RT-qPCR analysis of the effect of different concentrations of LPS on the expression of inflammatory genes in placental explants
In order to investigate the expression of inflammatory factors in the placental explant IAI model induced by LPS, we co-cultured the placental explants with 1 μg/ml LPS and assessed the expression of inflammatory genes at various time points (2h, 6h, 12h, 18h, and 24h) using RT-qPCR. The pro-inflammatory factors TNF-α and IL-1β, along with the anti-inflammatory factors IL-10 and IL-1Ra, were analyzed. As depicted in Figure 1 (A1-D1), compared to the control group, LPS stimulation elicited an inflammatory response in the placental explants, resulting in a significant upregulation of pro-inflammatory factors during the early stages of stimulation (2h, 6h) [TNF-α expression increased by (10.1±2.6) fold and (14.4±5.4) fold compared to the control group, respectively, both p < 0.05; IL-1β expression increased by (31.0±9.9) fold and (36.0±11.4) fold compared to the control group, respectively, both p < 0.05]. However, with prolonged LPS stimulation, the expression of TNF-α and IL-1β exhibited a decreasing trend (6h vs. 24h, TNF-α increased by [(14.4±5.4) fold vs. (4.0±0.5) fold], and IL-1β increased by [(36.0±11.4) fold vs. (9.9±2.4) fold], both p < 0.05). At 24 hours of LPS induction, there was no statistically significant difference in the expression of pro-inflammatory factors when comparing the LPS-induced group to the control group. The expression of the anti-inflammatory factor IL-10 showed no statistically significant difference among the groups as the culture time changed. However, the expression of the anti-inflammatory factor IL-1Ra demonstrated a decreasing trend with prolonged culture time (6h vs. 12h, increased by [(5.1±2.2) fold vs. (1.2±0.2) fold], p < 0.05). Figure 1 (A2-D2) represents the trend lines generated for the respective indices in Figure 1 (A1-D1) using the Graphpad 7.0 software.
Subsequently, we co-cultured the placental explants with 10 μg/ml LPS and examined the expression of inflammatory genes at different time points using RT-qPCR. As depicted in Figure 1 (E1-H1), similar to the inflammatory gene expression observed with 1 μg/ml LPS stimulation, both LPS concentrations induced high expression of inflammatory genes during the early stages of stimulation, and after 18 hours of stimulation, there was a downward trend in the expression of inflammatory genes (p < 0.05). At 24 hours of LPS induction, the expression of inflammatory genes in the LPS-induced group did not show statistically significant differences compared to its own control group (both p > 0.05). Figure 1 (E2-H2) represents the trend lines generated for the respective indices in Figure 1 (E1-H1) using the Graphpad 7.0 software.
ELISA analysis of the effect of LPS on the expression of inflammatory cytokines in the supernatant of placental explant cultures
Based on the aforementioned RT-qPCR results, we proceeded to employ a treatment of 1 μg/ml LPS on placental explants and quantified the levels of inflammatory cytokines in the culture supernatant through the ELISA method. Different time points (12h, 24h, 36h, and 48h) were set to analyze the impact of LPS on the expression of inflammatory cytokines in the placental explants (n=10). As shown in Figure 2 (A1), after co-culturing placental explants with 1 μg/ml LPS, the expression of the pro-inflammatory factor TNF-α exhibited a declining trend with prolonged LPS exposure [(12h vs. 48h, (444.6±287.9) vs. (227.8±126.6) pg/ml; 24h vs. 48h, (433.5±202.9) vs. (227.8±126.6) pg/ml, both p < 0.05)]. On the other hand, the expression of the anti-inflammatory factor IL-10 showed an increasing trend [(12h vs. 24h, (128.1±88.0) vs. (306.2±224.3) pg/ml, with statistical significance, p = 0.031)].[see Figure 2 (B1)]
The ratio of pro-inflammatory to anti-inflammatory factors (TNF-α/IL-10) was used as an indicator of the pro-inflammatory direction. As shown in Figure 2 (C1), with increasing exposure time of placental explants to LPS, the ratio of TNF-α/IL-10 gradually decreased [(12h vs. 36h, respectively (5.3±3.9) vs. (1.8±1.2), p < 0.05; 12h vs. 48h, respectively (5.3±3.9) vs. (1.0±0.8), p < 0.001)]. As shown in Figures 2 (D1), the ratio of IL-10/TNF-α was used as an indicator of the anti-inflammatory direction, and with prolonged exposure time to LPS, the ratio gradually increased [(12h vs. 48h, respectively (0.5±0.2) vs. (1.4±0.3), p < 0.05)]. This indicates that with prolonged exposure of placental explants to LPS, the inflammatory response shifts from a pro-inflammatory direction to an anti-inflammatory direction. Figure 2 (A2-D2) represents the trend lines generated for the respective indices in Figure 2 (A1-D1) using the Graphpad 7.0 software.
