Transcriptome analysis of porcine endometrium after LPS-induced inflammation: Effects of the PPARγ ligands in vitro
Karol Mierzejewski1, Łukasz Paukszto2, Aleksandra Kurzyńska1, Zuzanna Kunicka1, Jan Paweł Jastrzębski2 , Iwona Bogacka1,*
1 University of Warmia and Mazury in Olsztyn, Faculty of Biology and Biotechnology, Department of Animal Anatomy and Physiology; Oczapowskiego 1a, 10-719 Olsztyn, Poland 2 University of Warmia and Mazury in Olsztyn, Faculty of Biology and Biotechnology, Department of Plant Physiology, Genetics and Biotechnology; Oczapowskiego 1a, 10-719 Olsztyn, Poland
Grant support: This research was supported by the National Science Centre of Poland, Grant No. 2015/17/B/NZ9/03596.
* Correspondence: Iwona Bogacka, [email protected]
Running title: PPARγ ligands affect the endometrial genes profile
Summary sentence: PPARg ligands regulate genes expression in the porcine endometrium during LPS-induced inflammation in vitro
Keywords: endometrium, pig, inflammation, PPARγ, transcriptome, alternative splicing
© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: [email protected]
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Abstract
Female fertility depends greatly on the capacity of the uterus to recognize and eliminate microbial infections, a major reason of inflammation in the endometrium in many species. This study aimed to determine the in vitro effect of PPARγ ligands on the transcriptome genes expression and alternative splicing in the porcine endometrium in the mid-luteal phase during LPS-stimulated inflammation using RNA-seq technology. The endometrial slices were incubated in vitro in the presence of LPS and PPARγ agonists – PGJ2 or pioglitazone and antagonist – T0070907. We identified 222, 3, 4 and 62 differentially expressed genes after
LPS, PGJ2, pioglitazone or T0070907 treatment, respectively. In addition, we detected differentially alternative spliced events: after treatment with LPS – 78, PGJ2 – 60, pioglitazone – 52 or T0070907 – 134. These results should become a basis for further studies explaining the mechanism of PPARg action in the reproductive system in pigs.
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Introduction
Female fertility depends greatly on the capacity of the uterus to recognize and eliminate microbial infections [1]. The initial response to the uterine infection occurs through the innate immunity and the mucosal defense systems [2]. Moreover, the Toll-like receptor (TLR) 4/CD14/MD2 complex signaling pathway can be involved in LPS-induced response in endometrial cells [2,3]. In turn, activated TLRs stimulate the synthesis of pro-inflammatory cytokines, chemokines and prostaglandins (PGs) [4,5].
Abnormalities associated with inflammation in the reproductive system are frequently observed in various species [7–9]. In women it is most manifested with endometriosis, which is a chronic inflammatory disease [8,10]. It has been reported that endometriosis affects oocyte maturation and quality what results in a decreased ability of embryo to implantation in an already altered endometrium by a pro-inflammatory environment [11]. Uterine infections are also a major problem in the swine industry. Several studies have shown that the peri- estrus and perinatal periods are critical in the development of the above processes [1]. The long-term inflammation consequently leads to destructive problems with fertility, which in turn are the most common reasons for culling sows [1]. A high anatomical and physiological similarity of woman and porcine uterus and course of bacterial infection, make the pig a good model for studying in vitro effects of infection on the immune response in the uterus [14].
Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors family and they act as transcription factors regulating e.g. inflammatory and reproductive processes [15,16]. To date, three isoforms of PPAR – α, β/δ and γ – have been described [17]. It was reported that inactivation of PPARγ in mice leads to embryo mortality due to abnormalities in placental vascularization and its severe developmental damage [18–20]. Moreover knockout of PPARg leads to defective trophoblast differentiation, which can affect the placental labyrinth zone development [21]. In recent years, the role of PPARs inmediating the response to inflammation has been of particular interest [5,22,23]. Data from clinical studies, involving endometriosis patients treated with rosiglitazone, imply the possibility of using thiazolidinediones (TZDs) as a treatment for endometriosis [24]. Generally, PPARg promotes the inactivation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) during the inflammatory process [5]. The mechanism of PPARg action can involve direct binding and inactivation of p65 NF-κB or ubiquitination through proteolytic degradation of p65 NF-κB [25]. PPARg can also stimulate the expression of numerous antioxidant enzymes, which in consequence leads to reduction of reactive oxygen species (ROS) concentration [26,27].
