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Eimeria coecicola: Spleen response of Oryctolagus cuniculus
Experimental Parasitology 133 (2013) 137–143
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Eimeria coecicola: Spleen response of Oryctolagus cuniculus Mohamed A. Dkhil a,b,⇑, Saleh Al-Quraishy a, Abdel-Azeem Abdel-Baki a,c, Denis Delic d, Frank Wunderlich d a
Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Department of Zoology and Entomology, Faculty of Science, Helwan University, Egypt c Department of Zoology, Faculty of Science, Beni-Suef University, Egypt d Department of Molecular Parasitology, Heinrich Heine University, Duesseldorf, Germany b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Rabbit coccidiosis caused by Eimeria
coecicola. " Structural reorganizations in the
non-target spleen. " Deregulation of gene expression in
spleen. " Granzyme H expressing NK cells
possibly involved in host response.
a r t i c l e
i n f o
Article history: Received 23 July 2012 Received in revised form 5 November 2012 Accepted 7 November 2012 Available online 1 December 2012 Keywords: Eimeria coecicola Coccidiosis Rabbit Spleen Gene expression Granzyme H
a b s t r a c t The apicomplexan protozoon Eimeria coecicola is an infectious agent of intestinal coccidiosis in rabbits, causing severe injuries in the appendix as the final target, but also in the liver though not being a target. Here, we investigated the effect of E. coecicola on the spleen of the rabbit Oryctolagus cuniculus with respect to structure and gene expression using 2-color Agilent whole rabbit genome oligo-microarray technology in combination with quantitative PCR. At maximal fecal output of E. coecicola oocyts on day 7 p.i., the spleen did not contain any parasites, but displayed moderate inflammatory changes evidenced as fused white pulp areas, diffuse appearance of the marginal zones, and increased number of macrophages in the red pulp, eventually resulting in an increased histological score. The infections induced 36 genes to be up-regulated and 156 genes to be down-regulated, among which were 139 genes encoding diverse regions of antibodies. The highest upregulated genes were those encoding granzyme H (10-fold), lim1 (10-fold), xanthine dehydrogenase (9-fold), whereas the downregulated genes could be majorly assigned to the immune response, as e.g. the genes encoding the macrophage cationic peptide-2 (5-fold). Quantitative PCR of genes encoding GZMH, XDH, HSD17B1, SULT3A1, SAA, BPI, MCP-2, and GST exhibited the same transcriptional level as that detected by microarray analysis. The E. coecicola-induced changes in gene expression of the spleen were totally different to those found previously in appendix and liver. Only the granzyme H gene became upregulated in all three organs. Our data indicate that the spleen, though not a final target of E. coecicola, responds to E. coecicola infections, suggesting that the spleen may be part of an orchestrated host defense against E. coecicola critically involving GZMH-expressing NK-cells. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction ⇑ Corresponding author at: Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Fax: +966 14678514. E-mail address: [email protected] (M.A. Dkhil). 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.11.017
Industrial rabbit farming has become an important protein source for human nourishment in different countries including India and Saudi Arabia (Bhat et al., 1996; Al-Mathal, 2008). However,
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rabbit production is permanently faced with enormous economic losses due to diverse infectious diseases. One of these diseases is eimeriosis that causes severe diarrhea, weight loss, septicemia, and even death of rabbits. Infectious agents of eimeriosis are parasitic protozoans of the genus Eimeria (Mehlhorn, 2001), among which 11 species are specific for rabbits including Eimeria coecicola (Bhat et al., 1996; Pakandl, 2009). The final target site of E. coecicola is the intestinal tract, in particular the caecum and appendix (Pakandl et al., 2006). The infected appendix of rabbits infected with E. coecicola exhibits serious inflammations and injuries, which is associated with high expression of the pro- and anti-inflammatory cytokine interleukin 6 (IL-6) (Dkhil et al., 2012). However, also the liver, though not a direct target site of E. coecicola, is strongly inflamed during E. coecicola infections, as indicated by increases in size, hemorrhage, malondialdehyde and catalase activity, as well as a decrease in glutathione (Al-Quraishy et al., 2012). Also, expressions of numerous hepatic genes are affected, in particular those involved in metabolism and immune response (Al-Quraishy et al., 2012). This is not surprising since the liver and intestinal tract are the two first-pass organs that are directly connected through the portal vein and the liver as a lymphoid organ is also able to generate liver-specific immune reactions (Häussinger et al., 2004). It is a peculiarity of E. coecicola that early sporozoites take an extra-intestinal route through mesenteric lymph nodes and the spleen (Pakandl et al., 1995; Pakandl, 2009). Only then the parasites penetrate cells of the intestinal tract where they mature to oocysts which are ultimately released with the faeces of hosts. The extraintestinal route appears to be essential to complete the life cycle of the parasite (Pakandl et al., 1993, 1996; Renaux et al., 2001). Thus, it is possible that, besides the liver, also other organs, though not being a final target site for E. coecicola multiplication, are affected by intestinal infections of E. coecicola. Since the spleen is a major lymphoid organ of the host defense, we have investigated the spleen of E. coecicola-infected rabbits with respect to possible changes in structure and gene expression using Agilent two-color oligo microarray technology in combination with quantitative PCR. 2. Materials and methods
moved from rabbits and rapidly cut into smaller pieces. Two pieces per rabbit were fixed with 10% neutral buffered formalin, whereas the other pieces were frozen in liquid nitrogen and finally stored at 80 °C until use. 2.4. Histology and histochemistry Pieces of spleens were formalin fixed at room temperature overnight and embedded in paraffin, and 5 lm sections were stained with hematoxylin and eosin. To evaluate structural changes, a semi-quantitative scoring system was used (Giamarellos-Bourboulis et al., 2006). Segments of spleen were scored for the enlargement of white pulp areas (0, absent; 1, slight; 2, moderate; and 3, pronounced) and for the increased numbers of apoptotic cells, macrophages, necrotic cells and presence of pigments (0, absent; and 1, present). Scoring of each tissue sample represented the mean score of high microscopic power fields of five different sections. For immunohistochemical localization of CD68 cells, paraffin sections were cleared in xylene, and then processed as detailed previously (Al-Quraishy et al., 2012). Briefly, sections were incubated in 0.3% H2O2/70% methanol for 20 min to inhibit endogenous peroxidase activity, finally blocked in 3% bovine serum albumin or in 5% goat or rabbit serum and incubated with anti-CD68 primary antibody. The sections were then incubated with 7.5 g/ ml of the biotinylated secondary antibody, followed by avidin/biotin amplification (ABC Elite), before developing with 3,3-diaminobenzidine peroxidase substrate. Sections were counterstained with Mayer hematoxylin for 2–5 min. Negative controls were performed with PBS instead of the primary antibody (Huang et al., 2010). 2.5. Isolation of total RNA Frozen spleens were homogenized in liquid nitrogen and total RNA was isolated with Trizol (Sigma–Aldrich). Quality and integrity of RNA were determined using the Agilent RNA 6000 NanoKit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A260 nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
2.1. Rabbits 2.6. RNA amplification and labeling New Zealand white rabbits (Oryctolagus cuniculus), 7–9 weeks old and weighing of 1.5–2.5 kg were obtained from the animal facilities of King Saud University. They were kept on a robenidine-supplemented commercial pelleted feed as detailed by Licois et al. (1994). On day 4 before infection with E. coecicola, non-supplemented pelleted feed was given to the rabbits (Coudert et al., 1988). The experiments were approved by the state authorities and followed internationally accepted rules on animal protection.
