The role of IL-6 in neurodevelopment after prenatal stress

Abstract

Prenatal stress exposure is associated with adverse psychiatric outcomes, including autism and ADHD, as well as locomotor and social inhibition and anxiety-like behaviors in animal offspring. Similarly, maternal immune activation also contributes to psychiatric risk and aberrant offspring behavior. The mechanisms underlying these outcomes are not clear. Offspring microglia and the pro-inflammatory cytokine interleukin-6 (IL-6), known to influence microglia, may serve as common mechanisms between prenatal stress and prenatal immune activation. To evaluate the role of prenatal IL-6 in prenatal stress, microglia morphological analyses were conducted at embryonic days 14 (E14), E15, and in adult mice. Offspring microglia and behavior were evaluated after repetitive maternal restraint stress, repetitive maternal IL-6, or maternal IL-6 blockade during stress from E12 onwards. At E14, novel changes in cortical plate embryonic microglia were documented-a greater density of the mutivacuolated morphology. This resulted from either prenatal stress or IL-6 exposure and was prevented by IL-6 blockade during prenatal stress. Prenatal stress also resulted in increased microglia ramification in adult brain, as has been previously shown. As with embryonic microglia, prenatal IL-6 recapitulated prenatal stress-induced changes in adult microglia. Furthermore, prenatal IL-6 was able to recapitulate the delay in GABAergic progenitor migration caused by prenatal stress. However, IL-6 mechanisms were not necessary for this delay, which persisted after prenatal stress despite IL-6 blockade. As we have previously demonstrated, behavioral effects of prenatal stress in offspring, including increased anxiety-like behavior, decreased sociability, and locomotor inhibition, may be related to these GABAergic delays. While adult microglia changes were ameliorated by IL-6 blockade, these behavioral changes were independent of IL-6 mechanisms, similar to GABAergic delays. This and previous work from our laboratory suggests that multiple mechanisms, including GABAergic delays, may underlie prenatal stress-linked deficits.

Keywords: Embryonic; Gaba; IL-6; Microglia; Prenatal stress.

Figure 1

The experimental design included seven experimental groups and multiple time points of assessment.

Figure 2

Increased density of multivacuolated microglia after prenatal stress at E14 but not at E15. A, Embryonic day 14 (E14) embryonic microglia morphologies of four major subtypes: 1) multivacuolated, with multiple, pyknotic nuclei and/or vacuoles, 2) amoeboid, with normal nucleus and no processes, 3) transitional with normal nucleus and one process, and 4) ramified with normal nucleus and two or more processes. B, Prenatal stress resulted in increased multivacuolated cells at E14 (p=0.00095), C, which was not present at E15 (p=0.45). The density of transitional microglia is increased at E15 only (p=0.00049). Means ± SEMs are shown. n= 6-8 animals per group. (* p<0.0005 by two-tailed student's t-test; Arrows indicate cell nuclei. Arrowheads indicate cell processes.)

Figure 3

Prenatal stress (PS) and IL-6 both resulted in an increased density of multivacuolated microglia at E14 which was not present with anti-IL6. A, Prenatal IL-6 exposure resulted in an increased density of multivacuolated microglia at E14 over nonstress controls (NS/Saline) (p=0.00049). The density of transitional microglia was also increased (p=0.033). B, Anti-IL-6 concurrent with stress corrected the increase in multivacuolated microglia density resulting from prenatal stress exposure (ANOVA, interaction, F= 27.432, df=1, p<0.00005), with no difference between nonstress (NS) and nonstress/anti-IL-6 (NS/Anti-IL-6) or prenatal stress/anti-IL-6 (PS/Anti-IL-6) density (p=0.58, p=0.91). PS/Anti-IL-6 density was significantly lower than prenatal stress (p=0.0004). Means ± SEMs are shown. n= 6-7 animals per group. (* p<0.05, ** p<0.0005, † p<0.0005 by two-tailed student's t-test; § p<0.00005 interaction by one-way ANOVA)

Figure 4

IL-6 and prenatal stress with anti-IL6 both resulted in delays to GABAergic progenitor migration as with prenatal stress alone. Dashed line indicates prenatal stress (PS) migration average (69%); solid line indicates nonstress (NS) migration average (83%), as previously reported (Stevens et al., 2013). IL-6 exposure resulted in similar delays to these progenitors at E14 as occurs with prenatal stress (one-way ANOVA, F=16.476, df=3, p<0.0001; t-test: NS/Saline vs NS/IL-6 p=0.033). PS delayed migration was not corrected by anti-IL-6 (NS/Anti-IL-6 vs PS/Anti-IL-6 p<0.0001). The NS/Saline control and PS/Anti-IL-6 rescue conditions showed a trend difference (p=0.087). Anti-IL-6 advanced migration above saline control levels (p<0.0001). Means ± SEMs are shown. n= 6-8 animals per group. (* p<0.05, ** p<0.0001, # p=0.087 by two-tailed student's t-test)

Figure 5

Changes to adult microglia morphology after prenatal stress, IL-6, and anti-IL6. A, Adult microglia were categorized among four subtypes. B, In adult brain, prenatal stress increased highly ramified microglia (vs NS, p<0.0001), as did NS/IL-6 (vs NS, p<0.0001). Prenatal anti-IL-6 concomitant with stress (PS/Anti-IL-6) corrected this increase (non-significant vs. NS p=0.20), with less highly ramified microglia in PS/Anti-IL-6 compared to PS alone (p=0.002). PS decreased levels of lowly ramified cells (vs NS p=0.03), as did NS/IL-6 (vs NS p=0.01), while PS/Anti-IL-6 increased levels of these cells compared to PS animals (p=0.005) and did not differ from controls (p=0.36). Finally, as with lowly ramified cells, NS/IL-6 alone (vs. NS p=0.02) and PS (vs. NS p=0.0004) decreased levels of amoeboid cells, while anti-IL-6 concomitant with PS restored levels of these cells to NS levels (p=0.13), significantly lower than PS levels (p=0.02). Means ± SEMs are shown. n= 3-5 animals per group. (for comparisons to NS: *p<0.05, **p<0.0005; for comparisons to PS: † p<.05, ‡ p<0.005 by two-tailed student's t-test; Arrows indicate cell processes.)

Figure 6

Anti-IL-6 did not alter prenatal stress-induced behavioral phenotypes. A, Prenatal stress and saline (PS/Saline) resulted in decreased time spent in the open arm of the elevated plus maze over nonstress, saline controls (NS/Saline) (ANOVA, main effect of prenatal stress, F=10.562, df=1, p=0.003), indicative of elevated anxiety-like behavior, which was not ameliorated with concurrent anti-IL-6 administration. B, Animal sociability was decreased by prenatal stress, regardless of anti-IL-6 exposure (ANOVA, main effect of prenatal stress, F= 5.591, df=1; p=0.0258). Sociability ratio indicated an increase (values >0) or decrease (values <0) in animal sociability over time. C, Inhibited locomotor activity resulting from prenatal stress, was also not affected by anti-IL-6 (repeated measures between-subjects ANOVA, trend effect of prenatal stress, F=3.882, df=1, p=0.063). Means ± SEMs are shown. n= 7-9 adult males. (*p<0.05 by t-test; #p<0.05 by one-way ANOVA; † trend effect by one-way ANOVA; ‡ trend effect by repeated measures ANOVA)

Figure 7

IL-6 did not alter behavioral phenotypes in an independent cohort. A, Prenatal interleukin-6 treatment in nonstressed (NS/IL-6) offspring did not change time spent in the open arm of the elevated plus maze over nonstress controls (p=0.24). B, Animal sociability was unchanged by IL-6 (p=0.72). C, Locomotor activity was also unchanged by IL-6 treatment (repeated measures between-subjects ANOVA, F=0.981, df=3.166, p=0.409). Means ± SEMs are shown. n=12-14 adult males.