Our recent study has shown that PPARγ is engaged in the synthesis of inflammatory mediators in the porcine endometrium during luteal phase of the estrous cycle and early pregnancy [22]. In view of the above, we hypothesized that PPARg regulates the expression of genes involved in the inflammatory process in the porcine endometrium. Therefore, in this study, we examined the in vitro effect of PPARγ ligands (natural or synthetic agonists and antagonist) on the transcriptome profile of the porcine endometrium during LPS-stimulated inflammation on days 10-12 of the estrous cycle. In addition, the effect of PPARγ ligands on alternative splicing events was also analyzed.
Materials and methods
Animals
The study was conducted on crossbred pigs (Large White × Polish Landrace) on days 10 to 12 of the estrous cycle (mid-luteal phase; n = 4) from a private breeding, aged 7–8 months and weighing about 100 kg. The stage of the estrous cycle was selected according to the reports describing that the resistance to infection in the porcine endometrial tissue is least during the luteal phase, when plasma P4 level is high and E2 is low, while it is greater atestrus, when E2 concentration is high and the P4 level is low [6,28]. After slaughter, uterine horns were dissected, and the collected tissues were transported on ice in phosphate‐buffered saline (PBS) with antibiotics: 100 IU/mL penicillin and 100 mg/mL streptomycin (Polfa Tarchomin, Warsaw, Poland). The experimental material was collected in accordance with the national guidelines for animal care, and all procedures were approved by the Animal Ethics Committee of University of Warmia and Mazury in Olsztyn, Poland.
In vitro experiment
The procedure of collecting and incubating porcine endometrial tissue was described previously [29]. In the laboratory, the endometrium was separated from the myometrium, washed with sterile PBS containing antibiotics, and placed on ice in a sterile Petri dish. Tissue slices (100 ± 10 mg, collected in one piece in duplicate from each animal) were incubated in M199 medium (Sigma‐Aldrich, St. Louis, MO, USA) and supplemented with 0.1% BSA fraction V (Roth, Germany) with antibiotics: nystatin (120 IU/mL, Sigma‐ Aldrich) and gentamicin (40 mg/mL; 40 mg/mL, Sigma‐Aldrich). The explants were pre‐ incubated for 2 hours at a rocking platform in a water bath at 37°C in an atmosphere 95% O2 and 5% CO2 and then treated for 24 hours without (control) or with LPS (100 ng/ml, from Escherichia coli). Then medium was removed and explants were incubated in LPS-free medium for 6 hours with PPARγ ligands: 15‐deoxy‐Δ12,14‐prostaglandin J2 (PGJ2; natural agonist; 10 μmol/L, Enzo Life Sciences Int., New York, NY, USA), pioglitazone (P; synthetic agonist; 1 μmol/L, Cayman Chemical Company, Ann Arbor, MI, USA), and T0070907 (T; antagonist; 1 μmol/L, Cayman Chemical Company). Additionally, the control containing dimethyl sulfoxide (DMSO, solvent for the tested PPAR ligands, total volume of 20 µl). The doses of the tested factors were selected based on our preliminary study and the literature data [2,30]. After incubation, tissue explants were washed with PBS and frozen at
RNA isolation, library preparation and sequencing procedure
Total RNA from 20 samples (4 pigs x 5 treatments) was isolated with the “RNeasy Mini Kit” (Qiagen, Germany) according to the manufacturer’s protocol. The purity and concentration of the isolated RNA was measured with the Tecan Infinite M200 plate reader (Tecan Group Ltd., Switzerland). Sample degradation was evaluated in the Agilent Bioanalyzer 2100 (Agilent Technology, USA). Twenty RNA samples with an RNA Integrity Number (RIN) >7 were selected to future analysis. The libraries were performed by the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. After RNA fragmentation, poly(dT) oligonucleotides were applied to transcribe RNA into cDNA. Next, the cDNA was processed to 3′ tail adenylation and adapter ligation. The reverse transcription during the library preparation was strand-specific. Finally, pooled libraries were followed to sequencing procedure. The Illumina NovaSeq 6000 platform have produced 2×150 bp paired-end reads with 300 bp insert size.