This was performed as detailed in the protocol for the two-Color Microarray-Based Gene Expression Analysis (version 5.5, part number G4140-90050). Briefly, 1 lg of total RNA was used for amplification and labeling using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies, Palo Alto, Calif.) in the presence of cyanine 3-CTP and cyanine 5-CTP (Perkin Elmer), respectively. Yields of cRNA and dye-incorporation were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
2.2. Eimeria coecicola infections Rabbits, individually caged in autoclaved isolators, were orally gavaged with 50,000 sporulated oocysts of E. coecicola suspended in 10 ml of sterile saline, as described recently (Al-Quraishy et al., 2012; Dkhil et al., 2012). Once every 24 h, the faeces were collected from the rabbits, before the bedding was re-newed to eliminate reinfection (Dkhil et al., 2012). Oocysts were counted according to Schito et al. (1996). 2.3. Preparation of spleen tissue Six infected and six non-infected rabbits were euthanized on day 7 p.i. as described recently (Dkhil et al., 2012). Spleens were re-
2.7. Hybridization of Rabbit Genome Oligo Microarray The hybridization procedure was performed according to the two-Color Microarray-Based Gene Expression Analysis protocol (version 5.5, part number G4140–90050) using the Agilent Gene Expression Hybridization Kit (Agilent Technologies, Palo Alto, Calif.). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled cRNA were combined and hybridized overnight at 65 °C to Agilent Whole Rabbit Genome Oligo Microarray 4 44 K containing 43,603 gene-specific oligo-spots using the hybridization chamber and oven recommended by Agilent. After hybridization, the microarrays were washed by three different washing steps with acetonitrile as the last washing step.
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2.8. Scanning and data analysis Fluorescence signals of the hybridized microarrays were monitored using Agilent’s Microarray Scanner System G2505B and the Scan Control Software (Agilent Technologies, Palo Alto, Calif.). The Agilent Feature Extraction Software (FES) version 10.2.1.3 was used to read out and process the microarray image files. For determination of differential gene expression, the FES derived output data files were further analyzed using the Rosetta ResolverÒ gene expression data analysis system (Rosetta Bio software). The local signal of each spot was measured and local background was then subtracted to calculate the net signal intensity and the ratio of Cy5–Cy3 as described previously (Dkhil et al., 2012). The ratios were normalized to the median of all ratios, considering only those spots with fluorescence intensities three times larger than that of the control herring sperm DNA and spotting buffer negative controls. The values represented the means of four single spots and standard deviations. 2.9. Quantitative PCR Total RNA freed from DNA using the DNA free kit (Applied Biosystem, Darmstadt, Germany) was used to synthesize cDNA using QuantiTect™ Reverse QuantiTect™ SYBRÒ Green PCR kit (Qiagen) was applied for amplifications in the ABI PrismÒ 7500HT Sequence Detection System (AppliedBiosystems, Darmstadt, Germany) with the primers listed in Table 1 and delivered by TIB Molbiol (Berlin, Germany). Real-time PCR reactions were performed and evaluated as detailed recently (Al-Quraishy et al., 2012). 2.10. Statistical analysis Student’s t test was used to determine significant differences. 3. Results The course of E. coecicola infections in the rabbit O. cuniculus was recently characterized in detail (Pakandl et al., 2006; Dkhil et al., 2012). The prepatent period lasted in all rabbits for 4 days, and the patent period began with shedding of E. coecicola oocysts in the faeces of all infected rabbits on day 5 p.i.. Maximal shedding of approximately 1.2 million oocysts took place on day 7 p.i., before oocysts shedding continuously declined (Dkhil et al., 2012). At maximal faeces output of oocysts on day 7 p.i., we compared structure and gene expression of the spleen with that of non-infected rabbits of the same age. In infected rabbits, the spleen was not vis-
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ibly changed its appearance, color, and size. Sections of the spleen did not reveal any parasites in the spleen of E. coecicola-infected rabbits. Nevertheless, the spleen exhibits structural reorganizations (Fig. 1). The spleen revealed larger white pulp areas, and the marginal zone normally separating white and red pulp areas was somewhat enlarged with a more diffuse appearance, in comparison to the spleen of non-infected rabbits (Fig. 1A and B). The capsule was decreased in thickness compared to that of the control spleen (Fig. 1C and D). Some splenocytes of E. coecicola-infected rabbit contained mitotic figures (Fig. 1E and F). The marginal zone as well as the surrounding red pulp was enriched in macrophages as detected by an increased staining with anti-CD68 antibody (Fig. 1G and H). The structural reorganization of the spleen of infected rabbits became also evident as an increased number of apoptotic cells. The histological score of the splenic tissue was significantly larger (2.8 ± 0.4) in infected rabbits than that (1.2 ± 0.3) in non-infected rabbits. To identify possible changes in gene expression induced in the spleen by E. coecicola infections, we isolated the total RNA from individual spleens of the six infected rabbits and that of six non-infected rabbits. Then, we pooled equal amounts of RNA from the six infected and six non-infected rabbits before subjecting samples to hybridizations with Agilent 2-color microarray. Among the total 43,588 oligo spots on the microarray, 2097 spots were up-regulated and 1651 spots were down-regulated (cf. scatter plot shown in Fig. S1). In the following, we concentrated our evaluations only on those genes whose expressions were changed more than 2.5fold. Tables 2 and 3 show that E. coecicola infections induced 36 genes to be upregulated, whereas the expressions of 156 genes were downregulated. Among the 156 downregulated genes, there were 139 genes encoding diverse regions of antibodies (Table S1). Only one gene encoding a variable heavy region of antibody was upregulated by approximately 7-fold (Table S1). Table 2 summarized those 36 genes which were upregulated in the spleen by E. coecicola infections. These genes could be categorized in metabolism, immune response, signaling, transport and miscellaneous. In these categories, the highest upregulated genes were those encoding granzyme H (GZMH) (approximately 10-fold), xanthine dehydrogenase (approximately 9-fold), pituitary adenylate cyclase activating polypeptide (approximately 8-fold), soluble carrier family 3 (approximately 4-fold), and Lim homeobox protein 5 (approximately 10-fold), respectively. In addition, 16 genes were down-regulated in the spleen by E. coecicola infections, which could be majorly assigned to the category immune response and the maximally depressed gene was that encoding the macrophage cationic peptide-2 by approximately 5-fold (Table 3). The microarray data could be confirmed for a few genes arbitrarily selected
Table 1 Sequences of oligonucleotide primers used for qRT-PCR analysis. Primer name
Sequence
PCR Product size (bp)
GZMH forward GZMH reverse XDH forward XDH reverse HSD17B1 forward HSD17B1 reverse SULT3A1 forward SULT3A1 reverse SAA forward SAA reverse BPI forward BPI reverse MCP-2 forward MCP-2 reverse GST forward GST reverse
50 -CCT GCC CAC AGG CAA ACC CC-30 50 -TGC ACT GCC AGC TTC ACC TCC-30 50 -CTG GGC GGG GAG AAC CCT GA-30 50 -GCTGGACGTTGGCTGGAGGG-30 50 -GGG GCT GCC CTT CAA CGA CG-30 50 -CCG AAG GGC GGC AGC AGA AT-30 50 -GGA TGC CAT TGT GAG GCA GG CT-30 50 -GCT CCC TCA TTA TGT CGT CTG CCA-30 50 -TGT CCC TGG GCG CTG ACT CT-30 50 -TGG CTG CTG ACT CCC AGG ACC-30 50 -CAC GAG CCC CAG CCA GTG TT-30 50 -TCA TCA CCC CCG AGG TGC AGT-30 50 -TTG CTC CAG GAG GCC TGG GT-30 50 -GCA GCA AGC AGA GCG AGG GT-30 50 -CCA GCG CGA TCA CCA GCT TCC-30 50 -GGC TTT CTCGGG CTT CCA GGC-30
120 100 90 108 101 92 111 104
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Fig. 1. Spleen histology of rabbits infected with Eimeria coecicola. (A, E, C) Architecture of spleens from non-infected rabbits. Red pulps (RP) and white pulp areas (WP) are separated; marginal zone and trabeculae are clearly observed. (B, D, F) Spleen of rabbit infected on day 7 p.i. White pulp is starting to fuse together. Capsule of spleen appears thinner than that of the control (white lines in C, D). Spleen sections labeled with the macrophage detecting anti-CD68 antibody from non-infected (G) and infected rabbits (H). Bar indicates 50 lm.
