Maternal immunoglobulin G affects brain development of mouse offspring

doi:10.1186/s12974-024-03100-z

. 2024 May 2;21(1):114.

doi: 10.1186/s12974-024-03100-z.

Maternal immunoglobulin G affects brain development of mouse offspring

Mizuki Sadakata¹, Kazuki Fujii^{2

3

4}, Ryosuke Kaneko⁵, Emi Hosoya⁶, Hisako Sugimoto⁶, Reika Kawabata-Iwakawa⁷, Tetsuhiro Kasamatsu⁸, Shoko Hongo⁴, Yumie Koshidaka⁴, Akinori Takase⁹, Takatoshi Iijima¹⁰, Keizo Takao^{2

3

4}, Tetsushi Sadakata¹¹

Affiliations

¹ Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan. m2200038@gunma-u.ac.jp.
² Department of Behavioral Physiology, Faculty of Medicine, University of Toyama, Toyama, 930-0194, Japan.
³ Research Center for Idling Brain Science, University of Toyama, Toyama, 930-0194, Japan.
⁴ Life Science Research Center, University of Toyama, Toyama, 930-0194, Japan.
⁵ Medical Genetics Research Center, Nara Medical University, Kashihara, Nara, 634-8521, Japan.
⁶ Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.
⁷ Division of Integrated Oncology Research, Gunma University Initiative for Advanced Research (GIAR), Gunma University, Maebashi, Gunma, 371-8511, Japan.
⁸ Department of Medical Technology and Clinical Engineering, Gunma University of Health and Walfare, Maebashi, Gunma, 371-0823, Japan.
⁹ Medical Science College Office, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
¹⁰ Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
¹¹ Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan. sadakata-1024@gunma-u.ac.jp.

PMID: 38698428
PMCID: PMC11064405
DOI: 10.1186/s12974-024-03100-z

Maternal immunoglobulin G affects brain development of mouse offspring

Mizuki Sadakata et al. J Neuroinflammation. 2024.

. 2024 May 2;21(1):114.

doi: 10.1186/s12974-024-03100-z.

Authors

Affiliations

¹ Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan. m2200038@gunma-u.ac.jp.
² Department of Behavioral Physiology, Faculty of Medicine, University of Toyama, Toyama, 930-0194, Japan.
³ Research Center for Idling Brain Science, University of Toyama, Toyama, 930-0194, Japan.
⁴ Life Science Research Center, University of Toyama, Toyama, 930-0194, Japan.
⁵ Medical Genetics Research Center, Nara Medical University, Kashihara, Nara, 634-8521, Japan.
⁶ Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.
⁷ Division of Integrated Oncology Research, Gunma University Initiative for Advanced Research (GIAR), Gunma University, Maebashi, Gunma, 371-8511, Japan.
⁸ Department of Medical Technology and Clinical Engineering, Gunma University of Health and Walfare, Maebashi, Gunma, 371-0823, Japan.
⁹ Medical Science College Office, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
¹⁰ Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
¹¹ Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan. sadakata-1024@gunma-u.ac.jp.

PMID: 38698428
PMCID: PMC11064405
DOI: 10.1186/s12974-024-03100-z

Abstract

Maternal immunoglobulin (Ig)G is present in breast milk and has been shown to contribute to the development of the immune system in infants. In contrast, maternal IgG has no known effect on early childhood brain development. We found maternal IgG immunoreactivity in microglia, which are resident macrophages of the central nervous system of the pup brain, peaking at postnatal one week. Strong IgG immunoreactivity was observed in microglia in the corpus callosum and cerebellar white matter. IgG stimulation of primary cultured microglia activated the type I interferon feedback loop by Syk. Analysis of neonatal Fc receptor knockout (FcRn KO) mice that could not take up IgG from their mothers revealed abnormalities in the proliferation and/or survival of microglia, oligodendrocytes, and some types of interneurons. Moreover, FcRn KO mice also exhibited abnormalities in social behavior and lower locomotor activity in their home cages. Thus, changes in the mother-derived IgG levels affect brain development in offsprings.

