Abstract

   Trypanosoma cruzi and Toxoplasma gondii are two parasites than can be
   transmitted from mother to child through the placenta. However,
   congenital transmission rates are low for T. cruzi and high for T.
   gondii. Infection success or failure depends on complex parasite-host
   interactions in which parasites can alter host gene expression by
   modulating non-coding RNAs such as miRNAs. As of yet, there are no
   reports on altered miRNA expression in placental tissue in response to
   either parasite. Therefore, we infected human placental explants ex
   vivo by cultivation with either T. cruzi or T. gondii for 2 h. We then
   analyzed the miRNA expression profiles of both types of infected tissue
   by miRNA sequencing and quantitative PCR, sequence-based miRNA target
   prediction, pathway functional enrichment, and upstream regulator
   analysis of differentially expressed genes targeted by differentially
   expressed miRNAs. Both parasites induced specific miRNA profiles. GO
   analysis revealed that the in silico predicted targets of the
   differentially expressed miRNAs regulated different cellular processes
   involved in development and immunity, and most of the identified KEGG
   pathways were related to chronic diseases and infection. Considering
   that the differentially expressed miRNAs identified here modulated
   crucial host cellular targets that participate in determining the
   success of infection, these miRNAs might explain the differing
   congenital transmission rates between the two parasites. Molecules of
   the different pathways that are regulated by miRNAs and modulated
   during infection, as well as the miRNAs themselves, may be potential
   targets for the therapeutic control of either congenital Chagas disease
   or toxoplasmosis.

   Keywords: Trypanosoma cruzi, Toxoplasma gondii, human placental
   explants, miRNA profile, host gene expression

Introduction

   More than one billion people worldwide are burdened by parasitic
   diseases ([37]1). Of these, Chagas disease (American trypanosomiasis)
   and toxoplasmosis are caused by Trypanosoma cruzi (T. cruzi) and
   Toxoplasma gondii (T. gondii), respectively ([38]2–[39]4). Chagas
   disease is a devastating but neglected health problem in Latin America.
   Due to the extensive global migration of asymptomatic individuals, this
   infection has become an emerging disease in non-endemic countries.
   Congenital transmission is partially responsible for the progressive
   globalization of Chagas disease ([40]5, [41]6). T. gondii is one of the
   most successful parasites on earth and is estimated to infect over one
   billion people worldwide ([42]7). Importantly, both parasites can be
   congenitally transmitted and cause perinatal morbidity and mortality
   ([43]2–[44]4) but present different transmission rates. T. cruzi has a
   low transmission rate (1–12%) ([45]6, [46]8) while T. gondii has a high
   transmission rate (22–72%) ([47]3). Moreover, both parasites elicit a
   different local placental immune response that might be related to
   infection susceptibility ([48]9, [49]10). Thus, T. cruzi and T. gondi
   infection is related to the expression and activation of different
   Toll-like receptors, which in turn mediate the secretion of different
   cytokines and chemokines in defense against both parasites in the
   placenta ([50]9, [51]11).

   However, the probability of congenital transmission depends on a
   variety of complex interactions between the pathogen and the host
   ([52]4, [53]12). In particular, parasite factors, placental factors,
   and maternal and developing fetal immune systems determine infection
   occurrence ([54]4, [55]13). In this context, both parasites display
   sophisticated strategies to avoid host defenses and virulence factors
   that increase the chance of establishing infection and long-term
   persistence. One of these strategies is the ability to modulate host
   cell gene expression ([56]14–[57]16) through small non-coding RNAs such
   as microRNAs (miRNAs) that repress mRNAs in a sequence-specific manner
   by either an mRNA degradation process or through mRNA translation
   inhibition ([58]17–[59]19). MiRNAs play a key role in fine tuning gene
   expression in multiple physiological and pathological conditions
   including T. cruzi ([60]20) or T. gondii ([61]21) infection.
   Interestingly, the largest miRNA cluster in humans is encoded in
   chromosome 19 (C19MC; 19q13.41) and is almost exclusively expressed in
   the placenta ([62]22). Both C19MC-derived and non-C19MC-derived miRNAs
   have been associated with placental development pathologies such as
   pre-eclampsia and infection ([63]23, [64]24). However, there is no
   report in the literature regarding altered miRNA expression in
   placental tissue in response to either parasite.

   Here, we infected human placental explants (HPE) ex vivo by 2 h of
   incubation with either T. cruzi or T. gondii, then analyzed both miRNA
   expression profiles by miRNA sequencing and quantitative PCR of
   selected miRNAs. In addition, we used sequence-based miRNA target
   prediction and performed pathway functional enrichment and upstream
   regulator analysis of differentially expressed genes targeted by
   differentially expressed miRNAs (DEMs).

Materials and Methods

Parasite Culture and Harvesting

   For T. cruzi, Y strain (T. cruzi II) trypomastigotes were obtained from
   previously infected Vero cells (ATCC^® CCL-81) grown in RPMI medium
   supplemented with 5% fetal bovine serum and 1% antibiotics
   (penicillin-streptomycin) at 37°C in a humid atmosphere with 5% CO[2.]
   Parasites invaded the cells and replicated intracellularly as
   amastigotes. After 48–72 h, amastigotes transformed back to
   trypomastigotes and lysed the host cells. The infective trypomastigotes
   were separated from cellular debris by low speed centrifugation (500 ×
   g) for 10 min. Parasites were isolated from the supernatant by
   centrifugation at 3500×g during 15 min, suspended in RPMI media
   (without fetal bovine serum, 1% (penicillin-streptomycin) (RPMI 1640^®,
   Biological Industries Ltd.), and quantified in a Neubauer chamber
   ([65]9).

   For T. gondii, semi-confluent HFF cells were infected with RH
   tachyzoites at a multiplicity of infection of 3 to 5 parasites per
   cell. After 40 h, the infected cells were washed, then monolayers were
   scraped from the flasks and passed through 20-, 23-, and 25-gauge
   needles. Tachyzoites were purified from host cell debris with a 3.0 μm
   Isopore filter (Merck Millipore^®) ([66]25).

