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Keywords:

  • fetal growth deficit;
  • inflammation;
  • maternal mortality;
  • neurodevelopmental assessment;
  • preterm birth;
  • rat model.

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Aim

To study the effect of blocking the inflammatory cascade with interleukin-6 receptor antibody (anti-IL-6R) on feto-maternal outcomes in a rat model.

Methods

Pregnant Sprague–Dawley rats (n = 38) were injected intraperitoneally (day 22) (control, anti-IL-6R 30 μg/kg, lipopolysaccharide [LPS] 250 μg/kg or 500 μg/kg alone or combined with anti-IL-6R) followed by preterm caesarian performed 12 h later. Resuscitated pups (n = 179) were given to surrogate mothers. Primary outcomes were maternal and pup mortality.

Results

Fifty percent of pregnant rats died after LPS 500 μg/kg + anti-IL-6R injection but none in other groups. Neonatal mortality at 24 h was 63% and 86% in LPS 500 μg/kg and LPS 500 μg/kg + anti-IL-6R groups, respectively (P < 0.05). Surviving pups in the latter group presented a severe growth deficit compared to the LPS 500 μg/kg group (P < 0.01) and showed no difference with controls for open field testing. Maternal cytokine analysis after LPS 500 μg/kg + anti-IL-6R injection showed a tendency for increased IL-1 production (P = 0.06).

Conclusion

Paradoxically, the association of pregnancy, inflammation and anti-IL-6R increases the inflammatory effects of LPS.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Preterm delivery is the leading cause of perinatal mortality and morbidity. Unfortunately, the rate of preterm birth has increased over the last decades despite improved perinatal care.[1]

Inflammation has been shown to play a major role in the pathogenesis of preterm birth as a cascade of events starting from the lower genital tract and involving contamination of choriodecidual space and placenta.[2] Over 30% of placentas from preterm deliveries have demonstrated microscopic inflammatory changes.[3, 4] Chorioamnionitis is an important risk factor for the development of perinatal brain damage and subsequent neurological disorders.[5] Evidence suggests that production of soluble inflammatory mediators arising from maternal infection is a direct cause of fetal brain damage.[6, 7] Only a few studies have demonstrated protection against placental and neurodevelopmental defects induced by maternal inflammation. Recently, Girard et al. demonstrated that the administration of interleukin (IL)-1 receptor antagonist in a murine model alleviated the effects of intraperitoneal injection of lipopolysaccharides, such as placental inflammation, white matter lesions and motor neurodevelopmental alterations in the offspring.[8]

Interleukin-6 is a key player in the inflammatory cascade. The fetal inflammatory response syndrome (FIRS), characterized by an elevation in fetal plasma IL-6, is part of this inflammatory process and its association with long-term neurological sequelae has been widely studied.[6, 9, 10] Moreover, Yoon et al. demonstrated that elevated amniotic fluid IL-6 was a major risk factor for the development of neonatal white brain lesions and cerebral palsy.[9-11] Two pathways enable IL-6 to modulate a broad spectrum of target cells and to regulate acute inflammation in vivo: the classic pathway and the trans-signaling pathway.[12] In the classic pathway, IL-6 binds to the membrane-bound IL-6 receptor (IL-6R) and to the signal-transducing glycoprotein, gp130, while in the trans-signaling pathway, a complex is formed between IL-6 and a soluble IL-6 receptor which then binds to gp130. The trans-signaling pathway confers IL-6 sensitivity to many cell types that do not express the membrane-bound IL-6 receptor, but rather express gp130. Blockade of IL-6 signaling is accomplished via the IL-6 receptor antibody (anti-IL-6R). Anti-IL-6R binds to the IL-6 receptor, prevents IL-6 binding to its receptor and inhibits the subsequent downstream signaling events in target cells. Recently, IL-6 blockade has shown a promising therapeutic perspective in various medical inflammatory diseases. For example, in rheumatology, several phase III clinical trials with anti-IL-6R have successfully demonstrated clinical efficacy for the treatment of chronic autoimmune and inflammatory diseases, such as rheumatoid arthritis.[13] To our knowledge, no reports have studied the protective role of anti-IL-6R on neonatal outcomes, like perinatal mortality and morbidity or neurological sequelae induced by maternal inflammation.

