Effect of Lipopolysaccharide-Induced Immune Responses on Pregnancy Loss in Ewes

An acute phase response induced by Gram-negative bacteria can reduce pregnancy rate. Early pregnant ewes were used to monitor effects of lipopolysaccharide (LPS), an endotoxin in the outer cell membrane of Gram-negative bacteria, on acute phase/innate immunity response. In Exp. 1, mixed breed ewes were assigned to receive either LPS or LPS and flunixin meglumine, an inhibitor of prostaglandin synthetase, intravenously on day 5 after mating. In Exp. 2, mixed breed ewes were assigned to receive an intravenous injection on day 5 after mating of either saline, LPS, recombinant human tumor necrosis factor (TNF)-α or LPS after pretreatment with dexamethasone. Pregnancy was diagnosed ultrasonographically on d 25, and live births were recorded at parturition. Challenge with LPS induced acute phase responses (fever, mucosal responses, lethargy and increased serum TNF, haptoglobin and serum amyloid A) and decreased pregnancy rates. Predictably, flunixin meglumine attenuated fever but did not increase pregnancy rate in LPS-treated ewes. Similarly, exogenous TNF alone induced mucosal and serum amyloid A responses but did not affect pregnancy. Pre-treatment with dexamethasone blocked fever and mucosal and lethargic responses and attenuated increases in TNF and haptoglobin but did not ameliorate LPS-induced pregnancy loss. In summary, acute challenge with LPS mimics bacterial-induced pregnancy losses in early pregnant ewes. Although pretreatment with dexamethasone decreased clinical signs and some innate immune responses, neither it nor flunixin meglumine prevented LPS-induced pregnancy loss. That exogenous TNF alone did not promote pregnancy loss indicates that other cytokines also contribute to LPS-induced embryonic loss.


Introduction
Infectious disease can reduce pregnancy rate. For example, conception rate in cattle decreased if mastitis occurred before first insemination, and more inseminations were required to establish pregnancy if cows were infected in early as opposed to later pregnancy [1]. The early immune response associated with bacterial challenges likely compromises establishment or early maintenance of pregnancy [2] [3].
Both lipopolysaccharide (LPS), a component of Gram-negative bacteria, and peptidoglycan-polysaccharide, a component of Gram-positive bacteria, have been used to investigate the mechanism by which bacterial infection leads to early embryonic failure. LPS induced luteolysis and reduced conception rate and early embryonic survival through release of inflammatory mediators [4]. Although LPS alone reduced embryonic development of cultured mouse embryos in one study [5], LPS reduced development only if mouse embryos also were exposed to TNF in another study [6]. Ewes inoculated with either peptidoglycan-polysaccharide [7] or a heat-killed Gram-positive bacterium, Streptococcus pyogenes, [8] on day 5 after mating had increased early embryonic losses. Injection of peptidoglycan-polysaccharide on day 5 after mating resulted in acute phase response [APR: fever and increased jugular plasma concentrations of TNF, haptoglobin (Hp) and serum amyloid A (SAA)], decreased concentrations of progesterone on days 14 and 21 and interfered with establishment of early pregnancy [9].
Two other molecules produced during inflammation, prostaglandin (PG) F2α and cortisol [7] might compromise embryo survival. In several tissues, including the endometrium, LPS, inflammatory cytokines such as TNF and interleukin-1β (IL-1β), and glucocorticoids increased synthesis of PGF2α [10]. Therefore, the present studies were conducted to characterize components of the mechanism through which LPS affects reproductive efficiency in early pregnant ewes and to determine if treatment with anti-inflammatory drugs, specifically flunixin meglumine, an inhibitor of prostaglandin synthetase, or dexamethasone, a synthetic corticoid, alters the LPS-induced acute phase response.

Assays
Concentrations of TNF were assayed by RIA [15], an assay used previously [9] to measure TNF in sheep plasma, in duplicate in a single assay with an intra-assay coefficient of variance (CV) < 10%. Although cross-reactivity of the assay with hTNF is <1%, the exogenous dose of rhTNF in Exp.

White Blood Cell Counts
Total WBC counts were obtained (Beckman Coulter, Pasadena, CA) immediate-Open Journal of Animal Sciences ly after collection. A WBC differential was determined by thinly spreading 5 µL of whole blood over a glass slide, air drying, and staining with Giemsa stain (Sigma-Aldrich, Inc., St. Louis, MO). One hundred cells were counted and classified among monocyte, lymphocyte, eosinophil, neutrophil or basophil cell types [16].

