RESEARCH
Open Acce
Mizuki Sadakata¹*, Kazuki Fujii²²,³⁴, Ryosuke Kaneko  5, Emi Hosoya¹, Hisako Sugimoto¹, Reika Kawabata-Iwakawa 6, Tetsuhiro Kasamatsu², Shoko Hongo², Yumie Koshidaka², Akinori Takase², Takatoshi Iijima², Keizo Takao²-3,4 and Tetsushi Sadakata ¹*
Abstract
Maternal immunoglobulin (lg)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 lgG levels affect brain development in offsprings. Keywords  Microglia, Immunoglobulin G, Brain development
Introduction
Microglia, originate as the first glial cells in the brain from the embryonic yolk sac [ 1 , 2 ], migrate along the course of the fibers of the corpus callosum to all parts of the brain [ 3 , 4 ]. Microglia act as the primary immune cell in the central nervous system (CNS), and form the first *Correspondence: Mizuki Sadakata m2200038@gunma-u.ac.jp Tetsushi Sadakata sadakata-1024@gunma-u.ac.jp 1Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan ²Department of Behavioral Physiology, Faculty of Medicine, University of Toyama, Toyama 930-0194, Japan 3Research Center for Idling Brain Science, University of Toyama, Toyama 930-0194, Japan 4 Life Science Research Center, University of Toyama, Toyama 930-0194, Japan line of defense by regulating immunological and inflam- matory responses [ 5 ]. Microglia exert an extensive array of physiological functions in the healthy brain, involv- ing refinement of synaptic networks [ 6 – 9 ], promotion of developmental apoptosis [ 10 ], positioning of neurons within developing cortex [ 1 ], and secretion of growth 5 Medical Genetics Research Center, Nara Medical University, Kashihara, Nara 634-8521, Japan 6 Division of Integrated Oncology Research, Gunma University Initiative for Advanced Research (GIAR), Gunma University, Maebashi, Gunma 371-8511, Japan 7 Department of Medical Technology and Clinical Engineering, Gunma University of Health and Walfare, Maebashi, Gunma 371-0823, Japan 8Medical Science College Office, Tokai University, Isehara, Kanagawa 259-1193, Japan 9 Department of Molecular Life Science,  Division of Basic Medical Science and Molecular Medicine,  School of Medicine, Tokai University, Isehara, Kanagawa 259-1193, Japan © The Author(s) 2024. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by/40/.The Creative Commons Public Domain Dedication walver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. factors for neuronal survival [ 12 ]. The activation of microglia has been implicated in the pathogenesis of var- ious disorders, ranging from neuropsychiatric disorders (such as schizophrenia) to neurodevelopmental disorders (such as autism spectrum disorder (ASD)) [ 13 , 14 ]. Breastfeeding is associated with long-term well-being, including low risks of infectious diseases and non-com- municable diseases during childhood, such as asthma, cancer, autoimmune diseases, and obesity [ 15 ]. Breast milk is rich in microbiota and non-immune and immune components that ensure protection against numerous diseases and support the maturation of the develop- ing immune system of an infant [ 16 , 17 ]. On the other hand, an association between breastfeeding duration and adult intelligence has been reported [ 18 , 19 ]. Long-term breastfeeding is also associated with fewer attention-defi- cit hyperactivity symptoms [ 20 ]. Moreover, social compe- tence is related to breastfeeding duration [ 20 ]. However, the factors that affect brain development in breast milk are not well understood. Immunoglobulins (Ig), such as IgG, IgA, and IgM, are present in breast milk [ 21 ]. We first discovered that maternal IgG binds specifically to microglia in some regions of the infant brain. Therefore, we examined how maternal IgG affects neonatal brain development by ana- lyzing the brains of mouse offsprings lacking maternal IgG transfer. Downstream signal analysis using primary cultures and behavioral analysis were also performed to comprehensively analyze the effects of maternal IgG on brain development in the offspring.
Materials and methods
Clustered regularly interspaced palindromic repeats/ CRISPR-associated protein 9 -mediated deletion of the neonatal Fc receptor gene in vivo Global FcRn (Fcgrt) KO mice were generated using CRISPR/Cas9 genome-editing technology in pronuclear stage embryos. The Cas9 protein, guide RNA, and single- stranded oligodeoxynucleotide (ssODN) were introduced into pronuclear stage embryos using the technique for animal knockout system by electroporation [ 22 ]. The Cas9 protein and guide RNA were purchased from Inte- grated DNA Technologies, Inc. (Coralville, IA, USA). Guide RNAs were designed to delete exons 2 and 3 of the Fcgrt gene of C57BL/6 mice (5’-AACAAGAGGCTG AGGGCCCA-3’ and 5’-GTGAGATGATACATCAGTG G-3'). Super electroporator NEPA21 (NEPA GENE Co. Ltd., Chiba, Japan) was used to introduce Cas9 protein, guide RNA, and ssODNs into embryos. On the following day, two-cell embryos were transferred into the oviduct ampulla (18–24 embryos per oviduct) of pseudopregnant ICR females (Japan SLC, Shizuoka, Japan). The mice were backcrossed to C57BL/6J mice (Japan SLC) for at least six generations, which was also expected to minimize genetic drift.
Immunocytochemistry
Immunocytochemistry was performed as previously described [ 23 ]. Briefly, cells were fixed with Zamboni's fixative (2% paraformaldehyde in 0.1 M phosphate buf- fer, pH 7.4, containing 0.2% picric acid) at room tem- perature (RT) for 15 min. After washing three times with phosphate-buffered saline (PBS), the cells were permea- bilized in PBS containing 0.02% Triton X-100 at RT for 5 min. After blocking with Image-iT FX signal enhancer (Thermo Fisher Scientific, MA, USA) at RT for 60 min, the cells were incubated with primary antibodies at 4 °C overnight, rinsed in PBS, incubated with Alexa-conju- gated secondary antibodies at RT for 1 h, and rinsed again in PBS. Immunoreactive cells were mounted in DAPI Fluoromount-G° mounting medium (Southern- Biotech, Birmingham, AL, USA) and observed under a fluorescence microscope (BX51; Olympus, Tokyo, Japan) equipped with a CCD camera (VB-7000; Keyence, Osaka, Japan). Researchers analyzing the images were blinded to the experimental conditions.