   The laboratory has been certificated as a Biosafety level 2 laboratory
   by the Biosafety Committee (“Unidad de Prevención de Riesgo”) of the
   “Facultad de Medicina, Universidad de Chile” (approval # 0403/2019).

HPE Infection

   Human term placentas were obtained from 3 women with uncomplicated
   pregnancies with vaginal or caesarean delivery. Informed consent for
   experimental use of the placenta was given by each patient as
   stipulated by the Code of Ethics of the “Servicio de Salud
   Metropolitana Norte” (approval number 0010/2019). Exclusion criteria
   consisted of the following: major fetal abnormalities, placental tumor,
   intrauterine infection, obstetric pathology, positive serology for
   Chagas disease, and any other maternal disease. Donor patients were
   negative for anti-T. gondii IgG/IgM antibodies. The organs were
   collected in cold, sterile, saline-buffered solution (PBS) and
   processed no more than 30 min after delivery. The dissected explants
   (approximately 50 mg of tissue) were washed with sterile PBS to remove
   the blood and co-cultivated with T. cruzi trypomastigotes or T. gondii
   tachyzoites (10^5 parasites/ml) in serum free RPMI media. After 2 h of
   co-cultivation, explants were collected in RNAlater™ solution
   (ThermoFisher Scientific^®), then stored at 4°C for 24 h and at -80°C
   for posterior RNA isolation ([67]9). Three independent experiments were
   carried out in triplicates; HPEs from each placenta were infected with
   either T. cruzi or T. gondii parasites. The parasite load in the HPEs
   was confirmed by real-time PCR as described previously by us ([68]9,
   [69]10, [70]26).

RNA Extraction

   Total RNA was extracted from HPE by mechanical disruption in 1.3 ml of
   RNA-solv^® reagent (Omega Bio-tek) and isolated using an E.Z.N.A^®
   total RNA kit I (Omega Bio-tek) according to manufacturer instructions.
   RNA was stored at -80°C until analysis. The concentration and quality
   of RNA was determined with a Qubit^® RNA HS Assay kit and an IQ Assay
   kit (Invitrogen), respectively. Only RNA samples with an IQ ≥8 were
   further analyzed for quality with an Agilent 2100 Bioanalyzer System
   (Agilent Technologies, USA) using an RNA Nano 6000 Assay Kit. RNA
   samples with RNA integrity numbers >5.0 were used for miRNA profiling
   analysis ([71]9).

Library Construction and Sequencing

   Small RNA-Seq libraries were constructed with an Illumina TruSeq Small
   RNA library preparation kit according to manufacturer protocols. To
   assess the quality of the libraries, a DNA High Sensitivity Chip was
   used in an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The
   libraries were sequenced on an Illumina NextSeq 500 platform. For each
   condition, three independent biological replicates were sequenced and
   paired-end reads were generated.

Data Analysis

   Raw read quality was evaluated using the FastQC tool
   ([72]https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw
   reads were analyzed with Trim Galore Cutadapt software ([73]27) and
   low-quality reads were removed (phred value < 30) in order to obtain
   clean reads. Clean reads with a length range of 18–35 nucleotides were
   chosen to perform all subsequent analyses. The sofware STAR ([74]28)
   was used to align all reads to the reference human genome sequence
   (Hg38). The read counts per coding sequence were determined using
   HTSeq-count ([75]29). To evaluate replicates, we used Principal
   Component Analysis, Pearson correlation and standardized median
   correlation analyses and box plots. The program EdgeR was used for
   differential expression analysis ([76]30). Differentially expressed
   genes were defined as genes with p-value <0.05. Target gene prediction
   performed by using miRDB, psRNA target, and TargetScan sofwares.

Enrichment Analyses

   miRNA set enrichment analysis was performed using the TAM 2.0 tool
   ([77]http://www.lirmed.com/tam2/). KEGG pathways and functional
   annotation of the predicted target genes
   ([78]https://www.genome.jp/kegg/kegg2.html) were also analyzed to
   determine the biological processes, molecular functions, cellular
   components, and associations with disease.

RT-qPCR

   RNA enriched in small RNAs was extracted from HPEs (approximately 50 mg
   of tissue) by mechanical disruption in 1 ml RNAzol^® RT (Sigma-Aldrich)
   according to manufacturer instructions and stored at -80°C until
   analysis. The concentration of the isolated miRNAs was determined using
   a Qubit^® Quant-iT™ microRNA Assay Kit (Molecular Probes). cDNA of
   mature miRNAs was synthesized with a MystiCq™ microRNA cDNA Synthesis
   Mix Kit (Sigma-Aldrich Merck) per manufacturer guidelines. The 25 µl
   RT-qPCR reaction contained 12.5 µl 2× MystiCq microRNA SYBR Green qPCR
   Ready Mix, 0.5 µl of 10 µM MystiCq Universal PCR Primer, 0.5 µl of 10
   µM of each specific MystiCq microRNA qPCR Assay Primer ([79]
   Supplementary Table 1 ), 10.5 µl nuclease-free water, and 1 µl cDNA.
   All RT-qPCR reactions were performed in triplicates. RT-qPCR was
   performed under the following cycling conditions: initial denaturation
   at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for
   30 s. Gene expressions were calculated using the 2^−ΔΔCT relative
   expression method and normalized to snRNA U6 (RNU6-1) expression levels
   ([80]31).

Results

T. cruzi and T. gondii Change the miRNA Expression Profile in HPE

   The effects of T. cruzi and T. gondii on placental tissue were assayed
   in HPE after a 2 h challenge with 10^5 parasites/ml. Total miRNA
   extracted from infected and non-infected control HPE was analyzed by
   miRNA Seq. Key characteristics of the obtained sequencing data are
   summarized in [81]Table 1 . A total of 680 and 686 DEMs were identified
   in T. cruzi and T. gondii infected HPE, respectively. Only 14 DEMs with
   a minimum 1.5-fold change in expression and a 95% probability of being
   differentially expressed (p ≤ 0.05) were identified in T. cruzi
   challenged samples ([82] Figure 1A ). In T. gondii challenged samples,
   the number of DEMs increased to 42 ([83] Figure 1B ). Comparison of T.
   cruzi infected HPE with non-infected control samples showed that five
   miRNAs were downregulated and nine were upregulated. In T. gondii
   infected tissues, 13 miRNAs were downregulated and 29 were upregulated.
   The Venn diagram in [84]Figure 1C shows the miRNAs that were
   differentially expressed in the presence of both parasites compared to
   non-infected control samples and in HPE infected with either parasite.
   The complete list of DEMs in response to ex vivo T. cruzi and T. gondii
   infection is shown in [85]Table 2 .