Quality controls and mapping to genome
The raw paired-end reads were quality controlled using the FastQC and Trimmomatic, and sequences were processed to (a) minimum length > 120 bp, (b) PHRED score > 20, (c) cropped to equal length. High-quality trimmed reads were aligned to the Sus_scrofa 11.1 genome assembly with reference ENSEMBL annotation (98 version) using Spliced Transcripts Alignment to a Reference (STAR) aligner. Mapping results were indexed and sorted by coordinates. Gene expression values (read counts) were reconstructed by
Differentially expressed genes
The analysis of differentially expressed genes (DEGs) and corresponding false discovery rate (FDR < 0.05) were determined using DESeq2. Gene expression patterns changes in the porcine endometrium on days 10-12 of the estrous cycle treated in vitro with LPS and/or PPAR ligands, agonists: 15d-prostaglandin J2 (PGJ2) or pioglitazone (P) and antagonist T0070907 (T), were performed by high-throughput transcriptome sequencing. The transcriptomic effects of LPS administration and PPAR ligands were examined in the four comparisons: LPS vs. untreated, PGJ2 vs. LPS, P vs. LPS and T vs. LPS. Additionally, fragments per kilobase of transcript per million mapped reads (FPKM) were calculated as a normalized expression measure, which depend on the sequencing depth and length of genomic features. The enrichment main biological processes and metabolic pathways in DEGs were identified by enrichGO, enrichKEGG methods implemented in ontology-based clusterProfiler R package [31,32]. For the functional enrichment analysis, the parameters (organism, pig; ont, CC, MF, or BP; P-adjust value cut-off, 0.05; P-adjust method, BH) were used as the cut-off criteria.
The STRING functional protein association networks v. 11.0 and PANTHER databases were used for the functional analysis based on the Homo sapiens reference applying FDR ≤ 0.05 correction and default parameters.
Differentially alternative spliced genes
Differences in alternative splicing were predicted by a super-fast pipeline for alternative splicing analysis (SUPPA v.2) [33]. Trimmed equal length (90 bp) paired-end reads wereused to calculate percent of splicing inclusion (PSI) for all alternative splicing (AS) events. Reads were remapped to the reference transcriptome using Salmon software. Differential alternative splicing (DAS) events for each of four comparisons (LPS vs. untreated, PGJ2 vs. LPS, P vs. LPS and T vs. LPS) were statistically tested (FDR < 0.05). Splicing events with ΔPSI > 0.1 were classified as significant. Alternative events were divided to seven types according SUPPA software: alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE), retention intron (RI), skipping exon (SE), alternative first (AF) and alternative last (AL). AS events were visualized in the maser package and rmats2sashimiplot.py scripts.
Real-time PCR validation
Differentially expressed genes were validated by Real-time PCR with the use of the AriaMx Real-time PCR System (Agilent Technology, USA). Primer sequences (S5 Table) for reference and target genes (CCL4, CCL8, IL-1b, OXTR, CSF3, ACKR1) were developed using Primer Express Software 3 (Applied Biosystems, USA). PCR reaction mixtures with a final volume of 25 ml consisted of cDNA (4 ng), 300 mM of the primers, 12.5 ml of the Power SYBR Green PCR Master Mix (Applied Biosystems, USA), and RNase-free water. The Real- time PCR reaction conditions were as follows: one cycle at 48oC for 15 minutes, followed by one cycle at 95oC for 10 minutes, 40 cycles at 95oC for 15 seconds, and one cycle at 60oC for 1 minute. The levels of the tested gene expression were calculated using standard curves, which were prepared by a serial dilution of a known amount of total RNA. Constitutively expressed ACTB and GAPDH genes were adopted as the reference genes and their geometrical means of the expression levels were applied for the analysis. The results of Real- time PCR were statistically processed in Statistica software (Statsoft Inc. Tulsa, USA) with
Results
Statistics of RNA sequencing
The overall statistics of RNA sequencing data were constructed for 20 cDNA libraries, including four samples incubated without LPS (untreated), four with LPS, four with 15d- prostaglandin J2 (PPARγ natural agonist; PGJ2), four with pioglitazone (PPARγ synthetic agonist; P) and four with T0070907 (PPARγ antagonist; T). The sequencing produced 1 118 471 092 raw paired-end reads, on average 55.92 million per sample. The filtered reads were mapped to the Ss11.1.98 version of the pig genome with unique mapped average rate 94.39%. The analysis of the distribution of mapped reads to genes structures indicated that nearly 50.6% of read pairs mapped to coding sequences, 7.2% mapped to introns, 27.9% aligned to untranslated regions and the remaining 14.3% mapped to intergenic regions. Summary of the sequencing depth and mapping reads were presented in Supplemental Table S1. The volcano plots depicted an overview of changes in the endometrial genes expression in the LPS-treated vs. untreated group, as well as in the tested ligands (PGJ2 or P or T) vs. LPS-treated group (Fig. 1). Visualization of the differentially expressed genes (DEGs) was presented on Fig. 2. The volcano plots depicted the difference in the percent of splicing inclusion (ΔPSI) values in each alternative splicing (AS) event in the LPS-treated vs. untreated group, as well as in the tested ligands (PGJ2 or P or T) vs. LPS-treated group were presented on Fig. 3.