from microarrays by quantitative RT-PCR (Fig. 2) using the primers listed in Table 1. Indeed, the genes encoding GZMH, XDH, HSD17B1, SULT3A1, SAA, BPI, MCP-2, and GST exhibit about the same transcriptional level by qRT-PCR as by microarrays (Fig. 2).
4. Discussion This study showed that primary infections of E. coecicola in rabbits at maximal shedding of the oocysts on day 7 p.i. induced responses in the spleen, though it did apparently not contain any parasites at that time-point. These responses became evident as moderate structural reorganizations including an increased number of macrophages and as differential gene expression detected by microarray technology in combination with quantitative PCR. There occurred, for example, significant changes in the expression of genes coding for proteins involved in innate immunity, as e.g. different genes expressed by macrophages and gene encoding the acute phase proteins SAA and PAI2. The spectrum of genes affected by E. coecicola infections largely differed among the spleen, the liver as an off-target (Al-Quraishy et al., 2012), and the appendix as the final on-target. Besides this apparent organ specificity, there were only a very few genes whose
expressions responded to E. coecicola infections in both spleen and liver as well as in both spleen and appendix, respectively. For instance, the gene coding for MSR1 (macrophage scavenger receptor 1) was upregulated in the appendix by approximately 14-fold and in the spleen by approximately 2.6-fold. This supported the view that a much higher phagocytic activity of macrophages occurred in the E. coecicola-infected appendix than in the spleen. Moreover, ENA-78 (neutrophil activating peptide 78) was down-regulated in the spleen by approximately 7-fold, while it was concomitantly upregulated by approximately 5-fold in the appendix, suggesting that neutrophils were recruited and activated in E. coecicola-infected appendices. In both spleen and liver, there was an upregulation of genes encoding the acute phase protein SAA, the HPX (hemopoxin), and the XDH (xanthine dehydrogenase). The latter was structurally transformed to xanthine oxidase known to produce superoxide anion and hydrogen peroxide (Nishino et al., 2008). Such oxides might contribute to the oxidation burst recently observed in the liver (Al-Quraishy et al., 2012) and the moderate inflammatory changes found here in the spleen. The glycoprotein HPX might contribute to inactivation and breakdown of heme (Altruda and Tolosano, 2002), which was presumably released from the massively hemorrhagous E. coecicola-infected appendix (Dkhil et al., 2012). It is therefore reasonable to assume that the
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Table 2 Up-regulated expression of genes in the spleen of O. cuniculus infected with E. coecicola on day 7 p.i. Fold change Inf./C
Representative public ID
Functions
8.76 5.03 5.01 4.28 4.51 4.41 4.06 3.86 3.58
NM_001122938 NM_001082317 AF265661 NM_001101704 M29530 NM_001082210 NM_001082646 NM_001082395 NM_001082204
Involved in purine metabolism Lipid catabolic process Phase I metabolism Phase I metabolism Phase I metabolism Phase II metabolism Polysaccharide metabolic process Involved in blood pressure control Vitamin D metabolism
2.88 2.84 2.82 2.64
NM_001082297 NM_001082321 M15061 NM_001081987
Response to toxin Gluconeogenesis Phase I metabolism Phase II metabolism
Immune response GZMH Granzyme H ITGAV Alpha-v integrin SAA Serum amyloid protein A PAI2 Plasminogen activator inhibitor 2 MSR1 Macrophage scavenger receptor ALB Serum albumin precursor HPX Hemopexin
9.78 4.48 3.11 2.63 2.57 2.53 2.51
EF472898 EF472880 NM_001082327 EF472908 NM_001082248 NM_001082344 NM_001082760
Serine protease that is expressed in cytotoxic immune cells CD 51 antigen Acute phase response and acute inflammatory response
Signaling PACAP
Pituitary adenylate cyclase activating polypeptide
7.65
DQ421397
BDKRB1 VIPR2
Bradykinin B1 receptor Vasoactive intestinal peptide receptor 2
4.05 4.13
NM_001082347 DQ421400
Stimulates adenylate cyclase and subsequently increases the cAMP level in target cells G-protein coupled receptor protein signaling pathway Ontology G-protein coupled receptor protein signaling pathway Ontology
Solute carrier family 3, member 1 Potassium voltage-gated channel, Shal-related subfamily, member 2 Anion exchanger 4a
4.31 3.95
NM_001082242 NM_001082118
Cation binding Potassium ion transport
3.84
NM_001082005
Anion transport
AY575212
Gene symbol Metabolism XDH PNLIPRP2 HSD17B1 ADH1 M29530 SULT3A1 ITIH1 ACE DBP PON RBP4 CYP2E1l SULT2A1
Transport SLC3A1 KCND2 AE4A
Gene name
Xanthine dehydrogenase/oxidase Pancreatic lipase-related protein 2 17-beta-hydroxysteroid dehydrogenase type 1 Alcohol dehydrogenase Cytochrome P-450-ka2 Sulfotransferase family, 3A, member 1 Inter-alpha (globulin) inhibitor H1 Angiotensin-converting enzyme D site of albumin promoter (albumin D-box) binding protein (DBP) Paraoxonase Retinol binding protein 4, plasma Cytochrome P450 2E1-like Sulfotransferase family, cytosolic, 2A, dehydroepiandrosterone (DHEA)-preferring, member 1
Miscellaneous LHX5 Lim homeobox protein 5
10.12
Pattern recognition receptor activity Killing of cells of other organism Acute phase response and acute inflammatory response
SLN NEF3 ZP2 CER1
Sarcolipin Neurofilament protein M 75 kDa zona pellucida protein Cerberus-related protein 1
7.11 5.99 4.51 4.34
NM_001082387 Z47378 L12167 AY570542
KNG HRG MG72/ JP1 ZAN PAX6