In the present study, we hypothesized that blocking the inflammatory cascade with anti-IL-6R may prevent such neonatal outcomes associated with in utero inflammation. For this purpose, an established rat model of maternal inflammation was used to determine the effect of anti-IL-6R.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Animals

Sprague–Dawley rats (n = 81) were obtained from Charles River Laboratories (Saint-Constant, Quebec, Canada) and allowed to acclimatize to our animal facility prior to experimental manipulations. Animals included pregnant rats at gestational day (G)15 (n = 38), non-pregnant rats (n = 12) and surrogate mothers (n = 31). They were housed in a calm environment with a 12:12-h light : dark cycle, with food and water available ad libitum. The experimental protocol (213-09) was approved by the Institutional Animal Research Ethics Review Board on December 2009. Animals were used in accordance with the Animal Care and Use Committee of the Université de Sherbrooke (Sherbrooke, Quebec, Canada).

Materials

Lipopolysaccharide (LPS; 1 mg/mL, from E. coli, serotype 0127-B8) was purchased from Sigma-Aldrich (Oakville, ON, Canada). An antibody blocking the membrane-bound portion of the IL-6 receptor was used (anti-IL-6R; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Reported doses used by different authors in animal studies have ranged from 17 μg/kg to 8 mg/kg.[14, 15] In order to minimize the potential impact of high doses of anti-IL-6R on physiological function, a dose of 30 μg/kg was selected. The same dose was chosen for immunoglobulin (Ig)G controls (Rabbit IgG; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Experimental procedure for pregnant rats

A mini-laparotomy was performed under general anesthesia (Isoflurane 2%), at G22 in order to introduce a plastic catheter for the intraperitoneal (i.p.) injection of: (i) anti-IL-6R; (ii) IgG; (iii) LPS in association with IgG; or (iv) LPS in association with anti-IL-6R. In all groups, Marcaine 0.25% was injected subcutaneously at the end of the procedure as a local anesthetic. Maternal serum was collected at 0, 1, 3 and 12 h following the i.p. injection. Caesarean section was performed 12 h after i.p. injection on mothers that did not die in the exposition period and four to six pups per rat were resuscitated (n = 179). Pups were given to surrogate mothers. To facilitate this process, pups were sprinkled with urine from biological pups born the day before. Mothers were killed with isoflurane (5%) after caesarian section. Pups were killed with isoflurane (5%) after open field analysis at day 21.

Outcomes

Maternal mortality was assessed between i.p. injection and caesarean section. Principal maternal pro-inflammatory cytokines (IL-6, tumor necrosis factor [TNF]-α and IL-1) from serum collected at 0, 1, 3 and 12 h after i.p. injection were quantified using enzyme-linked immunoassay kits (R&D Systems, Minneapolis, MN, USA; eBioscience, San Diego, CA, USA) following the manufacturer's instructions. To assess neurological developmental behavior, pups were followed: (i) daily to assess vital status between resuscitation and postnatal day (P)21; (ii) every week to control postnatal growth; and (iii) at P21 with an open field test.[16] The apparatus for the open field test consisted of a 40 cm × 40 cm × 40 cm Plexiglas enclosure. Pup displacements were recorded for 5 min with a camera connected to a computer running the Any-Maze Video Tracking System program (Stoelting, Wood Dale, IL, USA). The following rat displacements were analyzed: total distance traveled, mean speed, mobile time and lines crossed. Pups and surrogate mothers were killed by CO2 inhalation 22 days after caesarian section.

