Maternal antibodies regulate the establishment of murine oral and salivary mucosal immunity

Domain: neuroscience
Published: 2026-04-10
Editorial outcome: minor_revision

Abstract
Mucosal homeostasis after birth depends on coordinated interactions between the microbiota and the immune system, with barrier tissues employing distinct mechanisms. Here, we show that maternal Immunoglobulin G (IgG), transferred both in utero and via breastfeeding, reaches the murine neonatal salivary glands and is secreted into saliva, where it recognizes maternal oral microbes and regulates colonization. Offspring lacking in utero-derived IgG show heightened salivary T- and B-cell activation, followed by alterations in the IgG/IgA balance in adult saliva. While postnatal microbiota is essential for priming salivary adaptive immunity, perinatal IgG intrinsically restrains the adaptive responses independent of microbial load. In utero-derived IgG further shapes adult gingival immunity and limits destructive inflammation in murine periodontitis. By contrast, breast milk antibodies promote timely and proper epithelial maturation, a process dependent on microbial load. Thus, we uncover distinct yet complementary roles of pre- and postnatal maternal antibodies in governing oral immune development and long-term protection against oral disease.

Introduction
Following birth, the oral mucosa is exposed to a surge of microbes, which gradually declines as weaning progresses 1. Neutrophils, recruited by IL-17-producing γδT cells in a microbiota-dependent manner, protect the neonatal epithelium from bacterial infiltration. During this period, the microbiota promotes epithelial maturation, barrier sealing, and the acquisition of adult immunological functions 1. Weaning also triggers microbiota-dependent upregulation of the homeostatic regulator GAS6, supporting host-commensal interactions 2. The microbiota further promotes recruitment of Langerhans cells and γδT cells, which are essential for gingival and epithelial function 3, 4. These findings highlight the dynamic interplay between immune cells and microbiota in establishing oral mucosal homeostasis, though precise mechanisms remain to be fully elucidated.

The salivary system is also crucial for oral mucosal homeostasis. Saliva aids mastication, swallowing, and oral cleansing 5, and shapes the oral microbiota, as many bacteria rely on salivary glycoproteins for growth 6. Beyond these physiological roles, the saliva contains antimicrobial molecules and complement components that serve as part of the innate defense system 7, 8. Saliva also contains immunoglobulins such as IgA and IgG, which are key mediators of adaptive humoral immunity 9. Salivary IgA secretion depends on the microbiota and IL-17-producing CD4+ T cells 10. A recent report further reveals that salivary glands function as immune induction sites, where dendritic cells (DC) migrate from the oral mucosa to activate naïve T cells within the glands 10. However, whether salivary immunity contributes to the establishment of oral mucosal immunity during the postnatal period remains unclear.

During weaning, adaptive immunity develops in both the oral mucosa and salivary glands, initiating mechanisms that support lifelong mucosal homeostasis 1, 10, 11. In contrast, the neonatal immune system is immature and must respond effectively to initial microbial colonization. To protect neonates, maternal IgG is transferred in utero, while both IgA and IgG are delivered through breast milk 12, 13. In the intestine, breast milk-derived IgA and IgG support the development of appropriate anti-commensal responses in the neonatal gut 14, 15. Additionally, IgG protects against enteric pathogens and promotes the establishment of gut homeostasis in early life 16. Recent work has shown that maternal IgG is actively transported by the neonatal Fc receptor (FcRn) from the circulation to the salivary glands and subsequently secreted into saliva 10. While this points to a potential protective role for maternal IgG in the oral mucosa, its source, mechanism of action, and long-term impact remain unclear. This study addresses the role of maternal antibody transfer in orchestrating the maturation of oral mucosal immunity after birth. We demonstrate that maternal antibodies acquired both in utero and through breastfeeding play a pivotal role in shaping adaptive immunity in the salivary glands and oral mucosa. The lack of in utero-derived IgG elevates immune activation in the salivary glands and gingiva, alters the load and composition of the oral microbiota, leading to oral dysbiosis and exacerbated experimental periodontitis in adulthood. At the same time, breast milk-derived antibodies influence oral epithelial maturation and impair epithelial integrity. By demonstrating how maternal antibodies protect against oral diseases later in life, our work identifies this immune transfer as a critical window for early-life interventions aimed at programming long-term oral and systemic health.