Table 1.

   Statistics of the small RNA sequences obtained in this study.
   M reads (millions) M Aligned (millions) % aligned Mature microRNA reads
   # Mature MicroRNAs # Mature MicroRNA (single aligned)
   S1 Control 12,96 11,68 90,12 3489287 922 732
   S2 Control 15,17 13,87 91,43 4855933 938 760
   S3 Control 15,58 13,16 84,50 3030989 843 714
   S4 T. cruzi 14,35 12,60 87,77 3000672 907 712
   S5 T. cruzi 14,76 12,94 87,69 3910944 907 737
   S6 T. cruzi 16,13 13,15 81,55 3571933 914 720
   S7 T. gondii 13,57 11,82 87,07 3160630 916 737
   S8 T. gondii 14,40 12,79 88,77 4747358 962 757
   S9 T. gondii 13,51 12,16 89,99 4148647 931 763
   [86]Open in a new tab

   M reads, total of sequences in analysis after raw data processing
   (millions); M aligned, total of mapped sequences to the human genome
   (millions); % aligned, total of mapped sequences to the human genome
   (percentage); Mature microRNA reads, mapped reads counted as mature
   miRNAs; # Mature microRNAs, number of mature miRNAs; # Mature MicroRNA
   (single aligned), number of mature miRNAs associated to unique miRNA
   precursors in the reference.

Figure 1.

   [87]Figure 1
   [88]Open in a new tab

   Expression profiling of mature miRNAs in human placental explants
   (HPEs) following T. cruzi or T. gondii infection. (A) Heat-map of
   differentially expressed microRNAs (DEMs) in control vs. T. cruzi
   infected HPE and (B) control vs. T. gondii infected HPE. The filtered
   miRNA data were subjected to unsupervised hierarchical clustering
   analysis. The metric was set as the Euclidean distance. Control: C1,
   C2, C3; T. cruzi: Tc1, Tc2, Tc3; T. gondii: Tx1, Tx2, Tx3. (C) Venn
   diagram showing the number of commonly expressed and specifically
   expressed miRNAs between the infected HPE groups. Significant miRNAs
   for HPE with T cruzi infection vs. those with T. gondii infection are
   shown in the yellow circle. The blue circle represents the miRNAs that
   discriminate between the control (uninfected HPE) and T. cruzi infected
   HPE, while the green circle represents the miRNAs that distinguish the
   control (uninfected HPE) and T. gondii infected HPE.

Table 2.

   The top differentially expressed miRNAs (P < 0.05) in HPE challenged
   with 10^5 T. cruzi trypomastigotes or T. gondii tachyzoites during 2 h.
     miRNAs Fold Change p-value p-adjustment Expression
   Control vs T. cruzi hsa-miR-490-5p 1,47644601 0,011939305 1
   Up-regulated
   hsa-miR-497-5p -0,95822045 0,021380502 1 Down-regulated
   hsa-miR-146a-5p 0,9111643 0,024398238 1 Up-regulated
   hsa-miR-12135 2,76826832 0,025125553 1 Up-regulated
   hsa-miR-210-5p 1,17471038 0,038576256 1 Up-regulated
   hsa-miR-146b-5p 0,70985057 0,041583753 1 Up-regulated
   hsa-miR-877-5p -0,71572824 0,049301458 1 Down-regulated
   hsa-miR-1271-5p -1,48497011 0,049553349 1 Down-regulated
   T. cruzi (*treatment exclusive) hsa-miR-636 0,003652701 1 Up-regulated
   hsa-miR-4449 0,005197851 1 Up-regulated
   hsa-miR-449a 0,009529598 1 Up-regulated
   hsa-miR-2115-5p 0,013101594 1 Up-regulated
   hsa-miR-561-3p 0,023021063 1 Up-regulated
   hsa-miR-4446-3p 0,041796581 1 Up-regulated
   Control vs T. gondii hsa-miR-12136 1,79576197 0,00017626 0,120736654
   Up-regulated
   hsa-miR-335-5p 1,35817451 0,0007489 0,227732906 Up-regulated
   hsa-miR-10b-5p -1,51399053 0,00099737 0,227732906 Down-regulated
   hsa-miR-1271-5p -2,41452099 0,00206894 0,267855922 Down-regulated
   hsa-miR-409-5p -2,51430487 0,00225422 0,267855922 Down-regulated
   hsa-miR-27a-3p -0,97356696 0,00234618 0,267855922 Down-regulated
   hsa-miR-29a-3p -0,92877383 0,00344997 0,337603687 Down-regulated
   hsa-miR-214-3p 0,9642214 0,00435186 0,372627686 Up-regulated
   hsa-miR-379-5p 1,46136088 0,00619518 0,453658878 Up-regulated
   hsa-miR-3120-5p 1,05085403 0,00662276 0,453658878 Up-regulated
   hsa-miR-376a-5p -1,06317073 0,00928715 0,578335925 Down-regulated
   hsa-miR-542-3p 1,36466429 0,01456701 0,760274481 Up-regulated
   hsa-miR-195-5p 1,29637989 0,01520981 0,760274481 Up-regulated
   hsa-miR-3130-5p -2,64645685 0,01847345 0,760274481 Down-regulated
   hsa-miR-519d-3p -0,91993812 0,0188483 0,760274481 Down-regulated
   hsa-miR-490-5p 1,42743639 0,01886813 0,760274481 Up-regulated
   hsa-miR-450b-5p 1,11411811 0,02263153 0,763303449 Up-regulated
   hsa-miR-374b-5p 0,86524386 0,02512358 0,763303449 Up-regulated
   hsa-miR-374c-3p 0,87017312 0,02523843 0,763303449 Up-regulated
   hsa-miR-143-3p 0,78495077 0,02900217 0,763303449 Up-regulated
   hsa-miR-21-3p 0,79317052 0,0291757 0,763303449 Up-regulated
   hsa-miR-675-5p 1,1546043 0,03213608 0,763303449 Up-regulated
   hsa-miR-671-5p 1,76630085 0,03293839 0,763303449 Up-regulated
   hsa-miR-146a-5p 0,86261284 0,03359957 0,763303449 Up-regulated
   hsa-miR-489-3p 1,02467619 0,03372793 0,763303449 Up-regulated
   hsa-miR-96-5p -2,45982286 0,03878464 0,77095716 Down-regulated
   hsa-miR-509-3p 0,86545472 0,03909277 0,77095716 Up-regulated
   hsa-miR-190b-5p 2,26556495 0,04749263 0,77095716 Up-regulated
   hsa-miR-371b-5p -0,92943309 0,04875339 0,77095716 Down-regulated
   hsa-miR-520d-5p -0,6667811 0,04979001 0,77095716 Down-regulated
   hsa-miR-371a-3p -0,92563764 0,04993063 0,77095716 Down-regulated
   hsa-miR-412-5p 0,86533956 0,0514002 0,77095716 Up-regulated
   hsa-miR-324-5p -1,01010797 0,05178532 0,77095716 Down-regulated
   hsa-miR-410-3p 0,63475365 0,05378966 0,77095716 Up-regulated
   hsa-miR-139-5p 0,66901369 0,0543406 0,77095716 Up-regulated
   T. gondii (*treatment exclusive) hsa-miR-548l 0,01756271 0,760274481
   Up-regulated
   hsa-miR-449a 0,02617767 0,763303449 Up-regulated
   hsa-miR-6125 0,03454366 0,763303449 Up-regulated
   hsa-miR-4638-3p 0,03454366 0,763303449 Up-regulated
   hsa-miR-6860 0,03454366 0,763303449 Up-regulated
   hsa-miR-4660 0,03454366 0,763303449 Up-regulated
   hsa-miR-2115-5p 0,04354449 0,77095716 Up-regulated
   [89]Open in a new tab