To further confirm this hypothesis, we selected another pair of pro-inflammatory factor IL-1β and anti-inflammatory factor IL-1Ra to examine their expression in placental explants co-cultured with 1 μg/ml LPS. As shown in the Figure 2 (E1), with increasing co-culture time with LPS, there was no statistically significant difference observed in the expression of IL-1β among the different time points. However, the expression of the anti-inflammatory factor IL-1Ra showed an upward trend with prolonged culture time [(12h vs. 36h, respectively (969.3±121.0) vs. (1447.9±125.9) pg/ml; and 12h vs. 48h, respectively (969.3±121.0) vs. (1452.4±170.3) pg/ml], with statistically significant differences (both p < 0.05).[(see Figure 2 (F1)].
Using the IL-1β/IL-1Ra ratio as an indicator of the pro-inflammatory response, as shown in Figure 2 (G1), the inflammatory response of placental explants showed a downward trend after 36 hours of co-culture compared to 12 hours, although the difference did not reach statistical significance [(2.0±0.2) vs. (1.5±0.3), p = 0.2785]. Using the IL-1Ra/IL-1β ratio as an indicator of the anti-inflammatory direction, as shown in Figure 2 (H1), with prolonged co-culture time, placental explants exhibited a trend of inflammation inhibition, although the differences did not reach statistical significance [(12h vs. 36h, respectively (0.6±0.1) vs. (1.0±0.2), p = 0.0774; 24h vs. 36h, respectively (0.6±0.1) vs. (1.0 ±0.2), p = 0.0837]. Figure 2 (E2-H2) represents the trend lines generated for the respective indices in Figure 2 (E1-H1) using the Graphpad 7.0 software.
ELISA analysis of the changes in inflammatory trend of placental explants cultured with normal culture medium
In order to demonstrate that placental explants cultured in a complete medium without LPS did not exhibit significant inflammation, we conducted ELISA analysis on the expression of the four inflammatory factors in the culture supernatant of placental explants from four patients at different culture time points (12h, 24h, 36h, and 48h) (n=4). As shown in the Figure S1, there was no increasing trend in the expression of inflammatory factors in placental explants from the same patient with prolonged culture time.
Preliminary investigation of the molecular mechanisms underlying placental endotoxin tolerance
Based on the previous RT-qPCR analysis of gene expression and ELISA analysis of protein expression, we observed that with prolonged co-culture time with LPS, placental explants exhibited a shift in inflammatory expression from a pro-inflammatory response to an anti-inflammatory response. Specifically, during the early stages of co-culture with LPS, a significant pro-inflammatory reaction was evident, while in the later stages of culture, a distinct anti-inflammatory response was observed. The phenomenon of endotoxin tolerance exhibited by placental tissue may play a role in maintaining an anti-inflammatory environment at the maternal-fetal interface and promoting fetal health. However, the exact mechanisms underlying the regulation of this phenomenon remain unclear.
microRNAs (miRNAs) are important cellular regulatory factors in immune responses, but there are few free circulating miRNAs in the extracellular environment, which are mainly packaged within Exosomes and act as a vehicles to transport miRNAs to target tissues or cells [9,10].
We hypothesize that prolonged exposure of placental tissue to LPS stimulation leads to the generation of "signaling molecules" that inhibit the formation of inflammatory factors, primarily immune-regulatory miRNAs. These miRNAs are encapsulated within Pd-Exos and, upon internalization by receptor cells, exert anti-inflammatory effects on target cells, thereby maintaining an anti-inflammatory environment at the maternal-fetal interface. Next, we will further investigate the possibility that this phenomenon is mediated by Pd-Exos and enclosed miRNAs, which may play a critical role in placental endotoxin tolerance at the maternal-fetal interface.
