Differential roles of inflammation and apoptosis in initiation of mid-gestational abortion in malaria-infected C57BL/6 and A/J mice

Abstract

Introduction Plasmodium chabaudi AS-infection in pregnant A/J and C57BL/6J mice results in mid-gestational pregnancy loss. Although associated with increased systemic and placental pro-inflammatory responses and coagulopathy, the molecular mechanisms that underlie poor pregnancy outcomes in these mice are not yet fully understood. This study investigates the relationships between inflammation, apoptosis and malaria-induced pregnancy loss. Methods Infection with Plasmodium chabaudi AS in early murine pregnancy and term human placental tissues from an endemic setting were assessed by histology, immunohistochemistry, TUNEL staining, real-time PCR, flow cytometry, western blot, and ELISA. Results Quantitative PCR reveals accumulation of lymphocytes and monocytes and upregulation of chemokines that attract these cell types in malaria-exposed mid-gestational A/J conceptuses. Monocyte accumulation is confirmed by flow cytometry and placental immunohistochemistry. Concurrent with initiation of malaria-induced abortion, markers of apoptosis are evident in the junctional zone, but not the labyrinth, of A/J placentae. In contrast, mid-gestation conceptuses in infected C57BL/6J lack evidence for monocyte accumulation, exhibiting low or no in situ placental staining despite trophoblast immunoreactivity for the monokine, CCL2. Additionally, placental apoptosis is not consistently observed, and when evident, appears after malaria-induced abortion typically initiates. Similarly, trophoblast apoptosis in term human placental malaria is not observed. Of those studied, a sole common feature of malaria-induced abortion in A/J and C57BL/6J mice is elevation of plasma tumor necrosis factor. Discussion Consistent with our previous observations, tumor necrosis factor is likely to be a central driver of malaria-induced pregnancy loss in both strains, but likely operates through mechanisms distinct from placental apoptosis in C57BL/6J mice.

Oxidative Stress: A Potential Therapeutic Target in Placental Malaria

“…Given that excessive or uncontrolled production of HNE interferes with cellular signaling, and a role in the pathogenesis of several diseases, including severe malaria, has been suggested (44), placental lipid peroxidation may be central to PM-associated compromise of pregnancy, Indeed, at very high levels, HNE reportedly induces apoptosis or necrosis (45). Although we previously demonstrated that the former is unlikely to mediate embryo loss in infected B6 mice (17), necrotic placental damage is observed (D. Sarr, J. Ecker, J.M. Moore, unpublished observations).…”

“…Plasma and tissues were collected as described below. Mice were dissected for evaluation of embryo status and isolation of tissues as previously described (14, 15, 17, 18). Briefly, resorptions or nonviable embryos were identified by their necrotic and/or smaller size compared with viable normal embryos as they appear in uninfected pregnant (UP) mice.…”

“…H&E-stained organ sections were used by a pathologist to assess malaria-associated histopathology and the effects of TPL treatment. Unstained 7 mm tissue sections (3) were used for immunohistochemistry (IHC) according to previously reported methods (17) with a Polink-HRP kit for rabbit Abs following the manufacturer’s instructions (GBI Mukilteo, WA). Samples were stained the same day with the same reagents to avoid interassay variability.…”

“…Cytokines and chemokine levels were assessed by ELISA using reference standard curves with known amounts of recombinant cytokines (TNF, IL-6, and IFN) and chemokine (CCL2) following the manufacturer’s instructions (BD and R&D as appropriate). For measurement of CCL2 in mouse conceptuses, total protein was isolated as previously described (17). Total proteins were quantified by CBQCA Protein Quantification Kit and diluted in assay diluent at the concentration of 1 mg/ml for use in ELISA.…”

“…To address this issue, an alternative murine model (3, 14–18) in which P. chabaudi chabaudi AS ( P. chabaudi AS) infection in early gestation induces placental damage and pregnancy loss in C57BL/6 and A/J mice (3, 17, 18) was used. Because apoptosis does not seem to play a universal role in this placental damage (17), we hypothesized that, consistent with the P. berghei model of PM (9, 11), oxidative stress participates in the pathogenesis of P. chabaudi AS infection during early pregnancy. To this end, oxidative stress was measured by quantitative PCR of markers of an antioxidant response and by immunohistochemistry of a reactive lipid mediator generated during oxidative stress, 4-hydroxynonenal (HNE).…”