   *Treatment Exclusive: refers to miRNAs sequenced only in the T. cruzi
   or T. gondii infected condition (treatment), they are not expressed in
   control explants.

Functional Annotation and KEGG Pathway Enrichment Analysis of miRNA Target
Genes

   To better understand the roles of the miRNAs identified in HPE in
   response to ex vivo infection with both parasites, the target genes of
   the miRNAs were identified using miRDB, psRNA target, and TargetScan.
   GO and KEGG enrichment analyses used to identify the biological
   functions of the DEMs (p < 0.05) during ex vivo T. cruzi and T. gondii
   infection revealed 679 best scored target genes of the 14 miRNAs from
   T. cruzi vs. control, 1970 best scored target genes of the 42 miRNAs
   from T. gondii vs. control, and 2011 best scored target genes of the 42
   miRNAs from T. cruzi vs. T. gondi. The target genes of the DEMs are
   shown in [90]Supplementary Table 2 . Among the significantly enriched
   GO terms in T. cruzi vs. control samples, DEMs were significantly
   enriched in regulation of NFκB pathways, chondrocyte development, cell
   death including apoptosis, peritoneal cavity homeostasis, angiogenesis,
   cell cycle, megakaryocyte differentiation, Toll-like receptor signaling
   pathway, and immune response including innate immunity ([91] Figure 2A
   ). Among the significantly enriched GO terms in T. gondii vs. control
   samples, DEMs were significantly enriched in cell proliferation, cell
   migration, osteoblast differentiation, oxidative stress, lipid
   metabolism, regulation of stem cells including embryonic stem cells,
   hepatotoxicity, DNA damage response, regulation of NFκB pathways,
   smooth muscle proliferation, T-helper 17 cell differentiation, T-cell
   activation, and response to estrogen ([92] Figure 2B ).

Figure 2.

   [93]Figure 2
   [94]Open in a new tab

   KEGG analysis of the differentially expressed miRNAs (P < 0.05)
   revealed significant enrichment in immune-related and cell cycle
   pathways. The top 15 enriched pathways of the differentially expressed
   miRNAs are presented for (A) control vs. T. cruzi and (B) control vs.
   T. gondii. The sizes and colors of the circles represent the number of
   predicted gene targets of the differentially expressed miRNAs (P <
   0.05) and the q-value, respectively.

   In addition, we performed GO and KEGG analyses to identify different
   pathologies in which the T. cruzi- and T. gondii-induced DEMs were
   related. In T. cruzi vs. control samples, significantly enriched DEMs
   were related to metabolic syndrome, IgA-nephropathy, acute childhood
   lymphoblastic leukemia, atherosclerosis, oral lichen planus, human
   papilloma virus infection, psoriasis, neuropsychiatric disorders, heart
   diseases, pancreatic carcinoma, Löfgren’s syndrome, Mycobacterium
   tuberculosis infection, male infertility, and gastric carcinoma ([95]
   Figure 3A ). In T. gondii vs. control samples, the significantly
   enriched DEMs were related to ankylosing spondylitis, type 2 diabetes
   mellitus, hypertrophic cardiomyopathy, congenital heart disease, fetal
   alcohol syndrome, pulmonary hypertension, ulcerative colitis, cystic
   fibrosis, vascular diseases, human cytomegalovirus infection, muscular
   dystrophy, liver diseases, coxsackievirus infection, and diabetic
   retinopathy ([96] Figure 3B ).

Figure 3.

   [97]Figure 3
   [98]Open in a new tab

   KEGG analysis of the differentially expressed miRNAs (P < 0.05)
   revealed significant enrichment in cancer-related, infections, and
   inflammatory disease pathways. The top 15 enriched pathways of the
   differentially expressed miRNAs are presented for (A) control vs. T.
   cruzi and (B) control vs. T. gondii. The sizes and colors of the
   circles represent the number of predicted gene targets of the
   differentially expressed miRNAs (P < 0.05) and the q-value,
   respectively.