Identification of the two groups of placenta-derived exosomes (Pd-Exos) through transmission electron microscopy (TEM), western blot (WB), and nanoparticle tracking analysis (NTA) techniques
The LPS-induced group and its respective control group were each subjected to the extraction of Pd-Exos from the culture supernatant of four pairs of samples. Following purification, the exosomes were stained with 2% uranyl acetate for observation under transmission electron microscopy (TEM). Both groups displayed typical exosome structures characterized by a double-layered "saucer-shaped" membrane [Figure 3 (A-B)]. Western Blot analysis revealed that the extracted Pd-Exos expressed specific markers of classic exosomes, including the tetraspanin protein CD63 and the endosomal transport protein marker TSG101, while lacking expression of the endoplasmic reticulum-specific molecule calnexin (used as a negative control). Additionally, the placenta-specific marker placental alkaline phosphatase (PLAP) showed positive expression, confirming the origin of these exosomes from placental sources [Figure 3 (C)]. Nanoparticle tracking analysis (NTA) revealed that the diameter of exosomes in the control group was (137.6±9.8) nm, and in the LPS-induced group was (135.9±5.9) nm, both falling within the characteristic diameter range of exosomes (30~150 nm). Collectively, this evidence indicates successful enrichment of Pd-Exos through ultracentrifugation [Figure 3 (D-E)].
Interaction between Pd-Exos and inflammatory cells at the maternal-fetal interface
Human monocytic leukemia cell line THP-1 was induced with 100 ng/ml PMA for 24 hours to differentiate into adherent macrophages, which served as the cellular model for inflammatory cells at the maternal-fetal interface. We used this model to investigate the impact of Pd-Exos on the expression of inflammatory genes in maternal-fetal interface macrophages.
PKH67-labelled exosomes internalize by THP-1 cells
THP-1 cells were selected and cultured in complete medium containing 100 ng/ml PMA for 24 hours to induce their differentiation into macrophages. PKH67-labeled Pd-Exos (green fluorescent) were added to the culture and co-cultured for 6 hours. Immunofluorescence analysis confirmed the internalization of Pd-Exos by THP-1 cells. As shown in Figure 4 (A-D), confocal microscopy imaging demonstrated the internalization of PKH67-labeled green fluorescent Pd-Exos by THP-1 cells.
Impact of Pd-Exos treatment on the expression of inflammatory genes in THP-1 cells
(1) THP-1 treated with Pd-Exos
RT-qPCR analysis was performed to assess the expression of inflammatory genes in THP-1 cells after co-culturing with Pd-Exos for 2 hours and 6 hours. The pro-inflammatory factors TNF-α and IL-10, as well as the anti-inflammatory factors IL-1β and IL-1Ra, were investigated. Comparing with the control group of THP-1 cells, after 2 hours of co-culturing with Pd-Exos, there was a significant upregulation of TNF-α mRNA expression (1.95±0.23-fold). However, there were no statistically significant differences in the expression of IL-10, IL-1β, and IL-1Ra. After 6 hours of co-culturing, Pd-Exos promoted the expression of IL-1β (2.93±0.89-fold, p <0.01) and significantly upregulated the expression of the anti-inflammatory factors IL-10 (1.85±0.20-fold) and IL-1Ra (6.02±3.31-fold). The expression of TNF-α showed no statistically significant difference[see Figure 5 (A1-D1)].The TNF-α/IL-10 ratio, as an indicator of pro-inflammatory direction, showed a decreasing trend after 2 hours and 6 hours of co-culturing Pd-Exos with THP-1 cells, from (1.73±0.18) to (1.03±0.29), although the difference did not reach statistical significance (p =0.066) [Figure 5 (E1)]. On the other hand, the IL-1β/IL-1Ra ratio, as an indicator of pro-inflammatory direction, exhibited a significant decrease after 2 hours and 6 hours of co-culturing, from (1.57±0.19) to (0.70±0.10) (p <0.01)[Figure 5 (F1)]. Our results indicate that Pd-Exos can mildly promote inflammation in THP-1 cells after 2 hours of interaction, but this pro-inflammatory effect gradually weakens with prolonged co-culturing up to 6 hours.