Kininogen Histidine-rich glycoprotein precursor Mitsugumin72/junctophilin type1
3.62 3.59 2.86
EF472900 U32189 NM_001081996
Encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development Regulation of calcium ion transport Structural molecule activity Involved in sexual reproduction Bone morphogenetic protein (BMP) antagonists that can bind directly to BMPs and inhibit their activity Precursor of kinin Peptidase inhibitor activity Muscle organ development
Zonadhesin Paired box protein PAX6 isoform b
2.85 2.83
NM_001082081 NM_001082217
Involved in cell adhesion Transcription factor involved in cell morphogenesis
E. coecicola-induced changes of gene expression in the appendix are predominantly induced by parasites, while host responses are majorly responsible for at last some of the changes observed in the off- target liver and spleen. Remarkably, we also detected one gene whose expression was affected in all three organs. Indeed, the gene encoding GZMH was significantly upregulated in the spleen by approximately 10fold, in the liver by 13-fold, and in the appendix by 21-fold, respectively. GZMH belongs to the multi-membered family of granzymes (Ewen et al., 2012). These are serine-proteases that are expressed predominantly in cytoplasmic granules of cytotoxic T cells and NK cells, and that induce apoptotic processes (Ewen et al., 2012). Granzymes have been well characterized in mice and humans, but not yet in the rabbit O. cuniculus (Andrade, 2010; Ewen et al., 2012). Besides the granzymes A, B, K, and M, granzyme H has been found hitherto only in humans (Waterhouse and Trapani, 2007; Andrade, 2010; Bovenschen and Kummer, 2010; Tang et al.,
2012; Wang et al., 2012). Mice, however, do not contain granzyme H, though their granzyme repertoire is twice as large as that of humans and encompasses the granzymes A, B, C, D, E, F, G, K, and M (Grossman et al., 2003; Andrade, 2010). The granzyme H of O. cuniculus we identified here in the spleen, as well as previously in the appendix (Dkhil et al., 2012) and in the liver (Al-Quraishy et al., 2012) by Agilent’s whole rabbit genome oligo-microarrays corresponded to a partial cDNA sequence of O. cuniculus, whose alignment revealed a sequence identity of 78% with the human granzyme H. Human NK cells constitutively express granzyme H suggesting a critical role of GZMH in innate immune mechanisms mediated by NK cells (Sedelies et al., 2004; Andrade, 2010). NK-cells also play a central role in the host response against E. papillata-infected intestinal cells of mice as previously reported by Schito and Barta (1997) and Schito et al. (1998). It is therefore plausible to assume that GZMH-expressing NK-cells are also involved in the host defense against E. coecicola-infected host cells
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Table 3 Down-regulated expression of genes in the spleen of O. cuniculus infected with E. coecicola on day 7 p.i. Gene symbol
Gene name
Fold Change Inf/C
Representative Public ID
Functions
2.53 2.72 3.88
M26234 S83370 NM_001122935
Blood coagulation Downregulated by IL-1b and TGFb Mediates fever generation
4.55 5.13
NM_001082325 L47283
CAP18 protein Bactericidal Neutrophil-activating peptide 78 Defensin NP-3a Defensin NP-4 Macrophage cationic peptide 2
5.63 6.38 7.22 7.89 8.09 9.44
NM_001082305 U61270 AF123437 NM_001082298 NM_001082299 M28884
Defense response Central tolerance regulates B cells reactive with goodpasture antigen 3(IV)NC1 collagen1 Defense response Defense response Defense response Defense response Defense response Defense response
PAPS synthase 2 Ankyrin repeat domain 1 Transmembrane tyrosine kinase receptor (c-kit) Epithelial calcium channel Indolethylamine N-methyltransferase
2.59 2.61 2.64
NM_001082173 NM_001082054 DQ356267
Sulfur compound metabolic process Regulation of transcription Proto-Oncogene
2.75 2.84
NM_001082657 NM_001082043
Calcium ion transport Catalyzes the N-methylation of tryptamine and structurally related compounds
Immune response F9 Factor IX COL2A1 Alpha1 type II collagen PTGER3 Prostaglandin E receptor 3 (subtype EP3) LOC100009166 Leukocyte protein COL4A3 Alpha 3 type IV collagen NC1 LOC100009142 BPI ENA-78 LOC100009134 LOC100009135 MCP-2 Miscellaneous PASS2 ANKRD1 C-KIT ECAC INMT
remains completely unknown, the more as both organs are free of E. coecicola-infected host cells. Since NK cells are known to induce apoptosis (Ewen et al., 2012), it may be that the GZMH-NK cells are involved in apoptosis of both liver and spleen cells. For instance, cells of the B-cell lineage may disappear thus contributing to the massive down-regulation of antibody-encoding genes as observed in spleen and liver. Future work is required to unravel the role of GZMH in spleen, liver, and appendix as well as IL-6 trans-signaling, possibly as part of an orchestrated host defense against E. coecicola infections in rabbits. Acknowledgment The authors extend their appreciation to the Distinguished Scientist Fellowship Program at King Saud University, Saudi Arabia for funding this work. Appendix A. Supplementary material
Fig. 2. Quantitative RT-PCR analysis of GZMH, XDH, HSD17B1, SULT3A1, SAA, BPI, MCP-2, and GST in the spleen of rabbits infected with E. coecicola. Expression of mRNAs were determined in spleens from non-infected and infected rabbits on day 7 p.i., normalized to GAPDH mRNA expression, and relative expression is given as – fold change compared to the non-infected control mice. Values are means ± SD.