Strategies

To assess the impact of anti-IL-6R in animals without inflammation

Rats were injected i.p. with: (i) anti-IL-6R 30 μg/kg + saline 0.5 mL/kg (n = 5); or with (ii) IgG 30 μg/kg + saline 0.5 mL/kg (n = 5) (control groups without inflammation).

To assess the impact of anti-IL-6R in animals with inflammation

Rats were injected i.p. with: (i) LPS 250 μg/kg (n = 5) or 500 μg/kg (n = 6) + IgG 30 μg/kg (control groups with inflammation); or (ii) LPS 250 μg/kg (n = 5) or 500 μg/kg (n = 12) + anti-IL-6R 30 μg/kg.

Experimental procedure for non-pregnant rats

Non-pregnant animals (n = 12) were injected by mini-laparotomy, using the same procedure as described above, with: (i) LPS 500 μg/kg + IgG 30 μg/kg (n = 6); or (ii) LPS 500 μg/kg + anti-IL-6R 30 μg/kg (n = 6). Cytokines and mortality were assessed as described above and rats were killed 24 h after i.p. injection.

Data analysis

Data are presented as median (25th–75th interquartile range). Comparison for mortality was performed using the χ2-test and Fisher's exact test, with P < 0.05 considered statistically significant. Comparison for weight, cytokine expression and neurological development was performed using the Kruskal–Wallis and Mann–Whitney U-tests, with P < 0.05 considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Maternal mortality following i.p. injection

The effect of anti-IL-6R alone or in association with systemic inflammation induced by LPS was assessed in pregnant rats. No death occurred when rats were exposed to control (IgG), anti-IL-6R, LPS 250 μg/kg or LPS 500 μg/kg (Fig. 1a). Surprisingly, mortality was observed in animals injected i.p. with LPS 500 μg/kg + anti-IL-6R, leading to maternal death in 50% of cases (n = 6), while no deaths were observed in pregnant rats exposed to LPS 250 μg/kg + anti-IL-6R. Furthermore, no deaths were observed in non-pregnant animals injected with LPS 500 μg/kg + anti-IL-6R.

figure

Figure 1. Maternal and neonatal mortality. Anti-IL-6R, interleukin-6 receptor antibody; LPS 250, lipopolysaccharide 250 μg/kg; LPS 500, lipopolysaccharide 500 μg/kg. (a) Maternal mortality 12 h after intraperitoneal (i.p.) injection. Maternal mortality was observed in animals injected i.p. with LPS 500 μg/kg + anti-IL-6R, leading to maternal death in 50% of cases (n = 6), while no deaths were observed in the other groups. (b) Neonatal pup mortality at 24 h and 21 days after birth. Antenatal exposure to LPS 500 μg/kg significantly increased pup mortality at 24 h and 21 days when compared to controls (P < 0.01). Concomitant administration of anti-IL-6R in presence of high-dose LPS (500 μg/kg) further increased pup mortality at 24 h (P < 0.05). *P < 0.05; ***P < 0.01.

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Mortality among resuscitated pups

Neonatal death mostly occurred on the first day after birth with 80.1% of deaths observed within the first 24 h (Fig. 1b and Table 1). When compared to controls, mortality was significantly increased in the LPS 500 μg/kg group (P < 0.01). Maternal exposure to inflammation led to a dose-dependent increase in neonatal mortality at 24 h. In the group exposed to LPS 500 μg/kg, 63.3% of deaths were noted compared to 36.7% in the LPS 250 μg/kg group (P < 0.05). Anti-IL-6R combined with systemic inflammation (LPS 500 μg/kg) significantly increased neonatal mortality at 24 h to 86.1% compared to the LPS alone group (P < 0.05). At 21 days, only one death (4.3%) was observed in the group exposed to anti-IL-6R compared to five deaths in the control group (17.8%). However, this difference was not statistically significant (P = 0.2).

