Heat-killed Lactobacillus rhamnosus GG Modulates Urocortin and Cytokine Release in Primary Trophoblast Cells

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Collection of placenta
  • 2.2. Primary trophoblast cell culture
  • 2.3. L. rhamnosus strain GG (LGG)
  • 2.4. Lipopolysaccharide (LPS) treatment
  • 2.5. Hormone and cytokine assays
  • 2.6. RNA extraction and quantitative RT-PCR
  • 2.7. Statistical analysis
• 3. Results
  • 3.1. Hormonal secretion after LGG exposure
  • 3.2. LGG-induced secretion of IL-4, IL-10 and Ucn in human trophoblast primary cells
  • 3.3. LGG reverses LPS-induced TNF-α secretion in human trophoblast primary cells
• 4. Discussion
• Acknowledgements
• References
• Copyright

Abstract 

A number of studies are showing that probiotic treatment induces an anti-inflammatory state. Intrauterine infection can lead to preterm delivery by modulating immune function and efforts to prevent this condition are ongoing nowadays. Lactobacillus rhamnosus GG (LGG) is a probiotic known to ameliorate inflammation by increasing local anti-inflammatory mediators in urinary and gastrointestinal tracts. The present study then analyzed the effect of heat-killed LGG over β-hCG, progesterone, interleukins (IL) 4 and 10, tumor necrosis factor-α (TNF-α), corticotropin releasing hormone (CRH) and urocortin (Ucn) release by primary trophoblast cells. Normal human term placentas (n = 6) were collected and purified trophoblast cells were incubated in the presence of LGG, lipopolysaccharide (LPS) or either LGG + LPS during 3 h, after which the target substances were quantified by ELISA and real-time PCR. LGG did not affect β-hCG, progesterone, or CRH secretion. Conversely, LGG increased IL-4 protein and mRNA expression (P < 0.05) while IL-10 and Ucn secretion were increased in a dose dependent manner and the highest dose of LGG increased significantly IL-10 mRNA (P < 0.05). LGG did not alter TNF-α, while LPS exposure increased TNF-α protein (P < 0.001) and mRNA expression (P < 0.01). Conversely, LGG treatment reversed LPS-induced TNF-α release at both protein (P < 0.01) and mRNA levels (P < 0.05) in a dose dependent fashion. In conclusion, LGG stimulates IL-4, IL-10 and Ucn expression and reverses LPS-induced TNF-α release from trophoblast cells, with no change in β-hCG or progesterone release, suggesting that this probiotic may play a role as an immunomodulatory agent in human placenta without altering basic trophoblast functions.

Keywords: Placenta, Lactobacilli, Preterm delivery, Urocortin, Cytokines

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1. Introduction 

Probiotics are live microorganisms that promote host immunomodulation by colonizing and protecting tissues against microbial infection [1]. They act on both innate and adaptive immunity by modifying cytokine production of different cell populations [2] and are effective in colonizing the vagina and curing women with bacterial vaginosis, or at least preventing its recurrence [3]. Low concentrations or even absence of vaginal lactobacilli are correlated to bacterial vaginosis, a condition that may lead to intrauterine infection and is associated with a 40% increased risk of preterm delivery [4]. Intrauterine infection activates the innate immune system, which prematurely initiates the parturition mechanisms through the production of cytokines and chemokines at the reproductive tissues [5], [6]. Most of the cytokines are expressed in the placenta and associated membranes [7] and the anti-inflammatory cytokines, interleukins (IL) 4 and 10 are considered to have a protective role during pregnancy [8]. Tumor necrosis factor-α (TNF-α), conversely, synergizes with oxytocin by increasing prostaglandin E2 (PGE2) synthesis via cyclooxygenase-2 in the myometrium and thereby promotes myometrial contractility, leading to term or preterm labor [5], [6], [7].