in the appendix. However, liver and spleen were free from any E. coecicola-infected host cells at least on day 7 p.i.. The increase in GZMH expression in both liver and spleen might therefore be explained as an immigration of GZMH-NK cells from the E. coecicola-infected appendix. However, it is also conceivable that liver and spleen contain organ-inherent GZMH-NK cells which become activated by alarming host signals released from the heavily infected appendix. One candidate might be IL-6, that was massively upregulated by about 50-fold only in the appendix (Dkhil et al., 2012). Indeed, circulating complexes of IL-6/soluble interleukin six receptor a are able to communicate with all cells via IL-6 trans-signaling (Scheller et al., 2011; Wunderlich et al., 2012). Nevertheless, the functional role of GZMH-NK cells in liver and spleen
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Eimeria coecicola: Spleen response of Oryctolagus cuniculus Mohamed A. Dkhil a,b,⇑, Saleh Al-Quraishy a, Abdel-Azeem Abdel-Baki a,c, Denis Delic d, Frank Wunderlich d a
Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Department of Zoology and Entomology, Faculty of Science, Helwan University, Egypt c Department of Zoology, Faculty of Science, Beni-Suef University, Egypt d Department of Molecular Parasitology, Heinrich Heine University, Duesseldorf, Germany b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Rabbit coccidiosis caused by Eimeria
coecicola. " Structural reorganizations in the
non-target spleen. " Deregulation of gene expression in
spleen. " Granzyme H expressing NK cells
possibly involved in host response.
a r t i c l e
i n f o
Article history: Received 23 July 2012 Received in revised form 5 November 2012 Accepted 7 November 2012 Available online 1 December 2012 Keywords: Eimeria coecicola Coccidiosis Rabbit Spleen Gene expression Granzyme H
a b s t r a c t The apicomplexan protozoon Eimeria coecicola is an infectious agent of intestinal coccidiosis in rabbits, causing severe injuries in the appendix as the final target, but also in the liver though not being a target. Here, we investigated the effect of E. coecicola on the spleen of the rabbit Oryctolagus cuniculus with respect to structure and gene expression using 2-color Agilent whole rabbit genome oligo-microarray technology in combination with quantitative PCR. At maximal fecal output of E. coecicola oocyts on day 7 p.i., the spleen did not contain any parasites, but displayed moderate inflammatory changes evidenced as fused white pulp areas, diffuse appearance of the marginal zones, and increased number of macrophages in the red pulp, eventually resulting in an increased histological score. The infections induced 36 genes to be up-regulated and 156 genes to be down-regulated, among which were 139 genes encoding diverse regions of antibodies. The highest upregulated genes were those encoding granzyme H (10-fold), lim1 (10-fold), xanthine dehydrogenase (9-fold), whereas the downregulated genes could be majorly assigned to the immune response, as e.g. the genes encoding the macrophage cationic peptide-2 (5-fold). Quantitative PCR of genes encoding GZMH, XDH, HSD17B1, SULT3A1, SAA, BPI, MCP-2, and GST exhibited the same transcriptional level as that detected by microarray analysis. The E. coecicola-induced changes in gene expression of the spleen were totally different to those found previously in appendix and liver. Only the granzyme H gene became upregulated in all three organs. Our data indicate that the spleen, though not a final target of E. coecicola, responds to E. coecicola infections, suggesting that the spleen may be part of an orchestrated host defense against E. coecicola critically involving GZMH-expressing NK-cells. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction ⇑ Corresponding author at: Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Fax: +966 14678514. E-mail address: [email protected] (M.A. Dkhil). 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.11.017
Industrial rabbit farming has become an important protein source for human nourishment in different countries including India and Saudi Arabia (Bhat et al., 1996; Al-Mathal, 2008). However,
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rabbit production is permanently faced with enormous economic losses due to diverse infectious diseases. One of these diseases is eimeriosis that causes severe diarrhea, weight loss, septicemia, and even death of rabbits. Infectious agents of eimeriosis are parasitic protozoans of the genus Eimeria (Mehlhorn, 2001), among which 11 species are specific for rabbits including Eimeria coecicola (Bhat et al., 1996; Pakandl, 2009). The final target site of E. coecicola is the intestinal tract, in particular the caecum and appendix (Pakandl et al., 2006). The infected appendix of rabbits infected with E. coecicola exhibits serious inflammations and injuries, which is associated with high expression of the pro- and anti-inflammatory cytokine interleukin 6 (IL-6) (Dkhil et al., 2012). However, also the liver, though not a direct target site of E. coecicola, is strongly inflamed during E. coecicola infections, as indicated by increases in size, hemorrhage, malondialdehyde and catalase activity, as well as a decrease in glutathione (Al-Quraishy et al., 2012). Also, expressions of numerous hepatic genes are affected, in particular those involved in metabolism and immune response (Al-Quraishy et al., 2012). This is not surprising since the liver and intestinal tract are the two first-pass organs that are directly connected through the portal vein and the liver as a lymphoid organ is also able to generate liver-specific immune reactions (Häussinger et al., 2004). It is a peculiarity of E. coecicola that early sporozoites take an extra-intestinal route through mesenteric lymph nodes and the spleen (Pakandl et al., 1995; Pakandl, 2009). Only then the parasites penetrate cells of the intestinal tract where they mature to oocysts which are ultimately released with the faeces of hosts. The extraintestinal route appears to be essential to complete the life cycle of the parasite (Pakandl et al., 1993, 1996; Renaux et al., 2001). Thus, it is possible that, besides the liver, also other organs, though not being a final target site for E. coecicola multiplication, are affected by intestinal infections of E. coecicola. Since the spleen is a major lymphoid organ of the host defense, we have investigated the spleen of E. coecicola-infected rabbits with respect to possible changes in structure and gene expression using Agilent two-color oligo microarray technology in combination with quantitative PCR. 2. Materials and methods
moved from rabbits and rapidly cut into smaller pieces. Two pieces per rabbit were fixed with 10% neutral buffered formalin, whereas the other pieces were frozen in liquid nitrogen and finally stored at 80 °C until use. 2.4. Histology and histochemistry Pieces of spleens were formalin fixed at room temperature overnight and embedded in paraffin, and 5 lm sections were stained with hematoxylin and eosin. To evaluate structural changes, a semi-quantitative scoring system was used (Giamarellos-Bourboulis et al., 2006). Segments of spleen were scored for the enlargement of white pulp areas (0, absent; 1, slight; 2, moderate; and 3, pronounced) and for the increased numbers of apoptotic cells, macrophages, necrotic cells and presence of pigments (0, absent; and 1, present). Scoring of each tissue sample represented the mean score of high microscopic power fields of five different sections. For immunohistochemical localization of CD68 cells, paraffin sections were cleared in xylene, and then processed as detailed previously (Al-Quraishy et al., 2012). Briefly, sections were incubated in 0.3% H2O2/70% methanol for 20 min to inhibit endogenous peroxidase activity, finally blocked in 3% bovine serum albumin or in 5% goat or rabbit serum and incubated with anti-CD68 primary antibody. The sections were then incubated with 7.5 g/ ml of the biotinylated secondary antibody, followed by avidin/biotin amplification (ABC Elite), before developing with 3,3-diaminobenzidine peroxidase substrate. Sections were counterstained with Mayer hematoxylin for 2–5 min. Negative controls were performed with PBS instead of the primary antibody (Huang et al., 2010). 2.5. Isolation of total RNA Frozen spleens were homogenized in liquid nitrogen and total RNA was isolated with Trizol (Sigma–Aldrich). Quality and integrity of RNA were determined using the Agilent RNA 6000 NanoKit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A260 nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
2.1. Rabbits 2.6. RNA amplification and labeling New Zealand white rabbits (Oryctolagus cuniculus), 7–9 weeks old and weighing of 1.5–2.5 kg were obtained from the animal facilities of King Saud University. They were kept on a robenidine-supplemented commercial pelleted feed as detailed by Licois et al. (1994). On day 4 before infection with E. coecicola, non-supplemented pelleted feed was given to the rabbits (Coudert et al., 1988). The experiments were approved by the state authorities and followed internationally accepted rules on animal protection.