- THP-1 treated with 1-LPS-Pd-Exos
Using the same methodology as described above, we examined the impact of 1-LPS-Pd-Exos on the expression of inflammatory genes in THP-1 cells. Comparing with the control group, after 2 hours of co-culturing 1-LPS-Pd-Exos with THP-1 cells, there was a significant increase in TNF-α (6.00±1.26-fold), IL-10 (1.52±0.16-fold), and IL-1β (1.63±0.23-fold) mRNA expression. However, the expression of the anti-inflammatory factor IL-1Ra was downregulated (0.57±0.09-fold), and all differences were statistically significant (p <0.05). After 6 hours of co-culturing, 1-LPS-Pd-Exos continued to promote the expression of TNF-α (4.63±1.31-fold), IL-10 (2.90±0.23-fold), and IL-1β (6.18±1.51-fold). Interestingly, the expression of IL-1Ra changed (6.65±1.46-fold) from inhibition to significant promotion [see Figure 5 (A2-D2)]. when considering the TNF-α/IL-10 ratio as an indicator of pro-inflammatory direction, 1-LPS-Pd-Exos and THP-1 co-culturing for 2 hours and 6 hours resulted in a decreasing trend in this ratio, from (3.80±0.47) to (1.63±0.44), with statistically significant differences (p <0.01) [Figure 5 (E2)]. Similarly, when considering the IL-1β/IL-1Ra ratio as an indicator of pro-inflammatory direction, 1-LPS-Pd-Exos and THP-1 co-culturing for 2 hours and 6 hours showed a decreasing trend in this ratio, from (3.03±0.32) to (1.02±0.26), with statistically significant differences (p <0.001) [Figure 5 (F2)]. These results indicate that 1-LPS-Pd-Exos can induce a pro-inflammatory response in THP-1 cells after 2 hours of interaction, and this pro-inflammatory effect gradually diminishes with prolonged co-culturing up to 6 hours.
Screening of differentially expressed placenta-derived exosomal miRNAs related to placental endotoxin tolerance
To investigate the expression profiles of differentially expressed miRNAs associated with placental endotoxin tolerance (ET) in Pd-Exos, we selected 4 pairs of Pd-Exos samples for analysis. (Note: Each pair of samples was derived from placental explant cultures of the same pregnant woman under two different culture conditions, namely Pd-Exos, n=4; and 1-LPS-Pd-Exos, n=4). Total RNA was extracted and subjected to miRNA high-throughput sequencing and bioinformatics analysis. The internationally recognized algorithm DESeq2 was employed for differential screening, with the screening criteria set at Log2FC > 1 and p < 0.05. A total of 6 differentially expressed miRNAs were identified. The differentially expressed miRNAs, fold change, and expression levels are shown in Table 1 (Note: FC=fold change).
The miRNA sequencing results revealed 1025 differentially expressed miRNAs in 4 pairs of Pd-Exos samples. Among them, 21 miRNAs were consistently identified in all pairs (Figure 6-A). Hierarchical clustering analysis was performed for the 1025 differentially expressed miRNAs in the 4 pairs of Pd-Exos samples (Figure 6-B). Based on the fold change and expression levels, we selected miR-24-1-5p, which showed the most significant differential expression, for further investigation and subsequent functional validation (Figure 6-C). To elucidate the functions of miRNA-regulated genes in different biological processes, we conducted GO enrichment analysis on the mRNA targets potentially influenced by miRNAs in the Pd-Exos samples. The results indicated that the enriched miRNAs primarily functioned in processes related to protein binding, cytoplasm, nucleoplasm, and cytosol, among others (Figure 6-D).
Prediction of the target genes of miR-24-1-5p
Based on the previous miRNA high-throughput sequencing results, we selected miR-24-1-5p with the highest differential expression for further functional validation. Using the miRDB database, we predicted potential target genes of miR-24-1-5p and found TNFAIP8 (TNF alpha induced protein 8) as a potential target, which is expressed in human placental tissues and located on chromosome 5q23.1. Combining with the literature, we further hypothesize that placental-derived exosomal miR-24-1-5p may inhibit the transcription of TNFAIP8 mRNA, thereby suppressing the activation of the NF-κB inflammatory pathway and reducing the secretion of inflammatory factors TNF-α and IL-1β [13]. (The proposed mechanism of miR-24-1-5p involvement in placental Enodtoxin Tolerance is depicted in Figure 7.)