The peptides of the corticotropin releasing hormone (CRH) family have also been implicated in inflammatory processes. CRH and Urocortin (Ucn) are expressed in the placenta and fetal membranes [9], [10] and are involved in the mechanisms leading to preterm delivery. CRH displays pro-inflammatory effects in trophoblast cells by increasing lipopolysaccharide (LPS) induced TNF-α and IL-8 release [11]. Ucn, in turn, stimulates IL-4 and IL-10 secretion and reverses LPS-induced TNF-α release from trophoblast cells via CRH-R2 receptors [12], suggesting a possible role for Ucn as an anti-inflammatory agent in human trophoblasts [13], [14].

Lactobacillus rhamnosus GG (LGG) is a lactobacilli strain frequently integrated in the elaboration of fermented milks. In rats, LGG decreases LPS-induced systemic inflammation [15], [16], [17] and LGG effects may be mediated by two secreted proteins, p75 and p40; they promote epithelial cell growth, inhibit apoptosis and protect epithelial barriers from hydrogen peroxide damage [18]. The aim of the present study was to investigate whether LGG treatment would affect the production and release of hormones (β-hCG and progesterone) and immunomodulatory factors (IL-4, IL-10, CRH and Ucn) from cultured human trophoblast cells. In addition, we investigated if LGG would be able to attenuate or even reverse lipopolysaccharide-induced TNF-α expression in a primary trophoblast cell culture model.

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2. Materials and methods 

2.1. Collection of placenta 

Normal term placentas (>37 weeks of gestation) were collected after uncomplicated elective caesarean delivery in absence of labor (n = 6), in the Division of Obstetrics and Gynecology of the University of Siena (Siena, Italy). Approval from the Local Human Investigation Committee was obtained and all participants signed an informed written consent before being included in the study.

2.2. Primary trophoblast cell culture 

Syncytiotrophoblast was prepared using a modification of the method of Kliman et al [19]. After the removal of the decidual tissue and blood clots, approximately 60 g of placental tissue were digested with 0.125% trypsin (Sigma–Aldrich, Steinheim, Germany) and 0.02% deoxyribonuclease-I (Sigma–Aldrich) in phenol red free DMEM (Invitrogen, Paisley, UK), during three times for 30 min at 37 °C. The dispersed trophoblast cells were filtered through a 200-μm-pore-size nylon gauze and were loaded onto a discontinuous Percoll gradient of 5–75% (Sigma–Aldrich) followed by centrifugation at 2500g for 20 min. Cells between the density markers (Amersham Biosciences, Uppsala, Sweden) of 1.049 and 1.062 g/ml were collected. Cells (10⁷ per well) were plated in six-well plates with DMEM culture medium containing 10% charcoal stripped fetal calf serum (CSFCS – Invitrogen) and 1% antibiotic/antimycotic solution (Invitrogen). Purified trophoblast cells were cultured for 3 days at 37 °C under 5% CO₂/95% O₂ for 72 h. After 72 h, trophoblast cells aggregated to form a syncytium and were washed twice with Hank’s solution pre-warmed at 37 °C, and cultured for 16 h in DMEM free of CSFCS and antibiotics. Cell viability was assessed by trypan blue exclusion and biochemical viability was confirmed by the measurement of human chorionic gonadotropin (β-hCG) by a commercially available ELISA kit (Radim, Rome, Italy), while the purity of the trophoblast culture was confirmed by the immunostaining of more than 90% of the cells positive for cytokeratin and less than 1% of the cells positive for vimentin, using primary antibodies (Dako, UK) at a dilution 1:1000 (for both cytokeratin and vimentin).

2.3. L. rhamnosus strain GG (LGG) 

LGG powder (LGG ID 1271), was kindly donated by Anidral S.R.L. (Novara, Italy) with a cellular viability of 350 × 10⁹ UFC/g. Heat killed LGG was prepared as previously described [20], [21]. Briefly, bacteria at a concentration of 10¹⁰ CFU/mL in cell culture medium were heated at 80 °C for 20 min and heat-killed bacteria were centrifuged at 8000 rpm for 10 min, so supernatants could be collected and then diluted at 3 different concentrations in culture medium. Syncytiotrophoblast cells were incubated at 37 °C with LGG at 0, 10⁶, 10⁸ and 10¹⁰ CFU/mL in duplicate for 3 h and then culture medium was collected and stored at −80 °C until use. Protein concentrations of samples were determined by the method of Bradford using a protein assay kit (Bio-Rad, Milano, Italy) with bovine serum albumin as a standard.