This was performed as detailed in the protocol for the two-Color Microarray-Based Gene Expression Analysis (version 5.5, part number G4140-90050). Briefly, 1 lg of total RNA was used for amplification and labeling using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies, Palo Alto, Calif.) in the presence of cyanine 3-CTP and cyanine 5-CTP (Perkin Elmer), respectively. Yields of cRNA and dye-incorporation were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
2.2. Eimeria coecicola infections Rabbits, individually caged in autoclaved isolators, were orally gavaged with 50,000 sporulated oocysts of E. coecicola suspended in 10 ml of sterile saline, as described recently (Al-Quraishy et al., 2012; Dkhil et al., 2012). Once every 24 h, the faeces were collected from the rabbits, before the bedding was re-newed to eliminate reinfection (Dkhil et al., 2012). Oocysts were counted according to Schito et al. (1996). 2.3. Preparation of spleen tissue Six infected and six non-infected rabbits were euthanized on day 7 p.i. as described recently (Dkhil et al., 2012). Spleens were re-
2.7. Hybridization of Rabbit Genome Oligo Microarray The hybridization procedure was performed according to the two-Color Microarray-Based Gene Expression Analysis protocol (version 5.5, part number G4140–90050) using the Agilent Gene Expression Hybridization Kit (Agilent Technologies, Palo Alto, Calif.). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled cRNA were combined and hybridized overnight at 65 °C to Agilent Whole Rabbit Genome Oligo Microarray 4 44 K containing 43,603 gene-specific oligo-spots using the hybridization chamber and oven recommended by Agilent. After hybridization, the microarrays were washed by three different washing steps with acetonitrile as the last washing step.
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2.8. Scanning and data analysis Fluorescence signals of the hybridized microarrays were monitored using Agilent’s Microarray Scanner System G2505B and the Scan Control Software (Agilent Technologies, Palo Alto, Calif.). The Agilent Feature Extraction Software (FES) version 10.2.1.3 was used to read out and process the microarray image files. For determination of differential gene expression, the FES derived output data files were further analyzed using the Rosetta ResolverÒ gene expression data analysis system (Rosetta Bio software). The local signal of each spot was measured and local background was then subtracted to calculate the net signal intensity and the ratio of Cy5–Cy3 as described previously (Dkhil et al., 2012). The ratios were normalized to the median of all ratios, considering only those spots with fluorescence intensities three times larger than that of the control herring sperm DNA and spotting buffer negative controls. The values represented the means of four single spots and standard deviations. 2.9. Quantitative PCR Total RNA freed from DNA using the DNA free kit (Applied Biosystem, Darmstadt, Germany) was used to synthesize cDNA using QuantiTect™ Reverse QuantiTect™ SYBRÒ Green PCR kit (Qiagen) was applied for amplifications in the ABI PrismÒ 7500HT Sequence Detection System (AppliedBiosystems, Darmstadt, Germany) with the primers listed in Table 1 and delivered by TIB Molbiol (Berlin, Germany). Real-time PCR reactions were performed and evaluated as detailed recently (Al-Quraishy et al., 2012). 2.10. Statistical analysis Student’s t test was used to determine significant differences. 3. Results The course of E. coecicola infections in the rabbit O. cuniculus was recently characterized in detail (Pakandl et al., 2006; Dkhil et al., 2012). The prepatent period lasted in all rabbits for 4 days, and the patent period began with shedding of E. coecicola oocysts in the faeces of all infected rabbits on day 5 p.i.. Maximal shedding of approximately 1.2 million oocysts took place on day 7 p.i., before oocysts shedding continuously declined (Dkhil et al., 2012). At maximal faeces output of oocysts on day 7 p.i., we compared structure and gene expression of the spleen with that of non-infected rabbits of the same age. In infected rabbits, the spleen was not vis-
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ibly changed its appearance, color, and size. Sections of the spleen did not reveal any parasites in the spleen of E. coecicola-infected rabbits. Nevertheless, the spleen exhibits structural reorganizations (Fig. 1). The spleen revealed larger white pulp areas, and the marginal zone normally separating white and red pulp areas was somewhat enlarged with a more diffuse appearance, in comparison to the spleen of non-infected rabbits (Fig. 1A and B). The capsule was decreased in thickness compared to that of the control spleen (Fig. 1C and D). Some splenocytes of E. coecicola-infected rabbit contained mitotic figures (Fig. 1E and F). The marginal zone as well as the surrounding red pulp was enriched in macrophages as detected by an increased staining with anti-CD68 antibody (Fig. 1G and H). The structural reorganization of the spleen of infected rabbits became also evident as an increased number of apoptotic cells. The histological score of the splenic tissue was significantly larger (2.8 ± 0.4) in infected rabbits than that (1.2 ± 0.3) in non-infected rabbits. To identify possible changes in gene expression induced in the spleen by E. coecicola infections, we isolated the total RNA from individual spleens of the six infected rabbits and that of six non-infected rabbits. Then, we pooled equal amounts of RNA from the six infected and six non-infected rabbits before subjecting samples to hybridizations with Agilent 2-color microarray. Among the total 43,588 oligo spots on the microarray, 2097 spots were up-regulated and 1651 spots were down-regulated (cf. scatter plot shown in Fig. S1). In the following, we concentrated our evaluations only on those genes whose expressions were changed more than 2.5fold. Tables 2 and 3 show that E. coecicola infections induced 36 genes to be upregulated, whereas the expressions of 156 genes were downregulated. Among the 156 downregulated genes, there were 139 genes encoding diverse regions of antibodies (Table S1). Only one gene encoding a variable heavy region of antibody was upregulated by approximately 7-fold (Table S1). Table 2 summarized those 36 genes which were upregulated in the spleen by E. coecicola infections. These genes could be categorized in metabolism, immune response, signaling, transport and miscellaneous. In these categories, the highest upregulated genes were those encoding granzyme H (GZMH) (approximately 10-fold), xanthine dehydrogenase (approximately 9-fold), pituitary adenylate cyclase activating polypeptide (approximately 8-fold), soluble carrier family 3 (approximately 4-fold), and Lim homeobox protein 5 (approximately 10-fold), respectively. In addition, 16 genes were down-regulated in the spleen by E. coecicola infections, which could be majorly assigned to the category immune response and the maximally depressed gene was that encoding the macrophage cationic peptide-2 by approximately 5-fold (Table 3). The microarray data could be confirmed for a few genes arbitrarily selected
Table 1 Sequences of oligonucleotide primers used for qRT-PCR analysis. Primer name
Sequence
PCR Product size (bp)
GZMH forward GZMH reverse XDH forward XDH reverse HSD17B1 forward HSD17B1 reverse SULT3A1 forward SULT3A1 reverse SAA forward SAA reverse BPI forward BPI reverse MCP-2 forward MCP-2 reverse GST forward GST reverse
50 -CCT GCC CAC AGG CAA ACC CC-30 50 -TGC ACT GCC AGC TTC ACC TCC-30 50 -CTG GGC GGG GAG AAC CCT GA-30 50 -GCTGGACGTTGGCTGGAGGG-30 50 -GGG GCT GCC CTT CAA CGA CG-30 50 -CCG AAG GGC GGC AGC AGA AT-30 50 -GGA TGC CAT TGT GAG GCA GG CT-30 50 -GCT CCC TCA TTA TGT CGT CTG CCA-30 50 -TGT CCC TGG GCG CTG ACT CT-30 50 -TGG CTG CTG ACT CCC AGG ACC-30 50 -CAC GAG CCC CAG CCA GTG TT-30 50 -TCA TCA CCC CCG AGG TGC AGT-30 50 -TTG CTC CAG GAG GCC TGG GT-30 50 -GCA GCA AGC AGA GCG AGG GT-30 50 -CCA GCG CGA TCA CCA GCT TCC-30 50 -GGC TTT CTCGGG CTT CCA GGC-30
120 100 90 108 101 92 111 104
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Fig. 1. Spleen histology of rabbits infected with Eimeria coecicola. (A, E, C) Architecture of spleens from non-infected rabbits. Red pulps (RP) and white pulp areas (WP) are separated; marginal zone and trabeculae are clearly observed. (B, D, F) Spleen of rabbit infected on day 7 p.i. White pulp is starting to fuse together. Capsule of spleen appears thinner than that of the control (white lines in C, D). Spleen sections labeled with the macrophage detecting anti-CD68 antibody from non-infected (G) and infected rabbits (H). Bar indicates 50 lm.