Table 1. Neonatal mortality
ParametersIgG + NSAnti-IL-6R + NSLPS 250 + IgGLPS 250 + anti-IL-6RLPS 500 + IgGLPS 500 + anti-IL-6R
  1. Resulting effects of inflammation alone or in association with anti-IL-6R on neonatal mortality at 24 h, 7 days, 14 days and 21 days after birth. IgG, immunoglobulin G; LPS 250, lipopolysaccharide 250 μg/kg; LPS 500, lipopolysaccharide 500 μg/kg; anti-IL-6R, interleukin-6 receptor antibody; NS, normal saline.

Resuscitated pupsn282330323036
Mortality, 24 hn401171931
%14.3036.721.963.386.1
Mortality, 7 daysn5012112131
%17.8040.034.470.086.1
Mortality, 14 daysn5112122332
%17.84.340.037.576.788.9
Mortality, 21 daysn5112152333
%17.84.340.046.976.791.7

Neonatal growth

Maternal systemic administration of LPS altered neonatal growth development in a dose-dependent fashion (Fig. 2 and Supplementary Table 1). Inflammation (LPS 250 μg/kg and LPS 500 μg/kg) increased weight of pups at birth and at 21 days of life compared to controls (P < 0.01). In the same manner, antenatal administration of anti-IL-6R increased weight of pups at 21 days when compared to controls (P = 0.02). Surprisingly, when anti-IL-6R was combined with LPS 500 μg/kg, a striking growth deficit was observed in pups (P < 0.01). Figure 3 illustrates a pup after antenatal exposure to LPS 500 μg/kg and a pup after antenatal exposure to LPS 500 μg/kg + anti-IL-6R (Fig. 3).

figure

Figure 2. Effects of maternal exposure to inflammation alone or in association with interleukin-6 receptor antibody (anti-IL-6R) on neonatal growth. LPS 250, lipopolysaccharide 250 μg/kg; LPS 500, lipopolysaccharide 500 μg/kg. Antenatal exposure to LPS at the end of gestation altered pup growth development. Maternal exposure to LPS (250 μg/kg, 500 μg/kg) increased neonatal growth in a dose-dependent manner (P < 0.01). However, exposure to anti-IL-6R in association with LPS 500 μg/kg had a surprising impact on pup weight and produced a severe growth deficit (P < 0.01). ***P < 0.01.

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figure

Figure 3. Effect of concomitant antenatal exposure to inflammation and interleukin-6 receptor antibody (anti-IL-6R) on postnatal growth of pups. LPS, lipopolysaccharide 500 μg/kg. A major impact on growth development was observed after antenatal exposure to LPS 500 μg/kg alone or in association with anti-IL-6R. Pups exposed to LPS 500 μg/kg were bigger than controls (P < 0.01), whereas pups exposed to LPS 500 μg/kg + anti-IL-6R presented a severe growth delay when compared to the LPS 500 μg/kg group (P < 0.01). ***P < 0.01.

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Systemic cytokine expression

Pregnant rats exposed to LPS alone presented a sharp increase in their systemic inflammatory cytokine expression (Fig. 4). IL-6 was increased in a dose-dependent fashion 3 h after i.p. injection (P < 0.01) (Fig. 4a) while TNF- α was increased 1 h after LPS 500 μg/kg administration (P < 0.01) (Fig. 4c). A trend of augmentation for IL-1 was also observed 3 h after maternal exposure to LPS 500 μg/kg (P = 0.08) (Fig. 4b).

