Proinflammatory response of canine trophoblasts to Brucella canis infection

Abstract

Brucella canis infection is an important cause of late-term abortion in pregnant bitches. The pathophysiological mechanisms leading to B. canis–induced abortion are unknown, but heavily infected trophoblasts are consistently observed. As trophoblasts responses to other pathogens contribute to placental inflammation leading to abortion, the aim of the present study was to characterize the cytokine response of canine trophoblasts to B. canis infection. To achieve this, trophoblasts isolated from term placenta of healthy female dogs were infected with B. canis, culture supernatants were harvested for cytokine determinations, and the load of intracellular viable B. canis was determined at different times post-infection. Additionally, cytokine responses were assessed in non-infected trophoblasts stimulated with conditioned media (CM) from B. canis-infected canine monocytes and neutrophils. Finally, cytokine response and bacteria replication were assessed in canine placental explants infected ex vivo. B. canis successfully infected and replicated in primary canine trophoblasts, eliciting an increase in IL-8 and RANTES (CCL5) secretion. Moreover, the stimulation of trophoblasts with CM from B. canis-infected monocytes and neutrophils induced a significant increase in IL-8, IL-6 and RANTES secretion. B. canis replication was confirmed in infected placental explants and the infection elicited an increased secretion of TNF-α, IL-8, IL-6 and RANTES. This study shows that canine trophoblasts produce proinflammatory cytokines in response to B. canis infection and/or to stimulation with factors produced by infected monocytes and neutrophils. These cytokines may contribute to placental inflammation leading to abortion in B. canis-infected pregnant bitches.

HMGB1 release from trophoblasts contributes to inflammation duringBrucella melitensisinfection

“…As B. canis infection can lead to the increase of RANTES in primary canine trophoblasts, and the increased RANTES contribute to placentitis of Brucella ‐infected pregnant mice (Fernandez et al, ; Kim et al, ; Watanabe et al, ), we investigated if HMBG1 also modulates the RANTES secretion during B. melitensis infection. As shown in Figure c, the production of RANTES was increased after 48 hr of infection in isotype control IgY‐treated cells compared with uninfected cells.…”

“…At 24 hr of stimulation, the supernatants from stimulated cultures were harvested to measure cytokines. To calculate the specific secretion of each factor, levels already present in CM from trophoblasts were subtracted from levels measured after stimulation (Fernandez et al, ).…”

“…Mice trophoblasts were isolated from placenta of the pregnant mice following previously described procedures (Fernandez et al, ; Pennington et al, ). Briefly, coarsely minced placental tissue were washed thoroughly with PBS and incubated with buffer containing collagenase and trypsin at 37°C for 30 min.…”

“…At 2, 24, and 48 h.p.i, the supernatants of explants were collected to measure LDH release and cytokines. To investigate Brucella intracellular growth, infected trophoblasts were lysed with PBS with 0.1% Triton X‐100 at indicated time points, and the serial dilutions of lysates were plated immediately into TSA plates to count CFUs (Fernandez et al, ).…”

“…Inflammatory responses in placental tissues have been associated with Brucella infection‐induced abortion, and in many cases, trophoblasts have been demonstrated to promote such inflammation (Fernandez, Hielpos, Ferrero, Fossati, & Baldi, ; Keestra‐Gounder et al, ). In line with these findings, the trophoblasts infected with B. abortus or B rucella canis in vitro induces the secretion of several inflammatory cytokines such as TNF‐α, IL‐6, IL‐8, and RANTES.…”