Gene expression of the liver of vaccination-protected mice in response to early patent infections of Plasmodium chabaudi blood-stage malaria

Gene expression of the liver of vaccination-protected mice in response to early patent infections... Background: The role of the liver for survival of blood‑ stage malaria is only poorly understood. In experimental blood‑ stage malaria with Plasmodium chabaudi, protective vaccination induces healing and, thus, survival of other‑ wise lethal infections. This model is appropriate to study the role of the liver in vaccination‑ induced survival of blood‑ stage malaria. Methods: Female Balb/c mice were vaccinated with a non‑ infectious vaccine consisting of plasma membranes isolated in the form of erythrocyte ghosts from P. chabaudi‑ infected erythrocytes at week 3 and week 1 before infec‑ tion with P. chabaudi blood‑ stage malaria. Gene expression microarrays and quantitative real‑ time PCR were used to investigate the response of the liver, in terms of expression of mRNA and long intergenic non‑ coding (linc)RNA, to vaccination‑ induced healing infections and lethal P. chabaudi malaria at early patency on day 4 post infection, when parasitized erythrocytes begin to appear in peripheral blood. Results: In vaccination‑ induced healing infections, 23 genes were identified to be induced in the liver by > tenfold at p < 0.01. More than one‑ third were genes known to be involved in erythropoiesis, such as Kel, Rhag, Ahsp, Ermap, Slc4a1, Cldn13 Gata1, and Gfi1b . Another group of > tenfold expressed genes include genes involved in natural cyto‑ toxicity, such as those encoding killer cell lectin‑ like receptors Klrb1a, Klrc3, Klrd1, the natural cytotoxicity‑ triggering receptor 1 Ncr1, as well as the granzyme B encoding Gzmb. Additionally, a series of genes involved in the control of cell cycle and mitosis were identified: Ccnb1, Cdc25c, Ckap2l were expressed > tenfold only in vaccination‑ protected mice, and the expression of 22 genes was at least 100% higher in vaccination‑ protected mice than in non‑ vaccinated mice. Furthermore, distinct lincRNA species were changed by > threefold in livers of vaccination‑ protected mice, whereas lethal malaria induced different lincRNAs. Conclusion: The present data suggest that protective vaccination accelerates the malaria‑ induced occurrence of extramedullary erythropoiesis, generation of liver‑ resident cytotoxic cells, and regeneration from malaria‑ induced injury in the liver at early patency, which may be critical for final survival of otherwise lethal blood ‑ stage malaria of P. chabaudi. Keywords: Plasmodium chabaudi, Blood‑ stage malaria, Liver, Gene expression, Vaccination, Extramedullary erythropoiesis, Natural cytotoxicity *Correspondence: mohameddkhil@yahoo.com Department of Zoology, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Al‑Quraishy et al. Malar J (2018) 17:215 Page 2 of 16 lethal infections in vaccinated and non-vaccinated mice, Background respectively [18]. Moreover, early patency is associated Malaria is still one of the most life-threatening infec- with a dramatic decline in malaria-induced expression of tious diseases in tropical countries. The World Health multifunctional cytokines, such as IFNγ, TNF, IL-1β, and Organization (WHO) had estimated about 212 million IL-6 in the liver, which drive various programmes of host new cases and about 429,000 deaths globally in 2015, defense [10, 18, 21]. Although still unexplainable at pre- with about 70% of total deaths occurring in children aged sent, this decline suggests the occurrence of yet unknown under 5 years [1]. An effective anti-malarial vaccine is not processes in the liver that may be critical for vaccine yet commercially available [2–4]. efficacy and, thus, for the final outcome of blood-stage Morbidity and mortality from malaria are caused by malaria. To track these processes in the liver during mid- the blood stages of the malaria-causing agent, parasitic precrisis and the possible effects by vaccination, a reason - protozoans of the genus Plasmodium, which develop able initial approach is to analyse global gene expression within host erythrocytes. The spleen with its inher - profiles in the liver for malaria-responsive genes at early ent mechanism to remove senescent and other aberrant patent infections of P. chabaudi blood-stage malaria in erythrocytes from circulation is currently thought to be vaccination-protected mice in comparison with non-vac- the exclusive effector organ to eliminate Plasmodium - cinated unprotected mice. parasitized erythrocytes from circulation [5]. However, the liver is also equipped with effective mechanisms for Methods removing aberrant erythrocytes including Plasmodium- Mice infected erythrocytes [6–11]. The liver with its intrinsic Balb/c mice bred under specified pathogen-free con - immune system is therefore increasingly, though still ditions were obtained from the central animal facility hesitantly, recognized as an effector organ against blood- of the University of Düsseldorf. The experiments were stage malaria [10]. Plasmodium chabaudi infection in performed only with female mice aged 10–12  weeks. mice is an appropriate model to study the effector func - Mice were housed in plastic cages and received a stand- tions of the liver against blood-stage malaria without ard diet (Woehrlin, Bad Salzuflen, Germany) and water interfering with the preceding liver-stages of malaria ad libitum. parasites [12, 13]. The P. chabaudi model shares several characteristics with P. falciparum, which causes about Protective vaccination 99% of global malaria-related deaths in humans [1]. Vaccination was performed under identical experimental The P. chabaudi model also appears appropriate to conditions as described previously [18]. Host cell plasma study the uncomprehended mechanisms of host defense membranes, isolated in the form of erythrocyte ghosts that occur in the liver during vaccination-induced sur- from P. chabaudi-parasitized erythrocytes, were used as vival in blood-stage malaria. First, an effective procedure a non-infectious vaccine, which was prepared as detailed of blood-stage vaccination for P. chabaudi is available previously [14, 22, 23]. Approximately 10 ghosts were [14]. The non-infectious vaccine consists of erythrocyte suspended in 100  µl Freund’s complete adjuvant (FCA) plasma membranes isolated from P. chabaudi-infected and subcutaneously injected at week 3 and week 1 before erythrocytes, which contain auto-antigens and parasite- infection with P. chabaudi-parasitized erythrocytes. Con- synthesized neo-antigens ([15, 16]; cf. also [17]). Immu- trol mice were treated in parallel with only FCA. nization with this vaccine results in survival of more than 80% mice, which would have otherwise succumbed to Plasmodium chabaudi malaria lethal malaria by P. chabaudi [14, 18]. This vaccination Blood-stage infections of P. chabaudi were maintained induces a healing course of the infection and reduces in outbred mice under sterile conditions by weekly pas- peak parasitaemia by approximately 30% on day 8 post- sages of infected red blood cells. A non-clonal line of P. infection (pi) and generation of long-lasting resistance chabaudi has been used [18, 20, 24]. This line resembles against homologous re-infections [10]. Secondly, the liver Plasmodium chabaudi chabaudi AS in terms of restric- of mice has been shown to respond to protective vac- tion fragment length polymorphism analysis [25] as well cination evidenced, for instance, as alterations in gene as sequence identity for dihydrofolate reductase and expression, miRNA expression, and DNA methylation of for a cysteine protease [26] with only a single nucleo- gene promoters upon blood-stage infection [10, 18–20]. tide exchange [18]. As the AS clone, the line used here A critical phase of P. chabaudi blood-stage infections has self-healing potential. However, this is controlled is the mid-precrisis on day 4 pi, when parasitized eryth- by sex and sex hormones, respectively, genes of the H-2 rocytes begin to appear in peripheral blood. At this early complex and genes of the non-H-2 background of the patency, parasitaemia ranges between 1 and 5% vary- infected mouse strain [27]. Challenge of Balb/c mice with ing with mice and does not differ between healing and Al‑Quraishy et al. Malar J (2018) 17:215 Page 3 of 16 10 P. chabaudi-infected erythrocytes, evaluation of par- Scanning and analyses of microarrays asitaemia, and counting of erythrocytes were performed Agilent microarray scanner system (Agilent Technolo- as described previously [18, 28]. Besides the sacrificed gies) was used for detecting fluorescence signals on the mice on day 0 pi and on day 4 pi, both the vaccinated hybridized microarrays. The microarray image files were group and the non-vaccinated group contained 4 ‘control’ processed with the Agilent feature extraction software mice, which were not sacrificed. In the non-vaccinated (FES), which determines feature intensities including group, all four mice succumbed to infection during crisis, background subtraction, rejects outliers, and calculates whereas only one mouse succumbed to infection during statistical confidences. Three different biological repli - crisis in the vaccinated group, but three mice survived cates were performed for each sample type, i.e., 12 micro- the infection for at least 3 weeks, in accordance with our arrays for four samples in toto. The expression variance previous results [18]. was stabilized through the lo g transform. Microarrays were normalized by the quantile method. The heat map of the most highly variable transcripts, the hierarchical clustering dendrograms (calculated using the unweighted RNA isolation pair group method with arithmetic mean and Euclidean Livers were aseptically removed from sacrificed mice, distance measure), and the Principal component analy- rapidly frozen in liquid nitrogen, and stored at − 80  °C sis were performed using in-home functions developed until use. For isolation of total RNA, livers were individ- in Matlab (MathWorks). The microarray data have been ually ground in a mortar under liquid nitrogen and ali- deposited at both the EMBL-EBI Array Express reposi- quots were subjected to standard RNA extraction using tory (Array accession number: E-MTAB.6494) and the Trizol. An additional RNA clean-up was followed using NCBI’s Gene Expression Omnibus (GEO) database with the miRNeasy Kit (Qiagen, Hilden, Germany). RNA accession number GSE111110 (https ://www.ncbi.nlm. integrity and quality was checked on the Agilent 2100 nih.gov/geo/query /acc.cgi?acc=GSE11 1110). Bioanalyzer platform (Agilent Technologies). The RIN values of all RNA samples ranged between 8.7 and 9.1. Quantitative real‑time PCR Quantitative real-time PCR was performed under RNA labelling experimental conditions identical to those described Each RNA sample was used to produce Cy3-labeled recently [28], using High Capacity cDNA Reverse Tran- cRNA. Equivalents of 100 ng from individual RNA sam- scription Kit (Life Technologies) and TaqMan mRNA ples were amplified and labelled using the Agilent Low assays (Life Technologies) for reverse transcription of Input Quick Amp Labelling Kit (Agilent Technologies) mRNAs encoding the following proteins: ERMAP (assay according to the manufacturer’s instructions. Yields of ID: Mm00469273_m1), CLDN13 (Mm00491038_s1), cRNA and dye-incorporation were determined using the CD163 (Mm00474091_m1), GZMB (Mm00442837_m1), ND-1000 Spectrophotometer (NanoDrop Technologies). KLRB1A (Mm00726548_s1), KLRC3 (Mm00650941_ The incorporations were between 18 and 23 fmol Cy3/ng m1), KLRD1 (Mm00495182_m1), NCR1 (Mm01337324_ cRNA. g1), KLRG1 (Mm00516879_m1), and GAPDH (Mm99999915_g1). Fold change of expression was calcu- −ΔΔct lated using the comparative Ct method (2 ) [29] and Hybridization of gene expression oligo microarrays data sets were analysed for statistical significance using Agilent mouse whole genome 8 × 60  K gene expres- a two-tailed unpaired heteroscedastic Student’s t test sion oligo microarrays (design 028005) were used for (*p < 0.01). hybridization. Each microarray displayed 39,430 Entrez Gene RNAs and 16,251 long intergenic non-coding Results (linc)RNAs. The Agilent Gene Expression Hybridization Identification of malaria‑inducible genes in the liver Kit was used for hybridization as detailed in the Agi- of vaccinated and non‑vaccinated mice lent processing protocol (Agilent technologies). In brief, To identify malaria-induced vaccination-responsive 600  ng of Cy3-labelled fragmented cRNA in hybridiza- genes in the liver at early patency, vaccinated and non- tion buffer was hybridized to the microarrays overnight vaccinated Balb/c mice were concomitantly infected with at 65  °C using the recommended hybridization chamber P. chabaudi, and livers prepared from three vaccinated and oven. Finally, the microarrays were washed with the mice on day 4 pi (Vd4 group) were individually analysed Agilent Gene Expression Wash Buffer 1 (1 min at 23 °C) with Agilent’s 8 × 60 K oligo microarrays for global gene and then with preheated Agilent Gene Expression Wash expression in relation to those of three non-infected Buffer 2 (1 min at 37 °C). Al‑Quraishy et al. Malar J (2018) 17:215 Page 4 of 16 vaccinated mice on day 0 pi (Vd0 group). Corresponding heatmap of the most highly variable RNA expression analyses were performed with livers prepared from three profiles of the four different groups, each with three rep - non-vaccinated mice on day 4 pi (Nd4 group) in relation licates. A group of transcripts is specifically expressed to those of three non-vaccinated mice