Results
Maternal IgG antibodies are transferred to the neonatal salivary glands and saliva
To study the influence of maternal antibodies on the development of postnatal immunity in the offspring, a crossbreeding experiment was conducted using J H T-/- mice, which failed to produce functional B cells as they lack the gene for the heavy chain joining region 17. J H T+/+ males were crossed with J H T-/- females to produce offspring with functional B cells but without maternal antibodies (mIgneg). The control group consisted of mating between J H T-/- males and J H T+/+ females, ensuring functional B cells in the offspring who received maternal antibodies (mIg+) (Fig. 1A). To identify the type of antibodies present in the salivary glands post-birth, we conducted a Western blot and ELISA analysis on the parotid glands of 2-week-old mice. As illustrated in Fig. 1B-C, we observed IgG antibodies in the parotid glands of mIg+ offspring, while IgA antibodies were absent. Additionally, IgG antibodies were detectable in the saliva collected from the oral cavity (Fig. 1D) and serum (Fig. 1E) of 3-week-old mIg+ but not mIgneg mice. To assess the persistence of maternal IgG in neonatal parotid glands, we crossed J H T-/+ males with J H T-/+ females, and collected homozygous J H T-/- offspring, which lack B cells, at various time points after birth (Fig. 1F). High levels of maternal IgG were detected in both the salivary glands (Fig. 1F) and serum (Fig. 1G) shortly after birth. These levels declined significantly during weaning but remained detectable up to four weeks postnatally. IgA antibodies were not detectable in the salivary glands of J H T-/- offspring at any tested time point (Supplementary Fig. 1A). In addition, J H T-/- offspring born to J H T-/+ dams exhibited no B cells in their parotid glands, suggesting that B cells are not maternally transferred to the offspring (Supplementary Fig. 1B). To assess the contribution of in utero- and breastfeeding-derived IgG to the salivary glands, we conducted a fostering experiment in which the dams of mIg+ and mIgneg offspring were exchanged after birth. As shown in Supplementary Fig. 1C, low but detectable levels of in utero-derived IgG were present in 3-week-old fostered mIg+ offspring, while higher levels were detected in fostered mIgneg offspring, indicating that IgG from both in utero transfer and breast milk reaches the neonatal salivary glands. We then examined whether the levels of IgG and IgA antibodies in weaned mIg⁺ (J H T-/+ ) offspring reflect those found in wild-type (WT) mice. Analysis of the parotid gland and saliva revealed comparable IgG levels, whereas IgA was undetectable in both groups, indicating that the two groups exhibit similar salivary antibody isotype profiles (Supplementary Fig. 1D). Next, we examined the presence of maternal antibodies in the parotid glands of 4-week-old WT mice under specific pathogen-free (SPF) and germ-free (GF) conditions. IgG antibodies were detected exclusively in SPF mice, whereas IgA antibodies were absent in both groups (Fig. 1H and Supplementary Fig. 1E). These findings suggest that during neonatal life, the salivary glands and saliva contain substantial levels of maternal IgG antibodies derived from both in utero transfer and breastfeeding. In addition, since GF mice harbor reduced IgG levels 18, while FcRn expression remains intact postnatally 10, the lack of IgG in GF offspring is likely attributable to the limited availability of maternal antibodies for transfer, rather than to a defect in FcRn-mediated translocation.

Absence of in utero-derived maternal IgG drives elevated adaptive immunity in early-life salivary glands
To examine the influence of maternal IgG on the development of adaptive immunity in the salivary glands, mIg+ and mIgneg mice were analyzed at four weeks of age, a time point that coincides with weaning and the onset of adaptive immune development in these glands 10. As illustrated in Fig. 2A and Supplementary Fig. 2A-BA, flow cytometry analysis indicated that while the frequencies and numbers of total CD45+ leukocytes were not changed, the frequencies and counts of CD4+ T cells, CD8+ T cells, and B cells were significantly elevated in the mIgneg offspring. Notably, the Ly6C+ CD4+ and CD8+ T cells, representing short-lived activated T cells (DeLong et al., 2018), were also increased in mice lacking maternal antibodies. In contrast, mIgneg offspring display reduced frequencies and numbers of macrophages (Fig. 2A and Supplementary Fig. 2A-B). To determine the origin of maternal antibodies shaping adaptive immunity in the salivary glands, we conducted a fostering experiment in which dams were swapped between experimental groups after birth, and the offspring were assessed four weeks later. As illustrated in Fig. 2B, increased frequencies and numbers of B cells were observed in the salivary glands of offspring that did not receive maternal antibodies in utero, regardless of whether they received maternal antibodies through breast milk. Additionally, higher frequencies and numbers of T cells were detected in offspring that received maternal antibodies in utero compared to those that did not (Fig. 2B). Notably, fostered offspring exhibited an overall elevation in the T cells compared to the unfostered control group, suggesting that the fostering process may enhance cell-mediated immunity in these mice. To further investigate the impact of maternal antibodies on salivary immunity, we examined the ability of oral DCs to migrate to the salivary glands, given their reported role in priming T cells in this context 10. First, as depicted in Supplementary Fig. 2C, DCs (both CD11b+ and CD103+ DC subsets) were present in the oral mucosa of 3-week-old mice, while Langerhans cells, the APCs of the epithelium, were not yet developed. Subsequently, we applied FITC solution to the oral mucosa of 3-week-old mice and, three days later, quantified the frequencies of FITC-labeled cells in the salivary glands, indicative of migratory DCs. Upon gating on MHCII+CD11c+B220neg cells, representing DCs and macrophages, and further on CD11c+CD64neg (DCs) and CD64+ (macrophages) cells, higher frequencies of FITC+ DCs were found in mIgneg offspring compared to mIg+ mice (Fig. 2C). In fact, the frequencies of DCs among the total APCs were elevated in the salivary glands due to the absence of maternal antibodies (Fig. 2D). Moreover, increased production of IFN-γ was observed by CD4+ and CD8+ T cells of the mIgneg group, whereas IL-17 production was not detected (Fig. 2E). Consistently, CD4⁺ T cells from mIgneg offspring exhibited elevated expression of the early activation marker CD69 (Fig. 2F), along with a reduced frequency of FOXP3⁺ CD4⁺ regulatory T cells (Tregs) (Fig. 2G). It is worth mentioning that at this early age, the salivary glands contain CD11b+ DCs and CD103+ DCs (Supplementary Fig. 2D). The former expresses CD301a, which has been shown to present commensal antigens for generating Treg cells in neonatal life 19. In conclusion, these results demonstrate that maternal IgG antibodies transferred in utero are essential for downregulating adaptive immunity in the salivary glands of offspring during early life.

