Global transcriptomics analyses reveal modifications by vaccination of malaria-induced hepatic gene expression on days 4 and 11 p.i.
The effect of protective vaccination on the time course of erythroid gene expression in the liver induced by P. chabaudi blood-stage malaria was analyzed using mouse whole genome 8x60K oligo microarrays. In toto, 30 microarrays were used for 30 livers prepared from the 10 different groups of vaccination-protected and un-protected non-vaccinated mice at the different phases of infections, i.e., upon infection on day 0 p.i., at early prepatency on day 1 p.i., at early patency on day 4 p.i., at peak parasitaemia on day 8 p.i., and towards the end of crisis on day 11 p.i..
Figure 1a shows the heatmap of gene expression profiles of those probes whose range or variation across all samples were at least 7 in the 10 different groups. In general, there is a good replicability among the three replicates of each analyzed condition. The heatmap also shows a gradual change in gene expression across time both in non-vaccinated and vaccinated samples with an abrupt change in gene expression at peak parasitaemia on day 8 p.i.. As the heatmap, the hierarchical clustering of the 30 different individual samples, shown as a dendogram in Figure 1b, also reveals a good replicability among the three replicates of each analyzed condition and the abrupt change in gene expression on day 8 p.i.. There are two main branches splitting the samples taken on days 0, 1, and 4 p.i. (red branches in Fig. 1b) from the samples taken on days 8 and 11 p.i. (blue branches in Fig. 1b). The Principal Component Analysis (PCA) of gene expression (Fig. 1c) shows that the 1st principal component (PC1) accounts for 40% of the gene expression variability and the 2nd PC2 captures 10% of the variability. The 1st principal axis, that is the most informative, separates between those liver samples taken before day 8 p.i. (positive coordinates) and those taken after day 8 p.i. samples (negative coordinates).
Collectively, the global transcriptomics analysis results indicate (i) that constitutive expression of the vast majority of hepatic genes is not essentially changed by protective vaccination, (ii) that P. chabaudi infections induce changes in hepatic RNA expression, being usually highest at peak parasitaemia on day 8 p.i. and towards the end of crisis on day 11 p.i., and (iii) that the malaria-induced changes in hepatic gene expression are modified by protective vaccination, particularly at early patency on day 4 p.i. and towards the end of crisis on day 11 p.i..
Erythropoiesis-involved genes are expressed in the liver of non-infected mice
Figure 2 shows the expression profiles of the selected 23 genes known to encode important non-hemoglobin constituents of erythrocytes and the EPO encoding gene known to stimulate erythropoiesis. The numbers in the heatmap indicate the probe-detected expression levels of genes which were log2 transformed for variance stabilization. In the liver of non-vaccinated mice on day 0 p.i., the expression of a given gene is similar among the 3 microarrays prepared from the 3 mice (Nd0 in Fig. 2) and also with the corresponding probes on the microarrays of the 3 vaccinated mice on day 0 p.i. (Vd0 in Fig. 2). However, the 23 genes reveal varying constitutive expression levels, which, however, are approximately the same at both Nd0 and Vd0. For instance, low constitutive expression levels reveal the genes Epb4.2, Epb4.9, Rhag, Ermap, Spta, and Sptb at both Nd0 and Vd0, whereas higher expression levels are found for the genes Epor, Slc4a1, Gata1, Klf1, and Tal1 in both vaccinated and non-vaccinated mice (Fig. 2). This supports the view that constitutive expression of erythroid genes in non-infected mice is not essentially affected by vaccination.
Erythroid genes respond to infections with increasing expressions in non-vaccinated mice
The heatmap in Figure 2 also shows that blood-stage infections of P. chabaudi malaria induce increasing expressions of the 23 erythroid genes in the liver, but with differences among vaccinated and non-vaccinated mice. To make these differences clearer and better comparable, the expression profiles were plotted in a linear scale as time series with the corresponding trajectories presented in Figures 3 - 6.
Blood-stage infections with P. chabaudi induce only a slight increase in the expression of all the 23 erythropoiesis-involved genes between days 0 p.i. and 4 p.i.. Thereafter, an abrupt increase in expression is observed at peak parasitaemia on day 8 p.i., and the increase even continues reaching still higher mRNA expression levels towards the end of the crisis phase on day 11 p.i.. Among genes encoding membrane-associated proteins [45, 46], the gene Slc4a1 coding for the major integral multi-pass membrane protein band 3 and the gene Gypa encoding the major one-pass membrane protein of the Gyp family reach maximal mRNA expression levels of approximately 180 and 50 above the normalization level, respectively (Fig. 3). The genes Kel and Rhd coding for blood groups and Rhag coding for the Rh-blood group associated transmembrane glycoprotein reveal maximal expressions of approximately 100, 120, and 90, respectively, on day 11 p.i. (Fig. 3). Ermap known to encode a protein associated with the outer surface of erythroblasts displays maximal expression of approximately 90 on day 11 p.i.. A very similar time-course of expression with a maximum level of approximately 100 reveals Cldn13 (Fig. 3), whose encoded protein has been previously suggested to be associated with erythroblastic islands . Lower maximal expressions on day 11 p.i. exhibit those genes, whose encoded proteins are known to be associated with the cytoskeletal meshwork on the inner surface of the erythrocyte plasma membrane (Fig. 4). Among them are Spta, Sptb, Epb4.1, Epb4.2, Epb4.9, and Tmod1 [45, 46], whose maximal expressed mRNA levels vary between 15 and 50 above the normalization level, respectively. Only the genes Add2 involved in the assembly of the spectrin-actin cytoskeletal network and Ank1 involved in the linkage of integral membrane proteins to the underlying cytoskeletal meshwork display higher expression levels between 80 and 70, respectively (Fig. 4).
