Maternal immune activation by poly I:C induces expression of cytokines IL-1β and IL-13, chemokine MCP-1 and colony stimulating factor VEGF in fetal mouse brain

Abstract

Background: Maternal viral infection during pregnancy is associated with an increase in the incidence of psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The enhanced risk for developing mental illness appears to be caused by deleterious effects of innate immune response-associated factors on the development of the central nervous system, which predispose the offspring to pathological behaviors in adolescence and adulthood. To identify the immune response-associated soluble factors that may affect central nervous system development, we examined the effect of innate immune response activation by polyriboinosinic-polyribocytidylic acid (poly(I:C)), a synthetic analogue of viral double-stranded RNA, on the expression levels of pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors in fetal and postnatal mouse brain 6 h and 24 h after treatment.

Methods: C57BL/6J pregnant mice (gestational day 16) or newborn mice (postnatal day 4) received a single intraperitoneal injection of the synthetic analogue of viral double-stranded RNA poly(I:C) (20 mg/kg). Thirty-two immune response-associated soluble factors, including pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors, were assayed 6 h and 24 h after poly(I:C) injection using multiplexed bead-based immunoassay (Milliplex Map) and processed in a Luminex 100 IS instrument.

Results: Maternal exposure to poly(I:C) at gestational day 16 induced a significant increase in cytokines interleukin (IL)-1β, IL-7 and IL-13; chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, interferon gamma-induced protein (IP)-10 and monokine induced by IFN-gamma (MIG); and in the colony stimulating factor vascular endothelial growth factor (VEGF) in the fetal brain. IL-1β showed the highest concentration levels in fetal brains and was the only cytokine significantly up-regulated 24 h after maternal poly(I:C) injection, suggesting that IL-1β may have a deleterious impact on central nervous system development. In contrast, poly(I:C) treatment of postnatal day 4 pups induced a pronounced rise in chemokines and colony stimulating factors in their brains instead of the pro-inflammatory cytokine IL-1β.

Conclusions: This study identified a significant increase in the concentration levels of the cytokines IL-1β and IL-13, the chemokine MCP-1 and the colony stimulating factor VEGF in the developing central nervous system during activation of an innate immune response, suggesting that these factors are mediators of the noxious effects of maternal immune activation on central nervous system development, with potential long-lasting effects on animal behavior.

Figure 1

Analysis of immune response-associated soluble factors expression in fetal brain homogenates after maternal innate immune activation. Pregnant mice received a single i.p. injection of poly(I:C) or PBS on GD16 and were killed 6 h and 24 h after treatment. IRSF concentrations in fetal brain homogenates were determined by multiplexed bead-based immunoassay (Milliplex Map). Only factors detected at least at one time-point are plotted. (A) Comparison of the expression levels of pro- and anti-inflammatory cytokines between saline and poly(I:C) injected animals. IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12(p40), IL-12(p70), IFN-γ, TNF-α and LIF were not detected at both time-points. At 6 h after treatment, IL-7 and IL-13 showed significantly higher concentrations as compared to PBS-treated animals. At 24 h post-treatment, only IL-1β showed significantly higher expression levels compared to saline-treated animals. Circadian variations in control animals in the concentration levels of IL-1β and IL-9 were also detected. (B) Comparison of chemokine expression levels showed increased concentration levels of IP-10 and MIG 6 h after treatment while MCP-1, IP-10 and MIP-1α were increased at 24 h post-treatment. Significant changes in control animals between the two times of the day were observed for eotaxin, MCP-1, MIP-1α and MIG. LIX and MIP-2 were not detected. (C) Analysis of changes in CSF expression levels showed that only VEGF was significantly up-regulated 6 h after poly(I:C) treatment. VEGF expression was also significantly different between the 4 p.m. and 10 a.m. time-points. G-CSF was below detection limits. The numbers of animals analyzed were as follows: PBS 6 h, n = 10; poly(I:C) 6 h, n = 10; PBS 24 h, n = 10; and poly(I:C) 24 h, n = 9. Values are presented as the mean ± standard error of the mean. Results obtained in pg/mL were normalized to pg/100 μg of total protein. *P <0.05 between PBS and poly(I:C) groups at 6 h and 24 h post-injection. ⁺P <0.05 between 6 h and 24 h after PBS injection. Statistical significance was based on Student’s t-test comparisons between groups. ND: not detected.

Figure 2

Comparison of immune response-associated soluble factors concentrations between brain homogenates and maternal serum in poly(I:C) treated animals 6 h after injection. Values were normalized to total protein content of each tissue. (A) Pro- and anti-inflammatory cytokines. IL-1β concentration was significantly lower in maternal serum compared with fetal brain homogenates, while IL-13 and IL-10 were significantly higher. IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12(p40), IL-12(p70), IL-15, IL-17, IFN-γ, TNF-α and LIF were not detected in fetal brain. (B) Chemokines. Maternal serum showed significantly higher concentration levels of eotaxin, MCP-1, IP-10 and MIG. MIP-1β, RANTES, KC, LIX and MIP-2 were not detected in fetal brain. (C) CSF. Only VEGF was detected in both tissues but no significant differences in their concentration levels were observed. *P <0.05 based on Student’s t-test comparisons between tissues. Values are expressed as the mean ± standard error of the mean; fetal brain, n = 10; maternal serum, n = 3. ND, not detected.

Figure 3

Comparison of immune response-associated soluble factors expression levels between GD17 and PND5 brain homogenates in phosphate-buffered saline-treated animals show a similar expression profile. (A) Pro- and anti-inflammatory cytokine expression levels were very similar in pre- and postnatal animals. IL-7, IL-13 and IL-15 were significantly higher in PND5 brain homogenates while IL-1β and IL-17 showed lower concentration levels compared with prenatal brains. IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12(p40), IL-12(p70), IL-15, IL-17, IFN-γ, TNF-α and LIF were not detected in GD17 brain. (B) Chemokines MIP-1α and MIG had lower concentration levels in postnatal compared with prenatal brains, while MIP-1β and KC showed higher concentration levels. LIX and MIP-2 were not detected in GD17 brain homogenates. (C) CSF, GM-CSF and M-CSF were significantly higher in postnatal brains while VEGF was significantly lower. G-CSF was not detected in GD17 brain homogenates. Values are expressed as the mean ± standard error of the mean; GD17, n = 10; PND5, n = 11. ND: not detected.

Figure 4

Analysis of the effect of poly(I:C) treatment on immune response-associated soluble factors expression levels between gestational day 17 and postnatal day 5 brain homogenates. (A) Comparison of IRSF concentrations normalized to total protein content. A significantly lower concentration of the cytokine IL-1β was detected in postnatal brain while IL-13 and IL-15 were significantly higher. The chemokine MIP-1α and the CSF VEGF were reduced in postnatal brain while IP-10, KC, MIG, GM-CSF and M-CSF were significantly higher. (B) Comparison of the percentage change in IRSF concentration levels induced by poly(I:C) between pre- and postnatal brain homogenates 24 h after treatment. Note the higher percentage changes in chemokines and CSF in the postnatal brain compared with less pronounced differences in cytokines. Values are mean ± standard error of the mean. *P <0.05 based on Student’s t-test comparisons. GD17, n = 9; PND5, n = 14. ND, not detected.