from microarrays by quantitative RT-PCR (Fig. 2) using the primers listed in Table 1. Indeed, the genes encoding GZMH, XDH, HSD17B1, SULT3A1, SAA, BPI, MCP-2, and GST exhibit about the same transcriptional level by qRT-PCR as by microarrays (Fig. 2).
4. Discussion This study showed that primary infections of E. coecicola in rabbits at maximal shedding of the oocysts on day 7 p.i. induced responses in the spleen, though it did apparently not contain any parasites at that time-point. These responses became evident as moderate structural reorganizations including an increased number of macrophages and as differential gene expression detected by microarray technology in combination with quantitative PCR. There occurred, for example, significant changes in the expression of genes coding for proteins involved in innate immunity, as e.g. different genes expressed by macrophages and gene encoding the acute phase proteins SAA and PAI2. The spectrum of genes affected by E. coecicola infections largely differed among the spleen, the liver as an off-target (Al-Quraishy et al., 2012), and the appendix as the final on-target. Besides this apparent organ specificity, there were only a very few genes whose
expressions responded to E. coecicola infections in both spleen and liver as well as in both spleen and appendix, respectively. For instance, the gene coding for MSR1 (macrophage scavenger receptor 1) was upregulated in the appendix by approximately 14-fold and in the spleen by approximately 2.6-fold. This supported the view that a much higher phagocytic activity of macrophages occurred in the E. coecicola-infected appendix than in the spleen. Moreover, ENA-78 (neutrophil activating peptide 78) was down-regulated in the spleen by approximately 7-fold, while it was concomitantly upregulated by approximately 5-fold in the appendix, suggesting that neutrophils were recruited and activated in E. coecicola-infected appendices. In both spleen and liver, there was an upregulation of genes encoding the acute phase protein SAA, the HPX (hemopoxin), and the XDH (xanthine dehydrogenase). The latter was structurally transformed to xanthine oxidase known to produce superoxide anion and hydrogen peroxide (Nishino et al., 2008). Such oxides might contribute to the oxidation burst recently observed in the liver (Al-Quraishy et al., 2012) and the moderate inflammatory changes found here in the spleen. The glycoprotein HPX might contribute to inactivation and breakdown of heme (Altruda and Tolosano, 2002), which was presumably released from the massively hemorrhagous E. coecicola-infected appendix (Dkhil et al., 2012). It is therefore reasonable to assume that the
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Table 2 Up-regulated expression of genes in the spleen of O. cuniculus infected with E. coecicola on day 7 p.i. Fold change Inf./C
Representative public ID
Functions
8.76 5.03 5.01 4.28 4.51 4.41 4.06 3.86 3.58
NM_001122938 NM_001082317 AF265661 NM_001101704 M29530 NM_001082210 NM_001082646 NM_001082395 NM_001082204
Involved in purine metabolism Lipid catabolic process Phase I metabolism Phase I metabolism Phase I metabolism Phase II metabolism Polysaccharide metabolic process Involved in blood pressure control Vitamin D metabolism
2.88 2.84 2.82 2.64
NM_001082297 NM_001082321 M15061 NM_001081987
Response to toxin Gluconeogenesis Phase I metabolism Phase II metabolism
Immune response GZMH Granzyme H ITGAV Alpha-v integrin SAA Serum amyloid protein A PAI2 Plasminogen activator inhibitor 2 MSR1 Macrophage scavenger receptor ALB Serum albumin precursor HPX Hemopexin
9.78 4.48 3.11 2.63 2.57 2.53 2.51
EF472898 EF472880 NM_001082327 EF472908 NM_001082248 NM_001082344 NM_001082760
Serine protease that is expressed in cytotoxic immune cells CD 51 antigen Acute phase response and acute inflammatory response
Signaling PACAP
Pituitary adenylate cyclase activating polypeptide
7.65
DQ421397
BDKRB1 VIPR2
Bradykinin B1 receptor Vasoactive intestinal peptide receptor 2
4.05 4.13
NM_001082347 DQ421400
Stimulates adenylate cyclase and subsequently increases the cAMP level in target cells G-protein coupled receptor protein signaling pathway Ontology G-protein coupled receptor protein signaling pathway Ontology
Solute carrier family 3, member 1 Potassium voltage-gated channel, Shal-related subfamily, member 2 Anion exchanger 4a
4.31 3.95
NM_001082242 NM_001082118
Cation binding Potassium ion transport
3.84
NM_001082005
Anion transport
AY575212
Gene symbol Metabolism XDH PNLIPRP2 HSD17B1 ADH1 M29530 SULT3A1 ITIH1 ACE DBP PON RBP4 CYP2E1l SULT2A1
Transport SLC3A1 KCND2 AE4A
Gene name
Xanthine dehydrogenase/oxidase Pancreatic lipase-related protein 2 17-beta-hydroxysteroid dehydrogenase type 1 Alcohol dehydrogenase Cytochrome P-450-ka2 Sulfotransferase family, 3A, member 1 Inter-alpha (globulin) inhibitor H1 Angiotensin-converting enzyme D site of albumin promoter (albumin D-box) binding protein (DBP) Paraoxonase Retinol binding protein 4, plasma Cytochrome P450 2E1-like Sulfotransferase family, cytosolic, 2A, dehydroepiandrosterone (DHEA)-preferring, member 1
Miscellaneous LHX5 Lim homeobox protein 5
10.12
Pattern recognition receptor activity Killing of cells of other organism Acute phase response and acute inflammatory response
SLN NEF3 ZP2 CER1
Sarcolipin Neurofilament protein M 75 kDa zona pellucida protein Cerberus-related protein 1
7.11 5.99 4.51 4.34
NM_001082387 Z47378 L12167 AY570542
KNG HRG MG72/ JP1 ZAN PAX6
Kininogen Histidine-rich glycoprotein precursor Mitsugumin72/junctophilin type1
3.62 3.59 2.86
EF472900 U32189 NM_001081996
Encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development Regulation of calcium ion transport Structural molecule activity Involved in sexual reproduction Bone morphogenetic protein (BMP) antagonists that can bind directly to BMPs and inhibit their activity Precursor of kinin Peptidase inhibitor activity Muscle organ development
