493l s at 1424305 at 1460423 x at 1427870 x at 1425247 a at

81.41 26.28 15.17 10.92 36.95 22.04 12.16 11.83 10.36 8.25 5.30 10.31 8.98 7.46 3.65

0.0001 0.0012 0.0018 0.0025 0.0003 0.0001 0.0001 0.0008 0.0001 0.0036 0.0011 0.0007 0.0001 0.001 0.0012

0.89 0.93 1.11 0.83 1.03 1.12 1.00 1.09 0.99 0.82 1.22 1.04 1.31 0.96 1.07

0.06 0.77 0.05 0.20 0.93 0.25 0.86 0.64 0.30 0.93 0.14 0.36 0.17 0.66 0.66

First, the liver has acquired the ability to express T-unresponsive mRNAs encoding diverse regions of heavy and light chains of IgG-antibodies. Though our data cannot discriminate between intrahepatic and/or circulating B cells, they are compatible with previous findings showing that antibodies, when once produced by plasma B cells, are T-insensitive [27,28] and that adoptive transfer of serum antibodies from mice immune to malaria can protect Ttreated naïve mice from a lethal outcome of blood-stage malaria [8]. Also, expression of IgM-encoding mRNAs has been here found to be T-unresponsive. This suggests that T impairs development of protective antibodies at a rather early stage. This view is also supported by previous findings that T promotes the TH 1-response as indicated by increased IFN␥ [7] and impairs the TH 2-response preceding antibody formation as indicated by suppressed IL-10 [3]. In this context, our present finding is also noteworthy that expression of the gene encoding euchromatic histone lysine N-methyltransferase 2 (Ehmt2) is identified to have become T-unresponsive in immune mice, whereas its expression is significantly decreased by T in naïve mice. This gene is thought of mediating epigenetic changes in those genes which are involved in the switch of TH 1-response to TH 2response [29]. Second, the liver of immune mice expresses less T-responsive genes than that of naïve mice. Among those genes, whose hepatic expression has lost their T-responsiveness with acquired immunity, we have identified Stomatin (Stom). This encodes the erythrocyte membrane protein band 7.2b. The deregulation of band 7.2b is known to be associated with stomatocytosis, in which erythrocyte membrane permeability is changed leading finally to hemolytic anemia [30]. The non-responsiveness of Stom to T in the liver of immune mice is consistent with previous findings suggesting that erythrocytes of immune mice are potentially less

penetrable by malaria parasites in immune mice than in naïve mice [31]. Third, the liver of immune mice still contains genes whose expression has remained T-responsive. In particular, a number of gender-specific genes of liver metabolism have retained their T-responsiveness during acquisition of T-unresponsive immunity. Indeed, T upregulates the expression of genes encoding the male-prevalent enzymes CYP2D9, CYP7B1, UGT2B1, HSD3B5 and HSD3B2, whereas it downregulates that of genes encoding the female-prevalent enzymes such as CYP2B9, CYP2B13, CYP3A41, CYP3A44, CYP7A1, FMO3 and SULT2A2, respectively, in both immune and naïve mice. Incidentally, the gene encoding HNF4␣, which has been previously described to be involved in the control of sexually dimorphic gene expression in the female mouse liver [32,33] has lost its T-responsiveness in immune mice. This supports our previous view that HNF4␣ is not directly involved in the control of gender-specific gene expression in the liver of female mice [7]. Remarkably, the expression of gender-specific genes reveals approximately the same T-responsiveness in immune and naïve mice, with one spectacular exception: this is the gene encoding HSD3B5 which is even 5-fold more upregulated by T in immune than naïve mice. Since this enzyme is involved in the metabolism of steroid hormones [34], it is reasonable to assume that increased expression of this enzyme contributes to lower concentrations of effective T in the liver of immune mice in comparison to naïve mice, which may contribute to the increased number of T-unresponsive genes in immune mice. Finally, our finding is noteworthy that genes encoding important inflammatory cytokines such as IL-1␤, IL-6, TNF␣ and IFN␥, as well as iNOS are neither inducible by P. chabaudi infections nor responsive to T, respectively, in the liver transcriptome of immune

Table 5 Effect of infection with P. chabaudi for 1 day on hepatic gene expression in vehicle- and T-treated mice immune to P. chabaudi malaria. Gene symbol

Gene name

Affymetrix probe ID

Tinf /Timm

p-Value

Cinf /Cimm

p-Value

Saa2 Saal Saal Orm2 Acnat2 Tend Adamts5

Serum amyloid A 2 Serum amyloid A 1 Serum amyloid A 1 Orosomucoid 2 Acyl-coenzyme A amino acid N-acyltransferase 2 Tensin like Cl domain-containing phosphatase A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5 5 nucleotidase, ecto Atonal homolog 8 (Drosophila) Lipase, endothelial Lipase, endothelial Thioredoxin-like 1

1449326 1419075 1450788 1420438 1425150 1452264 1422561

x at s at at at at at at

8.79 4.28 3.83 2.89 2.69 0.46 0.49

0.0013 0.0026 0.0011 0.0014 0.0036 0.0017 0.0012

7.64 4.48 4.37 4.23 0.88 0.92 0.91

0.003 0.004 0.002 0.002 0.474 0.637 0.386

1422974 1426418 1421262 1450188 1459817

at at at s at at

0.48 0.48 0.38 0.39 1.03

0.0009 0.0074 0.0057 0.0009 0.8843

0.89 0.84 0.56 0.52 0.41

0.492 0.296 0.048 0.025 0.003

Nt5e Atoh8 Lipg Lipg Txnll

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at. doi:10.1016/j.steroids.2011.05.013 References

Fig. 3. Quantitative RT-PCR of cytokines IL-1␤, IL-6, TNF␣, IFN␥ and iNOS in vehicletreated immune (−T) and T-treated immune mice (+T) as well as vehicle-treated (−T) and T-treated naïve mice (+T) infected with 106 P. chabaudi-parasitized erythrocytes on days 0, 1, and 8 p.i., respectively. Values represent means ± SD (n = 3). * indicate significant differences (p < 0.01) with respect to the corresponding control immune and naïve mice on day 0 p.i. (−T), respectively.

mice, in contrast to naïve mice. Collectively, our data support the view that the liver as an anti-malaria effector site has acquired a selective T-unresponsiveness in its gene expression which may contribute to the acquired T-unresponsive protective immunity to blood-stage malaria of P. chabaudi.

Conflict of interest None declared.

Acknowledgements This work was supported by Deutsche Forschungsgemeinschaft through GRK1427 and the Centre of Excellence for Biodiversity Research, College of Science, King Saud University, Riyadh, SaudiArabia.

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