Maternal IgG recognizes the maternal oral microbiota, enhances phagocytosis, prevents epithelial adhesion, and delays the expression of adult epithelial markers
Maternal IgG antibodies shape the neonatal microbiota and support intestinal homeostasis 14, 16, while also protecting against pathogens 20. Given that the microbiota plays a crucial role in shaping oral immunity during early life 1, we sought to investigate whether maternal IgG antibodies affect the neonatal oral microbiota. We first assessed whether these antibodies recognize the oral microbiota by performing an ELISA on serum from SPF and GF neonates using microbiota samples from various sources. As illustrated in Fig. 3A, the serum from SPF neonates exhibited strong recognition of the oral microbiota derived from their mothers, surpassing the recognition levels observed when exposed to oral microbiota from unrelated mothers. The serum from GF neonates displayed no recognition of the microbiota. Maternal IgG antibodies from SPF neonates also recognized the intestinal microbiota collected from their mothers (Fig. 3A). The subtypes of maternal IgG antibodies were then examined in the serum of 2-week-old offspring, revealing the presence of IgG1, IgG2b, and IgG3 antibodies (Fig. 3B). IgM was also detected in the neonatal serum, while IgA antibodies were undetectable, similar to IgG2a, a subtype that is not generated by B6 mice. Analysis of IgG subtypes in the parotid glands and saliva of 2-week-old offspring revealed the presence of all IgG isotypes, with IgG2b being the most abundant in the glands and saliva (Supplementary Fig. 3A). Among these isotypes, IgG2b demonstrated the highest affinity for the maternal oral microbiota, with IgG1 showing moderate binding (Supplementary Fig. 3B). Next, the oral microbial load of mIg+ and mIgneg neonates was quantified, revealing a higher load of cultivated aerobic and anaerobic bacteria, as well as elevated bacterial ribosomal 16S DNA, in the absence of maternal antibodies (Fig. 3C). To determine the origin of maternal antibodies that regulate the oral microbiota in early life, a fostering experiment was conducted. As shown in Fig. 3D, oral microbial levels were reduced in both fostered groups, with no significant differences between them, indicating that antibodies transferred via both the placenta and breast milk contribute to this effect. Notably, in addition to the presence of breast milk-derived IgG in the salivary system (Supplementary Fig. 1C), breast milk IgA was also detected in the saliva of WT neonates, albeit at very low levels, as salivary IgG exceeded IgA by roughly three orders of magnitude (Supplementary Fig. 3C), suggesting that IgA is unlikely to play a significant role in shaping the neonatal oral microbiota. To identify oral maternal bacteria recognized by maternal IgG, we performed IgG-seq analysis with modifications. Because the sampled oral microbiota was too sparse for direct analysis, maternal oral samples were first plated under aerobic and anaerobic conditions, and the cultivated bacteria were subjected to IgG-seq using serum from 2-week-old mIg+ and mIgneg offspring. As demonstrated in Fig. 3E, anaerobic bacterial families that were over-represented upon incubation with mIg+ serum compared to the input microbiota (the total cultivated oral microbiota) consist of Pasteurellaceae, Burkholderiaceae, and Corynebacteriaceae. Additionally, although Bacteroidaceae and Muribaculaceae were not enriched relative to the input microbiota, their abundance was higher than that observed after incubation with serum from mIgneg offspring, suggesting lower affinity binding by maternal IgG. Within the aerobic bacteria, the families Mycoplasmataceae, Betaproteobacteriales, and Corynebacteriaceae were over-represented compared to the input microbiota (Fig. 3E). We further assessed the functional role of maternal IgG and found that it inhibited bacterial attachment to oral epithelial cells (Fig. 3F) and enhanced phagocytosis by RAW264.7 cells or freshly isolated neutrophils (Fig. 3G-H). These findings suggest that maternal IgG recognizes specific maternal oral microbial taxa and can limit their epithelial adhesion while promoting clearance via phagocytosis postnatally.
Next, we investigated whether the elevated oral microbial load detected in mIgneg neonates impacts gingival immunity. Flow cytometry analysis revealed increased frequencies of neutrophils and inflammatory Ly6Chi monocytes in the oral mucosa of 2-week-old mIgneg mice compared to mIg+ mice (Fig. 3I). Consistently, elevated mRNA levels of Cxcl1 and Il17a, both involved in neutrophil recruitment, as well as Cramp, which is expressed by neutrophils, were detected in the oral tissues of mIgneg mice (Fig. 3J). To determine whether the neonatal oral epithelium is permeable to IgG antibodies, allowing them to opsonize bacteria for phagocytosis by intraepithelial neutrophils, we applied anti-CD45 IgG antibodies to the oral epithelium of 1- and 8-week-old mice. Positive staining of CD45-positive cells was observed in 1-week-old mice, whereas reduced staining was observed in 8-week-old mice (Supplementary Fig. 4A). Western blot analysis and immunofluorescence staining confirmed the presence of maternal IgG antibodies in the neonatal oral epithelium (Supplementary Fig. S4B-C). Next, the expression level of GAS6, a key immunological factor upregulated during weaning and signifying the transition of the neonatal oral epithelium to an adult phenotype, was examined 2. GAS6 expression was significantly elevated at both the RNA and protein levels in the gingiva of 2-week-old mIgneg mice compared to the mIg+ control group (Fig. 3K). Consistently, the levels of Tlr4, Myd88, and Defa14, genes associated with postnatal maturation of the oral epithelium 1, were also upregulated in the mIgneg mice (Fig. 3L). Collectively, these findings indicate that maternal IgG antibodies, acquired both in utero and through breastfeeding, recognize maternal oral bacteria and can restrict their epithelial adherence, promote phagocytic clearance, and modulate gingival immune responses and epithelial development during early life.

