Heat-killed Lactobacillus rhamnosus GG Modulates Urocortin and Cytokine Release in Primary Trophoblast Cells E. Bloise a , M. Torricelli a , R. Novembri a , L.E. Borges a , P. Carrarelli a , F.M. Reis b, c , F. Petraglia a, * a Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, “S. Maria alle Scotte”, viale Bracci 53100 Siena, Italy b Department of Obstetrics & Gynecology, Federal University of Minas Gerais, Belo Horizonte, Brazil c Department of Physiology and biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil article info Article history: Accepted 9 April 2010 Keywords: Placenta Lactobacilli Preterm delivery Urocortin Cytokines abstract A number of studies are showing that probiotic treatment induces an anti-inflammatory state. Intra- uterine 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 b-hCG, progesterone, interleukins (IL) 4 and 10, tumor necrosis factor-a (TNF-a), corticotropin releasing hormone (CRH) and urocortin (Ucn) release by primary trophoblast cells. Normal human term placentas (n ¼ 6) were collected and purified trophoblast cells were incubated in the presence of LGG, lipopolysaccharide (LPS) or either LGG þ LPS during 3 h, after which the target substances were quantified by ELISA and real-time PCR. LGG did not affect b-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-a, while LPS exposure increased TNF-a protein (P < 0.001) and mRNA expression (P < 0.01). Conversely, LGG treat- ment reversed LPS-induced TNF-a 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-a release from trophoblast cells, with no change in b-hCG or progesterone release, sug- gesting that this probiotic may play a role as an immunomodulatory agent in human placenta without altering basic trophoblast functions. Ó 2010 Published by Elsevier Ltd. 1. Introduction Probiotics are live microorganisms that promote host immu- nomodulation by colonizing and protecting tissues against micro- bial 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-inflam- matory cytokines, interleukins (IL) 4 and 10 are considered to have a protective role during pregnancy [8]. Tumor necrosis factor- a (TNF-a), 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 [5e7]. 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-a and IL-8 release [11]. Ucn, in turn, stimulates IL-4 and IL-10 secretion and reverses LPS-induced TNF-a 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, * Corresponding author. Tel.: þ39 0 577 233 453; fax: þ39 0 577 233 454. E-mail address: petraglia@unisi.it (F. Petraglia). Contents lists available at ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta 0143-4004/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.placenta.2010.04.007 Placenta 31 (2010) 867e872