Host-parasite interaction: changes in human placental gene expression induced by Trypanosoma cruzi

Background Chagas disease is caused by Trypanosoma cruzi, a parasite endemic to Latin America. Most infections occur in children by vector or congenital transmission. Trypanosoma cruzi establishes a complexity of specific molecular parasite-host cell interactions to invade the host. However, most studies have been mainly focused on the interaction between the parasite and different cell types, but not on the infection and invasion on a tissue level. During congenital transmission, T. cruzi must cross the placental barrier, composed of epithelial and connective tissues, in order to infect the developing fetus. Here we aimed to study the global changes of transcriptome in the placental tissue after a T. cruzi challenge. Results Strong changes in gene expression profiling were found in the different experimental conditions, involving the reprogramming of gene expression in genes involved in the innate immune response. Conclusions Trypanosoma cruzi induces strong changes in genes involved in a wide range of pathways, especially those involved in immune response against infections. Electronic supplementary material The online version of this article (10.1186/s13071-018-2988-0) contains supplementary material, which is available to authorized users.


Background
Chagas disease is a zoonotic disease caused by Trypanosoma cruzi, a parasite endemic to Latin America. Most infections occur in children by vector or congenital transmission. The prevalence of Chagas disease in pregnant women in Latin America ranges between 5-40% depending on the geographical area and the rate of congenital transmission is estimated to be 1-12% [1]. In addition, due to population mobility, Chagas disease has been increasingly detected in other non-endemic countries and continents (where the vector does not exist) such as the USA, Canada, Australia, Europe and Asia [2,3]. Congenital transmission, in spite of its low transmission rates, is partially responsible for the progressive globalization of the disease [2,4,5]. Importantly, congenital infection is responsible for an estimated 22% of new infections in 2010, making this form of transmission epidemiologically relevant [6].
The parasite presents a complex life-cycle that occurs in both vertebrate and invertebrate hosts, where three major developmental stages are observed: epimastigotes, trypomastigotes and amastigotes. Trypomastigotes constitute the extracellular infective form in mammals where they are able to infect a wide range of nucleated mammalian cells [7]. Interestingly, T. cruzi has co-evolved with mammals to establish a complexity of specific molecular parasite-host cell interactions to invade host cells and tissues, to evade the host immune system and to undergo intracellular replication [8]. Key steps in parasite infection include its host cell penetration and replication of the protozoa in the cytoplasm of infected cells. The application of oligonucleotide and cDNA microarray technologies in the study of host-parasite interactions have permitted rapid and unbiased examination of changes in expression of a large number of genes at the level of transcription [9,10]. However, these studies have been mainly focused on the interaction between the parasite and different cell types, but not on the infection and invasion on a tissue level.
During congenital transmission, T. cruzi must cross the placental barrier in order to infect the developing fetus [3,11]. This anatomical barrier is formed by the trophoblast, a two-layer epithelium which is in direct contact with maternal blood, the fetal connective tissue (villous stroma), the endothelium of fetal vessels and the basal laminae that support the epithelia [3,12]. Interestingly, the congenital transmission rate for T. cruzi is low [4,13] and it has been proposed that the placenta might play an important role avoiding parasite infection [3].
The study of gene expression profiles during infection constitutes a very powerful tool to analyze global responses of several kinds of cells and tissues, allowing the identification of new genes and/or pathways implicated in the establishment of the infection and pathogenesis as well as possible local tissue responses [3,10]. Therefore, here we aimed to study the global changes of transcriptome in the placental tissue after T. cruzi challenge.

Parasite harvesting
Trypomastigotes from T. cruzi (Y Strain, T. cruzi II) were obtained from previously infected Vero cells (ATCC® CCL-81) grown in RPMI medium supplemented with 5% fetal bovine serum (FBS) and antibiotics (penicillin-streptomycin) at 37°C in a humid atmosphere at 5% CO 2. Parasites invaded the cells and replicated intracellularly as amastigotes, after 48-72 h; amastigotes transformed back to trypomastigotes and lysed host cells. The infective trypomastigotes were separated from cellular debris by low speed centrifugation (500× g). From the supernatant, the parasites were isolated by centrifugation at 3500× g, suspended in RPMI media (without FBS, 1% antibiotics; RPMI 1640, Biological Industries Ltd., Kibbutz Beit Haemek, Israel) and quantified in a Neubauer Chamber [14,15].