miR-24-1-5p is highly expressed in 1-LPS-Pd-Exos released from LPS-induced placental explants compared to Pd-Exos from the control group
In order to validate the accuracy of the sequencing results, we additionally selected 5 samples of term, cesarean delivery placental explant tissues from pregnant women without pregnancy complications. Using the same placental explant culture method as described earlier, we cultured the explants with either complete medium or complete medium containing 1μg/ml LPS. After 18 hours of culture, a total of 10 exosome samples were extracted from the culture supernatant of placental explants (including Pd-Exos, n=5, and 1-LPS-Pd-Exos, n=5). RT-qPCR analysis showed that the expression of miR-24-1-5p in 1-LPS-Pd-Exos released from LPS-induced placental explants was (2.74±0.71) fold higher than the control group, and this difference was statistically significant (p < 0.001), which is consistent with the sequencing results.(Figure S2)
miR-24-1-5p directly targets the 3'UTR region of TNFAIP8
Using the dual luciferase reporter vector pmiR-RB-ReportTM, which simultaneously expresses firefly luciferase and Renilla luciferase activities, we constructed the wild-type TNFAIP8 gene 3'Untranslated Region (UTR) recombinant plasmid h-TNFAIP8-WT, containing the binding site of miR-24-1-5p. Additionally, we constructed the 3'UTR mutant plasmid h-TNFAIP8-MUT, with 7 nucleotide mutations at the miR-24-1-5p binding site. Using luciferase activity assay, we observed the effect of miR-24-1-5p on the firefly luciferase activity of h-TNFAIP8-UTR plasmids. The results showed that co-transfection of miR-24-1-5p mimic with h-TNFAIP8-WT significantly downregulated reporter vector luciferase activity (61% downregulation) compared to the miR-24-1-5p NC group, with statistical significance (p =0.0002). In contrast, no significant changes in luciferase activity were observed in the co-transfection group with h-TNFAIP8-MUT (p =0.18), suggesting that miR-24-1-5p likely regulates gene expression through its action on the 3'UTR region of the TNFAIP8 gene. [Figure 8 (A-B)]
Effect of miR-24-1-5p overexpression in recipient cells on target genes and downstream inflammatory cytokine expression
(1)Transfection of miR-24-1-5p into THP-1 cell line (Paracrine mechanism)
RT-qPCR Analysis
To investigate the impact of miR-24-1-5p on inflammation in the maternal-fetal interface, we transfected well-differentiated THP-1 cells with 100 nmol/L of miR-24-1-5p mimic, mimicking the effect of miR-24-1-5p on maternal-fetal interface inflammatory cells. Our results showed that, compared to the negative control (NC) group, transfection with miR-24-1-5p mimic led to a downregulation of the target gene TNFAIP8 in THP-1 cells by approximately 83% ± 2% (corresponding to a decrease of approximately 17%). Furthermore, the expression of downstream inflammatory cytokines TNF-α and IL-1β was downregulated to approximately 54% ± 8% and 47% ± 7%, respectively. All observed differences were statistically significant. [Figure 9 (A1)]
Western blot Analysis
After transfecting THP-1 cell line with miR-24-1-5p mimic/inhibitor for 72 hours, cells were collected, and total proteins were extracted. Western Blot analysis was conducted to assess the protein levels of the target gene TNFAIP8, as well as the expression of downstream inflammatory factors TNF-α and IL-1β. The results demonstrated that compared to the negative control group (NC), transfection with miR-24-1-5p mimic for 72 hours resulted in a reduction in the protein expression of the target gene TNFAIP8, as well as a downward trend in the expression of IL-1β precursor and TNF-α, all with statistical significance (p < 0.05). Conversely, when compared to the negative control group, transfection with miR-24-1-5p inhibitor led to an increase in the expression of IL-1β precursor and TNF-α, both showing statistically significant differences (p < 0.05) (relative grayscale values are indicated in the bar chart, represented as mean ± standard error).[Figure 9 (B1)]
ELISA