2.4. Lipopolysaccharide (LPS) treatment 

With the purpose of investigating LGG effects on pro-inflammatory cytokine secretion in the presence of an inflammatory stimulus, LPS from Escherichia coli serotype 0111:B4 (Sigma–Aldrich) was used in a concentration (100 ng/mL) known to induce cytokine secretion by trophoblast cells in primary culture [11], [12], [22]. Thirty min after LGG treatment at 0, 10⁶, 10⁸ and 10¹⁰ CFU/mL, trophoblast cells were challenged with LPS for 3 h and the supernatants were collected and kept frozen at −80 °C until assayed for the content of the pro-inflammatory cytokine TNF-α.

2.5. Hormone and cytokine assays 

Measurement of the hormones and cytokines in the cell culture supernatants was performed by ELISA using commercially available kits in accordance with the manufacturer’s instructions. IL-4 (range: 1.1–58 pg/mL) and IL-10 (range: 12.5–400 pg/mL) kits were purchased from Abcam, UK. TNF-α (range: 39.0–250 pg/mL), CRH (range: 0–100 ng/mL) and Ucn (range: 0–100 ng/mL) kits were purchased from Phoenix peptides USA, while β-hCG (range: 0.0–2000 mIU/l) and progesterone (range: 0.05–40 ng/mL) assays were acquired from Radim-Italy.

2.6. RNA extraction and quantitative RT-PCR 

Trophoblast primary cells were disrupted and homogenized by passing the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter) fitted to a RNase-free syringe, and total RNA was digested with RNase-free DNase while the resulting RNA was cleaned up and concentrated according to the instructions of the manufacturer (RNase protect Micro Kit Qiagen, Hilden, Germany). We performed reverse transcription (RT) using the high-capacity cDNA RT kit (Applied Biosystems, Foster City, CA) with 100 ng RNA. Subsequently, TaqMan real-time PCR was carried out for all the genes analyzed and the size of amplification products was confirmed by electrophoresis on 2% agarose gel stained with ethidium bromide.

We used the TaqMan gene expression assays (Applied Biosystems) reported in Table 1 and the following thermal cycle protocol was applied: initial denaturation at 95 °C for 20 s, followed by 40 cycles of 95 °CC for 1 s and 60 °C for 20 s using 100 ng cDNA in a final reaction volume of 20 μl. Blank samples for each reaction consisted of amplifications performed in the absence of the RT enzyme. All experiments were done in triplicate and standard curves were constructed for all target genes by serial dilution of a standard sample starting from 200 ng of cDNA. The results were then normalized to the housekeeping gene 18S in order to correct differences in concentration of the starting template.

Table 1. TaqMan gene expression assays (Applied Biosystems) used to perform the real time PCR.

2.7. Statistical analysis 

After a normality test showed that values were normally distributed, data (the average of all six placenta, each one in duplicate) are expressed as means ± standard error (SE), and the statistical significance was assessed using the one-way ANOVA, followed by the post hoc Tukey’s test for multiple comparisons. Statistical significance was assumed for P < 0.05.

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3. Results 

LGG preparation was tested for the presence of nonspecific immunoreactivity for all hormones, cytokines and peptides that were assayed here and no immunoreactivity was found for any of the substances analyzed. Real time PCR control reactions were negative, ruling out genomic DNA contamination.

3.1. Hormonal secretion after LGG exposure 

To study the effect of LGG on basic placental endocrine functions, cultures of primary trophoblast cells were treated with different concentrations of LGG. The concentrations of β-hCG, progesterone and CRH were determined by specific ELISA assays following LGG exposure at 10⁶, 10⁸ and 10¹⁰ CFU/mL. No significant changes on β-hCG (Fig. 1A), progesterone (Fig. 1B) and CRH (Fig. 1C) secretion were found.

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• Fig. 1 

  Analysis of placental endocrine functions after LGG exposure in human trophoblast primary cells. LGG at 10⁶; 10⁸ and 10¹⁰ CFU/mL does not alter (A) β-hCG, (B) progesterone or (C) CRH secretion. Results are taken from six independent cultures. Data are mean ± SE.