Keywords: Brain development; Immunoglobulin G; Microglia.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig. 1**
Immunoreactivity of IgG in microglia of the brains of infant mice. (**A–E**) Sagittal sections of C57BL/6J mice at postnatal day 8 (P8) were immunolabeled with an anti-Iba1 antibody (green) and anti-mouse IgG antibody (red). Scale bars, 50 μm. (**F, G**) Sagittal sections of mice at P8 were immunolabeled with anti-Iba1 antibody (green) and anti-CD16/32 antibody (red). Scale bars, 100 μm. (H) Flow cytometry data showing staining of microglia with specific antibodies (red) and isotype control (black) at P8. (I) Flow cytometry quantification of the expression of CD16, CD32, and CD64 in microglia is indicated by fluorescence intensity. Ctrl, isotype control. The p-values from Student’s t-test are indicated. (J) Representative plots of flow cytometry data showing staining of microglia with anti-mouse IgG antibodies and isotype control at P8, with or without fixation and permeabilization. (K) Quantification of the percentage of microglia stained with anti-mouse IgG, with (red) or without (blue) fixation and permeabilization, at P0, P4, P8, and P12 [P0 with permeabilization (n = 3); P0 without permeabilization (n = 3); P4 with permeabilization (n = 4); P4 without permeabilization (n = 3); P8 with permeabilization (n = 7); P8 without permeabilization (n = 3); P12 with permeabilization (n = 3); P12 without permeabilization (n = 3)]. Two-way analysis of variance revealed a significant effect on localization (p < 0.0001) and stage (p < 0.0001). P-values from Fisher’s protected least-significant difference (PLSD) post hoc tests for pairwise comparisons are indicated

**Fig. 2**
Comprehensive analysis of microglial genes differentially expressed by IgG stimulation. (**A, B**) Primary cultured microglia were immunolabeled with anti-mouse IgG antibody (green) and anti-CD16/32 antibody (red) without IgG addition (A) and 24 h after IgG addition to the culture media (B). Scale bars, 50 μm. (C) Volcano plots of DEGs between the control and primary cultured microglia stimulated with 10 µg/ml IgG for 24 h. Representative IFN-I-related genes are shown in red. (D) Graphical summary of the pathways regulated after IgG treatment generated by ingenuity pathway analysis (IPA). Orange and blue indicate activation and inhibition, respectively. The solid line indicates a direct interaction, the dashed line indicates an indirect interaction, and the dotted line indicates an inferred correlation from machine-based learning. (E) Significant pathways identified by IPA. Positive and negative z-scores, colored in orange and blue, represent the activation and inhibition of pathways, respectively. (F) Significant activation of the IFN-I signaling pathway was predicted using IPA. Orange indicates activation

**Fig. 3**
IgG activates the IFN-I feedback loop via Syk. (A) Immunoblot analysis of primary cultures of IgG-stimulated microglia. Protein lysates were immunoblotted using anti-phosphorylated Stat1 (pY701), anti-Stat1, and anti-actin antibodies. Whole-cell extracts of microglia stimulated for 6 h with mouse IgG were analyzed. (B) Whole-cell extracts of microglia stimulated for 24 h with mouse IgG were analyzed. (C) Whole-cell extracts of microglia stimulated with 10 µg/ml mouse IgG were analyzed. (D) Whole-cell extracts of microglia stimulated with 10 µg/ml mouse IgG for 24 h and 3 pg/ml IFN-β for 30 min were analyzed. (E) IFN-β secreted from primary cultured microglia in response to 10 µg/ml mouse IgG stimulation was measured using enzyme-linked immunosorbent assay (ELISA) (n = 3 each). One-way analysis of variance showed a significant difference (p = 0.00545). The p-values from Fisher’s PLSD post hoc tests are indicated. (F) Whole-cell extracts of microglia stimulated with 10 µg/ml mouse IgG with 1 or 5 µM ruxolitinib for 6 h were analyzed. (G) Whole-cell extracts of microglia stimulated with 10 µg/ml mouse IgG for 6 h with 1 µM PRT062607 and 5 or 10 µM BAY61-36061 were analyzed. PRT062607 and BAY61-36061 were added to the medium 1 h before the addition of IgG. (H) Whole-cell extracts of microglia stimulated with 20 µg/ml mouse IgG for 6 h with 2 µg/ml anti-IFNAR, 1 µM PRT062607, or 10 µM BAY61-3606 were analyzed. PRT062607 and BAY61-36061 were added to the medium 1 h before the addition of IgG