Maternal IgG antibodies modulate structural, functional, and immunological parameters of the salivary glands
To gain further insight into the impact of maternal antibodies on the salivary glands, we conducted a comprehensive analysis of global gene expression in the parotid glands of mIg+ and mIgneg offspring 4 weeks after birth. Hierarchical clustering (Fig. 4A) and principal component analysis (PCA) (Fig. 4B) demonstrated a significant distinction between the transcriptomic profiles of the two groups. Gene set enrichment analysis (GSEA) revealed that cellular pathways associated with cell growth and differentiation, such as the EF2 target, G2M checkpoint, mitotic spindle, MYC targets, and KRAS signaling, were upregulated in the mIgneg group 21 (Fig. 4C). Additionally, pathways involved in tissue regeneration, including epithelial-mesenchymal transition, myogenesis, and angiogenesis 22, 23, were found to be increased. Conversely, pathways related to metabolism, such as oxidative phosphorylation, glycolysis, protein secretion, heme metabolism, fatty acid metabolism, xenobiotic metabolism, and adipogenesis 24, 25, were downregulated in the mIgneg group. Many of these pathways are known to be regulated by P53 and TGF-β, which were also downregulated in the mIgneg offspring 26, 27. Moreover, IFN-α-associated responses, a cytokine known to induce hypoactivation of the salivary glands upon immune activation 28, were reduced in the mIgneg group. Given the altered transcriptomic signature observed in the parotid glands of mIg+ and mIgneg offspring, indicating potential structural and functional changes, we conducted further investigations. As illustrated in Fig. 4D, histological cross-sections of the parotid glands revealed higher numbers of excretory ducts in mIgneg offspring compared to mIg+. Moreover, the saliva flow generated by the salivary glands was higher in the mIgneg offspring (Fig. 4E). Together, these findings indicate that maternal antibodies modulate the structural and functional characteristics of the salivary glands.
Next, since maternal antibodies are directed against the microbiota, we asked if they regulate the microbial load in the salivary glands and whether the microbiota affects local immunity. First, using fluorescence in situ hybridization (FISH) for visualization of bacterial 16S rRNA, heightened bacterial load was observed in the parotid glands of 2-week-old neonatal mIgneg mice (Fig. 4F). Next, the leukocytes of the salivary glands were examined in GF and SPF mice 4 weeks after birth using flow cytometry. As demonstrated in Fig. 4G-H, the parotid glands of GF mice contained substantially reduced frequencies of total leukocytes, affecting all types of immune cells. Consistent with this finding, the expression of key immunological genes such as Il17a, Foxp3, Il6, and Ifna was reduced in the GF parotid gland, except for Il10, which was upregulated (Fig. 4I). To determine whether the capability of maternal antibodies to modulate mucosal immune responses is affected by the bacterial load, we administered broad-spectrum antibiotics to pregnant dams and their offspring until weaning. As shown in Fig. 4J, perinatal antibiotic treatment reduced the early-life oral bacterial burden in both mothers and pups, allowing a comparison of immune responses under comparable microbial load. Despite similarly reduced bacterial loads, flow cytometry analysis of the salivary glands in 4-week-old offspring revealed increased frequencies of CD4⁺ T cells and B cells in the mIgneg group compared to the mIg⁺ group (Fig. 4K). These findings suggest that although the microbiota is required to initiate salivary adaptive immunity, maternal IgG antibodies modulate this response through a distinct immunoregulatory mechanism that is not affected by the microbial load.