The genes Gata1, Gfi1b, Klf1, and Tal1 encode transcription factors which are known to be critically involved in erythropoiesis [47-49]. These genes also reach high expressions between approximately 80 and 110 above the normalization level towards the end of the crisis phase (Fig. 5). Among all erythroid genes responding to malaria, Ahsp, encoding the α hemoglobin stabilizing protein, exhibit the highest mRNA expression of approximately 190 towards the end of the crisis phase (Fig. 5). However, a maximal expression level of only approximately 35 reveals the gene Acyp1 encoding the erythrocytic acylphosphatase.
Erythropoiesis, in particular erythroblastosis, is critically dependent on EPO signaling through its receptor EPOR [23, 24]. Epor and Epo display a time-course of mRNA expression, which differs from that determined for the other erythroid genes in two aspects (Fig. 6). Epor reveals a sharp decline in its relative expression level from about 75 on day 0 p.i. to about 25 on day 1 p.i.., before it continuously increases almost linearly reaching its maximal level of about 120 towards the end of crisis on day 11 p.i.. By contrast, Epo expression is impaired during the first 4 days of infection, then it sharply increases reaching its maximum expression of approximately 8 at peak parasitaemia on day 8 p.i., before declining to a level of about 3 towards the end of the crisis phase on day 11 p.i..
Expression of erythroid genes is accelerated in the liver of vaccination-protected mice
In contrast to un-protected non-vaccinated mice, the expression of the vast majority of erythroid genes in the liver of vaccination-protected mice is accelerated during infections, as evidenced by three facts. First, the erythroid genes are significantly much higher expressed at early patency on day 4 p.i. than the corresponding genes in the liver of non-vaccinated mice (cf. Figs. 3-6). This significant higher expression is confirmed when the corresponding genes are directly compared between vaccinated and non-vaccinated mice on day 4 p.i. or when the increases in expression levels between day 1 p.i. and day 4 p.i. is compared in vaccinated mice vs non-vaccinated mice. The only exception is Epb4.1 (Fig. 4): its expression is significantly higher on day 1 p.i. in vaccinated mice, then it decreases to approximately the same low level as that of non-vaccinated mice on day 4 p.i., before it increases again reaching maximal expression at peak parasitaemia on day 8 p.i., which is about the same as that towards the end of the crisis phase on day 11 p.i.. Secondly, the majority of erythroid genes reach their maximal expression levels already at peak parasitaemia on day 8 p.i. and keep about the same level during crisis, in contrast to non-vaccinated mice in which the corresponding genes reach their maximal expression levels on day 11 p.i.. Thirdly, the final expression levels on day 11 p.i. are mostly lower with lower standard deviations in vaccination-protected mice than those of the corresponding genes in the liver of non-vaccinated un-protected mice (cf. Figs. 3-6). A few genes in the liver of vaccinated mice, which still show a minor increase of mRNA expressions after peak parasitaemia on day 8 p.i., slow down their expressions towards the end of the crisis phase on day 11 p.i.. Some genes, as e.g. Rhag, Epb4.1, Sptb, Tmod1 and Acyp1, have even significantly declined their expression levels on day 11 p.i..
Remarkably, the time course of expression of Epor in the liver of vaccinated mice is very similar to that of other genes proceeding from a level of about 25 on day 1 p.i. and reaches maximal levels of almost 100 on days 8 and 11 p.i. (Fig. 6). Also, the increase in expression of Epor between day 1 p.i. and day 4 p.i. as well as the expression on day 4 p.i. is significantly higher in vaccinated mice than in non-vaccinated mice. Remarkably, the time course of Epor expression significantly differs to that of Epo. Indeed, Epo expression is delayed during the first 4 days of infections, i.e., Epo is expressed at approximately the same very low level between day 0 p.i. and 4 p.i. in vaccinated mice as in non-vaccinated mice. Moreover, the maximal levels of Epo expression at peak parasitaemia on day 8 p.i. are significantly higher in the liver of vaccinated mice than in non-vaccinated mice (Fig. 6), though vaccination decreases maximum parasitaemia by approximately 30% in comparison with non-vaccinated mice [8, 43].
Quantitative PCR validates the response of erythroid genes to infections
Quantitative PCR (qPCR) was used to re-examine, in both non-vaccinated and vaccinated mice, the time courses of expression of some arbitrarily selected erythroid genes, whose expression was identified by microarrays to respond to P. chabaudi blood-stage malaria. The data are summarized in Figure 7. The trajectories of Ermap expression in both vaccinated and non-vaccinated mice during infection take a similar course as those detected by the microarray probes (Fig. 3). Moreover, the qPCR-determined trajectories for expression of Gata1, Add2, Slc4a1, Rhd, and Cldn13 take a similar course as those determined by microarrays (cf. Fig. 7 with Figs. 3 - 5). In particular, there a higher increase in expression was observed at early patency on day 4 p.i. and a lower expression towards the end of the crisis phase on day 11 p.i. in vaccinated mice than in non-vaccinated mice. Furthermore, correspondence largely exists between microarray and qPCR data with respect to the tendency of increasing expression for Epb4.2, Epb4.9, and Acyp1 (Fig. 7). Similarly to microarray data, the 9 genes examined by qPCR reveal maximal expression levels in the liver of vaccinated mice at peak parasitaemia on day 8 p.i., whereas, in the liver of non-vaccinated mice, maximal expression of the majority of these genes is reached towards the end of crisis phase on day 11 p.i.. Furthermore, the expressions of Epor and Epo measured by qPCR take very similar courses in vaccinated and non-vaccinated mice as those determined by microarrays (Fig. 6).