**Fig. 4**
Differences in the IgG subclass and immunoglobulin class for Stat1 phosphorylation. (A) Immunoblot analysis of primary cultures of IgG-stimulated microglia. Whole-cell extracts of microglia stimulated with 20 µg/ml mouse IgG for 4 h were analyzed. (B) Whole-cell extracts of microglia stimulated with mouse IgG Fc secreted from COS7 cells for 4 h were analyzed (see Materials and Methods). (C) Flow cytometry quantification of IgG subclass immunosignals in microglia is indicated using fluorescence intensity. Primary microglia culture were incubated for 2 h with Mouse IgG1, IgG2a, IgG2b, and IgG3 (20 µg/ml) [IgG3 (n = 369); others (n = 494)]. A two-way analysis of variance revealed a significant effect of IgG stimulation (p < 0.0001) and IgG subclass (p < 0.0001). The p-values from Fisher’s partial least significant difference (PLSD) post hoc tests are indicated. (D) Primary cultured microglia were immunolabeled with anti-mouse IgG antibody (green). Mouse IgG1, IgG2a, IgG2b, and IgG3 (20 µg/ml) were added to the culture media of primary cultured microglia 2 h before fixation. Scale bar, 40 μm. (**E, F**) Immunoblot analysis of primary cultures of microglia stimulated with immunoglobulin. Whole-cell extracts of microglia stimulated for 6 h (E) and 24 h (F) with 10 µg/ml mouse IgG, IgM, IgE, and IgA (monomer) were analyzed. (G) Primary cultured microglia were immunolabeled with anti-mouse IgA (green) and anti-Iba1 (red) antibodies. Images were capture before IgA addition and 2 h after the addition of 10 µg/ml IgA to the culture media. Scale bar, 40 μm. (H) Sagittal sections of C57BL/6J mice corpus callosum at P8 were immunolabeled with anti-IgA (green) and anti-Iba1 (red) antibodies. Scale bars, 50 μm. (I) Flow cytometry data showing staining of all CD45-positive cells, including microglia, with anti-CD45 and anti-mouse IgA antibodies or an isotype control at P8 after fixation and permeabilization

**Fig. 5**
Decreased IgG immunoreactivity in neonatal Fc receptor knockout mice. (**A–D**) Sagittal sections of WT (**A, C**) and FcRn KO (**B, D**) corpus callosum (**A, B**) and cerebellar white matter (**C, D**) at P8 were immunolabeled with an anti-Iba1 (green) and anti-mouse IgG (red) antibodies. Scale bars, 50 μm. (**E–H**) Sagittal sections of WT (**E, G**) and FcRn KO (**F, H**) pia mater (**E, F**) and choroid plexus (**G, H**) at P8 were immunolabeled with an anti-Iba1 antibody (green) and anti-mouse IgG antibody (red). Scale bars, 50 μm. (I) Representative flow cytometry plots of P8 microglia stained with anti-mouse IgG antibodies after fixation and permeabilization. (J) Flow cytometry quantification of the percentage of P8 microglia stained with anti-mouse IgG after fixation and permeabilization (n = 3 for each genotype). The p-values from Student’s t-test are indicated