Based on the analysis of DEGs (FDR < 0.05%), the total number of 222 protein-coding genes, 6 lncRNAs and 4 other transcript active regions (TARs) were found in the LPS-treated vs. untreated group comparison. This analysis identified 109 overexpressed and 113 underexpressed DEGs. The 194 DEGs were assigned to functional ontology annotations. Gene ontology (GO) annotations for biological processes (BP) encompassed 104 terms. The nine ontology terms were enriched to molecular function (MF) category and the four annotations were assigned to the cellular components (CC) category (Fig. 4). All detailed DEGs and GO results were described in Supplemental Table S2 and S3, respectively. The endometrial response to LPS administration indicated 11 genes (NFKBIA, TGFB1, TNFAIP3, AMCF-II, CXCL2, IL-6, LBP, ACP5, IRF8, WNT5A, SLC11A1) which were directly engaged in the response to lipopolysaccharide. The above DEGs, and additionally other five genes (PCK1, BMP2, SLPI, NLRC5, ISG15), encode proteins which are involved in the response to bacteria. The LPS treatment of the endometrial tissue induced changes in the expression of genes regulating defense (36 DEGs) and inflammatory (23 DEGs) response.
Arouse toxicity within the endometrium involved production of cytokines, which triggered transcription machinery regulating inflammatory effects. Within genes regulating response to cytokines, 21 DEGs were upregulated (NFKBIA, CXCL12, NLRC5, MX2, WNT5A, IRF8, CXCL2, ISG15,
IL-6, MSC, CSF2, CSF3, etc.) while 10 DEGs were downregulated (CCL8, CTH, SPOCK2, ALOX15, IL6R, GAS6, etc.). Moreover, 7 DEGs (CSF2, LDLR, LBP, VSIG4, WNT5A,
TREM2, SLC11A1) were involved in macrophages activation, whereas 18 DEGs (TGFB1, CTH, PLG, SFRP2, TBX3, FOSL1, IHH, RAMP1, IL6R, LDLR, GPR4, etc.) were engaged in
vasculature development. The 21 DEGs were assigned to four significant signaling pathways (KEGG). Among them, NFKBIA, TNFAIP3, MMP9, AMCF-II, CXCL2, MMP3, PTGS2, IL-6
and CSF2, were encompassed the TNF-signaling pathway while TGFB1, PIGR, IL-6,
Differentially expressed genes after PPARγ agonists treatment
This part of the study determined the influence of two PPARγ agonists on genes expression profile in the LPS-treated endometrium. The incubation of the tissue with PGJ2 or pioglitazone upregulated the expression of three (SELE, IL-1b, CCL8) and four (SELE, IL-1b, MUC13, MEOX1) genes, respectively, in comparison with the LPS-treated group (Table 1). According to the GO analysis, these DEGs were engaged in e.g. granulocyte chemotaxis or neutrophil migration (Fig. 4). The altered expression of SELE and IL-1b, involved in the TNF-signaling pathway (KEGG:04668, Supplemental Fig. S1), was noted in case of both groups treated with the agonists. All detailed DEGs and GO results were described in Supplemental Table S2 and S3, respectively.