on day 0 pi (Nd0 at intermediate level I in the Vd4 group, which is not group). Figure  1a shows the global expression analysis expressed in the Vd0, Nd0 and Nd4 groups. Figure  1b Fig. 1 Transcriptomic global analysis of the liver from vaccination‑protected Balb/c mice before infection with Plasmodium chabaudi on day 0 pi ( Vd0 group) and after infection on day 4 pi ( Vd4 group) as well as in non‑ vaccinated non‑protected mice in the Nd0 and Nd4 groups, respectively. a Heatmap. The colour bar at the top codifies the gene expression in the log2 scale. Higher RNA expression corresponds to increased intensity of the colour red. b Hierarchical clustering of samples. c PCA of RNA expression data. The PC1 captures 40% of the RNA expression variability and the PC2 captures 13% of the variability Al‑Quraishy et al. Malar J (2018) 17:215 Page 5 of 16 shows the hierarchical clustering (dendrogram) of the different samples performed using the correlation metric and the average linkage method. The dendrogram reveals a clean cluster of all the three replicates in the Vd4 group and another major cluster of the data from the Nd0, Vd0, and Nd4 group, with the subclusters of samples in the Vd0 and Nd0 groups clustering closer. Principal compo- nent analysis (PCA) of the gene expression data is shown in Fig. 1c, where the 1st principal component (PC1) cap- tures 40% of the gene expression variability and the 2nd principal component (PC2) captures 13% of the variabil- ity. The PCA indicates that the replicates of case cluster together, and that PC1, since capturing the higher per- centage of gene expression variability, clearly separates all the replicates of the Nd0, Nd4, Vd0 groups from those of the Vd4 group. Taken together, these results indicate that protective vaccination does not essentially affect consti - tutive RNA expression of livers on day 0 pi, whereas early patent infections with P. chabaudi blood-stage malaria induce changes in hepatic RNA expression, differing between the Vd4 and the Nd4 group. Malaria-induced changes in gene expression were then found in livers of both the Vd4 and Nd4 groups, when mRNAs were evaluated, which displayed > threefold Fig. 2 Number of genes expressed more than threefold (p < 0.01) in changed expressions at a stringent level of significance the liver of vaccinated mice infected with Plasmodium chabaudi on (p < 0.01) at both d4 groups in relation to correspond- day 4 pi ( Vd4 group) in relation to the corresponding constitutive expressions day 0 pi ( Vd0 group). Nd4 group vs. Nd0 group ing constitutive expressions of the Vd0 and Nd0 group, indicates the number of significantly expressed genes in the liver respectively. The total numbers of genes with signifi - of non‑ vaccinated mice. Numbers in brackets: genes with > tenfold cantly changed expression are summarized in the Venn changed expression diagrams shown in Fig. 2. Early patent infections induced upregulation of 329 genes in the Vd4 group, but only 173 genes in the Nd4 group, and 274 genes in both d4 groups. were changed by > tenfold in the Vd4 group. Table  1 A remarkably lower number of genes was found to be summarizes the 24 genes and their annotated func- downregulated, i.e., only 19 in the Vd4 group and still tions, among which 23 were upregulated and one gene fewer, namely 10 in the Nd4 group. The Additional file  1: was downregulated. Conspicuously, one-third of the 23 Tables S1 and S2 summarize the genes whose expression upregulated genes are known to be involved in erythroid is up- and downregulated by > threefold and < tenfold development. This group of genes includes Ahsp (48-fold) in the Nd4 group with p < 0.01, respectively. Additional and Kel (66-fold), which encode the alpha haemoglobin file  1: Table  S3 shows 16 genes whose expression is stabilizing protein and the Kell blood group, respectively. changed by > tenfold in the Nd4 group with 15 upregu- Other genes in this group encode erythroid constituents lated genes and only one downregulated gene. In the such as the erythroblast membrane-associated protein Vd4 group, 306 genes were identified to be upregulated Ermap, the Rhesus blood group-associated A glycopro- (Additional file  1: Table  S4) and 18 genes were down- tein, Rhag, and the soluble carrier family 4 Slc4a1, which regulated by > threefold and < tenfold, respectively (Addi- is also termed band 3 and is one of the major erythroid tional file  1: Table  S5). Additional file  1: Table  S6 shows integral multi-pass surface membrane proteins. The all genes that were significantly upregulated by > three- Slc4a1-encoded band 3 is known to function as a chlo- fold in both Vd4 and Nd4 groups. ride/bicarbonate exchanger transporting carbon dioxide and to associate with glycophorin A (GYPA), which is Characterization of genes changed by > tenfold in the Vd4 another major integral one-pass red cell membrane pro- group tein. Remarkably, the expression of Gypa gene is almost To further restrict the number of candidate genes of tenfold higher in vaccination-protected mice (Additional potential importance for early patency and final survival, file  1: Table  S4), similar to Car13 encoding carbonate the analysis concentrated on genes, whose expressions Al‑Quraishy et al. Malar J (2018) 17:215 Page 6 of 16 Table 1 Genes expressed more or  less  than tenfold (p < 0.01) in  the  liver of  vaccinated mice infected with  P. chabaudi on day 4 p.i. (Vd4) in comparison to constitutive expression on day 0 p.i. (Vd0) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Function (annotated according to www. genec ards.org) Erythropoiesis Ahsp Alpha hemoglobin stabilizing protein NM_133245 48.18 0.0095 Acts as a chaperone to prevent the harmful aggregation of alpha‑hemoglobin dur ‑ ing normal erythroid cell development. Specifically protects free alpha‑hemo ‑ globin from precipitation. It is predicted to modulate pathological states of alpha‑hemoglobin excess such as beta‑ thalassemia Cldn13 Claudin 13 NM_020504 43.50 0.0070 Plays a major role in tight junction‑specific obliteration of the intercellular space, through calcium‑independent cell‑ adhesion activity Ermap Erythroblast membrane‑associated protein NM_013848 21.59 0.0036 The protein encoded by this gene is a cell surface transmembrane protein that may act as an erythroid cell receptor, possibly as a mediator of cell adhesion Gata1 GATA binding protein 1 NM_008089 29.73 0.0015 Transcriptional activator or repressor which probably serves as a general switch fac‑ tor for erythroid development Gfi1b Growth factor independent 1B NM_008114 20.50 0.0092 Essential proto‑ oncogenic transcriptional regulator necessary for development and differentiation of erythroid and mega‑ karyocytic lineages Kel Kell blood group NM_032540 65.89 0.0010 This gene encodes a type II transmem‑ brane glycoprotein that is the highly polymorphic Kell blood group antigen Rhag Rhesus blood group‑associated A glyco ‑ NM_011269 17.37 0.0001 The protein encoded by this gene is eryth‑ protein rocyte‑specific and is thought to be part of a membrane channel that transports ammonium and carbon dioxide across the blood cell membrane Slc4a1 Solute carrier family 4 NM_011403 11.74 0.0067 Major integral membrane glycoprotein of the erythrocyte membrane; required for normal flexibility and stability of the erythrocyte membrane and for normal erythrocyte shape via the interactions of its cytoplasmic domain with cytoskel‑ etal proteins, glycolytic enzymes, and hemoglobin Cell cycle and mitosis Ccnb1 Cyclin B1 NM_172301 17.76 < 0.0001 The protein encoded by this gene is a reg‑ ulatory protein involved in mitosis. The gene product complexes with p34(cdc2) to form the maturation‑promoting factor (MPF) Cdc25c Cell division cycle 25C NM_009860 12.41 0.0009 Cdc25 activates cdk complexes that drive the cell cycle. Cdc25 is involved in the DNA damage checkpoints and is known as a key mediator of cell cycle progres‑ sion Ckap2 l Cytoskeleton associated protein 2‑like NM_181589 10.72 0.0030 Microtubule‑associated protein required for mitotic spindle formation and cell‑ cycle progression in neural progenitor cells Al‑Quraishy et al. Malar J (2018) 17:215 Page 7 of 16 Table 1 (continued) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Function (annotated according to www. genec ards.org) Innate immunity Abcg4 ATP‑binding cassette, sub ‑family G ( WHITE), NM_138955 26.79 0.0035 The protein encoded by this gene is member 4 included in the superfamily of ATP‑ binding cassette (ABC) transporters. May be involved in macrophage lipid homeostasis Ccl7 Chemokine (C‑ C motif ) ligand 7 NM_013654 34.97 0.0013 Chemotactic factor that attracts mono‑ cytes and eosinophils, but not neutro‑ phils Socs1 Suppressor of cytokine signaling 1 NM_009896 11.32 0.0070 This gene encodes a member of the STAT‑ induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling (SOCS), family. SSI family members are cytokine‑inducible negative regulators of cytokine signaling. The expression of this gene can be induced by a subset of cytokines, including IL2, IL3 erythropoi‑ etin (EPO), CSF2/GM‑ CSF, and interferon (IFN)‑ gamma Treml2 Triggering receptor expressed on myeloid NM_001033405 10.92 0.0001 Cell surface receptor that may play a role cells‑like 2 in the innate and adaptive immune response. Acts as a counter‑receptor for CD276 and interaction with CD276 on T‑ cells enhances T‑ cell activation Cd163 CD163 antigen NM_053094 0.03 0.0018 The protein encoded by this gene is a member of the scavenger receptor cysteine‑rich (SRCR) superfamily, and is exclusively expressed in monocytes and macrophages. It functions as an acute phase‑regulated receptor involved in the clearance and endocytosis of hemoglobin/haptoglobin complexes by macrophages, and may thereby protect tissues from free hemoglobin‑mediated oxidative damage Cytotoxicity Gzmb Granzyme B NM_013542 35.66 0.0085 The encoded preproprotein is secreted by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) and proteolyti‑ cally processed to generate the active protease, which induces target cell apoptosis. This protein also processes cytokines and degrades extracellular matrix proteins, and these roles are implicated in chronic inflammation and wound healing Klrb1a Killer cell lectin‑like receptor subfamily B NM_010737 10.43 0.0083 Plays an inhibitory role on natural killer (NK) member 1A cells cytotoxicity. Activation results in specific acid sphingomyelinase/SMPD1 stimulation with subsequent marked elevation of intracellular ceramide. Activation also leads to AKT1/PKB and RPS6KA1/RSK1 kinases stimulation as well as markedly enhanced T‑ cell proliferation induced by anti‑ CD3 Klrc3 Killer cell lectin‑like receptor subfamily C, NM_021378 11.38 0.0002 KLRC3 is a member of the NKG2 group member 3 which are expressed primarily in natural killer (NK) cells and encodes a family of transmembrane proteins characterized by a type II membrane orientation (extra‑ cellular C terminus) and the presence of a C‑type lectin domain Al‑Quraishy et al. Malar J (2018) 17:215 Page 8 of 16 Table 1 (continued) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Function (annotated according to www. genec ards.org) Klrd1 Killer cell lectin‑like receptor, subfamily D, NM_010654 16.33 0.0067 Several genes of the C‑type lectin super ‑ member 1 family, including members of the NKG2 family, are expressed by NK cells and may be involved in the regulation of NK cell function. Plays a role as a receptor for the recognition of MHC class I HLA‑E mol‑ ecules by NK cells and some cytotoxic T‑ cells Ncr1 Natural cytotoxicity triggering receptor 1 NM_010746 16.97 0.0084 Cytotoxicity‑activating receptor that may contribute to the increased efficiency of activated natural killer (NK) cells to medi‑ ate tumor cell lysis Miscellaneous Hist1h3g Histone cluster 1, H3g NM_145073 18.90 0.0075 Core component of nucleosome Htr7 5‑Hydroxytryptamine (serotonin) receptor 7 NM_008315 12.53 < 0.0001 Serotonin 5‑HT7 receptors are located primarily in the thalamus, hypothala‑ mus and hippocampus. The function of these receptors includes the regulation of circadian rhythms, thermoregula‑ tion, learning and memory and smooth muscle relaxation Nxpe5 Neurexophilin and PC‑ esterase domain fam‑ NM_001013773 22.61 0.0089 Unknown ily, member 5 anhydrase (besides Car2 and Car9), which is known to 22 genes are apparently involved in mitosis, particularly interconvert carbon dioxide and bicarbonate to maintain in the formation and function of the mitotic spindle. The the acid–base balance in blood and to help transport car- two genes Prr11 and Sapcd2 are critically involved in cell bon dioxide out of tissues. Moreover, the group of genes cycle progression. The gene Klrb1f encodes a killer cell upregulated by > tenfold also lists the transcription fac- lectin-like receptor, which plays an inhibitory role in NK tors Gata1 and Gfi1b as well as Cldn13 upregulated by cell cytotoxicity. Birc5 is still remarkable because it codes even > 43-fold. for a negative regulatory protein preventing apoptotic Furthermore, a group of five genes encoding proteins, cell death. known to be involved in cellular cytotoxicity, such as Among the four genes involved in innate immu- Gzmb encoding granzyme B (36-fold), Ncr1 encod- nity, expression of the suppressor of cytokine signal- ing the natural cytotoxicity triggering receptor 1, and ing 1, Socs1, was upregulated approximately by 11-fold Klrb1a, Klrc3, and Klrd1 encoding three different killer (Table  1). Cd163 is the only gene whose expression was cell lectin-like receptors, were upregulated by > tenfold significantly downregulated by > tenfold in the Vd4 group (Table  