**Fig. 6**
Decreased density of microglia, oligodendrocytes, and neurons in neonatal Fc receptor knockout mice. Body weight (A) and wet weight of the telencephalon (B) and cerebellum (C) at P21 (n = 10 WT males; n = 10 KO males; n = 13 WT females; n = 15 KO females). (D) Sagittal sections of WT and FcRn KO neocortices at P8, immunolabeled with an anti-Iba1 antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 50 μm. (E) Density of cells positive for both Iba1 and DAPI in the neocortex [6–8 images were taken per animal; P8 WT (n = 24); P8 KO (n = 30); P15 WT (n = 28); P15 KO (n = 24); P21 WT (n = 27); P21 KO (n = 24)]. A two-way analysis of variance revealed a significant effect of genotype (p = 0.0363) and stage (p < 0.0001). The p-values from Fisher’s PLSD post hoc tests are indicated. (F) Sagittal sections of WT and FcRn KO corpus callosum at P6 immunolabeled with an anti-myelin basic protein (MBP) antibody. Scale bar, 50 μm. (G) Density of cells positive for MBP (P4 and P6) or myelin-associated glycoprotein (MAG) (P8) in the corpus callosum [Five to eleven images were taken per animal; P4 WT (n = 24); P4 KO (n = 21); P6 WT (n = 41); P6 KO (n = 41); P8 WT (n = 21); P8 KO (n = 25)]. A two-way analysis of variance revealed a significant effect of genotype (p = 0.00108) and stage (p < 0.0001). The p-values from Fisher’s PLSD post hoc tests are indicated. (H) Sagittal sections of WT and FcRn KO retrosplenial cortices (RSC) at P21 immunolabeled with an anti-vasoactive intestinal peptide (VIP) antibody. Scale bar, 100 μm. (I) Densities of cells positive for VIP in layers 2/3 and layers 4–6 of the RSC [5–7 images were taken per animal; WT layers 2/3 (n = 24); KO layers 2/3 (n = 26); WT layers 4–6 (n = 22); KO layers 4–6 (n = 25)]. (J) Sagittal sections of WT and FcRn KO RSC at P21 immunolabeled with an anti-calbindin antibody. Scale bar, 100 μm. (K) Density of calbindin-positive cells in layer 5 of the RSC [15–19 images were taken per animal; WT (n = 66), KO (n = 72)]. The p-values from Student’s t-test are indicated. (L) Sagittal sections of WT and FcRn KO RSC at P21, immunolabeled with an anti-parvalbumin (pvalb) antibody. Scale bar, 100 μm. (M) Density of cells positive for pvalb in layers 2–6 of the RSC [20–24 images were taken per animal; WT (n = 85); KO (n = 88)]. The p-values from Student’s t-test are indicated. (N) Sagittal sections of WT and FcRn KO RSC at P21 immunolabeled with an anti-VGAT antibody. Scale bar, 20 μm. (O) Density of puncta positive for VGAT in layers 5–6 of the RSC [20–33 images were taken per animal; WT (n = 110); KO (n = 100)]. The p-values from Student’s t-test are indicated

**Fig. 7**
Increased apoptotic neurons and oligodendrocytes and decreased proliferative microglia in neonatal Fc receptor knockout brain. (**A, B**) Apoptotic neurons (CD200+/CD45−/CD31− cells) (A) and O4-positive oligodendrocytes (B) at P12. (C) The percentage of early apoptotic (annexin+/7-AAD−) neurons, oligodendrocytes, and microglia at P12. Cell debris was eliminated by initial gating, and 7-AAD-positive cells were excluded because neurons and oligodendrocytes are easily damaged during the process of isolation from the brain. WT neuron (n = 8); KO neuron (n = 8); WT oligodendrocyte (n = 5); KO oligodendrocyte (n = 5); WT microglia (n = 8); KO microglia (n = 8). A two-way analysis of variance revealed a significant effect of genotype (p < 0.0001) and stage (p < 0.0001). The p-values from Fisher’s PLSD post hoc tests are indicated. (**D, E**) Sagittal sections of WT (D) and FcRn KO (E) RSC at P8 immunolabeled with anti-GAD67 (green) and anti-cleaved caspase3 (red) antibodies. White arrows point to the double-positive cells. Scale bar, 50 μm. (F) The percentage of apoptotic cells in GAD67-positive interneurons in the P8 RSC [4–5 images were taken per animal; WT (n = 17); KO (n = 20)]. The p-values from Student’s t-test are indicated. (**G, H**) Sagittal sections of WT (G) and FcRn KO (H) corpus callosum at P8 immunolabeled with anti-cleaved caspase3 (green) and anti-MAG (red) antibodies. White arrows point to the double-positive cells. Scale bar, 50 μm. (I) Percentage of apoptotic cells in MAG-positive oligodendrocytes in the P8 corpus callosum [5–7 images were taken per animal; WT (n = 21); KO (n = 25)]. The p-values from Student’s t-test are indicated. (**J, K**) Sagittal sections of the WT (J) and FcRn KO (K) corpus callosum at P8 immunolabeled with anti-Ki67 (green) and anti-Iba1 (red) antibodies. White arrows point to the double-positive cells. Scale bar, 50 μm. (L) The percentage of Ki67-positive proliferating cells in microglia in the P8 corpus callosum [5–7 images were taken per animal; WT (n = 23), KO (n = 26)]. The p-values from Student’s t-test are indicated