Differentially expressed genes after PPARγ antagonist treatment
This part of the study was designed to determine the effect of PPARγ antagonist on genes expression profile in the LPS-treated endometrium. A total of 62 DEGs, with adjusted p- value < 0.05, displayed altered expression levels. Among them, 30 genes were upregulated while 32 were downregulated. A functional clustering analysis revealed that 52 DEGs were annotated to 18 BP terms and one to the MF term in the GO analysis as well as 4 signaling pathways in KEGG database. These DEGs were engaged in e.g. inflammatory response, cellular response to interleukin-1, cell migration and granulocyte chemotaxis (Fig. 4). Genes involved in cellular response to interleukin-1 (CCL3L1, CCL4, HPS5, CCL8) and inflammatory response (THBS1, IDO1, LBP, ACKR1, IL-1b, etc.) were mostly overexpressed. In addition, four upregulated genes (CCL3L1, LBP, CCL4, IL-1b) wereimplicated in the TLR-signaling pathway (KEGG:04620, Supplemental Fig. S2) and Salmonella infection (KEGG:05132). Only THBS1 and PTGIS, immune-related genes, were downregulated. The top 15 up- and downregulated DEGs are presented in Table 2. All detailed DEGs and GO results were described in Supplemental Table S2 and S3, respectively.
Differentially alternative spliced genes after LPS treatmentdescribed in Supplemental Table S4. Gene enrichment analysis revealed that arachidonic acid metabolism signaling pathway was regulated by alternative splicing machinery. There were three (PTGES, PTGS2, PLAAT3) differentially alternative spliced genes involved in this signaling pathway. The prostaglandin E synthase (PTGES; ΔPSI=-0.10; AF) and
prostaglandin-endoperoxide synthase 2 (PTGS2; ΔPSI=-0.28; AF) indicated a decrease of inclusion level of the AF within both genes. Moreover, SUPPA results disclosed that more transcripts of phospholipase A and acyltransferase 3 (PLAAT3; ΔPSI=0.25; AF) were expressed with alternative form of promoter/first exon.
Differentially alternative spliced genes after PPARγ agonists treatment
The study revealed 60 and 52 DASEs in the endometrial tissue treated with PGJ2 and pioglitazone, respectively. Summarizing, these changes in alternative exon inclusion occupied 68 protein coding genes. The natural agonist, PGJ2 (ΔPSI>0.1), participated in 24splicing events with a higher inclusion level. In the other site, 36 DASEs tended to be highly included in the LPS-treated samples (ΔPSI<-0.1). The agonists, pioglitazone and PGJ2, revealed the same 26 DASEs with a higher percentage of inclusion. The thirteen of those events share the common tendency of PSI values in comparison with both the agonists- and LPS-treated groups. The DAS genes from both comparisons did not enrich to any significant GO and KEGG annotations. However, there were certain important DAS genes, which play a
crucial role in the female reproductive system. Our findings revealed that the alternative AF event of PTGS2 (differed in the LPS-treated vs. untreated group comparison) also changed
after the incubation of the endometrial tissue with pioglitazone. Firstly, the PSI within PTGS2 transcripts decreased (ΔPSI=-0.28; AF) in the LPS-treated group in relation to the untreated, and next increased (ΔPSI=0.18; AF) after pioglitazone treatment (Fig. 5A). All detailed DAS results were described in Supplemental Table S4.
Differentially alternative spliced genes after PPARγ antagonists treatment
The present results showed 106 DAS genes and one lncRNA which occupied 134 DASEs, including 4 – A3, 4 – A5, 39 – AF, 4 – AL, 2 – MX, 19 – RI and 62 – SE. The RI of
ENSSSCG00000045200 (lncRNA) was a part of regulatory machinery engaged in the response to PPARγ antagonist. The highest alternative splicing ratio changes were described
in SEC24B (ΔPSI=0.75; SE), whereas the smallest inclusion level was detected in range of
EIF4E3 (ΔPSI= -0.69; AL). It is worth mentioning that EIF4E3 possessed the second DAS
event (ΔPSI= 0.69; SE) in the middle of gene structure. An enrichment analysis revealed that
DAS genes, linked with PPARγ antagonist influence, were engaged only in binding function (75 DAS genes) and were components of transcription factor complex (9 DAS) and intercellular spaces (75 DAS). The PPARγ antagonist affected the promoter exon (ΔPSI=0.32; AF) of PTGS2, similarly to PPARγ agonist (pioglitazone) influence. Our
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findings highlighted also a higher percentage of inclusion within the AF of PTGIS (ΔPSI=0.59; AF) implicated in the synthesis of prostaglandins. The INSR gene, next to PTGS2, was another steroidogenic regulator tended to be highly included (ΔPSI=0.21; SE)
after PPARγ antagonist treatment (Fig. 5B). Three differentially spliced sites, two skippingexon and one alternative 3’ splicing events, were identified in ACADM (ΔPSI=0.48; SE), SCD (ΔPSI=0.30; SE and ΔPSI=0.25; A3SS), genes engaged in the PPAR-signaling pathway. In addition to the PTGIS, PTGS2, mentioned above, three other – HIF1A, ARNT, ADAM19 –
indicated modification in splicing. The HIF1A (ΔPSI=-0.19; AF), ARNT (ΔPSI=-0.40 andΔPSI=-0.19; both AF) harbored the higher percentage of the AF usage in the LPS- vs. PPARγ antagonist-treated group. All detailed DAS results were described in Supplemental Table S4.