1). Remarkably, among the six genes significantly (Table 1; Fig. 3). upregulated by > tenfold in the liver in both Vd4 and Finally, quantitative PCR was used to reexamine the Nd4 groups (Additional file  1: Table S7), one gene, Klrg1, expression of some of the genes, which were identified to encoding the killer cell lectin-like receptor subfamily be expressed by > tenfold in the Vd4 group in the micro- G, was overexpressed by at least 100% in the Vd4 group arrays, particularly Ermap, Cldn13, Cd163, Gzmb, Ncr1, compared to the Nd4 group, i.e., 39.5-fold vs. 14.4-fold. Klrb1a, Klrd1, Klrc3, and Klrg1. Figure  3 shows that the Another group of genes in Table 1 includes the 3 genes constitutive expression of these genes was not signifi - Ccbn1, Cdc25c, and Ckap 21 that were upregulated cantly affected by protective vaccination. However, early between 10- and 18-fold, and are known to be involved patent infections of P. chabaudi significantly changed in cell cycle control including mitosis (Table  1). Table  2 the expression of these genes in the Vd4 group, which is also shows 22 genes extracted from Additional file  1: comparable with the result of the microarrays. Table S6, whose expression is significantly upregulated by > tenfold in the Vd4 group and, concomitantly, by at least LincRNAs expressed in vaccination‑protected mice 100% more than the corresponding genes significantly There is increasing evidence that long non-coding (lnc) expressed in the Nd4 group. Conspicuously, 15 of these RNAs including long intergenic non-coding (linc)RNAs Al‑Quraishy et al. Malar J (2018) 17:215 Page 9 of 16 Table 2 Hepatic expression of genes up-regulated by more than tenfold (p < 0.01) at Vd4 and by 100% more than at Nd4 Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Nd4 vs. Nd0 p value Function (annotated according to www.genec ards.org) Mitosis Bub1 Budding uninhibited by NM_009772 20.11 0.0049 5.88 0.0021 Serine/threonine‑protein kinase benzimidazoles 1 homolog that performs two crucial func‑ tions during mitosis: it is essential for spindle‑assembly checkpoint signaling and for correct chromo‑ some alignment Espl1 Extra spindle poles‑like 1 NM_001014976 12.35 0.0013 4.98 0.0020 Caspase‑like protease, which plays a central role in the chromo‑ some segregation by cleaving the SCC1/RAD21 subunit of the cohesin complex at the onset of anaphase Mki67 Antigen identified by monoclonal NM_001081117 13.08 0.0004 4.29 0.0014 Required to maintain individual antibody Ki 67 mitotic chromosomes dispersed in the cytoplasm following nuclear envelope disassembly Mxd3 Max dimerization protein 3 NM_016662 10.52 0.0031 3.84 0.0066 This gene encodes a member of the Myc superfamily of basic helix‑loop ‑helix leucine zip ‑ per transcriptional regulators. The encoded protein forms a heterodimer with the cofactor MAX which binds specific E‑box DNA motifs in the promoters of target genes and regulates their transcription Ndc80 NDC80 homolog, kinetochore NM_023294 11.19 0.0004 4.74 0.0005 Acts as a component of the complex component essential kinetochore‑associated NDC80 complex, which is required for chromosome segre‑ gation and spindle checkpoint activity Nusap1 Nucleolar and spindle associated NM_133851 14.19 0.0002 4.89 0.0005 NUSAP1 is a nucleolar‑spindle ‑asso ‑ protein 1 ciated protein that plays a role in spindle microtubule organization Plk1 Polo‑like kinase 1 NM_011121 14.14 0.0035 4.13 0.0018 Serine/threonine‑protein kinase that performs several important functions throughout M phase of the cell cycle, including the regulation of centrosome matura‑ tion and spindle assembly, the removal of cohesins from chro‑ mosome arms, the inactivation of anaphase‑promoting complex/ cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis Prc1 Protein regulator of cytokinesis 1 NM_145150 15.38 < 0.0001 4.28 0.0003 The protein is present at high levels during the S and G2/M phases of mitosis but its levels drop dramatically when the cell exits mitosis and enters the G1 phase Racgap1 Rac GTPase‑activating protein 1 NM_001253809 12.67 0.0042 3.83 0.0003 Component of the centralspin‑ dlin complex that serves as a microtubule‑ dependent and Rho‑mediated signaling required for the myosin contractile ring formation during the cell cycle cytokinesis Al‑Quraishy et al. Malar J (2018) 17:215 Page 10 of 16 Table 2 (continued) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Nd4 vs. Nd0 p value Function (annotated according to www.genec ards.org) Ska1 Spindle and kinetochore associ‑ NM_025581 23.19 0.0004 5.46 0.0039 Component of the SKA1 complex, a ated complex subunit 1 microtubule‑binding subcomplex of the outer kinetochore that is essential for proper chromosome segregation Ska3 Spindle and kinetochore associ‑ NM_198605 15.24 0.0027 4.68 0.0013 This gene encodes a component ated complex subunit 3 of the spindle and kinetochore‑ associated protein complex that regulates microtubule attach‑ ment to the kinetochores during mitosis Ticrr TOPBP1‑interacting checkpoint NM_029835 14.22 0.0008 4.66 0.0093 Regulator of DNA replication and and replication regulator S/M and G2/M checkpoints. Regulates the triggering of DNA replication initiation via its inter‑ action with TOPBP1 by participat‑ ing in CDK2‑mediated loading of CDC45L onto replication origins Tpx2 TPX2, microtubule‑associated NM_028109 16.11 0.0032 4.28 0.0011 Spindle assembly factor required protein homolog for normal assembly of mitotic spindles. Required for normal assembly of microtubules during apoptosis Ube2c Ubiquitin‑ conjugating enzyme NM_026785 12.49 < 0.0001 4.32 0.0010 Essential factor of the anaphase E2C promoting complex/cyclosome (APC/C), a cell cycle‑regulated ubiquitin ligase that controls progression through mitosis Cell cycle/cell signaling Klrb1f Killer cell lectin‑like receptor NM_153094 10.63 0.0005 5.32 0.0003 Plays an inhibitory role on natural subfamily B member 1F killer (NK) cells cytotoxicity. Activation results in specific acid sphingomyelinase/SMPD1 stimu‑ lation with subsequent marked elevation of intracellular ceramide Birc5 Baculoviral IAP repeat‑ containing NM_009689 16.68 0.0005 3.47 0.0006 This gene is a member of the 5 inhibitor of apoptosis (IAP) gene family, which encode negative regulatory proteins that prevent apoptotic cell death Cd7 CD7 antigen NM_009854 13.52 0.0022 4.54 0.0005 Plays an essential role in T‑ cell inter‑ actions and also in T‑ cell/B‑ cell interaction during early lymphoid development Kif11 Kinesin family member 11 NM_010615 11.80 0.0065 3.62 0.0077 Motor protein required for estab‑ lishing a bipolar spindle during mitosis Prr11 Proline rich 11 NM_175563 12.13 0.0001 4.52 0.0001 Plays a critical role in cell cycle progression Sapcd2 Suppressor APC domain contain‑ NM_001081085 11.96 0.0001 3.30 0.0040 Plays a critical role in cell cycle ing 2 progression Miscellaneous Iqgap3 IQ motif containing GTPase acti‑ NM_001033484 16.36 0.0002 3.88 0.0019 Unknown vating protein 3 Raet1c Retinoic acid early transcript NM_009018 15.69 0.0001 4.83 0.0011 Unknown gamma Al‑Quraishy et al. Malar J (2018) 17:215 Page 11 of 16 Fig. 3 Quantitative PCR of mRNAs in the liver of Balb/c mice in comparison with the microarray data. Livers were isolated from three vaccinated mice before infection with Plasmodium chabaudi on day 0 pi ( Vd0 group), from three vaccinated mice after infection on day 4 pi ( Vd4 group), and from three non‑ vaccinated mice at Nd0 group and at Nd4 group. Means of duplicate determinations, performed with liver aliquots from three different mice, with half SEM. Stars and hashtags indicate significant differences (p < 0.01) between Vd4 and Vd0 groups as revealed by qRT‑PCR and microarrays, respectively are critical for the course and outcome of different dis - upregulated in the Vd4 group in common with the Nd4 eases, including diverse liver diseases [30]. These non- group and 1 lincRNA is downregulated (Additional file  1: coding RNAs range in size between 200 bp and ~ 100 kb. Table S9). LincRNAs do not overlap with annotated coding regions Remarkably, the two lincRNA species lincRNA per definitionem, though an increasing number of lincR - :chr10:117021051-117038683 forward strand and NAs has been recently identified to contain small open lincRNA:chr2:84344350-843770075 reverse strand are reading frames coding for small functional peptides [31]. more highly expressed in the Vd4 group (by > 100%) Moreover, lincRNAs are widely transcribed in mamma- than in the Nd4 group. LincRNA species that are signifi - lian cells, though at lower levels and in a more cell-type cantly up- and down-regulated in non-vaccinated mice specific manner than mRNA [32–34]. The used micro - in the Nd4 group (Additional file  1: Table S9) differ from arrays contain 16,251 lincRNA probes and have been those identified in vaccination-protected mice in the Vd4 evaluated for lincRNA expression using the same strin- group. Annotated functions are not yet available for any gent conditions as those used for mRNAs. Early patent of these differentially regulated lincRNAs. infections of P. chabaudi blood-stage malaria induce changes in the expression of lincRNAs, which differ sig - Discussion nificantly (p < 0.01) between vaccination-protected and This study provides evidence that the hepatic response non-protected non-vaccinated mice (Fig.  4). In vacci- in terms of mRNA and lincRNA expression, to P. nation-protected mice, 7 lincRNAs are downregulated chabaudi blood-stage malaria at early patency differs and 19 lincRNAs are upregulated by > threefold (Addi- between vaccination-induced healing infections and tional file  1: Table  S8), among which one lincRNA is lethal infections in non-vaccinated mice. In particular, upregulated by > tenfold. In addition, 13 lincRNAs are 24 genes are altered by > tenfold at p < 0.01 in the liver Al‑Quraishy et al. Malar J (2018) 17:215 Page 12 of 16 Extramedullary erythropoiesis in the liver of vaccina- tion-protected mice has been recently shown to occur towards the end of the crisis phase on day 11 pi [28]. Crisis is characterized by much higher expression of erythroid-associated genes than that described here at early patency on day 4 pi For instance, the genes Ermap, Slca1, Gata1, and Gfi1b are expressed by > 100-fold at crisis. As previously shown under identical experimen- tal conditions, crisis in vaccination-protected mice is also characterized by a dramatic decrease in peripheral P. chabaudi-parasitized erythrocytes and, concomitantly, in a dramatic increase in the number of peripheral reticulo- cytes, the latter being impaired in non-vaccinated mice [18]. Incidentally, reticulocytes are not the preferred host cells for P. chabaudi [35]. At early patency, however, P. chabaudi-parasitized erythrocytes only begin to appear in the peripheral blood [18]. The number of periph - eral reticulocytes is still very low and not yet essentially changed [18]. These data suggest: (1) that extramedullary erythropoiesis occurring in the liver at early patency of the malaria blood-stage infections is still in an early state: and, (2) that extramedullary erythropoiesis in the liver is apparently accelerated in vaccination-protected mice in comparison to non-vaccinated mice. Fig. 4 Number of lincRNAs with expressions changed by > threefold There is evidence indicating that stress, including (p < 0.01) in the liver of non‑ vaccinated (N) and vaccinated mice ( V ) psychological stress, chemicals, and diverse viral and infected with Plasmodium chabaudi on day 4 pi in relation to their bacterial infections, can induce extramedullary eryth- constitutive expression in the Nd0 and Vd0 groups, respectively. ropoiesis in several organs, particularly in the spleen, of Numbers in brackets indicate the lincRNAs with > tenfold changed mice and even humans [36–46]. Even endo- and ecto- expressions parasites such as ticks [47] or Trypanosoma congolense [48] are able to induce extramedullary erythropoiesis in the spleen of their hosts. Remarkably, the latter has of vaccination-protected mice. In addition, there are 22 been found to be associated with increased expression genes > tenfold expressed in vaccination-protected mice of the apparent mouse-specific gene, Cldn13, encoding which are at least 100% higher induced than the corre- the most abundant claudin of the 26-membered clau- sponding genes significantly expressed in non-vaccinated din family in the spleen [49, 50]. Claudins are the main mice. These data indicate that, at early patency, critical constituents of tight junctions; however, CLDN13 has processes occur in the liver, which may contribute to vac- been predicted to be localized on the surface of erythro- cination-induced survival of blood-stage infections. blasts in the spleen [48]. Previously, a massively upregu- One of these processes may be extramedullary eryth- lated expression of Cldn13 by > 100-fold has been found ropoiesis in the liver. Indeed, approximately one-third of towards the end of the crisis phase of P. chabaudi blood- the 23 genes induced in vaccination-protected mice by stage infections in vaccination-protected mice and it was > tenfold are erythroid-associated genes, encoding the therefore suggested that Cldn13 is locally expressed in/ Kell blood group, the Rhesus blood group-associated gly- around erythroblast islands in the liver [28]. The present coprotein A, extrinsic and intrinsic membrane proteins data would then indicate that Cldn13 is already expressed such as ERMAP, SLC4A1 (band 3), and glycophorin A, at early extramedullary erythropoiesis in the liver of vac- as well as the transcription factors GATA1 and GFI1b, cination-protected mice. Another gene possibly involved which