HPE infection
HPE were obtained from healthy mothers with uncomplicated pregnancies by cesarean delivery. Placentas were processed in a class II laminar flow hood immediately after delivery. The maternal and fetal surfaces were discarded and villous tissue was obtained from the central part of the cotyledons. The dissected explants were washed with sterile PBS in order to get rid of the blood and co-cultivated with T. cruzi trypomastigotes in serum free RPMI media. HPE were challenged with 10 5 or 10 6 parasites/ml, since these concentrations have been proposed to correlate with low or high parasitaemia, respectively [16]. For validation experiments, LPS (10 ng/ ml) was used as positive control. After 2 or 24 h of infection (in order to study early and late placental responses [16][17][18], explants were collected in RNA later solution (Thermo Fisher Scientific, Waltham, Massachusetts, USA), stored at 4°C for 24 h and at -80°C for posterior RNA isolation [19].

RNA purification and microarray experiment
Total RNA was isolated with a Purelink RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA integrity was analyzed with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California, USA) obtaining RNA integrity numbers (RIN) above 8 for all samples (on a scale based on an rRNA 28S/18S ratio where a RIN of 1 corresponds to a totally degraded RNA and 10 to a totally non-degraded RNA). RNA concentration was quantified by spectrophotometry (Nanodrop, Thermo Fisher Scientific). One hundred nanograms of total RNA was reverse-transcribed into cDNA, then transcribed to cRNA and Cy3-labeled with a Low Input Quick Amp-One Color Labeling Kit (Agilent Technologies). The labeled cRNA was purified with an illustra RNAspin Mini Isolation Kit (GE Healthcare, Little Chalfont, UK) and the total yield was measured with a Qubit RNA HS Kit (Thermo Fisher Scientific). Hybridization, washing, assembling of the chips, and scanning were performed according to the manufacturers' instructions. Briefly, labeled samples were hybridized with SurePrint G3 Human GE 8x60K chips for 17 h at 60°C in an Agilent hybridization oven at 10× rpm. Posterior washing, stabilization and drying procedures were performed according to Agilent's Low Input Quick Amp Labeling Kit instructions [10].

Data analysis
Chips were scanned with an Agilent microarray scanner G2565BA; the software Agilent Feature Extraction (version 9.5.1), was used for quality control, data filtering and data normalization. Extracted data from the SurePrintG3 8x60K chips were analyzed using GeneSpring GX 13.0 software. Genes showing a 2-fold change in their expression (or more) with P ≤ 0.05 were considered differentially expressed using ANOVA and Benjamini-Hochberg false discovery rate correction for multiple testing. Analysis of interaction networks between upregulated genes in each experimental group was performed with Cytoscape network visualization and integration software and GeneMania open-source gene function prediction service plug-in (http://www.genemania.org/) and visualized by the corresponding Cytoscape software version 3.0.2 plug-ins [20] The weighting of the network attributes was set to Gene Ontology (GO)-based weighting for biological processes. Gene set enrichment analysis (GSEA) was performed with GSEA 3.0 software (Broad Institute, Cambridge, Massachussets, USA) [21]. Each gene set permutation was performed 1000 times, analyses were based on the GO pathways database (http://geneontology.org/) [22] and a normalized enrichment score (NES) was obtained for each gene set. An enrichment map from data obtained with GSEA was generated with the Enrichment Map plug-in for Cytoscape 3.0 [23]. NES and false discovery rate (FDR) q-value were considered as parameters for the analysis. The NES value allows the comparison of analysis results across gene sets because it considers differences in gene sets sizes corrected by the size of the expression dataset; the FDR represents the estimated probability that a gene set with a given NES represents a false positive finding [22].

RT-qPCR
One hundred picograms of total RNA was retro-transcribed to cDNA using an M-MLV Reverse Transcriptase system with Oligo(dT) primers (Thermo Fisher Scientific). For real-time reactions, 10 μl of Sensifast qPCR Master Mix (Bioline, London, UK) was mixed with 100 mM forward    of primers can be found in Table 1. The results were expressed as the mean ± SD. The significance of differences was evaluated using ANOVA followed by Dunnett's post-hoc test as indicated.

Results
Trypanosoma cruzi changes the gene expression profile in HPE The effect of the parasite on placental tissue was assayed in HPE after challenges with a low (10 5 parasites/ml) or a high (10 6 parasites/ml) concentration of trypomastigotes for 2 or 24 h. Total RNA extracted from infected and non-infected control HPE, was labeled and hybridized to a Human GE 60K Microarray, which allows the evaluation of the gene expression profile of 26,083 different human genes. Genes showing at least a 2-fold change in their expression and a 95% probability of being differentially expressed (P ≤ 0.05) were significantly regulated during parasite challenge. Figure 1 shows the total number of significant differentially expressed genes between infected and non-infected control HPE. A low parasite concentration induces the downregulation of 431 and 1474 genes as well as the upregulation of 210 and 469 genes after 2 and 24 h of parasite challenge, respectively (Fig. 1a). After a high parasite concentration challenge, 157 and 722 genes were downregulated, and 342 and 454 were upregulated after 2 and 24 h, respectively (Fig. 1b). Major changes occurred after 24 h of parasite challenge with the lowest parasite concentration. A selection of the most upregulated and downregulated genes (fold change range between 34.70 and 71.43) is shown in Table 2. Among the most upregulated genes are those involved in immune response such as CXCL9, TLR-7, TLR-8, CD46, C1qTNF3, HLA-DQB1 and CCL20; genes involved in extracellular matrix (ECM) remodeling (ADAM12, ADAMTSL3, MMP10) and related to pregnancy. The list of most downregulated genes also includes genes related to immunity such as LBP, CD14, DCD and IL-6.
The Venn diagrams in Fig. 2a show that 19 genes are upregulated in the four different experimental conditions compared to control non-infected samples, which are also shown in the corresponding heatmap (Fig. 2b) Table 3. Contrarily, only 5 genes are downregulated in the same conditions (Fig. 3a, b), which are listed in Table 3. Most of the upregulated genes are related to pregnancy processes.