Protein-protein interactions
A protein interaction network analysis was performed by submitting DEGs to the STRING software (v11.0). The analysis demonstrated a strong interaction network (Fig. 6) encompassed a total of 210 proteins (interaction score at least 0.7), which were clustered to a specified MCL inflation parameter (MCL = 3). Proteins not connected with any others were excluded.
According to the MCL clustering, 210 nodes were demonstrated as different color circles. Most of all analyzed proteins were mutual interactive or interactive with other proteins in a complex protein-protein interaction network. Among them, 192 interactions were shown in connection with co-expression, co-occurrence according to an information on interactions between those proteins in different databases. The line shape indicates the predicted mode of an action. The most complex nodes of this interaction network were related to response to stimulus, cytokine activity, receptor ligand activity and cytokine- cytokine receptor interaction.
Real-time PCR validation
To validate the obtained RNA-Seq results, six DEGs (CCL4, CCL8, IL-1b, OXTR, CSF3) in the PPARg- vs. LPS-treated groups and three DEGs (CCL8, ACKR1, CSF3) in the LPS- vs. untreated group were selected for Real-time PCR. The Real-time PCR expression patterns of the tested DEGs were in agreement with RNA-Seq results (Supplemental Fig. 3 and 4).
Discussion
During the past years, a lot of evidence highlighted the importance of inflammation in the development of different pathologies including those occurring in the reproductive system such as endometriosis, endometritis, recurrent implantation failure [11,34]. A growing number of researchers have discovered various signaling pathways that are associated with the initiation and progression of inflammation [35]. Referring to the current problem, we focused on possible mechanisms regulating inflammation through PPARγ in the porcine endometrium. This report provides a comprehensive transcriptome analysis concerning the in vitro impact of PPARγ ligands on the LPS-induced inflammation in the porcine endometrium during the mid-luteal phase of the estrous cycle. It is also the first study to indicate the effect This study has demonstrated the effects of PPARγ ligands on alternative splicing events in the LPS-treated tissue.
The present study revealed 194 genes that differentially express in the porcine endometrium after LPS administration to induce inflammation. We identified 109 genes overexpressed and 113 underexpressed, and gene ontology (GO) annotations for biological processes (BP) encompassed 104 terms. The nine ontology terms were assigned to molecular function (MF) category and the four annotations were assigned to the cellular components (CC) category. Only the most interesting genes and signore briefly discussed below.
We demonstrated that LPS influenced the expression of genes involved in the Tumor necrosis factor (TNF)-signaling pathway in the porcine endometrium during the mid-luteal phase of the estrous cycle – downregulates MMP9 expression while upregulates – NFKBIA, TNFAIP3, AMCF-II, CXCL2, MMP3, PTGS2, IL-6, CSF2. Furthermore PTGS2 and IL-6 areparticularly interesting in a context of the endometrial inflammation. The PTGS2 activation regulates prostaglandin E2 (PGE2) synthesis, which is involved in the regulation of a proper course of the estrous cycle, early pregnancy and the maternal recognition of pregnancy [36– 38]. Apart from regulating reproductive functions, PGs play a crucial role in inflammation, what was confirmed e.g. in knockout mouse studies [39].
Interleukin-6 is another pro-inflammatory mediator whose endometrial mRNA expression increased after LPS stimulation in the present study. The presence of IL-6 has been reported in the endometrial tissue od various species, including pigs [22,40]. A markedly higher concentration of IL-6 was noted in peritoneal fluid in women with endometriosis [41], as well as in the mouse endometrium treated with LPS [42]. Moreover, Paukszto and colleagues [43] demonstrated that LPS (Salmonella Enteritis subtype) induces transcriptomic changes in the porcine endometrium that might be associated with endometritis, immune response, angiogenesis and the tissue development. In summary, the above shortly presented results, indicate that the use of LPS in our studies induces inflammatory process in the proposed experimental model.