Offspring lacking maternal antibodies exhibit altered salivary immunity in adulthood
Next, we evaluated the impact of maternal antibody deficiency on salivary immunity in adult offspring. Flow cytometry analysis of 8-week-old mice showed similar frequencies of CD4⁺ T cells and B cells in both mIgneg and mIg⁺ groups (Fig. 5A). However, the antibody secretion profile differed, as mIgneg mice displayed higher IgG and lower IgA levels compared to mIg⁺ mice (Fig. 5B-C). Unlike CD4⁺ T cells, CD8⁺ T cell frequencies were elevated in the adult mIgneg group (Fig. 5A), accompanied by an increased neutrophil population (Fig. 5D). Microbiota analysis via FISH targeting bacterial 16S rRNA revealed higher bacterial loads in mIgneg mice (Fig. 5E). These findings suggest that the absence of maternal antibodies disrupts salivary B-cell antibody production also in adulthood. Furthermore, the microbiota of adult salivary glands increased in the absence of maternal antibodies, correlating with higher frequencies of neutrophils and CD8+ T cells.

In utero-derived maternal IgG antibodies regulate the development of oral microbial and immunological homeostasis in adult life
The impact of maternal antibodies on the adult salivary glands led us to examine whether oral mucosal homeostasis is also affected in adulthood. To test the oral microbiota, the oral cavity was sampled from 8-week-old mIgneg and mIg+ mice and subjected to taxonomic analysis. First, we quantified the bacterial load and found higher cultivated aerobic and anaerobic bacteria in mIgneg mice than in mIg+ mice (Fig. 6A). Second, alpha diversity analysis revealed higher taxa richness in mIgneg mice compared with mIg+ mice (Fig. 6B). Furthermore, the composition of taxa differed significantly between these groups, as demonstrated by PCA (Fig. 6C). Heat tree analysis further reveals an increased abundance of Proteobacteria, typically associated with inflammation, relative to Firmicutes in mIgneg mice (Fig. 6D). A single-factor analysis identified a significant expansion of the Pasteurellaceae family, which includes opportunistic oral pathogens, alongside a reduction of the Enterococcaceae family, which contains commensal species known to contribute to mucosal health (Fig. 6E). To determine the source of maternal antibodies that regulate the adult oral microbiota, we conducted a fostering experiment. As shown in Fig. 6F, adult offspring that lacked in utero exposure to maternal antibodies exhibited higher oral microbial loads, regardless of postnatal antibody transfer via breast milk. Next, we asked whether maternal antibodies also influence the adult oral mucosal immunity, specifically focusing on the gingiva as an important oral barrier. Analysis of the gingival leukocytes of adult mice revealed higher frequencies and numbers of CD4+ and CD8+ T cells in mIgneg mice compared to mIg+ mice (Fig. 6G-H and Supplementary Fig. 5). These results suggest that lacking maternal antibodies transferred in utero during early life disrupts the establishment of oral mucosal homeostasis in adulthood