3.2. LGG-induced secretion of IL-4, IL-10 and Ucn in human trophoblast primary cells 

In order to investigate if LGG drives trophoblast cells to an anti-inflammatory state, the anti-inflammatory cytokines IL-4 and IL-10 and the CRH-like peptide Ucn were assayed after heat-killed LGG exposure in different concentrations. LGG treatment increased IL-4 at both protein (P < 0.05 – Fig. 2A) and mRNA level (P < 0.05 – Fig. 2B). IL-10 protein secretion was increased in a dose dependent manner (P < 0.05 – Fig. 2C) while the highest dose of LGG increased significantly IL-10 mRNA expression (P < 0.05 – Fig. 2D). Similarly, LGG treatment also increased Ucn protein expression in a dose-dependent manner (P < 0.05 – Fig. 2E) while Ucn mRNA expression was not different after LGG exposure when compared to the control group (Fig. 2F).

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• Fig. 2 

  LGG-induced secretion of IL-4, IL-10 and Ucn in human trophoblast primary cells. Cultured cells were stimulated with LGG at different concentrations for 3 h. Cell culture supernatants were collected and assayed by ELISA and normalised to cell protein (A, C, E) while mRNA content was assayed by TaqMan real-time PCR and normalized to 18S expression (B, D, F). Results are taken from six independent cultures. Data are mean ± SE *p < 0.05; **p < 0.01; **p < 0.001.

3.3. LGG reverses LPS-induced TNF-α secretion in human trophoblast primary cells 

To further evaluate if the mechanism of LGG action includes regulation of a pro-inflammatory substance, the pro-inflammatory cytokine TNF-α was investigated at both protein and mRNA levels after LGG treatment. Heat-killed LGG exposure did not alter TNF-α protein secretion (Fig. 3A), neither TNF-α mRNA expression (Fig. 3B). Subsequently, since LGG did not change TNF-α expression per se, we investigated if LGG would play a protective role on trophoblast cells in the presence of a pro-inflammatory stimulus. TNF-α contents were then assayed after concomitant treatment of LGG and LPS. LPS exposure increased TNF-α protein (P < 0.001) and mRNA expression (P < 0.01) as expected. Conversely, LGG treatment reversed the effect of LPS on TNF-α expression at both protein (P < 0.01) and mRNA levels (P < 0.05) in a dose dependent fashion (Fig. 3).

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• Fig. 3 

  LGG reverses LPS-induced TNF-α secretion in human trophoblast primary cells. The cells were co-incubated with 100 ng/ml LPS, or a combination as indicated for 3 h. LGG at different concentrations does not alter TNF-α secretion at both protein (A) and mRNA (B) levels. Conversely, LGG reversed LPS-induced TNF-α secretion at both protein (C) and mRNA (D) levels. Cell culture supernatants were collected and assayed by ELISA and normalised to cell protein while mRNA content was assayed by TaqMan real-time PCR and normalized to 18S expression. Results are taken from six independent cultures. Data are mean ± SE *p < 0.05; **p < 0.01; **p < 0.001.

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4. Discussion 

The present study showed that the probiotic LGG significantly stimulates the release of Ucn, and IL-10 from trophoblast primary cells in a dose-dependent manner. The evidence of a specific effect of LGG on these two anti-inflammatory factors and also on IL-4, with no changes in CRH, β-hCG and progesterone secretion, suggests that probiotic treatment may have an anti-inflammatory effect on trophoblast cells via activation of specific anti-inflammatory factors. Furthermore, LGG treatment did not modify basal expression of the pro-inflammatory cytokine TNF-α, implying that even high dosages of LGG supernatants are not capable of inducing TNF-α related pro-inflammatory responses.