figure

Figure 4. Effects of maternal exposure to inflammation in association with interleukin-6 receptor antibody (anti-IL-6R) on cytokine production (interleukin [IL]-6, IL-1, tumor necrosis factor [TNF]-α). (a) Maternal exposure to lipopolysaccharide (LPS) (250 μg/kg; 500 μg/kg) significantly increased IL-6 concentration 3 h after intraperitoneal (i.p.) injection (P < 0.01). Administration of anti-IL-6R with either dose of LPS did not modify IL-6 levels. (b) Maternal exposure to LPS 500 μg/kg did not significantly modify IL-1 concentration (P = 0.08). However, LPS 500 μg/kg + anti-IL-6R resulted in a tendency toward an increase in IL-1 levels (P = 0.06) 3 h after i.p. injection. (c) TNF-α concentration was significantly increased 1 h after i.p. injection of LPS 500 μg/kg (P < 0.01). Administration of anti-IL-6R in combination with LPS 500 μg/kg resulted in a tendency toward a decrease in TNF-α levels (P = 0.08) 3 h after i.p. injection. ***P < 0.01.

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Anti-IL-6R alone did not modify maternal inflammatory status and no significant difference in cytokine concentration (IL-1, IL-6, TNF-α) was observed at 1, 3 and 12 h after i.p. injection between anti-IL-6R-injected animals and IgG-injected controls. Three hours after concomitant administration of anti-IL-6R and LPS 500 μg/kg, we observed a tendency toward a decrease in TNF-α (P = 0.08) (Fig. 4c) and a tendency toward an increase in IL-1 levels (P = 0.06) (Fig. 4).

Open field

Neurological developmental behavior was similar for pups in the anti-IL-6R group when compared to pups in the control group (Table 2). Among 30 pups from the LPS 500 μg/kg group, only seven survived until 21 days and were explored with open field tests. Maternal injection of LPS 500 μg/kg had no significant impact on neurological developmental parameters, spontaneous locomotor activities or exploratory behaviors of surviving pups when compared to controls. Similar results were observed with the lower dose of LPS. Only three pups survived, out of 36 resuscitated pups, following injection of the pregnant dams with a combination of LPS 500 μg/kg and anti-IL-6R. When tested at day 21, individual study of these three pups showed that: (i) one was more active (total distance traveled, 9.5 m; mean speed, 3.2 cm/s; mobile time, 223 s; line crossings, 102); (ii) one was less active (total distance traveled, 1 m; mean speed, 0.3 cm/s; mobile time, 37 s; line crossings, 2); and (iii) one presented similar results to the LPS 500 μg/kg group.

Table 2. Neurodevelopmental behavior of pups
ParametersIgG + NSAnti-IL-6R + NSLPS 250 + IgGLPS 250 + anti-IL-6RLPS 500 + IgGLPS 500 + anti-IL-6R
n = 22n = 22n = 18n = 16n = 7n = 3
  1. Resulting effects of inflammation alone or in association with interleukin-6 receptor antibody on spontaneous locomotor activities and exploratory behaviors of pups at postnatal day 21 using the open field test. No significant differences were observed between pups exposed to control, LPS alone or in association with anti-IL-6R with regard to total distance, speed, line crossings and total mobile time. However, results are most likely underpowered as only a few pups (LPS 500 μg/kg, n = 7; LPS 500 μg/kg + anti-IL-6R, n = 3) survived 21 days and were assessed in the open field. IgG, immunoglobulin G; LPS 250, lipopolysaccharide 250 μg/kg; LPS 500, lipopolysaccharide 500 μg/kg; anti-IL-6R, interleukin-6 receptor antibody; NS, normal saline.

Distance (m)Median4.85.25.57.05.43.5
Interval3.6–7.23.7–6.53.8–7.53.9–10.04.9–7.21.0–9.5
Speed (cm/s)Median1.61.71.92.41.81.2
Interval1.2–2.41.2–2.21.3–2.51.3–3.41.6–2.40.3–3.2
Lines crossedMedian61.560.565.582.070.046.0
Interval41.0–88.842.0–82.352.3–116.339.3–142.062.0–109.02.0–100.0
Time mobile (s)Median121.1100.0102.8123.1110.6122.0
Interval72.7–149.076.0–124.473.3–140.077.5–162.5102.2–123.936.8–223.3

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We demonstrated that administration of anti-IL-6R during rat pregnancy, accompanied by LPS-induced inflammation, resulted in increased maternal mortality, neonatal mortality and a severe growth deficit in the surviving pups. Blocking the IL-6 pathway also partially affected the inflammatory cascade and promoted a tendency toward an increase in maternal serum IL-1 levels.