   Specific GO and KEGG enrichment analyses focused on the functions of
   DEMs in response to both parasites that were related to parasitic
   diseases and/or placenta pathology. These DEMs are listed in [99]Table
   3 . In this context, we found that the significantly enriched DEMs were
   related to the regulation of apoptosis, wound healing, cardiomyocyte
   apoptosis, heart development, skeletal muscle cell differentiation,
   Toll-like receptor signaling pathway, innate immunity, epithelial to
   mesenchymal transition, chromatin remodeling, and nephrotoxicity ([100]
   Figure 4 ). Moreover, we analyzed the significantly enriched DEMs
   related to transcription activation and found that the following
   transcription factors or its activators were modulated by them: MYOG,
   calcineurin, AP-1, TNFSF12, NFκB1, myogenin, MYOD, MYF5, MRF4, and TP53
   ([101] Figure 5 ).

Table 3.

   Sequenced miRNA related to placental and/or parasitic diseases in HPE
   challenged with 10^5 T. cruzi trypomastigotes or T. gondii tachyzoites
   during 2 h.
   miRNAs Control vs T. cruzi Control vs T. gondii
   Expression p-value Expression p-value
   hsa-miR-3074 Up-regulated 0,282778175 Down-regulated 0,906716427
   hsa-miR-518e-5p Down-regulated 0,736493282 Up-regulated 0,655921033
   hsa-miR-127-3p Down-regulated 0,454473692 Up-regulated 0,899001399
   hsa-miR-512-3p Up-regulated 0,893095693 Up-regulated 0,554888541
   hsa-miR-516a-5p Down-regulated 0,573579895 Down-regulated 0,875664943
   hsa-miR-376a-3p Up-regulated 0,26239456 Up-regulated 0,890873441
   hsa-miR-523-5p Down-regulated 0,736493282 Up-regulated 0,655921033
   hsa-miR-517-5p Down-regulated 0,356215385 Down-regulated 0,131367096
   hsa-miR-523-3p Up-regulated 0,558965508 Down-regulated 0,070624754
   hsa-miR-519a-5p Down-regulated 0,902087668 Up-regulated 0,683786458
   hsa-miR-526a-5p Down-regulated 0,878621126 Up-regulated 0,975623088
   hsa-miR-519a-3p Up-regulated 0,855200309 Down-regulated 0,216046946
   hsa-miR-518e-3p Up-regulated 0,878596629 Down-regulated 0,062749763
   hsa-miR-520c-5p Down-regulated 0,878525243 Up-regulated 0,981710662
   hsa-miR-526a-3p Down-regulated 0,648457618 Down-regulated 0,194744348
   hsa-miR-29b-3p Up-regulated 0,688264908 Down-regulated 0,461394462
   hsa-miR-520c-3p Down-regulated 0,492741665 Down-regulated 0,321148465
   hsa-miR-133a-3p Up-regulated 0,800691009 Down-regulated 0,140142195
   hsa-miR-525-5p Down-regulated 0,892625222 Down-regulated 0,624806979
   hsa-miR-525-3p Up-regulated 0,896175408 Down-regulated 0,075998808
   hsa-miR-519c-5p Down-regulated 0,736493282 Up-regulated 0,655921033
   hsa-miR-518b Up-regulated 0,991656949 Down-regulated 0,078353573
   hsa-miR-519c-3p Down-regulated 0,262008953 Down-regulated 0,786169155
   hsa-miR-520e-5p Down-regulated 0,485165729 Up-regulated 0,996288443
   hsa-miR-520e-3p Up-regulated 1 Up-regulated 0,818473073
   hsa-miR-21-5p Up-regulated 0,783933507 Up-regulated 0,129614377
   hsa-miR-21-3p Up-regulated 0,749764521 Up-regulated 0,029175701
   hsa-miR-517a-3p Up-regulated 0,596271375 Down-regulated 0,461347018
   hsa-miR-519e-5p Up-regulated 0,105129034 Up-regulated 0,757374294
   hsa-miR-519e-3p Up-regulated 0,385269175 Up-regulated 0,69099671
   hsa-miR-518d-5p Down-regulated 0,878525243 Up-regulated 0,981710662
   hsa-miR-520g-5p Up-regulated 0,892300156 Up-regulated 0,751979117
   hsa-miR-518d-3p Up-regulated 0,55414416 Down-regulated 0,942071295
   hsa-miR-520b-5p Down-regulated 0,421187791 Down-regulated 0,477328076
   hsa-miR-520g-3p Up-regulated 0,728189158 Down-regulated 0,352658846
   hsa-miR-519a-2-5p Down-regulated 0,421187791 Down-regulated 0,477328076
   hsa-miR-520b-3p Up-regulated 0,530509712 Down-regulated 0,954450249
   hsa-miR-517c-3p Up-regulated 0,760586655 Down-regulated 0,675509383
   hsa-miR-524-5p Down-regulated 0,490615316 Down-regulated 0,115381834
   hsa-miR-210-5p Up-regulated 0,038576256 Up-regulated 0,576691951
   hsa-miR-204-5p Up-regulated 0,62227226 Down-regulated 0,583793777
   hsa-miR-524-3p Down-regulated 0,907890609 Down-regulated 0,099098809
   hsa-miR-519b-5p Down-regulated 0,736493282 Up-regulated 0,655921033
   hsa-miR-210-3p Up-regulated 0,497217414 Down-regulated 0,843820092
   hsa-miR-378a-5p Down-regulated 0,915728534 Down-regulated 0,504825517
   hsa-miR-526b-5p Down-regulated 0,501400731 Down-regulated 0,534886926
   hsa-miR-519b-3p Up-regulated 0,996536077 Up-regulated 0,986254335
   hsa-miR-518a-5p Down-regulated 0,396658878 Down-regulated 0,099042516
   hsa-miR-520d-5p Down-regulated 0,163923317 Down-regulated 0,049790011
   hsa-miR-526b-3p Up-regulated 0,690372369 Down-regulated 0,356016985
   hsa-miR-520d-3p Down-regulated 0,368956264 Down-regulated 0,595399092
   hsa-miR-30e-3p Up-regulated 0,708643074 Down-regulated 0,606142557
   hsa-miR-520h Up-regulated 0,420508027 Down-regulated 0,48103803
   hsa-miR-519d-5p Down-regulated 0,699132481 Down-regulated 0,210250996
   hsa-miR-515-5p Down-regulated 0,451420159 Down-regulated 0,479490879
   hsa-miR-519d-3p Up-regulated 0,795099209 Down-regulated 0,018848296
   hsa-miR-515-3p Down-regulated 0,961837732 Down-regulated 0,056222245
   hsa-miR-518c-5p Down-regulated 0,433629597 Down-regulated 0,461615584
   hsa-miR-155-5p Up-regulated 0,089556012 Up-regulated 0,307144182
   hsa-miR-518c-3p Up-regulated 0,825417746 Down-regulated 0,393062915
   hsa-miR-520a-5p Up-regulated 0,829237058 Down-regulated 0,786085152
   hsa-miR-376a-5p Down-regulated 0,101845272 Down-regulated 0,009287146
   hsa-miR-520a-3p Up-regulated 0,303331111 Down-regulated 0,733502488
   hsa-miR-144-5p Up-regulated 0,979441392 Down-regulated 0,781688613
   hsa-miR-204-5p Up-regulated 0,62227226 Down-regulated 0,583793777
   hsa-miR-424-5p Up-regulated 0,52797867 Down-regulated 0,318765339
   hsa-miR-346 Down-regulated 0,996223653 Up-regulated 0,484560011
   [102]Open in a new tab