Statistical Analysis
For each experiment, data for rectal temperature and concentrations of TNF, SAA, and Hp, and, for experiment 2, concentrations of white blood cells (total, lymphocytes, neutrophils, and monocytes) were analyzed by repeated measures using the ANOVA mixed procedure of SAS followed by the

Clinical Signs
Treatment with LPS increased rectal temperature (Exp. 1, mean increase 1.7˚C,

Assays
In both experiments, LPS induced increases in plasma concentrations of TNF, which peaked at 1.5 and 1 h, respectively. Jugular concentrations of TNF in ewes pre-treated with flunixin meglumine increased in comparable fashion to ewes treated only with LPS increasing from 0.13 ng/ml to a peak of 1.08 ng/ml ( Figure 2(a)). The response was attenuated (P < 0.05) in LPS and dexamethasone ewes (Figure 2

White Blood Cells
In Exp. 2, total WBC (Figure 3(a)) differed by treatment (P < 0.0001), hour (p < 0.0001), and treatment by hour (P < 0.0001). Total WBCs decreased the first hour after treatment with LPS, remained lower than control through 6 h, but exceeded the control values after 9 h. The pattern was comparable for the neutrophils with effects of treatment (P < 0.05), hour (P < 0.0001), and treatment by hour (P < 0.005, Figure 3

Discussion
In previous studies [7] [8] [9], mated ewes inoculated with peptidogly- fever but had no effect on serum concentrations of TNFα, which increased in a pattern comparable to that in ewes treated with LPS treatment alone, and tended to increase pregnancy rate indicative of a role of prostaglandin synthesis in pregnancy losses due to LPS. Treatment of cows with flunixin meglumine 14 days after insemination improved pregnancy rates compared to non-treated cows [18]. Because pregnancy rate was similar in TNF and control ewes but higher than in ewes treated with LPS or LPS and flunixin meglumine, the increased mucosal responses after TNF injection alone did not account for the observed changes in pregnancy rate in the LPS-challenged ewes. In fact, 6/13 ewes that displayed a mucosal response lambed compared to 15/25 of those that did not show a mucosal response. Treatment with TNF did not alter rectal temperature nor pregnancy rate, while LPS did; hence, change in rectal temperature Open Journal of Animal Sciences could be part of the mechanism for embryonic loss. Indeed, only 13/28 of the ewes showing increased rectal temperature lambed compared to 8/10 ewes that did not show increased rectal temperature.
Traditionally, administration of glucocorticoids has been associated with suppression of immune responses, particularly to LPS [19]. Dexamethasone reduced endotoxin levels, inflammatory mediators and down-regulated expression of NF-κB in ileal mucosa [20]. The dosage of dexamethasone used in Exp. 2 affected fetal hypothalamic maturation when administered to ewes on d 41 -42 of gestation [21]. Treatment of ewes with dexamethasone dampened both the LPS-induced increase in rectal temperature and incidences of mucosal responses and of lethargy; thus, the dosage of dexamethasone provided the expected anti-inflammatory response. Glucocorticoids down-regulate pro-inflammatory genes such as those encoding for COX-2 [22], which could explain why dexamethasone-treated ewes displayed less fever than ewes treated with just LPS.
Ewes treated with dexamethasone had the second highest TNF concentration at 0 h of the treatment groups. Enhanced TNF responses to LPS were observed in mice exposed to dexamethasone 24 -48 h before LPS [23], and pro-inflammatory mediators were up-regulated if glucocorticoids were administered prior (2 and 24 h) to a peripheral LPS challenge [24].
A decline in total WBCs, monocytes, neutrophils, Hp, and SAA was seen until

Conclusion
In summary, challenge with LPS resulted in an early innate immune response, which was followed by an apparently decreased pregnancy rate. Dexamethasone given prior to LPS elicited a "priming" effect on WBCs and decreased clinical Open Journal of Animal Sciences signs and some innate immune responses but did not inhibit the LPS-induced pregnancy loss. The cytokine TNF alone may not be responsible for early embryonic death, but in the context of a full innate immune response, such as that induced by LPS, TNF along with other pro-inflammatory cytokines may collectively contribute to early embryonic death.