After evaluating mRNA expression of Ucn, IL-4 and IL-10 we confirmed that LGG modifies both mRNA levels in cell lysates and protein levels in culture medium within a 3 h interval. Previous studies performed in our laboratory in endometrial cell cultures, primary trophoblast cells and placental explants (unpublished data) demonstrated that the optimal incubation period to evaluate changes in CRH and CRH like peptides in the culture medium is 3 h. Regarding cytokine secretion, we (in the present study) and others [22] were able to demonstrate the presence of cytokines in the culture medium of primary trophoblast cells within 2–3 h of incubation. This short term effect of LGG on mRNA and protein contents suggests that the stimulus quickly induces gene transcription and, presumably, the release of pre-existing protein pools, since novel protein synthesis would require more time to be completed.

The CRH-like peptide Ucn is involved in different processes of pregnancy [9]. It plays a protective role on neurons [23] and displays anti-oxidative [24] and anti-inflammatory effects [13], [14] in different tissues. Importantly, Ucn stimulates IL-4 and IL-10 secretion and reverses LPS-induced TNF-α release from trophoblast primary cells through action on CRH-R2 receptors [12], demonstrating an anti-inflammatory role of Ucn in trophoblast cells. Such role is further supported by the present observation that LGG does not affect CRH release, which is pointed as a pro-inflammatory mediator in trophoblast cells and has a role in the parturition pathway [5].

IL-4 and IL-10 are well known anti-inflammatory cytokines which play a role in the placenta [8]. In cultured decidual cells, IL-4 reduces LPS-stimulated PGE2 production [25], while IL-10 prevents LPS-induced PTD in rats and ameliorates LPS-induced TNF-α and PGE2 production in choriodecidual primary cells [26]. Recently, a potential evidence for a therapeutic benefit of lactobacilli in reducing PTD has been suggested since lactobacilli promote anti-inflammatory effects in trophoblast cells. L. rhamnosus GR-1 inhibited the LPS-stimulated TNF-α release and increased IL-10 secretion from trophoblast cultured cells [22]. The fact that LGG differentially stimulated IL-4 and IL-10 secretion, suggests that these cytokines may be separately activated during LGG immunological responses.

In the present study, LPS treatment increased TNF-α release, thus confirming previous findings that we [12] and others [11], [22] described, which validates the reproducibility of the present in vitro model. Moreover, here we also showed that LPS insult up-regulates TNF-α transcripts within a 3 h treatment, demonstrating that both TNF-α transcription and translation are up-regulated by LPS. Subsequently, concomitant treatment with LGG plus LPS was able to inhibit LPS-induced TNF-α expression in a dose-dependent manner, demonstrating a role for LGG supernatants in reversing the expression of an important pro-inflammatory cytokine in trophoblast primary cells.

Finally, a further mechanism to be hypothesized is that IL-4 and IL-10 may have a dual role in inflammatory/infection processes, since they have been correlated with the pathophysiology of infection-associated preterm delivery. In cultured human decidual cells, IL-4 induces macrophage inflammatory protein 1α (MIP-1α), an important chemokine that exhibits a variety of pro-inflammatory actions involving cell-mediated immunity [26]. Additionally, in amnion, chorion and decidual cells, IL-4 stimulates PGE2 production in a concentration-related manner [27], [28]. IL-10 also displays paradoxical pro-inflammatory actions in explant cultures of gestational membranes. It has been demonstrated that amnion IL-8 and PGE2 production was significantly increased following IL-10 treatment, even though a dual response to IL-10 seems to take place depending on which side of the maternal or fetal face the inflammatory insult is occurring [29]. Although the net result of IL-4 and IL-10 release in gestational tissues is not currently known, despite the paradoxical pro-inflammatory actions of these interleukins in gestational membranes, LGG-induced IL-4 and IL-10 release probably contributes to the global effect of these cytokines regulating different cell type responses; which remain to be elucidated, since it is not clear yet how these different cytokines contribute to the final maternal immune response.

In summary, the present study demonstrated that LGG supernatant stimulates IL-4, IL-10 and Ucn expression and reverses LPS-induced TNF-α release from trophoblast cells, with no change in β-hCG or progesterone release, suggesting that this probiotic may play a role as an immunomodulatory agent in human placenta without altering basic trophoblast functions.

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Acknowledgements 

This study was supported in part by FERRING S.P.A. We would like to thank to Dr Maurizio Acri and Anidral S.R.L. for donating LGG powder.

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