Trypanosoma cruzi alters a wide range of biological processes in HPE
GO and pathway analysis were performed using Gene-SpringGX 13.0 software (Agilent Technologies), comparing the different experimental conditions described above.
Our results indicate that a wide range of biological processes are altered at the different conditions in presence of the parasite ( Table 4). The different biological processes detected include immune response, pregnancy related processes and signaling. In order to understand the relationships between those biological processes, we performed a GSEA analysis of gene sets at different times and Fig. 2 Venn diagrams comparing common differentially upregulated genes. HPCVE were incubated for 2 and 24 h with 10 5 or 10 6 T. cruzi trypomastigotes/ml. All samples were compared to the respective uninfected control. The diagram in a shows the upregulated genes at both parasite concentrations and incubation times, b corresponds to the heatmap of the differentially expressed genes in the central intersection parasite load challenges. We analyzed biological processes pathways based on gene ontology results (Fig. 4). The biggest cluster is composed of pathways related with immune response, followed by development morphogenesis cluster, regulation of metabolic processes and signal transduction genes, metabolic processes, homeostasis, response to stimulus, cell death and endocytosis (Fig. 4).
Several gene sets grouped within the main cluster (immune response), are enriched at 2 or 24 h post-infection after parasite challenges of 10 5 trypomastigotes/ml but not of 10 6 parasites. Thus, immune system process pathways are positively regulated against 10 5   To understand the nature of the interaction between upregulated or downregulated genes, we performed a gene interaction analysis using the GeneMANIA plug-in for Cytoscape 3.0 software [24,25]. For each experimental condition, we analyzed co-expression, co-localization, physical interactions, genetic interactions shared protein domains and pathways amongst all the differentially expressed genes (Fc ≥ 2) in both upregulated or downregulated gene lists. The relative weight of each process between all interacting genes is depicted in Table 5. In all experimental conditions, co-expression (a category where two genes have similar expression levels) is the predominating interaction, co-localization (genes expressed in the same tissue) the second in the upregulated conditions; however, in downregulated groups physical interactions (when two gene products are found to interact in protein-protein interaction studies) is the second most common interaction. Shared protein domains, genetic and pathways interaction represent marginal interactions in all groups. A circular layout of the interaction network for each is shown in Additional file 1: Figure S1.