Effect of inflammation on pups

Cytokines are important mediators of inflammation and are widely interrelated in the inflammatory cascade. LPS increases TNF production which in turn upregulates IL-1 and IL-6 production.[17] Different LPS models have been used in pregnant animals along with varying doses and time of injection to study the inflammatory cascade. Following intrauterine injection, Chang et al. showed that LPS reaches the placental compartment as revealed by an increase in placental IL-6 concentration.[18] Similar results were also observed by Girard et al. who demonstrated an increase in IL-6 concentration in the placenta after i.p. LPS injection.[8] Other studies have shown that LPS also reaches the fetal compartment as witnessed by an increase in IL-6 concentration in fetal tissues, such as fetal brain[18] and fetal liver.[19] In the present model, antenatal inflammation induced by a single LPS injection (500 μg/kg) significantly increased neonatal mortality. Such increase in perinatal mortality rate has also been reported in animal models using multiple i.p. LPS injections: Girard et al. observed a reduction in fetal survival rate at birth with LPS 200 μg/kg from G18 to G20[8] while De-Xiang Xu et al. observed an increase in fetal death rate with LPS 75 μg/kg from G15 to G17.[20] Fetal death following antenatal LPS administration has previously been linked to structural abnormalities in the uteroplacental vasculature and deficient placental blood flow.[8, 21] Furthermore, antenatal inflammation has also been shown to result in alterations in fetal growth and development in several studies, whereby pups exposed to LPS presented an intrauterine growth deficit.[8, 20, 21] Surprisingly, in our model, pups exposed to LPS showed a significant increase in fetal and neonatal weight gain compared to controls. As suggested by previous studies which demonstrated that serum IL-6 levels are higher in patients with gestational diabetes,[22, 23] high IL-6 production may aggravate insulin resistance in pregnancy and participate in the pathogenesis of gestational diabetes and macrosomia.[24, 25] The Barker hypothesis may also account for the weight gain observed in our model. This hypothesis suggests that early-life metabolic adaptations help in survival by selecting an appropriate trajectory of growth in response to environmental cues and thus increase susceptibility to metabolic disorders, such as obesity and type II diabetes. Lastly, several studies have demonstrated that LPS-induced antenatal inflammation alters neurodevelopmental behavior in pups: Girard et al. showed diminished motor capacities[8] whereas others showed increased anxiety-related behavior.[26, 27] However, in our model, no difference was observed in spontaneous locomotor activities or in exploratory behaviors between pups exposed to LPS or LPS + anti-IL-6R and those exposed to control IgG. This could be accounted for by the fact that only a minority of animals, namely, the stronger pups, were alive at 21 days, while pups more likely to present a neurodevelopmental delay, namely, the sickest offspring, died before their neurological behavior could be assessed at 21 days of life.

Effect of IL-6 signaling blockade

In our model, blockade of the classic IL-6 signaling pathway in inflammatory and pregnant conditions had deleterious effects on the dams. The increase in maternal mortality following injection of LPS + anti-IL-6R could be explained by four factors: (i) the anti-inflammatory properties of IL-6; (ii) modification of the IL-6 cascade during pregnancy; (iii) amplification of the IL-1 cascade following blockade of IL-6 signaling; and (iv) the cumulative pro-inflammatory effect of inflammation, pregnancy and IL-6 signaling blockade.

