Activation of TLR3 in the trophoblast is associated with preterm delivery

Abstract

Problem: Toll-like receptors (TLRs) recognize conserved sequences on the surface of pathogens and trigger effector cell functions. Previously, we described the expression of TLR3 by human trophoblast and their ability to respond to (Poly[I:C]). Here we evaluate the effect of Poly[I:C] on mouse pregnancy and characterize the local and systemic response.

Method of study: C57B/6 wild type (wt) and TLR3 knockout (TLR3KO) mice were treated with Poly[I:C] at 16.5 dpc and pregnancy outcome recorded. Morphologic changes, cytokines and chemokines levels in blood and utero-placental tissue were determined. NF-kappaB pathway was evaluated in vivo and in vitro.

Results: Poly[I:C] in C57B/6 wt mice caused preterm delivery within 24 hr (4.5 mg/kg). No effect was observed in TLR3KO mice. In addition, we observed local (placenta) and systemic (serum) response characterized by increased production of proinflammatory cytokines and chemokines. The NF-kappaB pathway was activated by Poly[I:C] in human and mice trophoblast cells.

Conclusion: We report that Poly[I:C] induces preterm delivery via TLR3-dependent manner. Furthermore, we demonstrate that the trophoblast is able to recognize Poly[I:C] through TLR3 and respond to viral infection, modulating the immune system at the feto-maternal interface.

Figures

Fig. 1

Poly[I:C] injection induced morphologic change at feto-maternal interface; 4.5 mg/kg Poly[I:C] or PBS was injected i.p. to wild type mice on 16.5 dpc and killed after 4 hr. Hematoxylin and eosin staining was performed for histopathologic analysis. The feto-maternal interface from PBS-treated animals kept its morphologic integrity (a–c and i). On the contrary, the feto-maternal interface from Poly[I:C] treated mice lost the integrity (d–h and j). Necrosis (e*), inflammation (e–g), hemorrhage (h**) and edema (f and j) were apparent in the decidua, the yolk sac membrane, chorion and amniotic membrane of poly(I:C) treated animals. The inflammation is accompanied by polymorphonuclear leukocytes infiltration (e–g). V: blood vessel, am: amniotic membrane, ysm; yolk sac membrane, ch: chorion, dec: decidua, myo: myometrium. Original magnification; a, d, g: 40×, b, c, e, f, h, i, j: 400×. Data are representative of at least three mice from same treatments.

Fig. 2

Poly[I:C] injection induced NK cells and macrophages infiltration into the placenta; 4.5 mg/kg Poly[I:C] or PBS was injected i.p. to wild type mice on 16.5 dpc. Mice were killed after either 4 or 48 hr. Utero-placental units were collected. Immunohistochemical analysis of the feto-maternal interface using lectin (for uNK cells; brown) or anti f4/80 antibody (for Macrophages; pink) were performed. (A) In PBS-treated mice, uterine NK cells were restricted to the decidua (Ab) and no NK cells were seen in spongiotrophoblast and labyrinth, (a–c). The distribution of NK cells changed in Poly[I:C] treated mice (d). Fewer NK cells were found in decidua (e) and more cells were found in spongiotrophoblast (f) and labyrinth zone (g). Slides were counterstained with hematoxylin (purple). (B) Macrophages were identified mainly in the outer myometrium and rarely in the decidua and were never observed in spongiotrophoblast and labyrinth in PBS-treated control animals (a). Macrophage infiltration from the maternal side toward the placental zone was not observed up to 48 hr after Poly[I:C] injection (b and c). Instead, when we observed the fetal side of the placenta at 48 hr post injection, we found macrophages infiltrated from the fetal side (umbilical cord and yolk sac membrane) toward the placenta zone (e and f). No such infiltration was found in PBS-treated animals (d). Slides were counterstained with Hoechst (blue nuclei). ysm, yolk sac membrane; m, myometrium; dec, decidua; sp, spongio; trophoblast, lab, labyrinth; cord, umbilical cord. (C) Schematic localization for uterine NK cells and macrophages and the direction of their infiltrations toward placental zone. Original magnification; 2A a and d: 40X, 2Ab, c, e, f and g: 2B a–f; 400X. Data are representative of at least three mice from same treatments.

Fig. 3

Poly[I:C] injection induced robust local and systemic inflammatory response in wild type pregnant mice; 4.5 mg/kg Poly[I:C] or PBS was injected i.p. to wild type mice on 16.5 dpc. Mice were killed after 2 and 4 hr and placenta and sera were collected. Cytokine/chemokine concentrations in placental lysate (Placenta) and sera (Serum) were measured. Boxes represent the distance between the first (25%) and third (75%) quartiles and horizontal lines in the boxes represent medians. #P > 0.05. Data are representative of at least six mice per group.

Fig. 4

Poly[I:C] injection failed to induce local and systemic inflammatory response in TLR3 knockout pregnant mice; 4.5 mg/kg Poly[I:C] or PBS was injected i.p. to either wild type (wt) or TLR3 knockout (TLR3KO) mice on 16.5 dpc. Mice were killed after 2 and 4 hr and placenta and sera were collected. Cytokine/chemokine concentrations in placental lysate (Placenta) and sera (Serum) were measured. Boxes represent the distance between the first (25%) and third (75%) quartiles and horizontal lines in the boxes represent medians. #Significantly lower (P > 0.05) than wild type treated mouse, *Significantly higher (P > 0.05) than PBS-treated mouse.

Fig. 5

Poly[I:C] injection promoted NF-κB activation in murine placenta in vivo; 4.5 mg/kg Poly[I:C] or PBS was injected i.p. to wild type mice on 16.5 dpc. Mice were killed after 2 hr and placenta was collected. Immunohistochemistry with anti NF-κB p65 antibody was performed. Slides were counterstained with Hoechst (nuclear staining). In tissues from PBS-injected animals (c), NF-κB p65 was mainly detected in the cytoplasm of cells. On the contrary, a markedly higher number of stained nuclei was observed in tissue from Poly[I:C] treated animal (f). Arrows are indicating nuclear localization which has double color. Original magnification was 200×. Data are representative of at least three mice from same treatments.

Fig. 6

Poly[I:C] treatment induced cytokine/chemokine secretion from first trimester human trophoblast cells in a dose- and a time-dependent manner. First trimester trophoblast cell line HTR8 were treated with Poly[I:C] at the doses indicated. Supernatant were collected after either 24 or 48 hr. Cytokine/chemokine concentrations in supernatant were measured. Data are representative of 13 cytokine/chemokine data.

Fig. 7

Poly[I:C] promoted NF-κB activation in human trophoblast in vitro. HTR8 cells transfected with a NF-κB reporter construct were treated with Poly[I:C] (25 g/mL). NF-κB activity was increased at 4 hr post-treatment. *P < 0.05.