Zonadhesin Paired box protein PAX6 isoform b
2.85 2.83
NM_001082081 NM_001082217
Involved in cell adhesion Transcription factor involved in cell morphogenesis
E. coecicola-induced changes of gene expression in the appendix are predominantly induced by parasites, while host responses are majorly responsible for at last some of the changes observed in the off- target liver and spleen. Remarkably, we also detected one gene whose expression was affected in all three organs. Indeed, the gene encoding GZMH was significantly upregulated in the spleen by approximately 10fold, in the liver by 13-fold, and in the appendix by 21-fold, respectively. GZMH belongs to the multi-membered family of granzymes (Ewen et al., 2012). These are serine-proteases that are expressed predominantly in cytoplasmic granules of cytotoxic T cells and NK cells, and that induce apoptotic processes (Ewen et al., 2012). Granzymes have been well characterized in mice and humans, but not yet in the rabbit O. cuniculus (Andrade, 2010; Ewen et al., 2012). Besides the granzymes A, B, K, and M, granzyme H has been found hitherto only in humans (Waterhouse and Trapani, 2007; Andrade, 2010; Bovenschen and Kummer, 2010; Tang et al.,
2012; Wang et al., 2012). Mice, however, do not contain granzyme H, though their granzyme repertoire is twice as large as that of humans and encompasses the granzymes A, B, C, D, E, F, G, K, and M (Grossman et al., 2003; Andrade, 2010). The granzyme H of O. cuniculus we identified here in the spleen, as well as previously in the appendix (Dkhil et al., 2012) and in the liver (Al-Quraishy et al., 2012) by Agilent’s whole rabbit genome oligo-microarrays corresponded to a partial cDNA sequence of O. cuniculus, whose alignment revealed a sequence identity of 78% with the human granzyme H. Human NK cells constitutively express granzyme H suggesting a critical role of GZMH in innate immune mechanisms mediated by NK cells (Sedelies et al., 2004; Andrade, 2010). NK-cells also play a central role in the host response against E. papillata-infected intestinal cells of mice as previously reported by Schito and Barta (1997) and Schito et al. (1998). It is therefore plausible to assume that GZMH-expressing NK-cells are also involved in the host defense against E. coecicola-infected host cells
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Table 3 Down-regulated expression of genes in the spleen of O. cuniculus infected with E. coecicola on day 7 p.i. Gene symbol
Gene name
Fold Change Inf/C
Representative Public ID
Functions
2.53 2.72 3.88
M26234 S83370 NM_001122935
Blood coagulation Downregulated by IL-1b and TGFb Mediates fever generation
4.55 5.13
NM_001082325 L47283
CAP18 protein Bactericidal Neutrophil-activating peptide 78 Defensin NP-3a Defensin NP-4 Macrophage cationic peptide 2
5.63 6.38 7.22 7.89 8.09 9.44
NM_001082305 U61270 AF123437 NM_001082298 NM_001082299 M28884
Defense response Central tolerance regulates B cells reactive with goodpasture antigen 3(IV)NC1 collagen1 Defense response Defense response Defense response Defense response Defense response Defense response
PAPS synthase 2 Ankyrin repeat domain 1 Transmembrane tyrosine kinase receptor (c-kit) Epithelial calcium channel Indolethylamine N-methyltransferase
2.59 2.61 2.64
NM_001082173 NM_001082054 DQ356267
Sulfur compound metabolic process Regulation of transcription Proto-Oncogene
2.75 2.84
NM_001082657 NM_001082043
Calcium ion transport Catalyzes the N-methylation of tryptamine and structurally related compounds
Immune response F9 Factor IX COL2A1 Alpha1 type II collagen PTGER3 Prostaglandin E receptor 3 (subtype EP3) LOC100009166 Leukocyte protein COL4A3 Alpha 3 type IV collagen NC1 LOC100009142 BPI ENA-78 LOC100009134 LOC100009135 MCP-2 Miscellaneous PASS2 ANKRD1 C-KIT ECAC INMT
remains completely unknown, the more as both organs are free of E. coecicola-infected host cells. Since NK cells are known to induce apoptosis (Ewen et al., 2012), it may be that the GZMH-NK cells are involved in apoptosis of both liver and spleen cells. For instance, cells of the B-cell lineage may disappear thus contributing to the massive down-regulation of antibody-encoding genes as observed in spleen and liver. Future work is required to unravel the role of GZMH in spleen, liver, and appendix as well as IL-6 trans-signaling, possibly as part of an orchestrated host defense against E. coecicola infections in rabbits. Acknowledgment The authors extend their appreciation to the Distinguished Scientist Fellowship Program at King Saud University, Saudi Arabia for funding this work. Appendix A. Supplementary material
Fig. 2. Quantitative RT-PCR analysis of GZMH, XDH, HSD17B1, SULT3A1, SAA, BPI, MCP-2, and GST in the spleen of rabbits infected with E. coecicola. Expression of mRNAs were determined in spleens from non-infected and infected rabbits on day 7 p.i., normalized to GAPDH mRNA expression, and relative expression is given as – fold change compared to the non-infected control mice. Values are means ± SD.
in the appendix. However, liver and spleen were free from any E. coecicola-infected host cells at least on day 7 p.i.. The increase in GZMH expression in both liver and spleen might therefore be explained as an immigration of GZMH-NK cells from the E. coecicola-infected appendix. However, it is also conceivable that liver and spleen contain organ-inherent GZMH-NK cells which become activated by alarming host signals released from the heavily infected appendix. One candidate might be IL-6, that was massively upregulated by about 50-fold only in the appendix (Dkhil et al., 2012). Indeed, circulating complexes of IL-6/soluble interleukin six receptor a are able to communicate with all cells via IL-6 trans-signaling (Scheller et al., 2011; Wunderlich et al., 2012). Nevertheless, the functional role of GZMH-NK cells in liver and spleen
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