**Fig. 8**
Abnormal behavioral phenotypes of neonatal Fc receptor knockout mice. (A) Total distance traveled, vertical activity, time spent in the center area, and stereotypical behavior counts for WT (open circles, n = 20) and FcRn KO mice (closed circles, n = 20) are represented.(B) Social interaction test in novel environments, total duration of contacts, number of contacts, total duration of active contacts, mean duration per contact, and total distance traveled by WT (open columns, n = 10 pairs) and FcRn KO mice (closed columns, n = 10 pairs) are presented. The p-values indicate the genotype effects in one-way ANOVA. (**C, D**) Crawley’s sociability and social novelty preference test. Time spent around the cage and the rate of time spent around the stranger cage in the sociability (C) and social novelty preference (D) tests [WT (n = 20); KO (n = 20)]. The p-values indicate the genotype effects in one-way ANOVA. (E) Home-cage locomotor activity of WT (open circles, n = 17) and FcRn KO mice (closed circles, n = 19). The p-values indicate genotype effects in the two-way repeated-measures ANOVA. (F) Startle amplitude and percent of pre-pulse inhibition in WT (open columns, n = 20) and FcRn KO mice (closed columns, n = 20)

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References

1. Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–5. doi: 10.1126/science.1194637. - DOI - PMC - PubMed
1. Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L, Garner H, Trouillet C, de Bruijn MF, Geissmann F, Rodewald HR. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 2015;518:547–51. doi: 10.1038/nature13989. - DOI - PMC - PubMed
1. DiSabato DJ, Quan N, Godbout JP. Neuroinflammation: the devil is in the details. J Neurochem. 2016;139(Suppl 2):136–53. doi: 10.1111/jnc.13607. - DOI - PMC - PubMed
1. Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, Holscher C, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci. 2013;16:273–80. doi: 10.1038/nn.3318. - DOI - PubMed
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[1] Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–5. doi: 10.1126/science.1194637. - DOI - PMC - PubMed

[2] Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–5. doi: 10.1126/science.1194637. - DOI - PMC - PubMed

[3] Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L, Garner H, Trouillet C, de Bruijn MF, Geissmann F, Rodewald HR. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 2015;518:547–51. doi: 10.1038/nature13989. - DOI - PMC - PubMed

[4] Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L, Garner H, Trouillet C, de Bruijn MF, Geissmann F, Rodewald HR. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 2015;518:547–51. doi: 10.1038/nature13989. - DOI - PMC - PubMed

[5] DiSabato DJ, Quan N, Godbout JP. Neuroinflammation: the devil is in the details. J Neurochem. 2016;139(Suppl 2):136–53. doi: 10.1111/jnc.13607. - DOI - PMC - PubMed

[6] DiSabato DJ, Quan N, Godbout JP. Neuroinflammation: the devil is in the details. J Neurochem. 2016;139(Suppl 2):136–53. doi: 10.1111/jnc.13607. - DOI - PMC - PubMed

[7] Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, Holscher C, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci. 2013;16:273–80. doi: 10.1038/nn.3318. - DOI - PubMed

[8] Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, Holscher C, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci. 2013;16:273–80. doi: 10.1038/nn.3318. - DOI - PubMed

[9] Ransohoff RM, Perry VH. Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol. 2009;27:119–45. doi: 10.1146/annurev.immunol.021908.132528. - DOI - PubMed

[10] Ransohoff RM, Perry VH. Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol. 2009;27:119–45. doi: 10.1146/annurev.immunol.021908.132528. - DOI - PubMed

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Maternal immunoglobulin G affects brain development of mouse offspring

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Maternal immunoglobulin G affects brain development of mouse offspring

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Abstract

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