Figure 4.

   [103]Figure 4
   [104]Open in a new tab

   Bar plot illustrating the top 10 significant miRNA-function
   associations of the sequenced miRNA related to placental and/or
   parasitic diseases in human placental explants infected with T. cruzi
   or T. gondii.

Figure 5.

   [105]Figure 5
   [106]Open in a new tab

   Bar plot illustrating the top 10 significant miRNA transcription factor
   associations of the sequenced miRNA related to placental and/or
   parasitic diseases in human placental explants infected with T. cruzi
   or T. gondii.

Validation of miRNA Expression by RT-qPCR

   Validation of miRNAs associated with parasite infection or pregnancy
   related pathologies was performed by selecting six miRNAs [miR-3074
   ([107]26), miR-127-3p ([108]27, [109]28), miR-30e-3p ([110]29),
   miR-512-3p ([111]30), miR-515-5p ([112]31), and miR-190b ([113]32)] for
   confirmation by real time PCR to verify the DEM expression levels.
   Expression of miR-3074, miR-127-3p, and miR-30e-3p ([114] Figures 6A–C
   ) was analyzed in HPE in response to both parasites. miR-512-3p and
   miR-515-5p ([115] Figures 6D–E ) expression was determined in response
   to T. cruzi infection and miR-190b expression in response to T. gondii
   infection ([116] Figure 6F ). All selected miRNAs except for miR-30e-3p
   ([117] Figure 6C ) were differentially expressed. Thus, miR-3074
   expression ([118] Figure 6A ) was significantly decreased (T. cruzi:
   57.03 ± 19.99%, p ≤ 0.01; T. gondii: 69.84 ± 24.67%, p ≤ 0.01) with
   respect to the control but not the infected samples. Decreased miR-3074
   expression was expected in the T. gondii infected samples but not in
   the T. cruzi infected samples. According to the miRNA Seq data,
   miR-3074 was upregulated in T. cruzi challenged samples. Similar
   results were observed for miR-127-3p ([119] Figigure 6B ). Expression
   of miR-127-3p was significantly decreased in HPE infected with either
   parasite (T. cruzi: 68.218 ± 16.41%, p ≤ 0.01; T. gondii: 73.13 ±
   22.45%, p ≤ 0.01) compared to the control but not to infected samples;
   we expected an increase in miR-127-3p expression in the presence of T.
   gondii since in the miRNA Seq data this particular miRNA was increased
   ([120] Table 3 ). RT-qPCR validation results for miR-512-3p,
   miR-515-5p, and miR-190b confirmed the miRNA Seq data. Thus, miR-512-3p
   expression increased (40.83 ± 22.53%, p ≤ 0.01) ([121] Figure 6D ) and
   miR-515-5p expression decreased (21.44 ± 8.60%, p ≤ 0.01) ([122] Figure
   6E ) significantly in T. cruzi infected samples. In T. gondii infected
   HPE, miR-190b expression was significantly increased (59.02 ± 37.73%, p
   ≤ 0.01) ([123] Figure 6F ).

Figure 6.

   [124]Figure 6
   [125]Open in a new tab

   Validation of miRNAs related to placental and/or parasitic diseases
   using RT-qPCR. Human placental explants were challenged with 10^5 T.
   cruzi trypomastigotes or T. gondii tachyzoites for 2 h. The presence of
   miRNA was determined by real-time PCR. T. cruzi and T. gondii decreased
   miR-3074 and miR-127-3p expression, while no change was observed in
   miR-30e-3p expression (A–C). T. cruzi inhibited miR-515-5p expression
   and induced miR512-3p (D, E). T. gondii induced miR-190b-5p (F). All
   values are the mean ± S.D. and correspond to at least three independent
   experiments that were performed in triplicate. Data were normalized in
   terms of the control values and analyzed by Student’s t-test or ANOVA.
   *p ≤ 0.05; **p ≤ 0.01.

Discussion

   Pathogens have evolved strategies to exploit resources from their hosts
   to maximize their own survival, replication, and dissemination. Thus,
   different kinds of pathogens (including parasites) have developed
   sophisticated mechanisms that include hijacking host cellular machinery
   to modulate host gene expression to inhibit defense responses ([126]16,
   [127]32, [128]33). Both of the parasites studied here are able to
   manipulate host gene expression. For instance, during T. cruzi cell and
   tissue invasion, cell reprogramming affects cellular stress responses,
   host metabolism, and a significant number of transcription factors
   ([129]16, [130]34). T. gondii also reprograms host cells, primarily
   targeting cell-specific transcription factors that regulate host
   defenses (i.e., NF-κB, interferon regulatory factor, and JAK/STAT) by
   regulating their intrinsic activities and expression levels ([131]35).
   In addition, certain parasites including T. cruzi and T. gondii can
   alter host miRNA expression to favor both parasite clearance and
   infection ([132]18, [133]19). Moreover, different strains of T. gondii
   can induce specific miRNAs in mice that have been proposed as
   biomarkers for early infection ([134]19, [135]36).