are key regulators of erythroid development in liver erythropoiesis may be Cd163, which is the only [28]. These genes were not identified to be significantly gene found to be downregulated by > tenfold, since the (p < 0.01) expressed in non-vaccinated mice, except for encoded transmembrane scavenger receptor CD163 on Ermap, which was induced only by about fivefold in the the surface of Kupffer cells has been described to serve Nd4 group (Additional file 1: Table S1). not only in clearance and endocytosis of haemoglobin/ Al‑Quraishy et al. Malar J (2018) 17:215 Page 13 of 16 haptoglobin complexes [51–53], but also as an adhesion the end of the crisis phase in vaccination-protected mice factor for erythroblasts in erythroblastic islands [53, 54]. when there is a massive appearance of reticulocytes in the An increase in killer cells, i.e., NK cells, NKT cells, and peripheral blood [28]; concomitantly, the liver has been cytolytic CD8 cells [55], may also occur in the liver of shown to dramatically increase its uptake of particulate vaccination-induced healing infections at early patency. material [18] including P. chabaudi-parasitized erythro- This view is supported by the present finding that the cytes [68]. Thus, it is possible that the increased genera - granzyme b gene Gzmb, the natural cytotoxicity-trigger- tion of NK cells in the liver of vaccination-protected mice ing receptor 1 gene Ncr1, and the killer cell lectin–lectin at early patency may not fortuitously correlate with early like receptor genes Klrb1a, Klrc3, Klrd1, and Klrg1 are erythropoiesis in the liver. massively upregulated by > tenfold in vaccination-pro- Indeed, a recent report described that murine Cyto- tected mice. KLRs, GZMB inducing apoptosis in target megalovirus (MCMV) infections induce extramedul- cells via the caspase-mediated apoptotic pathway, and lary haematopoiesis in the spleen with a dominance of NCR1 are known to be predominantly expressed on NK the red blood cell lineage [62]. The development of this cells, though NCR1 is also expressed on type 1 innate extramedullary haematopoiesis requires the cytotoxic lymphoid cells [56–59]. The increased mRNA levels function of NK cells rather than their cytokine produc- encoded by Klrs, Gzmb, and Ncr1 might be interpreted as tion. This cytotoxic activity of NK cells is obviously to be due to an immigration of peripheral c(conventional) responsible for confining virus spread, thereby protect - NK cells [55] from circulation into the liver. On the other ing extramedullary haematopoietic niches and facilitat- hand, however, the major lymphocyte population in the ing extramedullary haematopoiesis, which otherwise is liver is presumably another subset of NK cells, namely suppressed by MCMV [69]. Depression of cytokine sign- liver-resident NK cells developing from progenitor cells aling in the liver of vaccination-protected mice at early in the liver [55]. Increasing evidence indicates that the patency is indicated by a dramatic decline in the expres- liver-resident NK cell subset differs in phenotype and sion of Ifnγ and Tnfα [18]. This is predictable because the function from cNK cells [55, 60], though both NK cell expression of Socs1 encoding the suppressor of cytokine subsets produce about the same high levels of GZMB signaling is increased by > tenfold in the liver of vaccina- − + [59]. In contrast to the CD49a DX5 cNK cells, the liver- tion-protected mice, but not in non-vaccinated mice at + − resident NK cells are CD49a DX5 and even differ, also early patency. It is, therefore, possible that Socs1 is criti- in terms of gene expression signatures, from cNK cells cally involved in the accelerated generation of liver-resi- and other tissue-resident NK cells, as e.g. those distinct dent killer cells, particularly NK cells. lineages of NK cells occurring in spleen, thymus, and Accelerated extramedullary erythropoiesis and genera- uterus [61]. Thus, it is more attractive to speculate that tion of killer cells in the liver may also explain why the the upregulated mRNA levels of the different NK cell present study identified a group of genes known to be markers found here to be induced by blood-stage malaria involved in cell cycle regulation and especially mitosis in the liver of vaccination-protected mice may reflect an i.e., Ccbn1, Cdc25c, and Ckap 21, at early patency in the intra-hepatic accelerated generation of liver-resident NK liver of vaccination-protected mice. Additionally, the vast cells. majority of the > tenfold expressed 22 genes, which are at Liver-resident NK cells have been described to exert least 100% higher induced in vaccination-protected mice numerous functions, but their predominant function is than the corresponding genes significantly expressed in killing of target cells using different apoptotic pathways non-vaccinated mice, is known to be involved in mitosis [55, 60, 62]. For instance, NK cells kill myofibroblasts, and cell cycle control. Even erythroblast enucleation dur- which are known to induce liver fibrosis, thus limiting ing erythroid development can be regarded as an asym- the spread of fibrosis in the liver [62]. There is evidence metric mitosis [28, 48]. However, several of these genes that NK cells are also able to kill Plasmodium-para- such as Bub1, Nusap1, Prc1, Ska1, and Ube2c, have also sitized erythrocytes thus contributing to protection from previously been found to be expressed at about the same murine and human malaria [63–67]. It is therefore plau- level, as here at early patency, towards the end of crisis in sible to assume that NK cells attack P. chabaudi-infected vaccination-protected mice [28]. u Th s, it is not unlikely erythrocytes in the liver thus transforming the normally that the changes in the expression of these cell cycle and tolerogenic milieu of the liver to an increasingly hostile mitosis controlling genes reflect accelerated extramed - parasite environment, at least at early patency when P. ullary erythropoiesis and generation of liver-resident chabaudi-infected erythrocytes begin to appear in the cytotoxic cells and may also be associated with acceler- peripheral blood. Remarkably, an increased NK cell activ- ated liver regeneration in general. Indeed, there is evi- ity, in terms of the here found upregulated genes of NK dence that the liver during the acute phase of P. chabaudi cell markers, has not been previously observed towards blood-stage malaria is pathologically damaged and even Al‑Quraishy et al. Malar J (2018) 17:215 Page 14 of 16 heavily injured with distant effects on other organs such more than tenfold at early patency and by more than 100- as in hepatoencephalopathy [9, 10]. Even Plasmodium fold at crisis in the liver of vaccination-protected mice [28], falciparum and Plasmodium vivax malaria in humans is is decreased by 80%. Specifically, Bloodlinc is located in the associated with massive liver dysfunction [70–73]. Accel- coordinates chr11:102,231,615–102,237,204 of the version erated liver regeneration may therefore contribute to mm9 of the mouse genome, which is also used for anno- accelerated recovery from the malaria-induced dysfunc- tation of our lincRNA containing arrays; incidentally, the tions of the liver [20, 28]. latter do not contain any specific probes for the lncRNA Finally, the present data demonstrate that P. chabaudi Bloodlinc. blood-stage malaria does not only alter gene expression in Collectively, the present data indicate that protective vac- the liver at early patency, but also affects the expression of cination changes the hepatic response in terms of mRNA lincRNAs, and this lincRNA expression has changed after and lincRNA expression, to early patent healing infections protective vaccination. The identified lincRNAs are not of P. chabaudi blood-stage malaria. These changes are sug - yet functionally annotated, as it is typical for most other gested to be associated with an accelerated occurrence of known lincRNAs [31]. In general, however, evidence is extramedullary erythropoiesis, generation of liver-resident increasing that lncRNAs including lincRNAs play a criti- cytotoxic cells, and liver regeneration. These accelerated cal role in nuclear organization and chromatin remode- processes at early patency may be of critical importance for ling, in cell-type specific activation and repression of gene the final vaccination-induced healing outcome of the oth - expression through diverse mechanisms, in tissue-specific erwise lethal blood-stage P. chabaudi malaria. fine-tuning of the expression of neighbouring genes, in reg - Additional file ulation of cell-lineage development, and in course and out- come of diverse diseases including liver diseases [31, 33, 34, Additional file 1: Table S1. Genes, whose expression is up ‑regulated 74–76]. Here, several lincRNA species have been identified more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of non‑ vac‑ in the liver, whose malaria-induced expression is increased cinated mice infected with P. chabaudi on day 1 p.i. (Nd1) in comparison to constitutive expression on day 0 p.i. (Nd0). Table S2. Genes, whose by protective vaccination during mid-precrisis on day 4 pi, expression is down‑regulated more than 3‑fold and less than 10‑fold and whose expression is still more increased in the liver of (p < 0.01) in the liver of non‑ vaccinated mice infected with P. chabaudi vaccination-protected mice towards the end of the crisis on day 1 p.i. (Nd1) in comparison to constitutive expression on day 0 p.i. (Nd0). Table S3. Genes up‑regulated and down‑regulated more than phase on day 11 pi as described recently [28]. For instance, 10‑fold (p < 0.01) in the liver of vaccinated mice infected with P. chabaudi expression of the lincRNA:chr12:32781477-32808567 on day 1 p.i. (Nd1) in relation to constitutive expression on day 0 p.i. is upregulated from 6.6 in the Vd4 group to 71.9 in the (Nd0. Table S4. Genes, whose expression is up‑regulated more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of both non‑ vaccinated Vd11 group, the lincRNA:chr5:77084398-77086144 (N) and vaccinated mice ( V ) infected with P. chabaudi on day 1 p.i. ( Vd1, reverse strand from 10.7 to 19.9, the Nd1) in comparison to constitutive expression on day 0 p.i. ( Vd0, Nd0). lincRNA:chr15:61984389-62102500 reverse strand from Table S5. Genes, whose expression is down‑regulated more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of both non‑ vaccinated (N) and 3.2 to 6.1, and the lincRNA:chr10:83980790-83986015 vaccinated mice ( V ) infected with P. chabaudi on day 1 p.i. ( Vd1, Nd1) in reverse strand from 3.1 to 9.7, respectively. At least these comparison to constitutive expression on day 0 p.i. ( Vd0, Nd0). Table S6. 4 lincRNA species in the liver may be speculated to con- Genes expressed more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of vaccinated mice infected with P. chabaudi on day 1 p.i. ( Vd1) in tribute to vaccination-induced healing of the otherwise comparison to constitutive expression on day 0 p.i. ( Vd0). Table S7. Genes lethal P. chabaudi malaria infections. Currently, the role down‑regulated more than 3‑fold and less than 10‑fold (p < 0.01) in the of lncRNAs is still poorly understood with respect to liver of vaccinated mice infected with P. chabaudi on day 1 p.i. ( Vd1) in comparison to constitutive expression on day 0 p.i. ( Vd0). Table S8. Lin‑ extramedullary erythropoiesis and/or generation of liver- cRNAs up‑regulated more than 3‑fold (p < 0.01) in the liver of vaccinated resident killer cells and/or hepatic regeneration [62] and/ mice infected with P. chabaudi on day 1 p.i. ( Vd1) in comparison to consti‑ or megakaryopoiesis in the liver [20]. Only erythropoie- tutive expression on day 0 p.i. ( Vd0).Table S9. LincRNAs down‑regulated more than 3‑fold (p < 0.01) in the liver of vaccinated mice infected with P. sis has been recently shown to be associated with diverse chabaudi on day 1 p.i. ( Vd1) in comparison to constitutive expression on lncRNAs [75, 76], particularly in steps of erythropoiesis day 0 p.i. ( Vd0). that are targeted by the transcription factor GATA1 [77, 78]. The expression of Gata1 in the liver was here detected to be upregulated by > tenfold at early patency during the Authors’ contributions MAD, SA, and FW designed the study. MAD, EMA, ASA, MJA and DD carried pre-crisis phase and, still more, by > 100-fold towards the out the experiments and analysed the data. SA prefinanced part of the experi‑ end of the crisis phase in vaccination-protected mice [28]. mental work. All authors wrote and revised the manuscript. All authors read One study has shown [77] that when the erythroid-specific and approved the final manuscript. lncRNA species alncRNA-EC7, also known as Bloodlinc, Author details is knocked down, the expression of the 10 kb away located 1 Department of Zoology, College of Science, King Saud University, P.O. Box: gene Slc4a1, which encodes the band 3 erythrocyte mem- 2455, Riyadh 11451, Saudi Arabia. Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt. Department of Zoology, brane protein and which is found here to be expressed by Al‑Quraishy et al. Malar J (2018) 17:215 Page 15 of 16 Faculty of Science, Beni‑Suef University, Beni‑Suef, Egypt. Group of Com‑ 12. Longley R, Smith C, Fortin A, Berghout J, McMorran B, Burgio G, et al. Host putational Biology and Systems Biomedicine, Biodonostia Health Research resistance to malaria: using mouse models to explore the host response. Institute, San Sebastián, Spain. 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Gene expression of the liver of vaccination-protected mice in response to early patent infections of Plasmodium chabaudi blood-stage malaria