After transfecting THP-1 cell line with miR-24-1-5p for 72 hours, cell culture supernatants were collected, and ELISA was performed to measure the expression levels of inflammatory factors TNF-α and IL-1β. The results showed that compared to the negative control group (mimic NC), transfection with miR-24-1-5p mimic for 72 hours led to a decrease in the expression of TNF-α and IL-1β in the cell culture supernatant, with TNF-α levels being [(373.2±52.3) pg/mL vs. (740.9±41.9) pg/mL] and IL-1β levels being [(85.3±24.5) pg/mL vs. (33.8±7.3) pg/mL], both with statistical significance (p < 0.05). Conversely, when compared to the negative control (Inhibitor NC), transfection with miR-24-1-5p inhibitor for 72 hours resulted in an upregulation of TNF-α and IL-1β expression in the cell culture supernatant, with TNF-α levels being [(116.5±17.4) pg/mL vs. (66.1±3.3) pg/mL] and IL-1β levels being [(25.9±5.9) pg/mL vs. (15.9±1.6) pg/mL], both with statistical significance (p < 0.05). [Figure 9 (C1)]
(2)Transfection of miR-24-1-5p into Swan 71 cell line (Autocrine mechanism)
RT-qPCR Analysis
Pd-Exos and their cargo of miRNAs can exert their effects through both paracrine mechanisms on maternal-fetal interface cells (such as macrophages) and autocrine mechanisms on placental tissue itself, including trophoblast cells [14].Subsequently, Pd-Exos utilize homologous surface markers to interact with target tissues/cells and release their cargo, thereby influencing various biological mechanisms, including protein biosynthesis and/or post-transcriptional regulation. To further investigate the inflammatory regulatory role of miR-24-1-5p in the placental tissue itself, we used 100nmol/L of miR-24-1-5p mimic to transfect Swan71 trophoblast cell line, simulating the interaction between Pd-Exos and trophoblast cells and their cargo. The results showed that compared to the NC group, after transfection with miR-24-1-5p mimic, the expression of the target gene TNFAIP8 in Swan71 cells was downregulated to (50.7% ± 8.3%, i.e., a decrease of 49.3%), and the downstream inflammatory factors TNF-α and IL-1β were downregulated to approximately (57.0% ± 5.2%) and (58.7% ± 10.8%), respectively, with all differences being statistically significant (all p < 0.05). [Figure 9 (A2)]
Western blot analysis
After transfecting Swan71 cells with miR-24-1-5p mimic for 72 hours, cells were collected, and total protein was extracted. Western blot analysis was performed to assess the protein levels of the target gene TNFAIP8, as well as the expression of downstream inflammatory factors TNF-α and IL-1β. The results showed that compared to the negative control group (mimic NC), transfection with miR-24-1-5p mimic for 72 hours led to a decrease in the protein expression of the target gene TNFAIP8 and TNF-α, with statistical significance (both p < 0.05). Additionally, the expression of IL-1β precursor showed a decreasing trend, although the difference did not reach statistical significance. On the other hand, compared to the negative control group (Inhibitor NC), transfection with miR-24-1-5p Inhibitor resulted in an upregulation of TNFAIP8 and TNF-α expression, with statistical significance (both p < 0.05). Similarly, the expression of IL-1β precursor showed an increasing trend, but the difference did not reach statistical significance. (Relative grayscale values are indicated in the bar charts, represented as mean ± standard error).[Figure 9 (B2)]
ELISA
After transfecting with miR-24-1-5p mimic for 72 hours, the cell culture supernatant was collected, and the expression of inflammatory factors TNF-α and IL-1β was detected using the ELISA technique. The results showed that compared to the negative control group (mimic NC), transfection with miR-24-1-5p mimic for 72 hours led to a decrease in the expression of TNF-α and IL-1β, with values of TNF-α [(14.5±0.15) pg/mL vs. (21.0±0.87) pg/mL] and IL-1β [(11.4±0.3) pg/mL vs. (13.6±0.8) pg/mL], respectively (both p < 0.05). Compared to the negative control, transfection with miR-24-1-5p Inhibitor for 72 hours resulted in an upregulation of TNF-α expression, with a value of TNF-α [(13.9±0.3) pg/mL vs. (16.6±1.3) pg/mL], while the expression of IL-1β showed an increasing trend, but the difference did not reach statistical significance, with values of [(8.5±0.8) pg/mL vs. (9.4±0.5) pg/mL], p = 0.135. [Figure 9 (C2)]