   Mature miRNAs regulate the expression of over 30% of fundamental genes;
   these are involved in key biological processes including development,
   cellular proliferation and differentiation, apoptosis, metabolism, and
   immune response ([136]18, [137]19, [138]37); all of these determine
   infection success or failure. Moreover, epigenetic and genetic defects
   in miRNAs and their processing machinery are a common hallmark of
   infection and diseases that include pregnancy-specific pathologies such
   as preeclampsia ([139]17, [140]38).

   Most of the transcriptomic studies as well as those analyzing miRNA
   profiles have focused on a single type of cell response ([141]34) or on
   tissues or organs in animal models ([142]39, [143]40); no studies have
   focused on human tissues. The present study is the first report on the
   miRNA profile of the human placenta in response to T. cruzi or T.
   gondii infection. We identified 680 and 686 DEMs, respectively, in T.
   cruzi and T. gondii infected samples. T. cruzi modulated only 14 DEMs
   with a minimum of a 1.5-fold change in expression and a 95% probability
   of being differentially expressed ([144] Figure 1A ). In contrast, the
   number of DEMs increased to 42 in T. gondii challenged HPE ([145]
   Figure 2B ). Our results showed that the DEMs identified here are
   related to the regulation of different fundamental cellular processes
   ([146] Figure 2 ) as well as different pathologies ([147] Figure 3 ).
   Moreover, fundamental cellular processes related to placenta
   pathologies and embryonic development are affected by the presence of
   both parasites ([148] Figures 4 –[149] 5 ). It is important to point
   out, that tissue response to infection is relevant during disease
   progression. The presence of the parasites leads to tissue damage as
   well as immune and regulatory/repair responses, which can lead to
   fibrosis and tissue dysfunction as observed in chagasic cardiomyopathy
   ([150]41) or Toxoplasma induced encephalitis in immune-compromised
   individuals ([151]42).

   Three miRNAs, miR-21, miR-146a/b, and miR-210, were overrepresented in
   most of the ontology terms ([152] Table 2 , [153]Supplementary Tables 3
   –[154] 4 ). Previous studies have implicated these miRNAs in immune and
   inflammatory response regulation via macrophage polarization controlled
   through transcription factor regulation in response to signals from the
   microenvironment ([155]43, [156]44). Concordantly, in T. cruzi-infected
   mice, increased miR-21 expression in the heart has been correlated with
   a parasitemia peak at 30 days post-infection ([157]39). In placenta,
   miR-21 has been associated with trophoblast differentiation and
   invasion and miR-21 dysregulation leads to placental pathology
   ([158]45). MiR-146a is a negative feedback regulator in TLR-4 signaling
   that acts by repressing TRAF6 to inhibit NFκB transcription factor
   activation ([159]46, [160]47). In macrophages, TRAF6 mediates the
   induction of the pro-inflammatory cytokine IL-12, which is essential to
   control T. gondii infection ([161]48). TRAF6 activation is also
   required for vacuole-lysosome fusion, a fundamental step during T.
   gondii infection ([162]49). Our results showed that in HPEs, T. gondii
   and T. cruzi infection increased miR-146a expression. Our previous
   studies showed that both parasites modulate placental immune response
   differentially through TLRs and NFκB pathways in HPEs ([163]9, [164]10)
   Interestingly, the inhibition of these pathways increased the DNA loads
   of both parasites in HPEs ([165]10). Increased T. gondii infection in
   placental tissue is also induced by TLR-4 inhibition ([166]9). In
   addition, increased levels of miR-146a have been reported in the brains
   of mice infected with T. gondii, moreover, miR-146a ablation affects
   early parasite burden and improves survival ([167]50). It was
   previously reported that miR-210 is induced by damage associated
   molecular patterns ([168]51). In preeclamptic placentas, miR-210 is
   increased ([169]52); in the present study, miR-210 was increased in HPE
   infected with T. cruzi but not with T. gondii. Expression of miR-210
   can be directly regulated by the specific binding of NF-κB p50 to its
   putative promoter ([170]53). In this context, it is important to
   mention that T. cruzi, but not T. gondii, infection of HPE activates
   both NF-κB signaling pathways ([171]10). Therefore, the increased level
   of miR-210 might be a placental response to signal transduction pathway
   activation.