Immunohistochemistry
Immunohistochemistry was performed as previously described [24]. Briefly, C57BL/6J male mice were deeply anesthetized with a combination of midazolam, medeto- midine, and butorphanol tartrate and transcardially perfused with PBS, followed by Zamboni’s fixative. The tissues were dissected, post-fixed in Zamboni's fixa- tive at 4 °C for 5 h, and cryoprotected by immersion in 15% sucrose in PBS overnight at 4 °C. After embedding in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan), the tissues were frozen and sectioned at a thick- ness of 15 μm using a cryostat (CM1950, Leica Microsys- tems, Frankfurt, Germany) at – 18 °C. The sections were air-dried for 1 h and rinsed in PBS three times. After blocking with 5% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS at room temperature for 1 h, the sections were incubated at 4 °C overnight with primary antibodies in immunoreaction buffer (2×PBS containing 0.3% Triton X-100 and 1% BSA). The sections were then washed in PBS, incubated at room temperature for 1 h with the appropriate secondary antibodies in the immu- noreaction buffer, and washed again in PBS. Stained sections were mounted in DAPI Fluoromount-G° mount- ing medium (SouthernBiotech) and observed by a fluo- rescence microscope (BX51; Olympus) equipped with a CCD camera (VB-7000; Keyence). Researchers analyzing the images were blinded to the experimental conditions.
Immunoblot analysis
Flow cytometry
Primary microglial culture
antibody (C247, Leinco Technology, St. Louis, MO, USA) was used for blocking. Intracellular antigens were stained using Fixation Buf- fer (420,801, BioLegend, San Diego, CA, USA) and Intra- cellular Staining Permeabilization Wash Buffer (421,002, BioLegend), according to the manufacturer's instruc- tions. Cell surface antigens were stained with unfixed cells before fixation. Initial gating was performed on the live and singlet cells. Annexin V/7-AAD staining was performed using the PE Annexin V Apoptosis Detection Kit with 7-AAD (640,934, BioLegend), according to the manufacturer's instructions. Staining was measured using a BD FACS- Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo (BD Biosciences). Cells were subjected to three washes with PBS and subse- quently lysed using SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 10% glycerol, and 0.025% bromophenol blue). High-molecular-weight DNA was sheared by passing the lysate through a 29-gauge needle (326,666, BD Biosciences). After centrifugation at 15,000 rpm for 5 min and heat treatment at 55 °C for 20 min, the samples were electrophoresed. Detection was performed using horseradish peroxidase (HRP)- conjugated secondary mouse or rabbit antibodies and chemiluminescence HRP substrate (WBKLS0100, Mil- lipore-Sigma), and the signals were captured using an image analyzer (LAS500, GE Healthcare, Chicago, IL, USA). Primary microglia were isolated from the brains of new- born mice (P0), following previously described proce- dures [ 26 ]. Briefly, the hippocampi and neocortices were mechanically dissociated. The meninges were removed, and the remaining tissue was placed in a 25-ml HBSS with 20 U/ml DNase I and 0.01% trypsin and then incu- bated in a rocking rotor at 37 °C for 15 min. Thereafter, 0.01% soybean trypsin inhibitor (17,075,029, Thermo Fisher Scientific) was added, and the tissues were cen- trifuged at 400 × g for 5 min and dissociated via gentle trituration with a pipette in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. Single- cell suspensions from five brains were pooled in three poly L-lysine-coated cell cultivation flasks (2-8589-05; Violamo, Osaka, Japan) in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml strepto- mycin (Thermo-Fisher Scientific). Subsequently, the medium was changed every four days. After 12 days of culture, the microglia were collected by shaking the flask at 200 rpm for 2 h. Detached microglia were then counted using a hemocytometer, plated at 4.5×10 5 cells/ ml in a poly L-lysine-coated 12-well plate, and placed in a 37 °C incubator. The primary microglia were maintained in these new plates for 3 days before treatment. For microglial stimulation with IgG Fc, COS7 cells in 6-well plates were transfected with either pFUSE-mIgG1- Fc2, pFUSE-mIgG2A-Fc2, pFUSE-mIgG2B-Fc2, pFUSE- mIgG3-Fc2 (InvivoGen, San Diego, CA, USA), or an empty vector. Transfection was performed for 72 h using Lipofectamine 3000 reagent (Thermo-Fisher Scientific). The medium was collected and centrifuged at 400 × g for 5 min, and the supernatant was added to the primary cul- tured microglia. Whole-cell extracts of microglia stimu- lated for 4 h with the media were analyzed. To measure the immunosignals of the IgG subclass using flow cytometry, primary cultured microglia stimu- lated for 2 h with 20 µg/ml of mouse IgG1, IgG2a, IgG2b, To identify microglia using flow cytometry, previously described protocols were modified [ 25 ]. The neocortices, hippocampi, and corpus callosum of C57BL/6J mice were dissected, minced, and incubated with 500 U of type II collagenase (4176, Worthington Biochemical Corp, Free- hold, NJ, USA) and 250 U of DNase (D4527, Millipore- Sigma, St. Louis, MO, USA) for 30 min at 37 °C in 10-ml Hanks’ Balanced Salt Solution (HBSS). The cells were triturated by repeated passage through 1-ml and 200-μL plastic micropipette tips every 5 min during incubation. After incubation, the cells were filtered through a 70-μm cell strainer (3-6649-02, ASONE, Osaka, Japan) and pel- leted by centrifugation (400 × g, 5 min, 18 °C). Cell pel- lets were resuspended in 37% solution of isotonic Percoll (SIP) (17,089,102, Cytiva, Marlborough, MA, USA), overlaid with PBS, and centrifuged (200 × g for 40 min at 18 °C). For the simultaneous identification of neurons, oli- godendrocytes, and microglia using flow cytometry, established protocols were adapted and modified [25]. The neocortices, hippocampi, and corpus callosum of C57BL/6j mice were dissected, minced, and ddgested with 45 U papain (PAPL, Worthington), 0.1 kU/ml DNase I, 0.02% DL-cysteine, 0.02% bovine serum albumin (BSA), and 0.5% glucose in PBS at 200 rpm for 10 min at 37 °C. Fetal bovine serum (FBS) was added to a final concentra- tion of 20%. The cells were triturated by repeated passage through 1-ml and 200-μL plastic micropipette tips, fil- tered through a 70-μm cell strainer (3-6649-02, ASONE), and pelleted in a centrifuge (300 × g, 3 min, 18 °C). Cell pellets were resuspended in 24% SIP (17,089,102, Cytiva, Marlborough, MA, USA), overlaid with PBS, and centri- fuged (400 × g for 25 min at 18 °C). After the centrifugation with SIP, erythrocytes were lysed using RBC lysing buffer (420,310, BioLegend), and 0.1% BSA–PBS containing 5 μg/ml of rat anti-CD16/32