The present study were undertaken to determine the effect of PPARγ ligands on the LPS-induced inflammation in the porcine endometrium. Surprisingly, a great impact of PPARγ antagonist (T0070907), whereas definitely less influence of both tested agonists (PGJ2 and pioglitazone), on the endometrial transcriptome profile was noted. This study revealed 62 genes that were differentially regulated after T0070907 treatment – 30 were upregulated while 32 were downregulated. In the group of differentially expressed high-impact genes, 10 genes (THBS1, IDO1, PTGIS, CCL3L1, LBP, CCL4, ACKR1, HPS5, IL-1b,
CCL8) are involved in the inflammatory response. In particular, these genes regulate response to IL-1 (PTGIS, CCL3L1, CCL4, HPS5, CCL8), neutrophil (ITGA1, CCL3L1, CCL4, IL-1b,CCL8) and eosinophil (CCL3L1, CCL4, CCL8) migration, Toll-like receptors signaling pathway (CCL3L1, LBP, CCL4, IL-1b). The present study revealed onlythree (SELE, IL-1b, CCL8) and four (SELE, MUC13, MEOX1, IL-1b) genes that were upregulated after PGJ2 (natural agonist) or pioglitazone (synthetic agonist) treatment, respectively. They are mostly involved in the TNF-signaling pathway. Only the most interesting genes, involved in inflammatory process and reproductive system functioning, are discussed below.
Toll-like receptors (TLRs) belong to a class of proteins that play an essential role in the innate immune system. Different types of TLRs are expressed on antigen presenting cells (APC) but TLR4 is mainly of particular interest in the endometrial tissue of different species, including the pig [3,44–46].
It is known that LPS binds to LPS-binding protein (LBP, acute- phase protein), initiates intracellular cascades through TLR4 activation stimulates innate immunity by inducing the synthesis e.g. IL-1b (pro-inflammatory cytokine) and chemotactic molecules like CCL3L1 and CCL4. It is worthy to underline that such relation was observed in our study after the treatment with PPARg antagonist. Based on these above, we could suggest that blocking of the PPARγ activity in vitro in the LPS-treated tissue deepens inflammation.
Based on the above results concerning pro-inflammatory impact of the antagonist, we would expect that the activation of PPARg by agonists should have an opposite effect. Surprisingly, we have demonstrated that PGJ2 or pioglitazone, did not change transcript level of LBP, CCL3L1 and CCL4 but upregulated the expression of IL-1b. In our previous work, we observed an increased expression of IL‐1β in the porcine endometrium treated with rosiglitazone during the luteal phase of the estrous cycle, but the experiment was performedin physiological stage [22]. In the current research, both PPARγ ligands also stimulated the endometrial expression of IL-1b during the LPS-induced inflammation.
Our observation is in a certain degree contradictory to the available results describing an association between PPARγ and inflammation in other experimental models. For instance, PGJ2 diminished the expression of IL-1β in the LPS-treated synovial fibroblasts of the rat [47], as well as the expression of TLR4 and IL-8 in the human HT-29 intestinal epithelial cells [48]. Moreover, rosiglitazone diminished LBP expression in the mice adipocytes [49]. The above discrepancy could be associated with different species, tissue/cell type, experimental protocol (incubation time and dose of the treatments) and various receptivity of the tested tissues on PPARg ligands. Furthermore, is also intriguing and often reported the fact that agonist and antagonist exerted a similar stimulatory effect on the endometrial expression of IL-1b during the LPS- induced inflammation. It can be explained by involvement of PPARg-independent activation of intracellular metabolic pathways which could modify the synthesis of IL-1b [50]. Moreover, certain molecules, like PGC1α, are capable of ligand-independent binding and activation of PPARγ [51]. A high expression of PGC1α was noted in the human endometrium during the mid-luteal phase of the menstrual cycle [52]. Taking into consideration the fact that the expression of Il-1b is high during the mid-luteal phase in physiological conditions [53], we can assume that blocking the receptor by the antagonist can trigger PPARg- independent pathway which led to an increased expression of IL-1b. There is also an evidence that some other molecules, like PTGS2 or MMP9, could modify signaling pathways and regulate the activity of the entire cytokine families [54,55]. Moreover, it should be pointed out that the activity of PPARγ is affected by phosphorylation process in a ligand- dependent or -independent manner [56]. The effects of phosphorylation depend on the cellular origin and physiological status, receptor subtype and residue metabolized which can influence PPAR activation, including ligand affinity, DNA binding, coactivator recruitmentand proteasomal degradation [56,57]. Therefore further research is required to understanding the intracellular mechanisms of the tested factors.