   In addition, several identified pathways, important, e.g. for
   chondrocyte development, megakaryocyte smooth and muscle cell
   differentiation, hepatotoxicity, and DNA damage response, are neither
   related to infection or with placental tissues ([172] Figure 3 ). This
   can be explained be the fact, that miRNAs target multiple genes, while
   individual genes are targeted by multiple miRNAs. Moreover, the same
   miRNA regulates different genes in different tissues and organs
   ([173]54, [174]55). Here, we chose to validate six miRNAs that were
   associated specifically with parasite infection and/or pregnancy
   related pathologies ([175] Figure 6 ). Deregulation of miR-30e-3p has
   been reported in mice that were experimentally infected with T. gondii
   ([176]40). This miRNA is also related to Chagas cardiomyopathy
   ([177]39) and is upregulated in placentas with intrauterine growth
   restriction ([178]56). Nonetheless, miR-30e-3p expression was
   unaffected by T. cruzi or T. gondii infection in HPE ([179] Figure 6C
   ). Increased miR-3074-5p expression has been described in placentas
   from recurrent miscarriages ([180]57) and in livers from T.
   gondii-infected cats ([181]42). However, miR-3074-5p expression was
   diminished in HPE infected either with T. cruzi or T. gondii ([182]
   Figure 6A ). The differences between our results and the reported data
   might be explained by differences in the studied organs (heart and
   liver versus placenta) and the complexity of the above mentioned
   placental pathologies. MiR-127 is a placenta-specific miRNA codified in
   the C14MC cluster ([183]58) and its levels are decreased in
   placenta-related pathologies such as recurrent miscarriage and
   small-for-gestational age ([184]59, [185]60); the downregulation of
   MiR-127 was also detected in babies infected congenitally with either
   parasite ([186]3, [187]4). Concordantly, our results showed that HPE
   infection with either T. cruzi or T. gondii led to the decrease of this
   miRNA ([188] Figure 6B ). Moreover, a decreased expression of
   miR-127-3p in non-placental tissues has been reported during T. gondii
   infection in mice and cats ([189]40, [190]42, [191]61), but there is no
   report regarding miR-127-3p expression in response to T. cruzi
   infection. Both miR-515-5p and miR-512-3p are placenta-specific miRNAs
   that are codified in the C19MC cluster ([192]62). Decreased miR-515-5p
   expression is related to fetal growth restriction ([193]63) and
   preeclampsia ([194]64). Importantly, this miRNA inhibits human
   trophoblast differentiation by directly repressing the aromatase P450
   (CYP19A1), frizzled 5 (FZD5), and glial cells missing 1 transcription
   factor (GCM1) genes ([195]65). Trophoblast differentiation is part of
   the trophoblast epithelial turnover and it has been proposed that this
   mechanism is part of an antiparasitic placental response against T.
   cruzi infection ([196]11, [197]13, [198]66, [199]67). Therefore, our
   reported decrease of miR-515-5p expression during ex vivo T. cruzi
   infection of HPE ([200] Figure 6D ) might be at least partially
   responsible for the parasite-induced trophoblast differentiation. In
   contrast, miR-512-3p was upregulated in HPE in response to T. cruzi
   infection ([201] Figure 6E ). Interestingly, miR-512-3p confers
   resistance to vesicular stomatitis virus in non-placental recipient
   cells ([202]68) and represses the caspase 8 inhibitor c-FLIP (cellular
   FLICE-like inhibitory protein); it consequently increases caspase 8
   activity ([203]69). Caspase 8 regulates trophoblast differentiation and
   apoptotic cell death and is activated by T. cruzi ([204]66). Therefore,
   miR-512-3p upregulation might also be a protective placental response
   to T. cruzi infection, as it is to viral infection. The upregulation of
   miR-190b in HPE during T. gondii infection was observed in the RNAseq
   analysis ([205] Figure 1B , [206]Table 3 ), then validated by qPCR
   ([207] Figure 6F ). Upregulation of miR-190b promotes cell
   proliferation and migration and reduces cell apoptosis in different
   types of cancer ([208]70, [209]71). Parasites modulate apoptotic
   responses in infected cells to avoid rapid clearance; T. gondii is
   particularly capable of blocking apoptosis by different mechanisms
   ([210]33). In neurons, increased miR-190b expression also increases
   cell viability, suppresses autophagy, and significantly decreases the
   levels of pro-inflammatory TNF-α, IL-6, and IL-1β cytokines ([211]72).
   In this context, we have shown that T. gondii, in contrast to T. cruzi,
   does not induce pro-inflammatory cytokines in HPE ([212]9). Therefore,
   it is postulated that the lack of pro-inflammatory cytokine secretion
   in response to T. gondii in HPE might be related to an increase in
   miR-190b expression and that, together with the modulation of the
   apoptotic pathway, it could allow parasite persistence and infection in
   the placenta.

   In conclusion, the present study provides a comparative analysis of RNA
   sequencing-based miRNA profiles in HPE in response to ex vivo T. cruzi
   and T. gondii infection ([213] Figure 7 ). Our findings provide new
   insight into the capacity of both parasites to modulate host gene
   expression. GO analysis revealed that the predicted targets of the DEMs
   were different cellular processes involved in development and immunity,
   and most of the identified KEGG pathways were related to chronic
   diseases and infection. Considering that the DEMs identified herein
   modulate crucial host cellular targets that participate in determining
   the success of infection, these miRNAs might explain the differences in
   congenital transmission rates. Molecules of the different pathways that
   are regulated by miRNAs and modulated during infection, as well as the
   miRNAs themselves, may be potential targets for the therapeutic control
   of either congenital Chagas disease or toxoplasmosis.

Figure 7.

   [214]Figure 7
   [215]Open in a new tab

   Schematic summary of the comparative analysis of RNA sequencing-based
   miRNA profiles in HPEs in response to ex vivo T. cruzi and T. gondii
   infection. Fourteen, and 42 DEMs were identified in T. cruzi and T.
   gondii infected samples. In T. cruzi infected HPEs, 5 miRNAs were
   downregulated and 9 were upregulated. In T. gondii infected HPEs, 13
   miRNAs were downregulated and 29 were upregulated. In addition, five
   miRNAs that are associated specifically with parasite infection and/or
   pregnancy-related pathologies were validated. GO analysis revealed that
   the predicted targets of the DEMs were different cellular processes
   involved in development and immunity, and most of the identified KEGG
   pathways were related to chronic diseases and infection. Considering
   that the DEMs identified herein modulate crucial host cellular targets
   potentially determining the success of infection, these miRNAs might
   explain the differences in the congenital transmission rates of the two
   parasites. This figure was created using BioRender.com.

Data Availability Statement

   The RNA-seq data reported in the present study have been submitted to
   the NCBI SRA database
   ([216]http://www.ncbi.nlm.nih.gov/bioproject/656620; accession number
   PRJNA656620). All other data supporting the findings can be found in
   the article/[217] Supplementary Material .

Ethics Statement

   The studies involving human participants were reviewed and approved by
   Ethical Committee of the “Servicio de Salud Metropolitano Norte”
   Santiago de Chile, Chile. The patients/participants provided their
   written informed consent to participate in this study.

Author Contributions

   LM and UK conceived of and planned experiments. LM, CC, MR-P, AL, and
   JG-M carried out experiments. JM and HP contributed to the
   interpretation of the results. UK and LM wrote the manuscript. All
   authors contributed to the article and approved the submitted version.

Funding

   UK received a grant from the Network of the European Union, Latin
   America and the Caribbean Countries on Joint Innovation and Research
   Activities (ERANet-LAC; grant number ERANet17/HLH-0142). UK, JM, and CC
   received grants from the National Fund for Scientific and Technological
   Development (FONDECYT; grant numbers 1190341, 1170126, and 3180452,
   respectively).

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgements