Offspring’s microglia show reduced proliferation and a Type-I interferon signature following maternal immune activation

In our paper by Matcovitch-Natan et al. 2016 [74], we demonstrated an early maturation of newborn’s microglia following MIA. The MIA was induced by injecting pregnant females, on embryonic day 14.5 (E14.5), with poly(I:C) (5 mg/kg) or PBS, as control (the paradigm used is schematically shown in Fig. 1a). Since a viral infection, or administration of the viral mimetic poly(I:C), results in a general upregulation of inflammatory cytokines [4, 9,10,11,12], including IFN-I [9, 15, 16], we envisioned that the effect of MIA on the newborn’s microglia might involve IFN-I signaling. To investigate this, we further analyzed RNA-seq data from our previous paper derived from microglia of newborn mice following MIA [74]. We found 594 genes with highly divergent expression between the treated and control groups (190 downregulated and 404 upregulated; Fig. 1b, Supplementary Table 1). GO analysis [77,78,79,80] revealed that newborn’s microglia derived from the poly(I:C) group showed lower expression levels of genes related to cell cycle and proliferation (Fig. 1c). In order to test whether the effect of MIA on microglia could be IFN-dependent, we analyzed the RNA-seq results using “Interferome”, the database of IFN-regulated genes [81]. This analysis revealed that approximately two-thirds of the differentially expressed genes (384 genes) are regulated by Type I and II interferons, out of which the majority are genes affected by Type I interferon (Fig. 1d). Specifically, the analysis showed that IFNβ had the greatest effect, compared with IFNα, on newborn’s microglial gene expression following MIA (Fig. 1d).

To confirm the RNA-seq results, we repeated the same MIA procedure and examined the newborn’s microglia for protein expression of the proliferation marker Ki67 by flow cytometry (Fig. 1a, e–g). Microglia from the brains of the dams were used as a negative control, due to their negligible Ki67 expression (Fig. 1e). We found that a significantly lower percentage of microglia derived from offspring of poly(I:C)-treated dams expressed Ki67, as compared with the percentage of microglia derived from offspring of PBS-treated animals (Fig. 1f). Cell quantification showed a trend toward a reduction in the total number of microglia following MIA (Fig. 1g). These results demonstrated that MIA resulted in reduced proliferation of newborn’s microglia.

Next, we assessed whether newborn’s microglia could be affected by IFN-I produced by the dams in response to poly(I:C), as a possible mechanism underlying the effect of MIA (Fig. 1b, d). Accordingly, we checked, using immunofluorescence, whether newborn’s microglia express IFN receptor 1 (IFNAR1). We found that CX3CR1+ microglia express IFNAR1 (Fig. 1h), and thus could respond to IFN-I. These results show that MIA downregulates microglial proliferation, and that this effect might involve IFN-I signaling.

Maternal immune activation elicits a Type-I interferon response in the embryonic yolk sac

The results above led us to hypothesize that if the effect on newborn’s microglia following MIA is mediated through maternal IFN-I, we might detect the IFN-I signature in the yolk sac, the source of microglial progenitors [82, 83], at an early time point after the MIA. To this end, pregnant females were injected on E14.5 with poly(I:C) or PBS, and 24 h later, the embryonic yolk sac was excised and analyzed by RTqPCR (Fig. 1i). We detected increased expression levels of IFN-I-dependent genes [including IFN-β 1 (ifnβ), IFN regulatory factor 3 (irf3), IFN regulatory factor 7 (irf7), IFN regulatory factor 9 (irf9), IFN-induced protein with tetratricopeptide repeats 1 (ifit1), and IFN-induced transmembrane protein 3 (ifitm3)] following MIA (Fig. 1j). Changes in other genes were detected as well, such as NF-kB-dependent inflammatory cytokines [interleukin 1 beta (il-1β), tumor necrosis factor alpha (tnfα), and interleukin 6 (il-6)] [84] and the myeloid markers cd45 and cd11b [85] (Fig. 1j). These results revealed an IFN-I signature in the offspring at an early time point following MIA.

IFN-I is an important regulator of microglial proliferation

To get a deeper insight into the role of IFN-I during development, we tested whether IFN-I has a homeostatic role in regulating microglial proliferation, using IFNARKO mice [56]. We first compared the basal microglial proliferation in WT and IFNARKO offspring of nontreated dams. Analysis of Ki67 by flow cytometry revealed that a significantly higher percentage of IFNARKO microglia expressed the proliferation marker, as compared with the WT microglia (Fig. 2a). A trend toward an increase in total numbers of microglia was also detected (Fig. 2b). These results suggested that IFN-I participates in the normal regulation of microglial proliferation, and raised the question of whether the newborn’s microglial response to MIA would be different in the absence of IFN-I signaling. To this end, pregnant IFNARKO dams were exposed to poly(I:C) or PBS, and flow cytometry analysis for expression of the proliferation marker Ki67 by newborn’s microglia was performed. We found that following MIA, a significantly higher percentage of IFNARKO newborn’s microglia expressed Ki67, as compared with the PBS-treated group (Fig. 2c). Cell quantification showed a significant increase in total number of microglia, as well (Fig. 2d). RNA-seq of sorted IFNARKO newborn’s microglia from poly(I:C) and PBS groups supported the flow cytometry results (Fig. 2e, f); comparison of poly(I:C) to PBS IFNARKO newborn’s microglia showed highly divergent expression of 405 genes (175 upregulated and 230 downregulated; Fig. 2e, Supplementary Table 2). GO analysis [77,78,79,80] showed enrichment of genes related to proliferation and cell cycle in IFNARKO newborn’s microglia following the poly(I:C) treatment, compared with the PBS group (Fig. 2f). The results supported the possibility that IFN-I signaling is involved in regulating newborn’s microglial proliferation in homeostasis. Notably, in the absence of IFN-I signaling, MIA caused an increase in proliferation of newborn’s microglia, and thus elicited a response opposite to that in WT mice. These results further suggest that additional factors, beyond IFN-Ι, participate in regulating microglial proliferation, such as the NF-kB-dependent inflammatory cytokines, IL-1β, TNFα, and IL-6 (Fig. 1j), that might be masked in the presence of IFN-I signaling.

Fig. 2 IFN-I regulates microglial proliferation. a Flow cytometry analyses showing percentage of C57 and IFNARKO newborn’s microglia expressing the proliferation marker Ki67 (Student’s t test: t (two tailed) = 4.524, df = 9, **p = 0.0014), and b normalized number of microglia (to 35,000 live-gate events. Student’s t test: t (one tailed) = 1.603, df = 9, p = 0.0717). Representative results of one of two independent experiments. Data are presented as means ± s.e.m. n = 5–6 newborns per group. c Flow cytometry analyses showing percentage of IFNARKO newborn’s microglia expressing the proliferation marker Ki67 (Student’s t test: t (two tailed) = 2.934, df = 22, **p = 0.0077), and d normalized number of microglia (to 40,000 live-gate events. Student’s t test: t (two tailed) = 3.3237, df = 22, **p = 0.0038) following MIA. Representative results of one of two independent experiments. Data are presented as means ± s.e.m. n = 12 newborns per group. e Volcano plot for RNA-seq data shows the fold change and significance of genes of IFNARKO newborn offspring of dams treated with either poly(I:C) or PBS [175 genes significantly upregulated (red) and 230 significantly downregulated (blue)]. f Gene ontology (GO) analysis [77,78,79,80] of the RNA-seq results showing enrichment of terms related to proliferation and cell cycle following the poly(I:C) treatment Full size image

Maternal IFNβ signaling downregulates newborn’s microglial proliferation

To validate the active involvement of IFN-I, elicited by the dams in response to MIA, in impairing newborn’s microglia, we tested whether blocking IFN-I signaling prior to poly(I:C) treatment would mitigate the effect of MIA on newborn’s microglial proliferation. For this purpose, we used antibodies to block the maternal-IFN-I signaling in WT dams. Specifically, pregnant C57 dams were injected 1 day prior to poly(I:C) treatment [on embryonic day 13.5 (E13.5)], with anti-IFN receptor 1 (αIFNAR; MAR1-5A3 [60], 400 µg) or IgG (IgG1; MOPC-21, 400 µg), to allow efficient blockade before the MIA-induced IFN-I effect on the offspring (Fig. 1i, j). Subsequently, the newborn’s microglia were isolated and tested (Fig. 3a). The control group was injected with IgG on E13.5 and with PBS on E14.5 (IgG–PBS group) to determine the basal levels of microglial proliferation and cell numbers (Fig. 3a). Flow cytometry analysis of newborn’s microglia for the proliferation marker Ki67 showed that maternal treatment with αIFNAR prior to MIA [αIFNAR-Poly(I:C)] diminished the negative effect of MIA on microglial proliferation, as compared with the group that was treated with IgG prior to the poly(I:C) [IgG-Poly(I:C); Fig. 3b]. No changes in the total number of microglia were apparent (Fig. 3c).

Fig. 3 Maternal IFNβ signaling downregulates newborn’s microglial proliferation. a Pregnant females were i.v. injected on E13.5 with 400 µg αIFNAR (αIFNAR; MAR1-5A3) or IgG (IgG1; MOPC-21), and 1 day later, on E14.5 with poly(I:C). Females from the control group were injected with IgG on E13.5 and with PBS on E14.5 (IgG-PBS) to determine the basal levels of microglial proliferation and cell numbers. The newborn’s microglia were analyzed by flow cytometry. b Flow cytometry analyses showing percentage of microglia expressing the proliferation marker Ki67 (Student’s t test: t (one tailed) = 1.735, df = 22, *p = 0.0484), and c normalized number of microglia (to 30,000 live-gate events). Dashed line represents the IgG–PBS control average. Combined results from two different experiments. Data are presented as means ± s.e.m. n = 12 newborns per group. d Pregnant dams were i.v. injected with IFNβ (22,700 U) or vehicle on E14.5, and newborn’s microglia were examined by RNA-seq and by flow cytometry. e Volcano plot for RNA-seq data shows the fold change and significance of gene expression between microglia of newborn offspring of dams treated IFNβ or vehicle [257 significantly downregulated (blue) and 57 significantly upregulated (red)]. f Flow cytometry analyses showing percentage of microglia expressing the proliferation marker Ki67 following maternal vehicle and IFNβ treatments (Student’s t test: t (two tailed) = 3.139, df = 10, *p = 0.0105), and g normalized number of microglia (to 20,000 live-gate events). Representative results of one of two independent experiments. Data are presented as means ± s.e.m. n = 6 newborns per group. h Flow cytometry analyses showing percentage of microglia expressing the proliferation marker Ki67 following maternal vehicle and IFNγ treatments, and i normalized number of microglia (normalized to 30,000 live-gate events. Student’s t test: t (two tailed) = 2.339, df = 14, *p = 0.0347). Representative results of one of two independent experiments. Data are presented as means ± s.e.m. n = 8 newborns per group. j Flow cytometry analyses showing percentage of microglia expressing the proliferation marker Ki67 following maternal vehicle and TNFα treatments, and k normalized number of microglia (to 60,000 live-gate events). Data are presented as means ± s.e.m. n = 13–16 newborns per group Full size image

These results prompted us to examine whether maternal elevation of IFN-I could, by itself, mimic the effect of MIA on microglial proliferation in the offspring. Since our “interferome” analysis showed that IFNβ had the greatest effect among the IFN-I family members on newborn’s microglial gene expression following MIA (Fig. 1d), we decided to use this cytokine for our subsequent experiments. Pregnant dams were injected at E14.5 with IFNβ (22,700 U) [61] or vehicle, as a control (see Materials and Methods), and the microglia of newborn offspring were examined (Fig. 3d). We first verified the signature of the maternal IFNβ treatment on the microglia of the offspring by RNA-seq. Analysis revealed 57 and 257 genes that exhibited at least 1.5-fold increased or decreased expression, respectively, in offspring of IFNβ-treated dams, compared with vehicle-injected controls (Fig. 3e, Supplementary Table 3). We detected upregulation of various genes related to inflammation, IFN signaling, and the viral response (e.g., ctla2b, lmln, ddit4, and gpr65). Next, we assessed the proliferation of newborn’s microglia by flow cytometry. A significantly lower percentage of microglia expressed Ki67 following maternal IFNβ injection, as compared with controls (Fig. 3f). No change in the total number of microglia was detected (Fig. 3g). These results demonstrated that the mere maternal elevation of IFNβ was sufficient to induce an effect on the microglia of the offspring, which was manifested by their reduced proliferation.

Because other factors are likely involved in the regulation of the newborn’s microglia (Figs. 1i, j and 2), and in order to determine whether maternal IFNβ uniquely reduces their proliferation, we assessed the effect of additional cytokines. To this end, we injected pregnant dams at E14.5 with interferon gamma (IFNγ; 5000 U [62]), a Type-II interferon member (Fig. 1d), or with TNFα [2.7 × 105 U (1 μg) [63]], an NF-kB-dependent inflammatory cytokine (Fig. 1j). Analyses by flow cytometry reveled that maternal treatment with IFNγ did not elicit a change in percentage of newborn’s microglia expressing Ki67 (Fig. 3h), however, a higher number of microglial cells was observed (Fig. 3i). Analyses of the newborn’s microglia following maternal TNFα treatment, did not show any change in percentage of microglia expressing Ki67, nor in number of microglial cells (Fig. 3j, k). These results suggest that although many factors could be involved in regulation of microglia, IFNβ seems to uniquely reduce their proliferation.

Elevated IFNβ during pregnancy imposes behavioral abnormalities in the offspring

The results above encouraged us to examine whether the prenatal exposure to IFNβ would have any effect on the behavior of the offspring at adolescence and adulthood (Fig. 4, Supplementary Fig. 1). For this purpose, pregnant dams were injected on E14.5 with vehicle or a high dose of IFNβ (45,400 U) to elicit a strong response, and behavior of the offspring was assessed. Specifically, at the age of 1 month we evaluated the repetitive behavior and anxiety of the offspring using marble burying and elevated plus maze tests [66, 67]. At the age of 3 months the offspring were tested for sociability by social preference test [68], and again by marble burying and elevated plus maze tests. At the age of 4 months the offspring were tested for anxiety by open field arena [69, 70] and for working memory by spontaneous alternation test in the Y-maze [71] (Fig. 4a). Overall, we found different response of the sexes to the maternal IFNβ treatment. In female offspring, we found at the age of 1 month that the IFNβ group buried a significantly higher number of marbles in the first 5 min of the marble burying test, as compared with the control group (Fig. 4b). At the age of 3 months, we found a significant reduction in social preference following maternal IFNβ treatment (Fig. 4c, d), and at 4 months we found a significantly higher percentage of spontaneous alternation, compared with the control group (Fig. 4e). In male offspring, we found at the age of 3 months, reduced exploration time of open arms in the elevated plus maze following maternal IFNβ treatment (Fig. 4f, g). This was not due to reduced distance covered by the animals (Fig. 4h). At the age of 4 months, we detected a trend toward reduced time spent at the center of the open field arena following the maternal treatment, which was not accompanied by change in total distance covered by the animals (Fig. 4i, j). These results demonstrate that maternal upregulation of IFNβ is manifested by changes in behavior of the offspring with different characteristics in females and males.

Fig. 4 Maternal elevation of IFNβ leads to behavioral abnormalities in offspring. a Pregnant females were i.v. injected on E14.5 with IFNβ (45,400 U) or vehicle. At the age of 1 month, the offspring were tested by marble burying and elevated plus maze tests. At the age of 3 months the offspring were tested by social preference, marble burying, and elevated plus maze tests. At the age of 4 months the offspring were tested by open field arena and by spontaneous alternation (Y-maze) tests. b Number of buried marbles by 1-month-old female offspring of dams treated with vehicle versus IFNβ, during the first 5 min of the marble burying test (Student’s t test: t (one tailed) = 1.903, df = 12, *p = 0.0406). c Percent stranger exploration time by 3-month-old female offspring in social preference test (Student’s t test: t (two tailed) = 2.446, df = 13, *p = 0.0294), and d representative heatmaps. STR -stranger mouse, OBJ -novel object. e Percent spontaneous alternation in Y-maze by 4-month-old female offspring (Student’s t test: t (one tailed) = 1.808, df = 13, *p = 0.0469). f Time spent in open arms of the elevated plus maze by 3-month-old male offspring (Student’s t test: t (two tailed) = 2.212, df = 13, *p = 0.0455), and g representative heatmaps. h Total distance of 3-month-old male offspring in the elevated plus maze arena. i Time spent in the center of the open field arena by 4-month-old male offspring (Student’s t test: t (one tailed) = 1.410, df = 13, p = 0.091), and j total distance covered in the arena. Data are presented as means ± s.e.m. n = 5–10 offspring of 2–3 dams per group Full size image