Neuromuscular strength test
RNA-sequencing analysis
Rotarod test
Hot plate test
Open field test
Behavioral tests
Social interaction test in a novel environment
and IgG3 were trypsinized, and intracellular IgG immu- nosignals were analyzed using Fixation Buffer (420,801, BioLegend) and Intracellular Staining Permeabilization Wash Buffer (421,002, BioLegend), according to the man- ufacturer’s instructions. To quantify the level of IFN-β in the supernatant, the culture media from IgG-stimulated microglia were col- lected and subjected to centrifugation at 400 × g for 5 min. The amount of IFN-β was measured using the LEGEND MAX Mouse IFN-β enzyme-linked immuno- sorbent assay Kit (439,407, BioLegend), according to the manufacturer’s instructions. To measure the immunosignal intensity, the mean gray value of each cell was measured using ImageJ Fiji [27]. (one to three for each genotype) in a room with a 12-hour light/dark cycle (lights on at 7:00 am), with access to food and water ad libitum. Behavioral tests were performed between 9:00 am and 6:00 pm. After each test, the testing apparatus was cleaned with super hypochlorous water to prevent bias due to olfactory cues unless otherwise noted. A grip strength meter (O’HARA & Co., Tokyo, Japan) was used to assess the forelimb grip strength. The peak force applied by the forelimbs of the mouse was recorded in Newtons (N). Each mouse was tested three times, and the highest value was used for data analysis. In the wire hang test, the mouse was placed on a wire mesh that was then slowly inverted so that it gripped the wire to pre- vent it from falling off. Latency to fall was recorded, with a 60 s cutoff time. RNA was extracted from microglia treated with PBS or IgG (10 μg/ml) at DIV15 for 24 h. After QC, mRNA was enriched using oligo(dT) beads, and rRNA was removed using the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA). First, the enriched mRNA was ran- domly fragmented using fragmentation buffer, followed by cDNA synthesis using the NEB Next Ultra RNA Library Kit (Illumina), according to the manufacturer's protocols. The qualified libraries were pooled and fed into NovaSeq6000 sequencers (Illumina) according to the recommended pooling protocol based on the effective concentration and anticipated data volume. The filtering process for cleaning reads included the removal of reads containing adapters, those with N>10% (where N repre- sents an undetermined base), and those with low-qual- ity bases (Q score≤5), accounting for >50% of the total bases. Clean reads were aligned with the mouse reference genome mm10 using hisat2 [ 28 ]. The p-value was calcu- lated using the population stability index distribution for each group as the input. The p-value for this event was <0.0001, considering the clear separation of the two dis- tributions. In total, 373 DEGs (261 upregulated and 112 downregulated genes) with p-values<0.01 and |logFC| > 0.5 were further analyzed using the Qiagen Ingenuity pathway analysis (IPA) software (version 81,348,237; Qia- gen, Hilden, Germany). Motor coordination and balance were assessed using the rotarod test. This test, which uses an accelerating rotarod (UGO Basile, Varese, Italy), was performed by placing mice on rotating drums (3 cm diameter) made of polyvi- nyl chloride (PVC) and measuring the time each animal was able to maintain its balance on the rod. e speed of the rotarod was accelerated from 4 to 40 rpm over a 5-minute period. All mice were subjected to the test without pre-test training. The hot plate test was used to evaluate the sensitivity to a painful stimulus. Mice were placed on a 55 °C hot plate with a black anodized aluminum floor (Columbus Instru- ments, Columbus, OH, USA), and the latency to the first fore- or hind paw response was recorded with a 15 s cut- off time. The paw response was defined as either a paw lick or foot shake. Each mouse was placed in the corner of an open field apparatus  (40×40×30  cm;  Accuscan  Instruments, Columbus, OH, USA), which consisted of a white plas- tic floor and transparent Plexiglas wall. The total dis- tance traveled (cm), vertical activity, time spent in the center area (20 cm × 20 cm), and beam-break counts for stereotyped behaviors were recorded. Immediately after the mice were placed in the arena, their behavior was recorded for 120 min. Animals were cared for and treated according to the 'Jap- anese Act on the Welfare and Management of Animals’ and the ‘Guidelines for the Proper Conduct of Animal Experiments’ issued by the Science Council of Japan. All experimental protocols were reviewed and approved by the Gunma University Animal Care and Experimentation Committee. Unless otherwise noted, most behavioral tests were performed as previously described [ 29 ]. Male mice older than 11 weeks were subjected to different behavioral tests. Mice were housed two to four per cage In the social interaction test, two mice of identical genotypes that were previously housed in different cages were placed together in a white PVC plastic box
Crawley’s sociability and social novelty preference test
Data analysis
Statistical analyses
Results
lgG immunoreactivity in the corpus callosum and
Locomotor activity monitoring in home cage
Startle response/prepulse inhibition (PPI) test
(40×40×30 cm) (O’HARA & Co.) and allowed to explore freely for 10 min. Behavior was recorded and analyzed automatically using Image SI software (see Section, “Data analysis"). The total number of contacts, total duration of active contact, total contact duration, mean duration per contact, and total distance traveled were measured. If the two mice contacted each other and the distance traveled by either mouse was longer than 10 cm, the behavior was classified as an “active contact.” startle stimulus for all the trial types. Startle response was recorded for 400 ms starting with the onset of the startle stimulus. The background noise level in each chamber was 70 dB. The intensity of the startle stimulus was 110 dB or 120 dB. The prepulse sound was presented 100 ms before the startle stimulus, and its intensity was 74 dB or 78 dB. Four combinations of prepulse and startle stimuli were used. Six blocks of the six trial types were presented in a pseudo-random order such that each trial type was presented once within a block. This test is a well-designed method for investigating the effects of complex genetics on sociability and preference for social novelty [ 30 ]. The testing apparatus consisted of a rectangular, three-chambered PVC plastic box and lid with an infrared video camera (O’HARA & Co.). We modified the method as follows: a habituation session was performed in the apparatus for 10 min the day before the sociability test, and the wire cages in the lateral com- partments were located in the corners of each compart- ment. In the sociability test, an unfamiliar C57BL/6J male mouse (stranger) that had no prior contact with the sub- ject mouse was placed in one of the side chambers. The subject mouse was first placed in the middle chamber and allowed to explore the entire test box for a 10-minute session. In the social novelty preference test, each mouse was tested in a 10-minute session to quantify social preference for a new stranger. After the first 10-minute session, a second unfamiliar mouse was placed in the chamber that had been empty during the first 10-minute session. Data acquisition and analysis were performed automatically using the Time CSI software (O’Hara & Co.). Behavioral data were obtained automatically through applications based on ImageJ software and were modified for each test by Tsuyoshi Miyakawa (available through O’HARA & Co.). e ImageJ plugins, and the precom- piled plugins for open field test (Image OF) and home cage test (Image HA) are freely available on the website of “Mouse Phenotype Database” (http://www.mouse- phenotype.org/software.html). Statistical analyses were performed using Excel Sta- tistics  (Statcel  3;  Social  Survey  Research  Information, Tokyo, Japan). All data are presented as the mean±SEM, unless otherwise noted. Differences between groups were analyzed using the Student’s t-test, Mann-Whitney U test, one-way analysis of variance (ANOVA), two- way repeated measures ANOVA, and chi-squared test according to each experimental design. cerebellar white matter microglia of the mouse infant brain We found that microglia exhibited IgG immunoreactiv- ity in the mouse corpus callosum (Fig. 1A) and cerebellar white matter (Fig. 1B) on postnatal day 8 (P8). Immuno- reactivity for IgG was either weak or absent in the hip- pocampus (Fig. 1C), midbrain (Fig. 1D), and medulla oblongata (Fig. 1E). IgG Fc receptors fall into three classes– CD16 (FcγRIII), CD32 (FcγRII) and CD64 (FcγRI). CD16 (FcγRIII) and CD32 (FcyRII) are low-to-medium affinity receptors and mediate signaling after cross-linking of the receptors on the cell surface, whereas CD64 (FcyRI) is a high affinity receptor for IgG [ 31 ]. Immunoreactivity of the antibody that recognizes both CD16 and CD32 was observed in the microglia of the corpus callosum (Fig. 1F) and cer- ebellar white matter (Fig. 1G). We also examined the expression of the three IgG receptor classes using flow cytometry. Microglia in the telencephalon were analyzed (see Materials and Methods). The results showed that all three IgG receptor classes were expressed in microglia (Fig. 1H and I). Locomotor activity monitoring in the home cage was performed using a system that automatically analyzed the locomotor activity of mice in their home cage. The sys- tem contained a home cage (29×18×12 cm), a filtered cage top, and an infrared video camera attached to the top of a stand. Each mouse was housed individually in a home cage. Five hours after the mice were placed in their home cages, the light was turned off, and the analysis was performed. Images from each cage were captured at a rate of one frame per second, and the distance traveled was measured automatically using Image HA software (see Section, “Data analysis”). A startle reflex measurement system (O’HARA & Co.) was used to measure acoustic startle response and PPI. The test session began by placing a mouse in a trans- parent PVC plastic cylinder, where it was left undis- turbed for 10 min. White noise (40 ms) was used as the
(See figure on previous page.) 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 lgG 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 interac- tion, 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 Border-associated macrophages (BAMs) reside at the interface between the brain and periphery, including the meninges and choroid plexus [ 32 ]. Immunoreactivity for IgG was also observed in BAMs at the choroid plexus (Fig. S1A) and pia mater (Fig. S1B). These BAMs also showed immunoreactivity for CD16/32 (Fig. S1C and S1D). Next, intracellular and cell surface IgG localizations were examined using flow cytometry. e percentage of microglia showing IgG immunoreactivity was higher when the cells were permeabilized than that without per- meabilization at P8 (Fig. 1J). The percentage of microg- lia showing IgG immunoreactivity peaked at P8 and was almost nonexistent at P12 (Fig. 1K). Accordingly, at P15, the immunoreactivity for IgG in microglia in the corpus callosum (Fig. S2A) and cerebellar white matter (Fig. S2D) was very weak. At P28 and P56, little immunore- activity for IgG in the microglia in the corpus callosum (Fig. S2B and S2C) and cerebellar white matter (Fig. S2E and S2F) was observed. In contrast, immunoreactivity for IgG in BAMs was higher at P28 (Fig. S2H) than at P15 (Fig. S2G) and P56 (Fig. S2I). Similarly, results for the pia mater were obtained (Fig. S2J,S2K, and S2L). The corpus callosum and cerebellar white matter of the infant brain, called the “fountain of microglia,” shows large amounts of amoeboid microglia and high expres- sion of Mac3 up to P10 [ 33 ]. Immunoreactivity for IgG in microglia in the corpus callosum was heterogeneous and particularly strong in the "fountain of microglia," where cells were densely packed when observed using 4,6-diamidino-2-phenylindole (DAPI) (Fig. S3A). CD11b immunoreactivity in microglia in the corpus callosum was also heterogeneous (Fig. S3B), and microglia with strong IgG immunoreactivity were observed in paral- lel with microglia with strong CD11b immunoreactiv- ity (Fig. S3C ). When the expression levels of CD11b and FcyR were examined by flow cytometry, the expression of CD16, CD32, and CD64 was positively correlated with that of CD11b (Fig. S3D, S3E and S3F).
Activation of type I interferon-related genes in primary
cultured microglia by lgG
To examine the effects of IgG binding on microglia, we prepared primary microglia cultures. Primary cultured microglia showed CD16/32 (Fig. 2A) and IgG immu- noreactivity after the addition of IgG to the culture medium (Fig. 2B). Immunostaining for IgG in microglia was strongly observed up to 24 h after addition to the medium; however, degradation was observed after 48 h (Fig. S4A and S4B). We identified differentially expressed genes (DEGs) between the control and IgG-treated primary cul- tured microglia based on RNA sequencing (RNA-seq) data. Functional annotation analysis showed significant enrichment of genes involved in the response to type I interferon (IFN-I), IFN-α, and IFN-β and the regulation of IFN-I production (Table S1 ). Volcano plots display the statistical significance of the difference relative to the magnitude of difference for each gene in the compari- son. In the volcano plots, IFN-I-related gene expression increased in response to IgG stimulation (Fig. 2 C). Fig- ure 2D shows the clusters generated by pathway analysis of DEGs between control and IgG-stimulated microg- lia. IFN-I genes (i.e., IFNA1, IFNA2, and IFNB1), recep- tor of IFN-I (IFNAR2), and interferon regulatory factors (i.e., IRF1, IRF3, and IRF7) were included in this gene network. Ingenuity pathway analysis of DEGs revealed altered canonical pathways that were either activated or inhib- ited (Fig. 2E). Cytokine storm signaling and phagosome formation pathways were also activated, in addition to the interferon signaling pathway (Fig. 2E). The IFN-I signaling pathway and upregulated genes are shown in Fig. 2F. Stat1 and Stat2 are key markers of IFN-I activa- tion and transcription of interferon-stimulated gene (ISG) expression [ 34 ]. Binding of IFN-I to its receptors initiates a signaling cascade that results in the phos¬ phorylation and heterodimerization of Stat1 and Stat2 to form the active transcription factor complex. Therefore, we examined whether Stat1 was phosphorylated in IgG- stimulated microglial cells. We found that, in primary cultured microglia, Stat1 was phosphorylated in a dose- dependent manner by 6 h IgG stimulation (Fig. 3A). IgG concentrations in mouse blood are generally in the range of 1 to 10 mg/ml [ 35 – 37 ]. Here, we found that Stat1 was phosphorylated at much lower concentrations as shown in Fig. 3A. IgG stimulation for 24 h similarly phosphory- lated Stat1 in a dose-dependent manner (Fig. 3B). How- ever, the Stat1 protein also increased in a dose-dependent manner, indicating that phosphorylated Stat1 increased proportionally (Fig. 3B). Long-term stimulation with interferon increases Stat1 protein levels and induces a larger response to subsequent stimulation by interferon, a phenomenon termed priming [ 38 ]. As shown in Fig. 3C, IgG-stimulated phosphorylation of Stat1 began after 6 h, and Stat1 protein levels also increased in a time-depen- dent manner. When IFN-β was added 24 h after IgG treatment, Stat1 phosphorylation was elevated compared with that after the addition of IFN-β alone, depending on the increase in the Stat1 protein (Fig. 3D). In Fig. 3E, IgG stimulation induced IFN-β secretion from primary cultured microglia. IFN-I production can be amplified using a positive feedback loop (Fig. S5). All IFN-I signals through the IFN-I receptors IFNAR1 and IFNAR2 dimerize upon ligand binding and activate Jak1 and Tyk2 receptor tyrosine kinases (Fig. S5 ). Acti- vated Jak1 phosphorylates the intracellular domain of the receptor and facilitates the recruitment and phosphoryla- tion of Stat [ 39 ]. Ruxolitinib, which is a selective inhibitor of Jak1 and Jak2, inhibited the phosphorylation of Stat1 in IgG-stimulated microglia (Fig. 3F). How does IgG stimulation activate the IFN-I feed- back loop? Syk is essential for signaling through immune receptors involved in the recognition of antigens, immu- noglobulins, and cell surface proteins (Fig. S5 ) [40]. Reports have shown that Syk is associated with the prim- ing of interferon signaling by phosphorylating the Stat1 protein [41] . Syk inhibitors—PRT062607 and BAY61- 3606— strongly suppressed the Stat1 phosphorylation in IgG-stimulated microglia (Fig. 3G). When feedback was inhibited by the IFN-I blocker anti-IFNAR alone, Stat1 phosphorylation in response to IgG stimulation was only partially reduced (lane 3, Fig. 3H). Co-treatment with Syk inhibitors (PRT062607, BAY61-3606) and anti-IFNAR further inhibited the Stat1 phosphorylation in response to IgG stimulation (lanes 5 and 6, Fig. 3H), indicating that Syk is upstream of the IFN-I feedback loop (Fig. S5). Mouse IgG can be divided into four subclasses: IgG1, IgG2a, IgG2b, and IgG3, with IgG2c being the equiva- lent of IgG2a in some mouse strains [ 42 ]. In primary cultures of microglia prepared from C57BL/6J mice, Stat1 was phosphorylated by IgG2a and IgG2b stimula- tion (Fig. 4A). Furthermore, the Stat1 protein was phos- phorylated only by stimulating the Fc (constant) region of IgG2a, IgG2b, and IgG3 (Fig. 4B). Flow cytometry results showed that the binding activity of IgG2a was higher than that of IgG2b (Fig. 4C). Immunocytochemistry also revealed the binding of IgG2a and IgG2b to primary cul- tured microglia (Fig. 4D). When the classes of immunoglobulins other than IgG, IgM, IgE, and IgA were examined, stimulation with monomeric IgA also phosphorylated Stat1 (Fig. 4E and F). When IgA was added to the culture media, IgA bind- ing was observed in a few microglia (Fig. 4G). However, IgA immunoreactivity was not observed in the brains of infant mice (Fig. 4H). Furthermore, IgA immunoreactiv- ity was not observed in all CD45-positive cells, including microglia, in the infant mouse brain using flow cytom- etry (Fig. 4I). A study reported that some mouse strains show IgG immunoreactivity in epithelial cells and microglia, whereas others, such as CBA/J and CBA/N mice [ 43 ], do not show IgG immunoreactivity. However, IgG immu- noreactivity was observed in the brains of CBA/N mice (Fig. S6A). Upon stimulation with IgG, Stat1 was phos- phorylated in primary cultured microglia prepared from CBA/N mice (Fig. S6B). Moreover, both the Syk and Stat1 proteins were phosphorylated by stimulation with human IgG in the human microglial HMC3 cell line (Fig. S6C).
Effects of lack of maternal IgG transfer on offspring brain
development
IFN-I plays a protective role in neuronal homeostasis, and a lack of IFN-I signaling causes neurodegenera- tion [44, 45]. To investigate the effects of maternal IgG on infant brain development, we utilized genetically engineered FcRn KO mice. Maternal IgG is transferred from maternal serum into the offspring by FcRn present on placenta and infant intestine [ 35 ]. FcRn is primarily expressed in endothelial cells and is also expressed in the respiratory tract upper airway and genitourinary system, and it is mainly involved in the turnover of IgG [35, 46]. Further, FcRn-related brain abnormalities are not found in FcRn KO mice [35, 46]. We compared neonatal brains in offsprings of wild-type (WT; offsprings that received maternal IgG) and FcRn KO mice (offsprings that did not receive maternal IgG). IgG immunoreactivity was observed in the microglia of the corpus callosum of WT mouse brains born from FcRn heterozygote mothers (Fig. 5A) and cerebellar white matter (Fig. 5C). Similarly, the BAMs in the pia mater (Fig. 5E) and choroid plexus (Fig. 5G) also showed IgG immunoreactivity. In the FcRn KO mouse brain, IgG immunoreactivity in these microg- lia (Fig. 5B and D) and BAMs (Fig. 5F and H) were not observed. e percentage of FcRn KO microglia that showed IgG immunoreactivity was lower than that of WT microglia (Fig. 5I and J). No significant changes in the appearance of FcRn KO mice were observed, and no differences in body weight (Fig. 6A) or wet weight of the telencephalon (Fig. 6B) and cerebellum (Fig. 6C) were observed. However, the den- sity of the microglia in the FcRn KO neocortex decreased only at P8 compared with that in the WT neocortex (Fig. 6D and E). The density of oligodendrocytes in the FcRn KO corpus callosum was significantly decreased at P6 and P8 compared with that in the WT corpus callo- sum (Fig. 6F and G). Microglia in the corpus callosum showed strong IgG binding activity (Fig. 1A). Because retrosplenial cor- tex (RSC) is the caudal subdivision of the strip of cortex around the corpus callosum, we immunohistochemically analyzed neurons in the RSC. Rodent studies consistently indicate that the RSC plays a crucial role in learning and navigation, thought to be orchestrated in collaboration with various thalamic and hippocampal systems [47, 48]. Moreover, ketamine treatment increases neural activity in the RSC and decreases social behaviors in mice [49]. We found that the density of vasoactive intestinal pep- tide (VIP)-positive interneurons did not differ among the cortical areas examined at P21 (Fig. 6H and I). In contrast, at P21, the density of calbindin- (Fig. 6J and K) and parvalbumin- positive interneurons (Fig. 6L and M) in RSC region of FcRn KO mouse brain was significantly decreased compared with that in RSC region of WT mouse brain. The density of the vesicular γ-Aminobutyric acid (GABA) transporter (VGAT), a marker of inhibi- tory synapses, also decreased in RSC region of FcRn KO mouse brain, at P21 (Fig. 6N and O). These changes were persistent at postnatal age of 4 months. In 4 month old mice, we found similar decreases in parvalbumin- and calbindin- positive interneurons, and VGAT puncta in RSC region of FcRn KO mice brain (Fig. S7 ). CD200 (OX2) is mainly expressed in central nervous system (CNS) neurons [50] . However, because it is also expressed in lymphocytes and endothelial cells [50], we investigated neural apoptosis using flow cytometry in the CD200+/CD45–/CD31–population. Because neu- rons and oligodendrocytes are intricately intertwined, cells are easily destroyed during the process of isola- tion for flow cytometric analysis. Therefore, only early apoptotic cells (7-AAD-negative and annexin V-positive cells) were quantified after gating out cell debris. e results showed that the density of apoptotic neurons and oligodendrocytes was higher in the FcRn KO infant brain than in the WT brain (Fig. 7A and B, and 7C). In contrast, no difference in the percentage of apoptotic microglia was observed between the control and FcRn KO brains (Fig. 7 C). The immunohistochemistry results showed that the percentage of apoptotic GAD67-positive interneurons in the FcRn KO neocortex was higher than that in the WT neocortex (Fig. 7D and E, and 7F). Simi- larly, the percentage of apoptotic oligodendrocytes in the FcRn KO corpus callosum was higher than that in the WT corpus callosum (Fig. 7G and H, and 7I). In contrast, the density of Ki67-positive proliferating microglia in the WT corpus callosum was higher than that in the FcRn KO corpus callosum (Fig. 7J and K, and 7L).
Behavioral phenotypes of FcRn KO offspring
To examine the effects of maternal IgG on brain develop- ment in offsprings, a comprehensive behavioral analysis of FcRn KO mice was performed. No significant differences in body temperature, grip strength, latency to fall off the wire grid, motor function assessed using the rotarod test, or pain sensitivity examined using the hot plate test were observed between WT and FcRn KO mice (Fig. S8 ). The locomotor activity of FcRn KO mice was evaluated using the open field test. No significant differences in the total distance traveled, vertical activity, time spent in the center area, or stereotypic behaviors were observed between the genotypes (Fig. 8A). In the novel environ- ment open field social interaction test, FcRn KO mice exhibited a significant increase in the total duration of contact (Fig. 8B). The mean duration per contact was also significantly increased in FcRn KO mice (Fig. 8B). The mice were also subjected to Crawley's sociability and social novelty preference test, which comprises a socia- bility test and a social novelty preference test. In the sociability test, social behavior is assessed based on the time spent around a wire cage with an unfamiliar mouse (stranger side) versus the time spent around an empty cage (empty side). Both WT and FcRn KO mice spent more time around the stranger-side cage than around the empty-side cage, and no difference in the time spent around the stranger side was observed between the geno- types (Fig. 8C). In the social novelty preference test, FcRn KO mice stayed longer around the stranger side of the cage than WT mice; however, the difference was statisti- cally insignificant ( p =0.0721) (Fig. 8D). Figure 8E shows that FcRn KO mice exhibited less locomotor activity than WT mice during the night in the home cage, which was different from that in the open field (Fig. 8A). Pre-pulse startle inhibition is an operational measure of sensorimotor gating; it is often impaired in patients with schizophrenia [ 51 ]. Acoustic startle responses were similar in WT and FcRn KO mice (Fig. 8F). Although the percentage of prepulse inhibition was higher in FcRn KO mice than in WT mice at 120 dB, the difference did not reach statistical significance ( p =0.0812) (Fig. 8F).
Discussion
Here, we showed that maternal IgG immunoreactivity is observed in the offspring brain microglial cells, peak- ing at P8 (Fig. 1 ). IgG stimulation of primary cultured microglia activated the IFN-I feedback loop by Syk (Figs. 2 and 3). IgG2a and IgG2b were found to exhibit these activities in the Fc region alone (Fig. 4 ). Analysis of FcRn KO mice showed that the density of microglia was decreased only in the infant brain (Fig. 6D), whereas the number of calbindin- and parvalbumin-positive inter- neurons decreased in later developmental stages (Fig. 6 and S7). FcRn KO mice also exhibited abnormalities in social behavior and locomotor activity in their home cages (Fig. 8 ). This study suggests that maternal IgG affects brain development in offspring. Parvalbumin cells have been suggested to be associated with sociability [ 52 , 53 ]. Mice with reduced (parvalbu- min+/−) or absent (parvalbumin−/−) parvalbumin expres- sion levels show behavioral deficits related to human autism spectrum disorder core symptoms [ 54 ]. Moreover, dysfunction in parvalbumin cells has been observed in various mouse models of autism [ 55 – 59 ]. Interestingly, the number of parvalbumin-expressing interneurons is decreased in the prefrontal cortex of patients with autism [60]. Moreover, stimulating parvalbumin cells has been shown to be sufficient to improve social behavior [ 56 , 61 ]. FcRn KO mice showed a decreased density of parvalbu- min-positive interneurons (Fig. 6L and M, S7E, and S7F) and increased duration of contact during the social inter- action test (Fig. 8B). However, it is difficult to conclude that the absence of IgG from the mother improves socia- bility because some cases in which autism mouse models exhibit behavior similar to that of FcRn KO mice have been reported. Shank2 KO mice show increased social inter- actions following isolation [ 62 ]. Mice with heterozygous Chd8 mutations exhibit prolonged social interaction dura- tion in tests [ 63 ]. Mice lacking intergenic Dlx enhancers also show increased social interaction [ 64 ]. These reports suggest that the increased duration of interaction in the social interaction test is a social behavior impairment. On the other hand, an increased duration of contact is gener- ally considered an increase in sociability. Is it better for the offspring’s brain if there is no IgG transfer from the mother to the infant? Epidemiological studies have shown an asso- ciation between maternal infection and a child's subse- quent risk of autism and schizophrenia [ 65 , 66 ]. Analyses of maternal immune activation (MIA) mouse models have shown that maternal interleukin (IL)-17a promotes autistic behavior in pups [ 67 ]. In MIA mouse models, it is prob- able that increased maternal IgG is transferred to pups. The effect of abnormally increased IgG levels in the mater- nal blood on the social behavior of offspring should also be elucidated in the future. The binding of IgG to neurons [ 68 ], oligodendrocytes [ 69 ], and microglia [ 43 ] has been previously reported. Nakahara et al. reported IgG immunoreactivity in oligo- dendrocytes [ 69 ] in the infant mouse brain. In contrast, Hazama et al. demonstrated IgG immunoreactivity in microglia and neurons in the adult mouse brain [43]. In this report, no IgG immunoreactivity was observed in cell types other than the microglia (Fig. 1 ). Hazama et al. also reported species differences in IgG immuno- reactivity in the brains of various rodents and no IgG immunoreactivity in CBA/N and CBA/J mice [43] . In contrast, we observed IgG immunoreactivity in the brains of infant CBA/N mice (Fig. S6A). Moreover, upon stimulation with IgG, Stat1 was phosphorylated in pri¬ mary cultured microglia prepared from CBA/N mice (Fig. S6B) and the human microglial HMC3 cell line (Fig. S6C). When observing IgG immunoreactivity using immunohistochemistry, "diffusion artifacts" can cause false-positive results [ 70 , 71 ]. The diffusion of compo- nents is usually caused by the translocation of serum pro- teins, such as immunoglobulins, from the extracellular matrix into the cytoplasm of some cells because ischemia during the preparation of tissue specimens is assumed to quickly change cell membrane permeability in animal organs. Therefore, we examined IgG immunoreactivity using flow cytometry and Stat1 phosphorylation by IgG stimulation and confirmed that IgG immunoreactivity in the microglia was not an artifact. We performed a detailed analysis of the effects of IgG in breast milk on the neonatal brain using immunohisto- chemistry, flow