Breast milk- and in utero-derived antibodies play distinct roles in maintaining oral epithelial integrity and regulating periodontal immunity
The altered homeostasis observed in the oral mucosa encourages us to ask whether the barrier function of the gingiva was also affected. For this, we profiled global gene expression in the gingiva of adult mIgneg and mIg+ mice. Hierarchical clustering identifies differential gene expression between the two groups of mice (Fig. 7A). GSEA revealed an elevation of immunological-associated pathways in mIgneg mice such as inflammatory response, allograft rejection, UV response, IL-2/STAT5 signaling, and complement (Fig. 7B). In addition, pathways associated with tissue homeostasis and remodeling were upregulated in this group and include epithelial to mesenchymal transition, Kras singling, mitotic spindle, apical junction, hedgehog signaling, coagulation, and angiogenesis 22. The GSEA also identified the down-regulation of various pathways related to tissue metabolism, such as oxidative phosphorylation, fatty acid metabolism, glycolysis, hypoxia, adipogenesis, myogenesis, and reactive oxygen species 25. Other reduced pathways were associated with cell proliferation, such as MYC target genes, G2M checkpoint, DNA repair, E2F targets, and the p53 pathway.
Since several of the above-dysregulated pathways imply an alteration in the function of the gingival epithelium, we subsequently examined the gingival epithelium. Initially, epithelial permeability was assessed by administering FITC solution onto the oral epithelium of anesthetized mice and then quantifying FITC staining intensity within the gingiva. As depicted in Fig. 7C, higher fluorescence intensity was observed in gingival tissues isolated from mIg+ mice compared to mIgneg mice, indicating greater epithelial sealing in the latter. Corresponding with this finding, the expression of the tight junction protein ZO-1 was elevated in the epithelium of mIgneg mice (Fig. 7D). qPCR analysis further revealed an upregulation of Cldn1 and Cldn4, other types of tight junction molecules, in mIgneg offspring, supporting their enhanced epithelial sealing (Fig. 7E). Agreeing with the upregulation of tissue remodeling pathways noted earlier, the gingival epithelial cells of adult mIgneg mice exhibited increased BrdU incorporation upon treatment, suggesting a faster proliferation rate compared to mIg+ mice (Fig. 7F). To assess the respective contributions of in utero versus breastfeeding-derived antibodies to epithelial development, a fostering experiment was performed. As shown in Fig. 7G-H, mice that received maternal antibodies via breastfeeding exhibited increased FITC penetration, reduced expression of tight junction genes, and downregulation of the proliferation-associated gene Ki67, suggesting a postnatal role in regulating oral epithelial maturation. Finally, perinatal antibiotic treatment, which reduced early-life microbial load, eliminated the differences in epithelial gene expression between mIg⁺ and mIg⁻ offspring (Supplementary Fig. 6), indicating that epithelial maturation depends on the ability of breast milk antibodies to limit microbial exposure during early life
Next, we assessed natural bone loss around the teeth (the alveolar bone) in each group, as this type of bone is sensitive to changes in gingival health. While no significant differences were found in levels of natural bone loss (as indicated by the values of bone volume and the exposed root area), the alveolar bone of mIgneg mice exhibited alterations in trabecular numbers and thickness (Fig. 7I). Accordingly, increased numbers of TRAP-positive cells, representing bone-resorbing osteoclasts, were observed in the maxilla of mIgneg mice (Fig. 7J). To determine whether the altered oral mucosal homeostasis in mIgneg mice increases their susceptibility to periodontal disease, we utilized the ligature-induced periodontitis (LIP) model, a well-established approach that triggers local inflammation and microbial dysbiosis leading to alveolar bone loss. As demonstrated in Fig. 7K, the mIgneg mice exhibited elevated bone loss, the hallmark of periodontitis. The elevated bone loss was associated with higher frequencies of neutrophils and CD8+ T cells (Fig. 7L and Supplementary Fig. 7), indicative of heightened periodontal immune responses. To dissect the respective contributions of in utero- versus breastfeeding-derived antibodies to gingival immunity, we performed LIP in fostered mice. As shown in Fig. 7M, mice lacking in utero maternal antibodies exhibited elevated bone loss, underscoring their pivotal role in gingival immune regulation. These results collectively suggest that maternal antibodies exert distinct effects on the adult gingival barrier. Antibodies transferred postnatally via breast milk regulate epithelial maturation, whereas those acquired in utero program gingival immunity and shape adult inflammatory responses and susceptibility to periodontitis. Fig. 8 provides a schematic summary of the respective contributions of breast milk- and placentally-derived antibodies to offspring oral immunity.

Methods
Mice
C57BL/6 (B6) (The Jackson Laboratory, strain #: 3000664)and J H T-/- (The Jackson Laboratory, strain #: 002438) mice were bred and maintained in the central animal facility at the Hebrew University Faculty of Medicine (Jerusalem, Israel). The mice were housed under specific pathogen–free (SPF) conditions in the institutional animal facility, maintained on a 12-hour light/12-hour dark cycle, at an ambient temperature of 22 ± 2 °C and relative humidity of 50–60%, with ad libitum access to food and water, and analyzed at various ages as described in the text. Control and experimental groups were housed separately, unless indicated otherwise in the text. All animal protocols were approved by the Hebrew University Institutional Animal Care and Use Committee (IIACUC). The Germ-free (GF) B6 mice were maintained in sterile isolators at the Weizmann Institute of Science; the GF studies were approved by the IACUC of the Weizmann Institute of Science.

Antibodies
All antibodies, their catalogue numbers, final dilutions, and source are documented in Supplementary Table 1.

Isolation of oral leukocytes
The gingiva and the parotid gland tissues were excised into wells containing PBS-FBS 2%. Tissues were then minced and treated with a Collagenase type II (2 mg/mL; Worthington Biochemicals) and DNase I (1 mg/mL; Sigma) solution in PBS plus 2% FCS for 25 min at 37 °C in a shaker bath (for the list of reagents used in this study, see Supplementary Table 3)A total of 20 μL of 0.5 M EDTA per 2 mL sample was added to the digested tissues and incubated for an additional 10 min. The cells were washed, filtered with a 70-μM filter, stained with antibodies, run in an Aurora (Cytek) flow cytometer, and further analyzed, generating plots and t-SNE plots using FlowJo software (BD Biosciences).

T-cell stimulation assay
For intracellular cytokine staining, purified gingival cells were stimulated with phorbol myristate acetate (PMA) (1 mg/mL)/ionomycin (1 mg/mL) and Golgi Plug (2 mL/mL) for 6 hr at 37°C. The cells were then stained with CD45, CD3, CD4, CD8, and αβTCR antibodies, permeabilized using BD Cytofix/Cytoperm™ kit (BD Biosciences), and then stained with IL-17A and IFN-γ antibodies. The samples were run in the Aurora (Cytek) flow cytometer and further analyzed using FlowJo software (BD Biosciences).

Immunofluorescence staining
For frozen section staining, the maxilla was fixed overnight at 4°C in 4% paraformaldehyde/PBS solution and then decalcified for 2-3 weeks in EDTA 0.5M/PBS. The tissue was cryopreserved in 30% sucrose (overnight at 4°C), embedded in OCT, and cryo-sectioned into 10-μm-thick sections. The cross-sections were washed 3 times in PBS, blocked in blocking buffer (5% FCS, 0.1% Triton X-100 in PBS) for 1 hr at room temperature, and incubated with a primary antibody overnight at 4°C. Following 3 washing steps in PBS, the samples were incubated with a secondary antibody diluted 1:200 in blocking buffer for 2 hr at room temperature, washed 3 times, stained with DAPI, and mounted. For paraffin sections, the maxilla was fixed overnight at 4°C in 4% paraformaldehyde/PBS solution and then decalcified for 2-3 weeks in EDTA 0.5M/PBS, which was changed every other day. The tissues were dehydrated using 70%, 80%, 90%, and 100% ethanol and then xylene to dissolve the alcohol. Next, the tissues were embedded in paraffin and micro-sectioned into 7μm-thick sections. Slides were deparaffinized with xylene, and 100%, 95%, 80%, and 70% ethanol washed 3 times with PBS, blocked in blocking buffer (PBS, 10%FCS, 10% BSA, 2% Triton X-100) for 1.5h at room temperature, and incubated with primary antibodies at 4°C. Following 3 washing steps in PBS, the samples were incubated with secondary antibodies diluted 1:200 in blocking buffer for 1.5 hr at RT, washed 3 times, stained with DAPI, and mounted. As a negative staining control, the primary antibody was omitted and replaced with the blocking buffer. Signals were visualized, and digital images were obtained using a Nikon spinning disk confocal microscope.

Fluorescent in Situ Hybridization for 16S ribosomal RNA
For fluorescent in situ hybridization (FISH) analysis, the tissues were excised and fixed overnight at 4°C in 4% paraformaldehyde/PBS solution. The tissues were dehydrated using 70%, 80%, 90%, and 100% ethanol and then xylene to dissolve the alcohol. Next, the tissues were embedded in paraffin and micro-sectioned into 7 μm-thick sections. Slides were deparaffinized with xylene, and 100%, 95%, 80%, and 70% ethanol and washed 3 times with PBS, dried, and stained with 1:100 with universal 16S probe in pre-warmed to 46°C hybridization solution (20 mM Tris–HCl, pH 7.4, 0.9 M NaCl, 0.1% (w/v) SDS). The slides were then sealed with a cover slip and incubated in a humid chamber overnight at 46°C. Following the 3 washing steps in PBS, the samples were stained with DAPI and mounted. As a negative staining control, a nonsense probe control was replaced with the 16S probe. Signals were visualized, and digital images were obtained using a Nikon spinning disk confocal microscope.