Elevated IFNβ during pregnancy increases the vulnerability of the offspring and their microglia to postnatal stress

Finally, we examined whether the exposure to the upregulated maternal IFNβ during pregnancy renders the offspring’s microglia less resilient to stressful conditions. We envisioned that this would be in line with the “Two-hit” model of neuropsychiatric disorders, according to which two “hits” are required for the pathology to occur; a first “hit” during prenatal life disrupts the offspring’s CNS development, thereby causes increased vulnerability to a second “hit”, which might occur later in life and would lead to the pathological onset of disease [19,20,21,22]. To this end, pregnant dams were injected on E14.5 with vehicle or a high dose of IFNβ (45,400 U) (first “hit”), and the newborn pups were subsequently subjected to a 2-week maternal separation (MS) protocol [on postnatal days 1–14 (P1–P14)] as a source of stress (second “hit”). Control groups were left with their mothers (Fig. 5a). We tested whether microglia of offspring that were subjected to MS would show a stronger inflammatory response if the dams were first injected with IFNβ during pregnancy. For this, microglia were harvested from the pups on P15, and were analyzed by flow cytometry (Fig. 5b–g) and by immunofluorescence (Fig. 5h, i) to detect changes in their inflammatory and activation state. We started by measuring microglial TNFα expression by flow cytometry. To analyze the entire microglial population, including activated cells, we analyzed all the CD11b+CD45+cells (Fig. 5b). Most of the cells were CD11bintCD45int, and thus mainly represented the microglial population. The effect of maternal IFNβ treatment on microglial TNFα expression was calculated as the expression ratio relative to the relevant vehicle-treated group (control or MS). In both control and MS groups, the microglial TNFα mean fluorescence intensity (MFI) was significantly increased in samples derived from pups of dams treated with IFNβ, relative to vehicle (Fig. 5c, d). Calculations of effect size using Cohen’s d showed an effect size of d = 0.9838 for the IFNβ treatment in the control groups, and an effect size of d = 2.71 for IFNβ treatment in the MS groups. To verify that the changes in TNFα occurred mainly in the microglia and were not due infiltration of monocyte-derived macrophages, expression of Ly6C and CX3CR1 was measured for the CD11b+CD45+TNFα+ cells (Fig. 5e). We found that, on average, 99.19 ± 0.037% of the cells were Ly6C−CX3CR1+, suggesting that TNFα was produced almost exclusively by microglia. In addition, we assessed microglial CD45 expression and found that following maternal treatment with IFNβ, the microglial CD45 MFI was higher only after MS, while expression in the control group remained unchanged (Fig. 5f). The results were not accompanied by any changes in the total number of microglia (Fig. 5g). Next, brains of the offspring from the MS groups (IFNβ and vehicle) were isolated, and analyzed using immunofluorescence to estimate microglial activation [86]. Analysis of IBA-1 immunoreactivity in hippocampi revealed increased expression in sections derived from brains of IFNβ–MS pups, as compared with the vehicle-MS group (Fig. 5h, i). These results showed that maternal elevation of IFNβ increased the sensitivity of the offspring-derived microglia to postnatal stress. Finally, we tested whether IFNβ exacerbates the effect of MS at the behavioral level. Offspring from the MS groups (IFNβ and vehicle) were examined with the same battery of tests as described above (Fig. 4a). Overall, we found different responses of the sexes to the treatment, with the main effect in females (Fig. 5j–n, Supplementary Fig. 2). At the age of 1 month, female offspring of the IFNβ–MS group buried a significantly higher number of marbles within the first 5 min of the test, relative to the vehicle-MS group (Fig. 5j). At the age of 3 months, IFNβ–MS female offspring showed a trend of burying a higher number of marbles, as compared with the vehicle-MS group (Fig. 5k). Those females also demonstrated a trend of spending more time in the open arms of the elevated plus maze (Fig. 5l), which may suggest increased risk-taking behavior, since they did not spend more time in closed arms, although they covered a significantly greater distance (Fig. 5m, n). These results demonstrate that maternal elevation of IFNβ renders the offspring less resilient to stressful conditions.