cytometry and behavioral analyses. No reports have shown the behavioral phenotypes of FcRn KO mice or the immunohistochemical phenotypes of FcRn KO brains. On the other hand, knockout of the γ-chain subunit decreases the number of myelin-associated glyco- protein (MAG)-positive oligodendrocytes in the brain of infant mice [ 69 ], which is similar to our results (Fig. 6G). It has also been reported that knockout of the γ-chain sub- unit reduces hippocampal pyramidal cell death induced by kainic acid [ 72 ]. IgG Fc receptors have three classes: FcyRI, FcyRII, and FcyRIII [ 73 ]. The γ-chain is a subunit of FcyRI and FcγRIII. γ-chains are also a shared subunit of FcεRI [ 73 ]. The differences in the deleted classes should be care- fully analyzed in the future to determine what phenotypic differences may result from the differences. How does maternal IgG reach the brains of pups? In the postnatal mouse brain, the blood–brain barrier (BBB) is considered complete [ 74 ]. However, not all CNS vas- cularities possess a BBB; exceptions include the pineal gland, neurohypophysis, area postrema, subfornical organ, median eminence, and vascular organ of the lamina ter- minalis [ 74 ]. These regions are referred to as circumven- tricular organs and permit the diffusion of blood-borne molecules [ 75 ]. On the other hand, incidental intracranial hemorrhages are relatively common among human infants born by vaginal delivery [ 76 ]. Although whether this phe- nomenon occurs in mice is unclear, it is thought to be one of the candidate pathways through which IgG migrates to the brain. It is also possible that FcRn transports IgG across blood–brain barrier (BBB). However, the research on this is not settled. Intracranially applied antibody is exported to the blood across the BBB by FcRn-mediated transcytosis [ 77 , 78 ]. In contrast, studies using the systemic application of antibodies suggest that FcRn is irrelevant for brain-to-blood export [ 79 – 82 ]. Further, a substantial anti- body deposition in brain was observed upon the systemic administration of a modified human IgG1 FcRn-high- binding variant [ 83 ]. Furthermore, this effect was abol- ished in FcRn KO mice and when the unmodified human IgG1 antibody was used. These findings suggest that when excess IgG is administered to the brain, IgG is exported from the brain in an FcRn-dependent manner, but oth- erwise, FcRn may be involved in bidirectional transport across the brain-blood barrier in all other conditions. In future, we will determine whether FcRn is involved in the bidirectional transport of IgG across BBB by using FcRn conditional KO mice. During postnatal development, microglia contribute to the survival of neocortical neurons by releasing IGF-1 as a trophic factor [ 12 ]. IGF-1 also directly acts on oli- godendrocytes and myelination, as demonstrated in oli- godendrocyte lineage-specific KO mice of the IGF-1R [84]. Other factors produced by microglia have also been found to support the health and maturation of oligoden- drocyte precursor cells and mature oligodendrocytes in the developing CNS [ 85 ]. For example, microglia-derived IGF-2 has been shown to prevent tumor necrosis factor- α-induced apoptosis of oligodendrocytes [ 86 ]. However, no difference in the expression of IGF-1 or IGF-2 was observed between the control and IgG-stimulated pri- mary cultured microglia in our study (Fig. 2 ). IFN-I also plays a protective role in neuronal homeostasis, and a lack of IFN-β signaling increases the expression of mul- tiple genes annotated as neuronal degeneration and increases neuronal apoptosis [44, 45] . IgG stimulation of primary cultured microglia activated the IFN-I feedback loop (Figs. 2 and 3 ). The effects of microglia on oligoden- drocytes and neurons via IFN-I and/or trophic factors should be examined in detail in future studies. A phagocytic/activated microglial phenotype, referred to as DAM/ARM/MGnD, has been recognized in mouse models of Alzheimer's disease (AD) featuring amyloid pathology [87–89]. Single-cell RNA-seq (scRNA-seq) has further resolved DAM/ARM/MGnD complexity, iden- tifying four microglial signatures in amyloid pathology, namely, DAM/ARM/MGnD proper (enriched for ApoE, Clec7a, Cst7, and Lpl), MHC class II microglia (enriched for MHC-II molecules), proliferating microglia, and IFN- I-responsive microglia (enriched for Ifit2, Irf7, Oasl2, and Stat1) [ 89 – 91 ]. In this study, we showed that Syk is upstream of the IFN-I feedback loop (Fig. 3H). Deletion of Syk gene in microglia reduces the proportion of IFN- I-responsive microglia, and DAP12 is an upstream can- didate for Syk in the AD brain [ 92 ]. Syk may also bind to the IgG receptor FcyR because disruption of the BBB may allow IgG to enter the brain in AD [ 93 ]. Whether Syk- mediated activation of the IFN-I feedback loop occurs in the AD brain would be interesting to examine. Here, we showed that changes in the mother-derived IgG levels affect neonatal brain development. Immune- related molecules, including major histocompatibility complex class I (MHC I), CD3zeta, and IgM, contribute to brain development [ 94 – 96 ]. The effects of immune- related molecules on brain development remains to be comprehensively elucidated. Abbreviations AD APC BAM BBB DEG IFN-I IL ISG MAG MBP PE PerCP PPI VGAT VIP Alzheimer's disease Allophycocyanin Border-associated macrophage Blood–brain barrier Differentially expressed gene Type I interferon Interleukin Interferon-stimulated gene Myelin-associated glycoprotein Myelin basic protein Phycoerythrin Peridinin chlorophyll protein Prepulse inhibition Vesicular γ-Aminobutyric acid (GABA) transporter Vasoactive intestinal peptide
Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s12974-024-03100-z Supplementary Material 1
Acknowledgements
We are grateful to Dr. Hiroshi Ohnishi for providing the flow cytometry protocol. We would also like to express our gratitude for Dr. Teiichi Furuichi, Dr. Yasuki Ishizaki, and Dr. Koji Shibasaki, who contributed with fruitful discussions. Author contributions MS and TS designed the experiments. MS, KF, RK, EH, HS, SH, YK, AT, TI, and TS performed experiments. MS, KF, RKI, TK, TI, KT, and TS analyzed the data. MS, RK, KT, and TS wrote the manuscript. MS, RKI, TK, TI, KT, and TS contributed to discussion. Funding This research was supported by grants provided by The Naito Foundation, The Mother and Child Health Foundation, The Food Science Institute Foundation (Ryoushoku-kenkyukai) (Grant No. 2023A09), and MEXT/JSPS KAKENHI Grant No. JP21H05611 and JP22H02714 (AdAMS). Data availability The data of RNA-Seq presented in this manuscript have been deposited in NCBI's Gene Expression Omnibus and are accessible through the GEO Series accession number GSE248310.
Declarations
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