The current study revealed a relation between PPARg and the expression of colony stimulating factor (CSF3) in the LPS-treated endometrium. It should be highlighted that majority of available literature concerning the role of this factor in the reproductive system comes from the last few years. Specific receptors for CSF3 are present in different immune and reproductive system cells, including placenta, trophoblastic, fetomaternal interface or
granulosa luteinized cells [58]. It has been reported that CSF3 regulates the embryo[60,61] and it used clinically in patient with implantation failure [62].There is only one report demonstrating a relation between PPARg and the expression of CSF3 [63]. Scirpo et al. [63] demonstrated an inhibitory effect of rosiglitazone or pioglitazone on the expression of CSF3 was observed in the LPS-stimulated mouse cholangiocytes. The above results could be partially consistent with ours, since the exposure of the endometrial slices to the PPARg antagonist, T0070907, significantly increased CSF3 expression. However, it should be pointed out that any changes in the CSF3 expression were noted after treatment with PPARg agonists. Thus, the next studies are needed to explain the specific role of the CSF3 in the regulation of the reproductive functions.
The current study provided a new information regarding alternatively spliced genes in the porcine endometrium treated with LPS and PPARγ ligands. We have shown that 218 genes differed in the alternative splicing events in the LPS-treated group in comparison with the control. The GO analysis revealed that these genes modulate intracellular transport, play a crucial role in GTPase activator activity and are cellular components of endoplasmicreticulum exit site. Moreover, the treatment with PPARγ agonists resulted in 227 (for PGJ2) and 270 (for pioglitazone) DAS events, which were located in 343 genes. In turn, the PPARγ antagonist (T0070907) was engaged in occurrence of 384 DAS events. The GO analysis concerning PPARγ ligands, revealed genes engaged in endopeptidase activity, RNA- dependent ATPase activity and transferase activity. Interesting results should be considered those regarding alternative splicing events occurring within genes coding prostaglandin synthases and insulin receptor (INSR). At present, very little is known about the role of INSR in the endometrium. There is evidence that INSR expression changes in the human endometrium during different phases of the menstrual cycle and the highest level was noted in the luteal phase [64]. In addition, it is proposed that signaling by INSR significantly contributes to cell growth and survival in the endometrial carcinoma [65]. However, deeper research is required to understanding the importance of the obtained alternative splicing genes.
Conclusions
This study provided a detailed description of the in vitro effect of PPARγ ligands on the transcriptome profile in the LPS-treated porcine endometrium during the mid-luteal phase of the estrous cycle. A great influence of PPARγ antagonist (T0070907), whereas certainly less impact of both tested agonists (PGJ2 and pioglitazone), on the endometrial transcriptome profile was noted. Generally, PPARγ antagonist displayed pro-inflammatory properties in the proposed experimental model. The results revealed the involvement of PPARγ ligands in the regulation of many intracellular pathways including inflammatory response, cellular response to interleukin-1, cell migration and granulocyte chemotaxis. Moreover, PPARγ ligands exerted an effect on alternative splicing genes which play a crucial role in the reproductive system, as well as in the immune response. These results should become a basis for further
Acknowledgments
We would like to thank to Anna Szydłowska (MSc), Jakub Adamowicz (MSc), Monika Golubska (MSc), Wiktoria Rosińska and Małgorzata Mierzejewska for technical assistance during performing the experiments and Karol G. Makowczenko (MSc) for bioinformatic support.
Author Contributions
Conceptualization: K.M., A.K., I.B.; Data curation: L.P. and J.P.J.; Formal analysis: K.M., L.P., J.P.J.; Funding acquisition: I.B.; Investigation: K.M., Z.K., A.K.; Methodology: K.M.,
I.B.; Project administration: I.B.; Resources: K.M., Z.K., A.K.; Software: L.P., J.P.J.; Supervision: I.B.; Validation: K.M., Z.K.; Visualization: L.P., J.P.J. K.M.; Writing – original draft: K.M.; Writing – review and editing: K.M., I.B., L.P. All authors have read and agreed to the published version of the manuscript.
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