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Biomedicine; Parasitology; Tropical Medicine; Infectious Diseases; Entomology; Microbiology; Public Health
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Abstract

Background: The role of the liver for survival of blood‑ stage malaria is only poorly understood. In experimental blood‑ stage malaria with Plasmodium chabaudi, protective vaccination induces healing and, thus, survival of other‑ wise lethal infections. This model is appropriate to study the role of the liver in vaccination‑ induced survival of blood‑ stage malaria. Methods: Female Balb/c mice were vaccinated with a non‑ infectious vaccine consisting of plasma membranes isolated in the form of erythrocyte ghosts from P. chabaudi‑ infected erythrocytes at week 3 and week 1 before infec‑ tion with P. chabaudi blood‑ stage malaria. Gene expression microarrays and quantitative real‑ time PCR were used to investigate the response of the liver, in terms of expression of mRNA and long intergenic non‑ coding (linc)RNA, to vaccination‑ induced healing infections and lethal P. chabaudi malaria at early patency on day 4 post infection, when parasitized erythrocytes begin to appear in peripheral blood. Results: In vaccination‑ induced healing infections, 23 genes were identified to be induced in the liver by > tenfold at p < 0.01. More than one‑ third were genes known to be involved in erythropoiesis, such as Kel, Rhag, Ahsp, Ermap, Slc4a1, Cldn13 Gata1, and Gfi1b . Another group of > tenfold expressed genes include genes involved in natural cyto‑ toxicity, such as those encoding killer cell lectin‑ like receptors Klrb1a, Klrc3, Klrd1, the natural cytotoxicity‑ triggering receptor 1 Ncr1, as well as the granzyme B encoding Gzmb. Additionally, a series of genes involved in the control of cell cycle and mitosis were identified: Ccnb1, Cdc25c, Ckap2l were expressed > tenfold only in vaccination‑ protected mice, and the expression of 22 genes was at least 100% higher in vaccination‑ protected mice than in non‑ vaccinated mice. Furthermore, distinct lincRNA species were changed by > threefold in livers of vaccination‑ protected mice, whereas lethal malaria induced different lincRNAs. Conclusion: The present data suggest that protective vaccination accelerates the malaria‑ induced occurrence of extramedullary erythropoiesis, generation of liver‑ resident cytotoxic cells, and regeneration from malaria‑ induced injury in the liver at early patency, which may be critical for final survival of otherwise lethal blood ‑ stage malaria of P. chabaudi. Keywords: Plasmodium chabaudi, Blood‑ stage malaria, Liver, Gene expression, Vaccination, Extramedullary erythropoiesis, Natural cytotoxicity *Correspondence: mohameddkhil@yahoo.com Department of Zoology, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Al‑Quraishy et al. Malar J (2018) 17:215 Page 2 of 16 lethal infections in vaccinated and non-vaccinated mice, Background respectively [18]. Moreover, early patency is associated Malaria is still one of the most life-threatening infec- with a dramatic decline in malaria-induced expression of tious diseases in tropical countries. The World Health multifunctional cytokines, such as IFNγ, TNF, IL-1β, and Organization (WHO) had estimated about 212 million IL-6 in the liver, which drive various programmes of host new cases and about 429,000 deaths globally in 2015, defense [10, 18, 21]. Although still unexplainable at pre- with about 70% of total deaths occurring in children aged sent, this decline suggests the occurrence of yet unknown under 5 years [1]. An effective anti-malarial vaccine is not processes in the liver that may be critical for vaccine yet commercially available [2–4]. efficacy and, thus, for the final outcome of blood-stage Morbidity and mortality from malaria are caused by malaria. To track these processes in the liver during mid- the blood stages of the malaria-causing agent, parasitic precrisis and the possible effects by vaccination, a reason - protozoans of the genus Plasmodium, which develop able initial approach is to analyse global gene expression within host erythrocytes. The spleen with its inher - profiles in the liver for malaria-responsive genes at early ent mechanism to remove senescent and other aberrant patent infections of P. chabaudi blood-stage malaria in erythrocytes from circulation is currently thought to be vaccination-protected mice in comparison with non-vac- the exclusive effector organ to eliminate Plasmodium - cinated unprotected mice. parasitized erythrocytes from circulation [5]. However, the liver is also equipped with effective mechanisms for Methods removing aberrant erythrocytes including Plasmodium- Mice infected erythrocytes [6–11]. The liver with its intrinsic Balb/c mice bred under specified pathogen-free con - immune system is therefore increasingly, though still ditions were obtained from the central animal facility hesitantly, recognized as an effector organ against blood- of the University of Düsseldorf. The experiments were stage malaria [10]. Plasmodium chabaudi infection in performed only with female mice aged 10–12  weeks. mice is an appropriate model to study the effector func - Mice were housed in plastic cages and received a stand- tions of the liver against blood-stage malaria without ard diet (Woehrlin, Bad Salzuflen, Germany) and water interfering with the preceding liver-stages of malaria ad libitum. parasites [12, 13]. The P. chabaudi model shares several characteristics with P. falciparum, which causes about Protective vaccination 99% of global malaria-related deaths in humans [1]. Vaccination was performed under identical experimental The P. chabaudi model also appears appropriate to conditions as described previously [18]. Host cell plasma study the uncomprehended mechanisms of host defense membranes, isolated in the form of erythrocyte ghosts that occur in the liver during vaccination-induced sur- from P. chabaudi-parasitized erythrocytes, were used as vival in blood-stage malaria. First, an effective procedure a non-infectious vaccine, which was prepared as detailed of blood-stage vaccination for P. chabaudi is available previously [14, 22, 23]. Approximately 10 ghosts were [14]. The non-infectious vaccine consists of erythrocyte suspended in 100  µl Freund’s complete adjuvant (FCA) plasma membranes isolated from P. chabaudi-infected and subcutaneously injected at week 3 and week 1 before erythrocytes, which contain auto-antigens and parasite- infection with P. chabaudi-parasitized erythrocytes. Con- synthesized neo-antigens ([15, 16]; cf. also [17]). Immu- trol mice were treated in parallel with only FCA. nization with this vaccine results in survival of more than 80% mice, which would have otherwise succumbed to Plasmodium chabaudi malaria lethal malaria by P. chabaudi [14, 18]. This vaccination Blood-stage infections of P. chabaudi were maintained induces a healing course of the infection and reduces in outbred mice under sterile conditions by weekly pas- peak parasitaemia by approximately 30% on day 8 post- sages of infected red blood cells. A non-clonal line of P. infection (pi) and generation of long-lasting resistance chabaudi has been used [18, 20, 24]. This line resembles against homologous re-infections [10]. Secondly, the liver Plasmodium chabaudi chabaudi AS in terms of restric- of mice has been shown to respond to protective vac- tion fragment length polymorphism analysis [25] as well cination evidenced, for instance, as alterations in gene as sequence identity for dihydrofolate reductase and expression, miRNA expression, and DNA methylation of for a cysteine protease [26] with only a single nucleo- gene promoters upon blood-stage infection [10, 18–20]. tide exchange [18]. As the AS clone, the line used here A critical phase of P. chabaudi blood-stage infections has self-healing potential. However, this is controlled is the mid-precrisis on day 4 pi, when parasitized eryth- by sex and sex hormones, respectively, genes of the H-2 rocytes begin to appear in peripheral blood. At this early complex and genes of the non-H-2 background of the patency, parasitaemia ranges between 1 and 5% vary- infected mouse strain [27]. Challenge of Balb/c mice with ing with mice and does not differ between healing and Al‑Quraishy et al. Malar J (2018) 17:215 Page 3 of 16 10 P. chabaudi-infected erythrocytes, evaluation of par- Scanning and analyses of microarrays asitaemia, and counting of erythrocytes were performed Agilent microarray scanner system (Agilent Technolo- as described previously [18, 28]. Besides the sacrificed gies) was used for detecting fluorescence signals on the mice on day 0 pi and on day 4 pi, both the vaccinated hybridized microarrays. The microarray image files were group and the non-vaccinated group contained 4 ‘control’ processed with the Agilent feature extraction software mice, which were not sacrificed. In the non-vaccinated (FES), which determines feature intensities including group, all four mice succumbed to infection during crisis, background subtraction, rejects outliers, and calculates whereas only one mouse succumbed to infection during statistical confidences. Three different biological repli - crisis in the vaccinated group, but three mice survived cates were performed for each sample type, i.e., 12 micro- the infection for at least 3 weeks, in accordance with our arrays for four samples in toto. The expression variance previous results [18]. was stabilized through the lo g transform. Microarrays were normalized by the quantile method. The heat map of the most highly variable transcripts, the hierarchical clustering dendrograms (calculated using the unweighted RNA isolation pair group method with arithmetic mean and Euclidean Livers were aseptically removed from sacrificed mice, distance measure), and the Principal component analy- rapidly frozen in liquid nitrogen, and stored at − 80  °C sis were performed using in-home functions developed until use. For isolation of total RNA, livers were individ- in Matlab (MathWorks). The microarray data have been ually ground in a mortar under liquid nitrogen and ali- deposited at both the EMBL-EBI Array Express reposi- quots were subjected to standard RNA extraction using tory (Array accession number: E-MTAB.6494) and the Trizol. An additional RNA clean-up was followed using NCBI’s Gene Expression Omnibus (GEO) database with the miRNeasy Kit (Qiagen, Hilden, Germany). RNA accession number GSE111110 (https ://www.ncbi.nlm. integrity and quality was checked on the Agilent 2100 nih.gov/geo/query /acc.cgi?acc=GSE11 1110). Bioanalyzer platform (Agilent Technologies). The RIN values of all RNA samples ranged between 8.7 and 9.1. Quantitative real‑time PCR Quantitative real-time PCR was performed under RNA labelling experimental conditions identical to those described Each RNA sample was used to produce Cy3-labeled recently [28], using High Capacity cDNA Reverse Tran- cRNA. Equivalents of 100 ng from individual RNA sam- scription Kit (Life Technologies) and TaqMan mRNA ples were amplified and labelled using the Agilent Low assays (Life Technologies) for reverse transcription of Input Quick Amp Labelling Kit (Agilent Technologies) mRNAs encoding the following proteins: ERMAP (assay according to the manufacturer’s instructions. Yields of ID: Mm00469273_m1), CLDN13 (Mm00491038_s1), cRNA and dye-incorporation were determined using the CD163 (Mm00474091_m1), GZMB (Mm00442837_m1), ND-1000 Spectrophotometer (NanoDrop Technologies). KLRB1A (Mm00726548_s1), KLRC3 (Mm00650941_ The incorporations were between 18 and 23 fmol Cy3/ng m1), KLRD1 (Mm00495182_m1), NCR1 (Mm01337324_ cRNA. g1), KLRG1 (Mm00516879_m1), and GAPDH (Mm99999915_g1). Fold change of expression was calcu- −ΔΔct lated using the comparative Ct method (2 ) [29] and Hybridization of gene expression oligo microarrays data sets were analysed for statistical significance using Agilent mouse whole genome 8 × 60  K gene expres- a two-tailed unpaired heteroscedastic Student’s t test sion oligo microarrays (design 028005) were used for (*p < 0.01). hybridization. Each microarray displayed 39,430 Entrez Gene RNAs and 16,251 long intergenic non-coding Results (linc)RNAs. The Agilent Gene Expression Hybridization Identification of malaria‑inducible genes in the liver Kit was used for hybridization as detailed in the Agi- of vaccinated and non‑vaccinated mice lent processing protocol (Agilent technologies). In brief, To identify malaria-induced vaccination-responsive 600  ng of Cy3-labelled fragmented cRNA in hybridiza- genes in the liver at early patency, vaccinated and non- tion buffer was hybridized to the microarrays overnight vaccinated Balb/c mice were concomitantly infected with at 65  °C using the recommended hybridization chamber P. chabaudi, and livers prepared from three vaccinated and oven. Finally, the microarrays were washed with the mice on day 4 pi (Vd4 group) were individually analysed Agilent Gene Expression Wash Buffer 1 (1 min at 23 °C) with Agilent’s 8 × 60 K oligo microarrays for global gene and then with preheated Agilent Gene Expression Wash expression in relation to those of three non-infected Buffer 2 (1 min at 37 °C). Al‑Quraishy et al. Malar J (2018) 17:215 Page 4 of 16 vaccinated mice on day 0 pi (Vd0 group). Corresponding heatmap of the most highly variable RNA expression analyses were performed with livers prepared from three profiles of the four different groups, each with three rep - non-vaccinated mice on day 4 pi (Nd4 group) in relation licates. A group of transcripts is specifically expressed to those of three non-vaccinated mice on day 0 pi (Nd0 at intermediate level I in the Vd4 group, which is not group). Figure  1a shows the global expression analysis expressed in the Vd0, Nd0 and Nd4 groups. Figure  1b Fig. 1 Transcriptomic global analysis of the liver from vaccination‑protected Balb/c mice before infection with Plasmodium chabaudi on day 0 pi ( Vd0 group) and after infection on day 4 pi ( Vd4 group) as well as in non‑ vaccinated non‑protected mice in the Nd0 and Nd4 groups, respectively. a Heatmap. The colour bar at the top codifies the gene expression in the log2 scale. Higher RNA expression corresponds to increased intensity of the colour red. b Hierarchical clustering of samples. c PCA of RNA expression data. The PC1 captures 40% of the RNA expression variability and the PC2 captures 13% of the variability Al‑Quraishy et al. Malar J (2018) 17:215 Page 5 of 16 shows the hierarchical clustering (dendrogram) of the different samples performed using the correlation metric and the average linkage method. The dendrogram reveals a clean cluster of all the three replicates in the Vd4 group and another major cluster of the data from the Nd0, Vd0, and Nd4 group, with the subclusters of samples in the Vd0 and Nd0 groups clustering closer. Principal compo- nent analysis (PCA) of the gene expression data is shown in Fig. 1c, where the 1st principal component (PC1) cap- tures 40% of the gene expression variability and the 2nd principal component (PC2) captures 13% of the variabil- ity. The PCA indicates that the replicates of case cluster together, and that PC1, since capturing the higher per- centage of gene expression variability, clearly separates all the replicates of the Nd0, Nd4, Vd0 groups from those of the Vd4 group. Taken together, these results indicate that protective vaccination does not essentially affect consti - tutive RNA expression of livers on day 0 pi, whereas early patent infections with P. chabaudi blood-stage malaria induce changes in hepatic RNA expression, differing between the Vd4 and the Nd4 group. Malaria-induced changes in gene expression were then found in livers of both the Vd4 and Nd4 groups, when mRNAs were evaluated, which displayed > threefold Fig. 2 Number of genes expressed more than threefold (p < 0.01) in changed expressions at a stringent level of significance the liver of vaccinated mice infected with Plasmodium chabaudi on (p < 0.01) at both d4 groups in relation to correspond- day 4 pi ( Vd4 group) in relation to the corresponding constitutive expressions day 0 pi ( Vd0 group). Nd4 group vs. Nd0 group ing constitutive expressions of the Vd0 and Nd0 group, indicates the number of significantly expressed genes in the liver respectively. The total numbers of genes with signifi - of non‑ vaccinated mice. Numbers in brackets: genes with > tenfold cantly changed expression are summarized in the Venn changed expression diagrams shown in Fig. 2. Early patent infections induced upregulation of 329 genes in the Vd4 group, but only 173 genes in the Nd4 group, and 274 genes in both d4 groups. were changed by > tenfold in the Vd4 group. Table  1 A remarkably lower number of genes was found to be summarizes the 24 genes and their annotated func- downregulated, i.e., only 19 in the Vd4 group and still tions, among which 23 were upregulated and one gene fewer, namely 10 in the Nd4 group. The Additional file  1: was downregulated. Conspicuously, one-third of the 23 Tables S1 and S2 summarize the genes whose expression upregulated genes are known to be involved in erythroid is up- and downregulated by > threefold and < tenfold development. This group of genes includes Ahsp (48-fold) in the Nd4 group with p < 0.01, respectively. Additional and Kel (66-fold), which encode the alpha haemoglobin file  1: Table  S3 shows 16 genes whose expression is stabilizing protein and the Kell blood group, respectively. changed by > tenfold in the Nd4 group with 15 upregu- Other genes in this group encode erythroid constituents lated genes and only one downregulated gene. In the such as the erythroblast membrane-associated protein Vd4 group, 306 genes were identified to be upregulated Ermap, the Rhesus blood group-associated A glycopro- (Additional file  1: Table  S4) and 18 genes were down- tein, Rhag, and the soluble carrier family 4 Slc4a1, which regulated by > threefold and < tenfold, respectively (Addi- is also termed band 3 and is one of the major erythroid tional file  1: Table  S5). Additional file  1: Table  S6 shows integral multi-pass surface membrane proteins. The all genes that were significantly upregulated by > three- Slc4a1-encoded band 3 is known to function as a chlo- fold in both Vd4 and Nd4 groups. ride/bicarbonate exchanger transporting carbon dioxide and to associate with glycophorin A (GYPA), which is Characterization of genes changed by > tenfold in the Vd4 another major integral one-pass red cell membrane pro- group tein. Remarkably, the expression of Gypa gene is almost To further restrict the number of candidate genes of tenfold higher in vaccination-protected mice (Additional potential importance for early patency and final survival, file  1: Table  S4), similar to Car13 encoding carbonate the analysis concentrated on genes, whose expressions Al‑Quraishy et al. Malar J (2018) 17:215 Page 6 of 16 Table 1 Genes expressed more or  less  than tenfold (p < 0.01) in  the  liver of  vaccinated mice infected with  P. chabaudi on day 4 p.i. (Vd4) in comparison to constitutive expression on day 0 p.i. (Vd0) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Function (annotated according to www. genec ards.org) Erythropoiesis Ahsp Alpha hemoglobin stabilizing protein NM_133245 48.18 0.0095 Acts as a chaperone to prevent the harmful aggregation of alpha‑hemoglobin dur ‑ ing normal erythroid cell development. Specifically protects free alpha‑hemo ‑ globin from precipitation. It is predicted to modulate pathological states of alpha‑hemoglobin excess such as beta‑ thalassemia Cldn13 Claudin 13 NM_020504 43.50 0.0070 Plays a major role in tight junction‑specific obliteration of the intercellular space, through calcium‑independent cell‑ adhesion activity Ermap Erythroblast membrane‑associated protein NM_013848 21.59 0.0036 The protein encoded by this gene is a cell surface transmembrane protein that may act as an erythroid cell receptor, possibly as a mediator of cell adhesion Gata1 GATA binding protein 1 NM_008089 29.73 0.0015 Transcriptional activator or repressor which probably serves as a general switch fac‑ tor for erythroid development Gfi1b Growth factor independent 1B NM_008114 20.50 0.0092 Essential proto‑ oncogenic transcriptional regulator necessary for development and differentiation of erythroid and mega‑ karyocytic lineages Kel Kell blood group NM_032540 65.89 0.0010 This gene encodes a type II transmem‑ brane glycoprotein that is the highly polymorphic Kell blood group antigen Rhag Rhesus blood group‑associated A glyco ‑ NM_011269 17.37 0.0001 The protein encoded by this gene is eryth‑ protein rocyte‑specific and is thought to be part of a membrane channel that transports ammonium and carbon dioxide across the blood cell membrane Slc4a1 Solute carrier family 4 NM_011403 11.74 0.0067 Major integral membrane glycoprotein of the erythrocyte membrane; required for normal flexibility and stability of the erythrocyte membrane and for normal erythrocyte shape via the interactions of its cytoplasmic domain with cytoskel‑ etal proteins, glycolytic enzymes, and hemoglobin Cell cycle and mitosis Ccnb1 Cyclin B1 NM_172301 17.76 < 0.0001 The protein encoded by this gene is a reg‑ ulatory protein involved in mitosis. The gene product complexes with p34(cdc2) to form the maturation‑promoting factor (MPF) Cdc25c Cell division cycle 25C NM_009860 12.41 0.0009 Cdc25 activates cdk complexes that drive the cell cycle. Cdc25 is involved in the DNA damage checkpoints and is known as a key mediator of cell cycle progres‑ sion Ckap2 l Cytoskeleton associated protein 2‑like NM_181589 10.72 0.0030 Microtubule‑associated protein required for mitotic spindle formation and cell‑ cycle progression in neural progenitor cells Al‑Quraishy et al. Malar J (2018) 17:215 Page 7 of 16 Table 1 (continued) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Function (annotated according to www. genec ards.org) Innate immunity Abcg4 ATP‑binding cassette, sub ‑family G ( WHITE), NM_138955 26.79 0.0035 The protein encoded by this gene is member 4 included in the superfamily of ATP‑ binding cassette (ABC) transporters. May be involved in macrophage lipid homeostasis Ccl7 Chemokine (C‑ C motif ) ligand 7 NM_013654 34.97 0.0013 Chemotactic factor that attracts mono‑ cytes and eosinophils, but not neutro‑ phils Socs1 Suppressor of cytokine signaling 1 NM_009896 11.32 0.0070 This gene encodes a member of the STAT‑ induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling (SOCS), family. SSI family members are cytokine‑inducible negative regulators of cytokine signaling. The expression of this gene can be induced by a subset of cytokines, including IL2, IL3 erythropoi‑ etin (EPO), CSF2/GM‑ CSF, and interferon (IFN)‑ gamma Treml2 Triggering receptor expressed on myeloid NM_001033405 10.92 0.0001 Cell surface receptor that may play a role cells‑like 2 in the innate and adaptive immune response. Acts as a counter‑receptor for CD276 and interaction with CD276 on T‑ cells enhances T‑ cell activation Cd163 CD163 antigen NM_053094 0.03 0.0018 The protein encoded by this gene is a member of the scavenger receptor cysteine‑rich (SRCR) superfamily, and is exclusively expressed in monocytes and macrophages. It functions as an acute phase‑regulated receptor involved in the clearance and endocytosis of hemoglobin/haptoglobin complexes by macrophages, and may thereby protect tissues from free hemoglobin‑mediated oxidative damage Cytotoxicity Gzmb Granzyme B NM_013542 35.66 0.0085 The encoded preproprotein is secreted by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) and proteolyti‑ cally processed to generate the active protease, which induces target cell apoptosis. This protein also processes cytokines and degrades extracellular matrix proteins, and these roles are implicated in chronic inflammation and wound healing Klrb1a Killer cell lectin‑like receptor subfamily B NM_010737 10.43 0.0083 Plays an inhibitory role on natural killer (NK) member 1A cells cytotoxicity. Activation results in specific acid sphingomyelinase/SMPD1 stimulation with subsequent marked elevation of intracellular ceramide. Activation also leads to AKT1/PKB and RPS6KA1/RSK1 kinases stimulation as well as markedly enhanced T‑ cell proliferation induced by anti‑ CD3 Klrc3 Killer cell lectin‑like receptor subfamily C, NM_021378 11.38 0.0002 KLRC3 is a member of the NKG2 group member 3 which are expressed primarily in natural killer (NK) cells and encodes a family of transmembrane proteins characterized by a type II membrane orientation (extra‑ cellular C terminus) and the presence of a C‑type lectin domain Al‑Quraishy et al. Malar J (2018) 17:215 Page 8 of 16 Table 1 (continued) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Function (annotated according to www. genec ards.org) Klrd1 Killer cell lectin‑like receptor, subfamily D, NM_010654 16.33 0.0067 Several genes of the C‑type lectin super ‑ member 1 family, including members of the NKG2 family, are expressed by NK cells and may be involved in the regulation of NK cell function. Plays a role as a receptor for the recognition of MHC class I HLA‑E mol‑ ecules by NK cells and some cytotoxic T‑ cells Ncr1 Natural cytotoxicity triggering receptor 1 NM_010746 16.97 0.0084 Cytotoxicity‑activating receptor that may contribute to the increased efficiency of activated natural killer (NK) cells to medi‑ ate tumor cell lysis Miscellaneous Hist1h3g Histone cluster 1, H3g NM_145073 18.90 0.0075 Core component of nucleosome Htr7 5‑Hydroxytryptamine (serotonin) receptor 7 NM_008315 12.53 < 0.0001 Serotonin 5‑HT7 receptors are located primarily in the thalamus, hypothala‑ mus and hippocampus. The function of these receptors includes the regulation of circadian rhythms, thermoregula‑ tion, learning and memory and smooth muscle relaxation Nxpe5 Neurexophilin and PC‑ esterase domain fam‑ NM_001013773 22.61 0.0089 Unknown ily, member 5 anhydrase (besides Car2 and Car9), which is known to 22 genes are apparently involved in mitosis, particularly interconvert carbon dioxide and bicarbonate to maintain in the formation and function of the mitotic spindle. The the acid–base balance in blood and to help transport car- two genes Prr11 and Sapcd2 are critically involved in cell bon dioxide out of tissues. Moreover, the group of genes cycle progression. The gene Klrb1f encodes a killer cell upregulated by > tenfold also lists the transcription fac- lectin-like receptor, which plays an inhibitory role in NK tors Gata1 and Gfi1b as well as Cldn13 upregulated by cell cytotoxicity. Birc5 is still remarkable because it codes even > 43-fold. for a negative regulatory protein preventing apoptotic Furthermore, a group of five genes encoding proteins, cell death. known to be involved in cellular cytotoxicity, such as Among the four genes involved in innate immu- Gzmb encoding granzyme B (36-fold), Ncr1 encod- nity, expression of the suppressor of cytokine signal- ing the natural cytotoxicity triggering receptor 1, and ing 1, Socs1, was upregulated approximately by 11-fold Klrb1a, Klrc3, and Klrd1 encoding three different killer (Table  1). Cd163 is the only gene whose expression was cell lectin-like receptors, were upregulated by > tenfold significantly downregulated by > tenfold in the Vd4 group (Table  1). Remarkably, among the six genes significantly (Table 1; Fig. 3). upregulated by > tenfold in the liver in both Vd4 and Finally, quantitative PCR was used to reexamine the Nd4 groups (Additional file  1: Table S7), one gene, Klrg1, expression of some of the genes, which were identified to encoding the killer cell lectin-like receptor subfamily be expressed by > tenfold in the Vd4 group in the micro- G, was overexpressed by at least 100% in the Vd4 group arrays, particularly Ermap, Cldn13, Cd163, Gzmb, Ncr1, compared to the Nd4 group, i.e., 39.5-fold vs. 14.4-fold. Klrb1a, Klrd1, Klrc3, and Klrg1. Figure  3 shows that the Another group of genes in Table 1 includes the 3 genes constitutive expression of these genes was not signifi - Ccbn1, Cdc25c, and Ckap 21 that were upregulated cantly affected by protective vaccination. However, early between 10- and 18-fold, and are known to be involved patent infections of P. chabaudi significantly changed in cell cycle control including mitosis (Table  1). Table  2 the expression of these genes in the Vd4 group, which is also shows 22 genes extracted from Additional file  1: comparable with the result of the microarrays. Table S6, whose expression is significantly upregulated by > tenfold in the Vd4 group and, concomitantly, by at least LincRNAs expressed in vaccination‑protected mice 100% more than the corresponding genes significantly There is increasing evidence that long non-coding (lnc) expressed in the Nd4 group. Conspicuously, 15 of these RNAs including long intergenic non-coding (linc)RNAs Al‑Quraishy et al. Malar J (2018) 17:215 Page 9 of 16 Table 2 Hepatic expression of genes up-regulated by more than tenfold (p < 0.01) at Vd4 and by 100% more than at Nd4 Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Nd4 vs. Nd0 p value Function (annotated according to www.genec ards.org) Mitosis Bub1 Budding uninhibited by NM_009772 20.11 0.0049 5.88 0.0021 Serine/threonine‑protein kinase benzimidazoles 1 homolog that performs two crucial func‑ tions during mitosis: it is essential for spindle‑assembly checkpoint signaling and for correct chromo‑ some alignment Espl1 Extra spindle poles‑like 1 NM_001014976 12.35 0.0013 4.98 0.0020 Caspase‑like protease, which plays a central role in the chromo‑ some segregation by cleaving the SCC1/RAD21 subunit of the cohesin complex at the onset of anaphase Mki67 Antigen identified by monoclonal NM_001081117 13.08 0.0004 4.29 0.0014 Required to maintain individual antibody Ki 67 mitotic chromosomes dispersed in the cytoplasm following nuclear envelope disassembly Mxd3 Max dimerization protein 3 NM_016662 10.52 0.0031 3.84 0.0066 This gene encodes a member of the Myc superfamily of basic helix‑loop ‑helix leucine zip ‑ per transcriptional regulators. The encoded protein forms a heterodimer with the cofactor MAX which binds specific E‑box DNA motifs in the promoters of target genes and regulates their transcription Ndc80 NDC80 homolog, kinetochore NM_023294 11.19 0.0004 4.74 0.0005 Acts as a component of the complex component essential kinetochore‑associated NDC80 complex, which is required for chromosome segre‑ gation and spindle checkpoint activity Nusap1 Nucleolar and spindle associated NM_133851 14.19 0.0002 4.89 0.0005 NUSAP1 is a nucleolar‑spindle ‑asso ‑ protein 1 ciated protein that plays a role in spindle microtubule organization Plk1 Polo‑like kinase 1 NM_011121 14.14 0.0035 4.13 0.0018 Serine/threonine‑protein kinase that performs several important functions throughout M phase of the cell cycle, including the regulation of centrosome matura‑ tion and spindle assembly, the removal of cohesins from chro‑ mosome arms, the inactivation of anaphase‑promoting complex/ cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis Prc1 Protein regulator of cytokinesis 1 NM_145150 15.38 < 0.0001 4.28 0.0003 The protein is present at high levels during the S and G2/M phases of mitosis but its levels drop dramatically when the cell exits mitosis and enters the G1 phase Racgap1 Rac GTPase‑activating protein 1 NM_001253809 12.67 0.0042 3.83 0.0003 Component of the centralspin‑ dlin complex that serves as a microtubule‑ dependent and Rho‑mediated signaling required for the myosin contractile ring formation during the cell cycle cytokinesis Al‑Quraishy et al. Malar J (2018) 17:215 Page 10 of 16 Table 2 (continued) Gene Gene description RefSeq ID Vd4 vs. Vd0 p value Nd4 vs. Nd0 p value Function (annotated according to www.genec ards.org) Ska1 Spindle and kinetochore associ‑ NM_025581 23.19 0.0004 5.46 0.0039 Component of the SKA1 complex, a ated complex subunit 1 microtubule‑binding subcomplex of the outer kinetochore that is essential for proper chromosome segregation Ska3 Spindle and kinetochore associ‑ NM_198605 15.24 0.0027 4.68 0.0013 This gene encodes a component ated complex subunit 3 of the spindle and kinetochore‑ associated protein complex that regulates microtubule attach‑ ment to the kinetochores during mitosis Ticrr TOPBP1‑interacting checkpoint NM_029835 14.22 0.0008 4.66 0.0093 Regulator of DNA replication and and replication regulator S/M and G2/M checkpoints. Regulates the triggering of DNA replication initiation via its inter‑ action with TOPBP1 by participat‑ ing in CDK2‑mediated loading of CDC45L onto replication origins Tpx2 TPX2, microtubule‑associated NM_028109 16.11 0.0032 4.28 0.0011 Spindle assembly factor required protein homolog for normal assembly of mitotic spindles. Required for normal assembly of microtubules during apoptosis Ube2c Ubiquitin‑ conjugating enzyme NM_026785 12.49 < 0.0001 4.32 0.0010 Essential factor of the anaphase E2C promoting complex/cyclosome (APC/C), a cell cycle‑regulated ubiquitin ligase that controls progression through mitosis Cell cycle/cell signaling Klrb1f Killer cell lectin‑like receptor NM_153094 10.63 0.0005 5.32 0.0003 Plays an inhibitory role on natural subfamily B member 1F killer (NK) cells cytotoxicity. Activation results in specific acid sphingomyelinase/SMPD1 stimu‑ lation with subsequent marked elevation of intracellular ceramide Birc5 Baculoviral IAP repeat‑ containing NM_009689 16.68 0.0005 3.47 0.0006 This gene is a member of the 5 inhibitor of apoptosis (IAP) gene family, which encode negative regulatory proteins that prevent apoptotic cell death Cd7 CD7 antigen NM_009854 13.52 0.0022 4.54 0.0005 Plays an essential role in T‑ cell inter‑ actions and also in T‑ cell/B‑ cell interaction during early lymphoid development Kif11 Kinesin family member 11 NM_010615 11.80 0.0065 3.62 0.0077 Motor protein required for estab‑ lishing a bipolar spindle during mitosis Prr11 Proline rich 11 NM_175563 12.13 0.0001 4.52 0.0001 Plays a critical role in cell cycle progression Sapcd2 Suppressor APC domain contain‑ NM_001081085 11.96 0.0001 3.30 0.0040 Plays a critical role in cell cycle ing 2 progression Miscellaneous Iqgap3 IQ motif containing GTPase acti‑ NM_001033484 16.36 0.0002 3.88 0.0019 Unknown vating protein 3 Raet1c Retinoic acid early transcript NM_009018 15.69 0.0001 4.83 0.0011 Unknown gamma Al‑Quraishy et al. Malar J (2018) 17:215 Page 11 of 16 Fig. 3 Quantitative PCR of mRNAs in the liver of Balb/c mice in comparison with the microarray data. Livers were isolated from three vaccinated mice before infection with Plasmodium chabaudi on day 0 pi ( Vd0 group), from three vaccinated mice after infection on day 4 pi ( Vd4 group), and from three non‑ vaccinated mice at Nd0 group and at Nd4 group. Means of duplicate determinations, performed with liver aliquots from three different mice, with half SEM. Stars and hashtags indicate significant differences (p < 0.01) between Vd4 and Vd0 groups as revealed by qRT‑PCR and microarrays, respectively are critical for the course and outcome of different dis - upregulated in the Vd4 group in common with the Nd4 eases, including diverse liver diseases [30]. These non- group and 1 lincRNA is downregulated (Additional file  1: coding RNAs range in size between 200 bp and ~ 100 kb. Table S9). LincRNAs do not overlap with annotated coding regions Remarkably, the two lincRNA species lincRNA per definitionem, though an increasing number of lincR - :chr10:117021051-117038683 forward strand and NAs has been recently identified to contain small open lincRNA:chr2:84344350-843770075 reverse strand are reading frames coding for small functional peptides [31]. more highly expressed in the Vd4 group (by > 100%) Moreover, lincRNAs are widely transcribed in mamma- than in the Nd4 group. LincRNA species that are signifi - lian cells, though at lower levels and in a more cell-type cantly up- and down-regulated in non-vaccinated mice specific manner than mRNA [32–34]. The used micro - in the Nd4 group (Additional file  1: Table S9) differ from arrays contain 16,251 lincRNA probes and have been those identified in vaccination-protected mice in the Vd4 evaluated for lincRNA expression using the same strin- group. Annotated functions are not yet available for any gent conditions as those used for mRNAs. Early patent of these differentially regulated lincRNAs. infections of P. chabaudi blood-stage malaria induce changes in the expression of lincRNAs, which differ sig - Discussion nificantly (p < 0.01) between vaccination-protected and This study provides evidence that the hepatic response non-protected non-vaccinated mice (Fig.  4). In vacci- in terms of mRNA and lincRNA expression, to P. nation-protected mice, 7 lincRNAs are downregulated chabaudi blood-stage malaria at early patency differs and 19 lincRNAs are upregulated by > threefold (Addi- between vaccination-induced healing infections and tional file  1: Table  S8), among which one lincRNA is lethal infections in non-vaccinated mice. In particular, upregulated by > tenfold. In addition, 13 lincRNAs are 24 genes are altered by > tenfold at p < 0.01 in the liver Al‑Quraishy et al. Malar J (2018) 17:215 Page 12 of 16 Extramedullary erythropoiesis in the liver of vaccina- tion-protected mice has been recently shown to occur towards the end of the crisis phase on day 11 pi [28]. Crisis is characterized by much higher expression of erythroid-associated genes than that described here at early patency on day 4 pi For instance, the genes Ermap, Slca1, Gata1, and Gfi1b are expressed by > 100-fold at crisis. As previously shown under identical experimen- tal conditions, crisis in vaccination-protected mice is also characterized by a dramatic decrease in peripheral P. chabaudi-parasitized erythrocytes and, concomitantly, in a dramatic increase in the number of peripheral reticulo- cytes, the latter being impaired in non-vaccinated mice [18]. Incidentally, reticulocytes are not the preferred host cells for P. chabaudi [35]. At early patency, however, P. chabaudi-parasitized erythrocytes only begin to appear in the peripheral blood [18]. The number of periph - eral reticulocytes is still very low and not yet essentially changed [18]. These data suggest: (1) that extramedullary erythropoiesis occurring in the liver at early patency of the malaria blood-stage infections is still in an early state: and, (2) that extramedullary erythropoiesis in the liver is apparently accelerated in vaccination-protected mice in comparison to non-vaccinated mice. Fig. 4 Number of lincRNAs with expressions changed by > threefold There is evidence indicating that stress, including (p < 0.01) in the liver of non‑ vaccinated (N) and vaccinated mice ( V ) psychological stress, chemicals, and diverse viral and infected with Plasmodium chabaudi on day 4 pi in relation to their bacterial infections, can induce extramedullary eryth- constitutive expression in the Nd0 and Vd0 groups, respectively. ropoiesis in several organs, particularly in the spleen, of Numbers in brackets indicate the lincRNAs with > tenfold changed mice and even humans [36–46]. Even endo- and ecto- expressions parasites such as ticks [47] or Trypanosoma congolense [48] are able to induce extramedullary erythropoiesis in the spleen of their hosts. Remarkably, the latter has of vaccination-protected mice. In addition, there are 22 been found to be associated with increased expression genes > tenfold expressed in vaccination-protected mice of the apparent mouse-specific gene, Cldn13, encoding which are at least 100% higher induced than the corre- the most abundant claudin of the 26-membered clau- sponding genes significantly expressed in non-vaccinated din family in the spleen [49, 50]. Claudins are the main mice. These data indicate that, at early patency, critical constituents of tight junctions; however, CLDN13 has processes occur in the liver, which may contribute to vac- been predicted to be localized on the surface of erythro- cination-induced survival of blood-stage infections. blasts in the spleen [48]. Previously, a massively upregu- One of these processes may be extramedullary eryth- lated expression of Cldn13 by > 100-fold has been found ropoiesis in the liver. Indeed, approximately one-third of towards the end of the crisis phase of P. chabaudi blood- the 23 genes induced in vaccination-protected mice by stage infections in vaccination-protected mice and it was > tenfold are erythroid-associated genes, encoding the therefore suggested that Cldn13 is locally expressed in/ Kell blood group, the Rhesus blood group-associated gly- around erythroblast islands in the liver [28]. The present coprotein A, extrinsic and intrinsic membrane proteins data would then indicate that Cldn13 is already expressed such as ERMAP, SLC4A1 (band 3), and glycophorin A, at early extramedullary erythropoiesis in the liver of vac- as well as the transcription factors GATA1 and GFI1b, cination-protected mice. Another gene possibly involved which are key regulators of erythroid development in liver erythropoiesis may be Cd163, which is the only [28]. These genes were not identified to be significantly gene found to be downregulated by > tenfold, since the (p < 0.01) expressed in non-vaccinated mice, except for encoded transmembrane scavenger receptor CD163 on Ermap, which was induced only by about fivefold in the the surface of Kupffer cells has been described to serve Nd4 group (Additional file 1: Table S1). not only in clearance and endocytosis of haemoglobin/ Al‑Quraishy et al. Malar J (2018) 17:215 Page 13 of 16 haptoglobin complexes [51–53], but also as an adhesion the end of the crisis phase in vaccination-protected mice factor for erythroblasts in erythroblastic islands [53, 54]. when there is a massive appearance of reticulocytes in the An increase in killer cells, i.e., NK cells, NKT cells, and peripheral blood [28]; concomitantly, the liver has been cytolytic CD8 cells [55], may also occur in the liver of shown to dramatically increase its uptake of particulate vaccination-induced healing infections at early patency. material [18] including P. chabaudi-parasitized erythro- This view is supported by the present finding that the cytes [68]. Thus, it is possible that the increased genera - granzyme b gene Gzmb, the natural cytotoxicity-trigger- tion of NK cells in the liver of vaccination-protected mice ing receptor 1 gene Ncr1, and the killer cell lectin–lectin at early patency may not fortuitously correlate with early like receptor genes Klrb1a, Klrc3, Klrd1, and Klrg1 are erythropoiesis in the liver. massively upregulated by > tenfold in vaccination-pro- Indeed, a recent report described that murine Cyto- tected mice. KLRs, GZMB inducing apoptosis in target megalovirus (MCMV) infections induce extramedul- cells via the caspase-mediated apoptotic pathway, and lary haematopoiesis in the spleen with a dominance of NCR1 are known to be predominantly expressed on NK the red blood cell lineage [62]. The development of this cells, though NCR1 is also expressed on type 1 innate extramedullary haematopoiesis requires the cytotoxic lymphoid cells [56–59]. The increased mRNA levels function of NK cells rather than their cytokine produc- encoded by Klrs, Gzmb, and Ncr1 might be interpreted as tion. This cytotoxic activity of NK cells is obviously to be due to an immigration of peripheral c(conventional) responsible for confining virus spread, thereby protect - NK cells [55] from circulation into the liver. On the other ing extramedullary haematopoietic niches and facilitat- hand, however, the major lymphocyte population in the ing extramedullary haematopoiesis, which otherwise is liver is presumably another subset of NK cells, namely suppressed by MCMV [69]. Depression of cytokine sign- liver-resident NK cells developing from progenitor cells aling in the liver of vaccination-protected mice at early in the liver [55]. Increasing evidence indicates that the patency is indicated by a dramatic decline in the expres- liver-resident NK cell subset differs in phenotype and sion of Ifnγ and Tnfα [18]. This is predictable because the function from cNK cells [55, 60], though both NK cell expression of Socs1 encoding the suppressor of cytokine subsets produce about the same high levels of GZMB signaling is increased by > tenfold in the liver of vaccina- − + [59]. In contrast to the CD49a DX5 cNK cells, the liver- tion-protected mice, but not in non-vaccinated mice at + − resident NK cells are CD49a DX5 and even differ, also early patency. It is, therefore, possible that Socs1 is criti- in terms of gene expression signatures, from cNK cells cally involved in the accelerated generation of liver-resi- and other tissue-resident NK cells, as e.g. those distinct dent killer cells, particularly NK cells. lineages of NK cells occurring in spleen, thymus, and Accelerated extramedullary erythropoiesis and genera- uterus [61]. Thus, it is more attractive to speculate that tion of killer cells in the liver may also explain why the the upregulated mRNA levels of the different NK cell present study identified a group of genes known to be markers found here to be induced by blood-stage malaria involved in cell cycle regulation and especially mitosis in the liver of vaccination-protected mice may reflect an i.e., Ccbn1, Cdc25c, and Ckap 21, at early patency in the intra-hepatic accelerated generation of liver-resident NK liver of vaccination-protected mice. Additionally, the vast cells. majority of the > tenfold expressed 22 genes, which are at Liver-resident NK cells have been described to exert least 100% higher induced in vaccination-protected mice numerous functions, but their predominant function is than the corresponding genes significantly expressed in killing of target cells using different apoptotic pathways non-vaccinated mice, is known to be involved in mitosis [55, 60, 62]. For instance, NK cells kill myofibroblasts, and cell cycle control. Even erythroblast enucleation dur- which are known to induce liver fibrosis, thus limiting ing erythroid development can be regarded as an asym- the spread of fibrosis in the liver [62]. There is evidence metric mitosis [28, 48]. However, several of these genes that NK cells are also able to kill Plasmodium-para- such as Bub1, Nusap1, Prc1, Ska1, and Ube2c, have also sitized erythrocytes thus contributing to protection from previously been found to be expressed at about the same murine and human malaria [63–67]. It is therefore plau- level, as here at early patency, towards the end of crisis in sible to assume that NK cells attack P. chabaudi-infected vaccination-protected mice [28]. u Th s, it is not unlikely erythrocytes in the liver thus transforming the normally that the changes in the expression of these cell cycle and tolerogenic milieu of the liver to an increasingly hostile mitosis controlling genes reflect accelerated extramed - parasite environment, at least at early patency when P. ullary erythropoiesis and generation of liver-resident chabaudi-infected erythrocytes begin to appear in the cytotoxic cells and may also be associated with acceler- peripheral blood. Remarkably, an increased NK cell activ- ated liver regeneration in general. Indeed, there is evi- ity, in terms of the here found upregulated genes of NK dence that the liver during the acute phase of P. chabaudi cell markers, has not been previously observed towards blood-stage malaria is pathologically damaged and even Al‑Quraishy et al. Malar J (2018) 17:215 Page 14 of 16 heavily injured with distant effects on other organs such more than tenfold at early patency and by more than 100- as in hepatoencephalopathy [9, 10]. Even Plasmodium fold at crisis in the liver of vaccination-protected mice [28], falciparum and Plasmodium vivax malaria in humans is is decreased by 80%. Specifically, Bloodlinc is located in the associated with massive liver dysfunction [70–73]. Accel- coordinates chr11:102,231,615–102,237,204 of the version erated liver regeneration may therefore contribute to mm9 of the mouse genome, which is also used for anno- accelerated recovery from the malaria-induced dysfunc- tation of our lincRNA containing arrays; incidentally, the tions of the liver [20, 28]. latter do not contain any specific probes for the lncRNA Finally, the present data demonstrate that P. chabaudi Bloodlinc. blood-stage malaria does not only alter gene expression in Collectively, the present data indicate that protective vac- the liver at early patency, but also affects the expression of cination changes the hepatic response in terms of mRNA lincRNAs, and this lincRNA expression has changed after and lincRNA expression, to early patent healing infections protective vaccination. The identified lincRNAs are not of P. chabaudi blood-stage malaria. These changes are sug - yet functionally annotated, as it is typical for most other gested to be associated with an accelerated occurrence of known lincRNAs [31]. In general, however, evidence is extramedullary erythropoiesis, generation of liver-resident increasing that lncRNAs including lincRNAs play a criti- cytotoxic cells, and liver regeneration. These accelerated cal role in nuclear organization and chromatin remode- processes at early patency may be of critical importance for ling, in cell-type specific activation and repression of gene the final vaccination-induced healing outcome of the oth - expression through diverse mechanisms, in tissue-specific erwise lethal blood-stage P. chabaudi malaria. fine-tuning of the expression of neighbouring genes, in reg - Additional file ulation of cell-lineage development, and in course and out- come of diverse diseases including liver diseases [31, 33, 34, Additional file 1: Table S1. Genes, whose expression is up ‑regulated 74–76]. Here, several lincRNA species have been identified more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of non‑ vac‑ in the liver, whose malaria-induced expression is increased cinated mice infected with P. chabaudi on day 1 p.i. (Nd1) in comparison to constitutive expression on day 0 p.i. (Nd0). Table S2. Genes, whose by protective vaccination during mid-precrisis on day 4 pi, expression is down‑regulated more than 3‑fold and less than 10‑fold and whose expression is still more increased in the liver of (p < 0.01) in the liver of non‑ vaccinated mice infected with P. chabaudi vaccination-protected mice towards the end of the crisis on day 1 p.i. (Nd1) in comparison to constitutive expression on day 0 p.i. (Nd0). Table S3. Genes up‑regulated and down‑regulated more than phase on day 11 pi as described recently [28]. For instance, 10‑fold (p < 0.01) in the liver of vaccinated mice infected with P. chabaudi expression of the lincRNA:chr12:32781477-32808567 on day 1 p.i. (Nd1) in relation to constitutive expression on day 0 p.i. is upregulated from 6.6 in the Vd4 group to 71.9 in the (Nd0. Table S4. Genes, whose expression is up‑regulated more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of both non‑ vaccinated Vd11 group, the lincRNA:chr5:77084398-77086144 (N) and vaccinated mice ( V ) infected with P. chabaudi on day 1 p.i. ( Vd1, reverse strand from 10.7 to 19.9, the Nd1) in comparison to constitutive expression on day 0 p.i. ( Vd0, Nd0). lincRNA:chr15:61984389-62102500 reverse strand from Table S5. Genes, whose expression is down‑regulated more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of both non‑ vaccinated (N) and 3.2 to 6.1, and the lincRNA:chr10:83980790-83986015 vaccinated mice ( V ) infected with P. chabaudi on day 1 p.i. ( Vd1, Nd1) in reverse strand from 3.1 to 9.7, respectively. At least these comparison to constitutive expression on day 0 p.i. ( Vd0, Nd0). Table S6. 4 lincRNA species in the liver may be speculated to con- Genes expressed more than 3‑fold and less than 10‑fold (p < 0.01) in the liver of vaccinated mice infected with P. chabaudi on day 1 p.i. ( Vd1) in tribute to vaccination-induced healing of the otherwise comparison to constitutive expression on day 0 p.i. ( Vd0). Table S7. Genes lethal P. chabaudi malaria infections. Currently, the role down‑regulated more than 3‑fold and less than 10‑fold (p < 0.01) in the of lncRNAs is still poorly understood with respect to liver of vaccinated mice infected with P. chabaudi on day 1 p.i. ( Vd1) in comparison to constitutive expression on day 0 p.i. ( Vd0). Table S8. Lin‑ extramedullary erythropoiesis and/or generation of liver- cRNAs up‑regulated more than 3‑fold (p < 0.01) in the liver of vaccinated resident killer cells and/or hepatic regeneration [62] and/ mice infected with P. chabaudi on day 1 p.i. ( Vd1) in comparison to consti‑ or megakaryopoiesis in the liver [20]. Only erythropoie- tutive expression on day 0 p.i. ( Vd0).Table S9. LincRNAs down‑regulated more than 3‑fold (p < 0.01) in the liver of vaccinated mice infected with P. sis has been recently shown to be associated with diverse chabaudi on day 1 p.i. ( Vd1) in comparison to constitutive expression on lncRNAs [75, 76], particularly in steps of erythropoiesis day 0 p.i. ( Vd0). that are targeted by the transcription factor GATA1 [77, 78]. The expression of Gata1 in the liver was here detected to be upregulated by > tenfold at early patency during the Authors’ contributions MAD, SA, and FW designed the study. MAD, EMA, ASA, MJA and DD carried pre-crisis phase and, still more, by > 100-fold towards the out the experiments and analysed the data. SA prefinanced part of the experi‑ end of the crisis phase in vaccination-protected mice [28]. mental work. All authors wrote and revised the manuscript. All authors read One study has shown [77] that when the erythroid-specific and approved the final manuscript. lncRNA species alncRNA-EC7, also known as Bloodlinc, Author details is knocked down, the expression of the 10 kb away located 1 Department of Zoology, College of Science, King Saud University, P.O. Box: gene Slc4a1, which encodes the band 3 erythrocyte mem- 2455, Riyadh 11451, Saudi Arabia. Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt. Department of Zoology, brane protein and which is found here to be expressed by Al‑Quraishy et al. Malar J (2018) 17:215 Page 15 of 16 Faculty of Science, Beni‑Suef University, Beni‑Suef, Egypt. Group of Com‑ 12. Longley R, Smith C, Fortin A, Berghout J, McMorran B, Burgio G, et al. Host putational Biology and Systems Biomedicine, Biodonostia Health Research resistance to malaria: using mouse models to explore the host response. Institute, San Sebastián, Spain. 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Malaria JournalSpringer Journals

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