Discussion
The interaction between the host and pathogens, including T. cruzi, is the most important factor in determining whether an infection is successful. Host-parasite interaction Fig. 3 Venn diagrams comparing common differentially downregulated genes. HPCVE were incubated for 2 and 24 h with 10 5 or 10 6 T. cruzi trypomastigotes/ml. All samples were compared to the respective uninfected control. The diagram in a shows the downregulated genes at both parasite concentrations and incubation times, b corresponds to the heatmap of the differentially expressed genes in the central intersection includes invasion of the host through primary barriers (such as the placental barrier), evasion of host defenses, pathogen replication in the host, and immunological capacity of the host to control or eliminate the pathogen [26]. Importantly, infected organisms are capable of sensing the intrusion by pathogens and react by triggering host defenses [17,26]. On the other hand, the parasite is equipped with multiple tools to establish a long-term relationship with the infected host. Tissue infection in particular is relevant during disease progression. The presence of the parasite provokes tissue damage as well as immune and reparatory responses, which can lead to fibrosis and tissue dysfunction as observed in chagasic cardiomyopathy [27]. Considering the temporary existence of the placenta, the effect on parasite infection on this particular tissue is relevant to understand the physiopathology of congenital transmission in order to obtain tools for diagnosis, prognosis and treatment of the disease.
Previous studies about transcriptomics related to T. cruzi and Chagas disease have been focused on a single type cell response [10,28] or on tissues or organs in animal models [24,29] but not on human tissues. Here, we describe for the first time, the transcriptomics of an ex vivo human placental tissue model in response to challenges with the parasite.
As expected, T. cruzi modifies an ample range of biological processes during tissue invasion and infection. As described before, the parasite dramatically changes the gene expression in single cells [10]. However, in tissue and organ samples a more complex change in gene expression can be expected since they are composed of different cell types or tissues. For instance, in HPE epithelial cells derived from the trophoblast and fetal capillaries as well as fibroblasts and macrophages in the fetal connective tissue can be found, between others [3]. In addition, ECM components, that are synthetized by the resident cells are also present in tissue and organ samples [18,19]. Similar results have been obtained in animal models, where important changes in murine myocardium metabolic pathways [29] and in murine placental response [24] are described. The increase of gene expression of proteases involved in ECM-remodeling (Table 2) is in concordance with our previous results showing that the parasite increased expression and activity of matrix metalloproteases (MMP-2 and MMP-9) in human placenta [19]. The profound changes in genes involved in signaling agrees with numerous previous studies that showed that T. cruzi activates or inhibits several signal transduction pathways [14,28,30].
The effect of T. cruzi on the immune system responses are particularly relevant since they are our main defense against the pathogen. The parasite dramatically changes host genes involved in the immune response. The expression of an important number of genes of innate immunity is increased. Thus, genes related to complement regulation and function such as CD46 and C1q are upregulated. We have previously shown, that during ex Fig. 4 Enrichment map of gene sets of biological processes pathways. HPCVE were incubated for 2 and 24 h with 10 5 or 10 6 T. cruzi trypomastigotes/ml. Genes with fold changes ≥ 2 (P ≤ 0.05) were considered for the analysis. Gene set analysis (GSEA) was performed based in pathways from GO biological processes. Nodes correspond to gene sets and connecting lines to overlapping member genes between nodes. Divided circles represent predicted pathways, each segment of the circle represents a different experimental group according to attached legend and colors depict upregulated (red) or downregulated (blue) genes. Clusters grouped by biological function were manually labeled  , TGF β (f) and IL-17 (g) was assayed. Samples were processed for RT-qPCR. The sequences of primers can be found in Table 1. The significance of differences was evaluated using Student's t-tests for paired data. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001

Fig. 5
Trypanosoma cruzi induce differential expression of pathogen pattern recognition receptors in HPE. Explants were co-incubated with 10 5 T. cruzi trypomastigotes/ml for 2 h and the expression of TLR-2 (a), TLR-4 (b), TLR-7 (c), TLR-9 (d), NOD-1 (e) and NOD-2 (f) were assayed. Samples were processed for RT-qPCR. The sequences of primers can be found in Table 1. The significance of differences was evaluated using Student's ttests for paired data. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 vivo infection of HPE, T. cruzi calreticulin (TcCRT) acts as a virulence factor since it binds maternal classical complement component C1q and increases parasite infectivity [31]. On the other hand, TLRs are also increased, particularly TLR-7 and TLR-8 which are increased over 20-fold. The validation experiments show that both mentioned TLRs as well as TLR-2, TLR-4 and TLR-9 are significantly increased (Fig. 5). However, TLR-2, the TLR whose inhibition increases parasite infection in HPE as well as parasite-induced tissue damage [17] showed no significant increase in the microarray analysis (Additional file 1: Figure S1). A similar contradictory result was obtained with IL-6; a high parasite concentration decreases the expression of this cytokine more than 60-fold (Table 2). However, a low parasite concentration does not change IL-6 expression (Additional file 1: Figure S1). However, our RT-qPCR data show a significant increase of IL-6 ( Fig. 5) that is in concordance with the increase of IL-6 protein in the culture media of HPE after parasite challenge in the same condition [17], suggesting regulation at post-transcriptional levels.
Another important group of genes that change their expression are those related to pregnancy. Most of the 19 upregulated genes in the four different experimental conditions compared to control samples are related to pregnancy processes (Table 3) and to the maintenance and development of the fetus such as pregnancy specific beta-1-glycoproteins, GH2 (growth hormone 2), CSH1 and CSH2 (chorionic somatomammotropin hormone 1 and 2). Given that the placenta is the sole interface between mother and fetus and that this organ not only protects the fetus from infection but also regulates important metabolic and other physiological processes [32], it appears easily explainable that different pregnancy related processes are affected.

Conclusions
Trypanosoma cruzi induces strong changes in genes involved in a wide range of pathways, especially those involved in immune response against infections.

Additional file
Additional file 1: Figure S1. Interaction networks in differentially expressed genes from each experimental group. HPCVE were incubated during 2 and 24 h with 10 5 or 10 6 T. cruzi trypomastigotes. Interaction networks from differentially expressed genes (FC ≥ 2) compared with uninfected control with GeneMANIA function prediction service plug-in in Cytoscape software. Co-expression, co-localization, physical interactions, genetic interactions shared protein domains and pathways are shown and each color represents specific interactions according to legend. (PDF 707 kb)