Interleukin-6 has traditionally been described as a pro-inflammatory cytokine given that it is frequently increased in acute inflammatory processes[28] as well as in chronic inflammatory and autoimmune diseases.[29] However, as demonstrated by Xing et al., IL-6 has an important anti-inflammatory role through downregulation of pro-inflammatory cytokines such as IL-1 and TNF-α.[30] During pregnancy, several studies have shown that IL-6 levels, in non-inflammatory conditions, are increased: Lashley et al. observed that peripheral blood cells of pregnant women produced more IL-6 than non-pregnant controls[31] while Unal et al. showed that maternal IL-6 levels in serum were further increased at onset of labor.[32] However, in inflammatory conditions, a particular interaction exists between pregnancy and the regulation of the inflammatory cascade. Denney et al. observed, as pregnancy is carried to term, a dampened IL-6 response in controls as well as following in vitro induced-inflammation, suggesting that the immune system modulates itself during pregnancy to allow tolerance of the fetal allograft.[33] This immune regulation diminishes the defense mechanisms and may be harmful for the mother, especially at the end of pregnancy. In the present model, blockade of IL-6 signaling with anti-IL-6R promoted a tendency toward an increase in IL-1 concentration, resulting in a pro-inflammatory state. Girard et al. previously demonstrated that IL-1 plays an important role in mediating severe placental damage and neonatal neurodevelopmental anomalies.[8] These authors also illustrated the pro-inflammatory properties of IL-1 by demonstrating better neonatal outcomes with blockade of IL-1 signaling.[8] No maternal mortality was reported in our model and in the published work with LPS doses of 500 μg/kg or less.[7, 8] However, Cai et al. observed significant maternal mortality upon i.p. injection of higher doses of LPS.[34] In our model, the maternal mortality following the administration of LPS + anti-IL-6R suggests that IL-6 signaling blockade amplifies the pro-inflammatory effects of inflammation and pregnancy, allowing these effects to reach a critical threshold. The deleterious outcomes observed are hence similar to the deleterious outcomes that would be observed at higher inflammation levels.

Blockade of the classic IL-6 signaling pathway in inflammatory conditions also had adverse effects on pups, as illustrated by increased neonatal mortality and postnatal growth deficit. The cumulative pro-inflammatory effects of IL-6 signaling blockade as demonstrated by the tendency toward the amplification of the IL-1 cascade and by the blockade of the anti-inflammatory properties of IL-6 are all factors that may explain these findings.

In a recent study, Lee et al. suggested that the trans-signaling IL-6 signaling pathway plays a critical role in preterm birth and premature rupture of membranes by demonstrating an increase in amniotic fluid concentration of soluble IL-6 receptor and a decrease in the concentration of soluble gp130, a natural inhibitor of the IL-6 trans-signaling pathway.[12] These findings combined with the present results raise the idea that the classic and the trans-signaling IL-6 pathways may modulate different aspects of inflammation. Lee et al. suggested that targeting the trans-signaling pathway may prevent complications such as premature rupture of membranes and preterm birth. Conversely, we observed that targeting the classic pathway is associated with deleterious maternal and neonatal outcomes when associated with in utero inflammation.

In conclusion, modulation of the inflammatory cascade could represent a new therapeutic strategy in preterm labor induced by inflammation, although blockade of the IL-6 classic signaling pathway is associated with high maternal mortality rate and poor fetal outcome. Further work, with a broader number of targeted cytokines, should be performed to better understand the complete inflammatory process and effects of such signaling pathway blocking.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

This work was supported by a grant from the Foundation of Stars and was performed at the Centre de Recherche Clinique Etienne-Le Bel, a research center funded by the Fonds de la Recherche en Santé du Québec (FRSQ). We are grateful to Dr Éric Rousseau (Université de Sherbrooke, Quebec, Canada), Dr Guillaume Sébire (Université de Sherbrooke) and Dr Sylvie Girard (Université de Sherbrooke) for helpful discussion and to Nathalie Carrier (Université de Sherbrooke) for statistical analysis. We thank Tanya Fayad and Simon Blouin for help in the final revision of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
jog12089-sup-0001-si.doc26K

Table S1 Average weight calculation.

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