Hematoxylin and tartrate-resistant acid phosphatase staining
The maxilla was fixed overnight at 4°C in 4% paraformaldehyde/PBS solution and then decalcified for 2-3 weeks in EDTA 0.5M/PBS, which was changed twice a week. The tissues were dehydrated using 70%, 80%, 90%, and 100% ethanol and then xylene to dissolve the alcohol. Next, the tissues were embedded in paraffin and micro-sectioned into 7 μm-thick sections. Slides were deparaffinized with xylene, and 100%, 95%, 80%, and 70% ethanol and washed three times with PBS. TRAP (Sigma-Aldrich, Rehovot, Israel) staining with hematoxylin counterstain according to the manufacturer’s instructions enabled quantification of the osteoclasts (TRAP-positive multinucleated cells at the alveolar bone surface). Images of TRAP-stained cross-sections were captured with a Nikon TL microscope.

BrdU incorporation assays
8-week-old mice were injected once intraperitoneally with BrdU, 10µL/gram of body weight (Sigma). Gingival tissues were collected after 16 hr and then embedded in OCT and cryo-sectioned into 10 μm-thick sections. The cross sections were washed 3 times in PBS, incubated for 1 hr with 1N HCl at 37°C, and blocked in blocking buffer (20% FCS, 0.1% Triton X-100, 0.02% NaN3, 0.05% Tween-20 in PBS) for 1 hr at RT, and incubated with anti-BrdU antibody (Abcam) overnight at 4°C. Following 3 washing steps in PBS, the samples were incubated with a secondary donkey anti-Sheep IgG H&L (Alexa Fluor 488) (Abcam), diluted 1:200 in dilution buffer (2% FCS, 0.1% Triton X-100, 0.02% NaN3, 0.05% Tween-20 in PBS) for 2 hr at RT, washed 3 times, stained with DAPI, and mounted. Signals were visualized, and digital images were obtained using a Nikon spinning disk confocal microscope.

Micro-computed tomography analysis
The hemi-maxillae were secured in an acrylic mold using orthodontic wax for stability during act scans, which were conducted at 8 µm resolution using a μCT instrument Skyscan1272 (Bruker microCT, Kontich, Belgium). Air served as the scan medium to optimize the contrast between the sample and the background. 3D reconstruction and analysis of the μCT images were executed using Dragonfly software [Version 2022.2 for Windows; Object Research Systems (ORS) Inc., Montreal, Canada] with TIFF files. Bone volume evaluation was conducted between the mesiobuccal and distal roots of M2, utilizing the Bone Analysis Wizard. A 0.2 mm radius sphere was chosen to measure properties in 3D. Otsu’s threshold method was employed to segment bone from non-bone; in our case, teeth were manually erased from the analysis since the software cannot distinguish between bone and teeth. The Bone Analysis module in Dragonfly was utilized to segment cortical and trabecular bone. Another adjustment made in the wizard was to disregard cortical bone, as it is very thin in mouse maxillae and cannot be distinguished by the software. After delineating all bone structures, bone morphometric measurements were computed, following the guidelines described by (Bouxsein et al., 58. Using the Bone Analysis module, trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th, µm), and trabecular number (Tb.N, 1/mm) were determined for trabecular bone. In the second method, DICOM files were extracted from the scanner and analyzed in the OnDemand3D Application. The area between the cementoenamel junction and the alveolar bone level was marked and measured on the buccal and palatal sides.

RNA extraction and qPCR
For RNA isolation, the excised gingiva was homogenized in 500 µl TRI reagent (Sigma) using an electric homogenizer (IKA labortechnik), and RNA was extracted according to the manufacturer’s instructions. cDNA synthesis was performed using the qScript cDNA Synthesis Kit (Quanta-BioSciences). qPCR reactions (20 µL volume) were performed using Power SYBR Green PCR Master Mix (Quanta-BioSciences) and specific primers to the examined gene. The following reaction conditions were used: 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 60 s at 60 °C. The samples were normalized to 18S as a control mRNA using the change in cycling threshold (ΔCT) method and calculated based on the 2-ΔΔCT formula. The sequences of all primers used in this study are provided in Supplementary Table 2.

RNA-Seq differential expression analysis
Raw reads were processed according to the QuantSeq User Guide recommendations. Reads were trimmed at their 5' end to remove the first 12 bases, then low-quality and technical bases were removed from the 3' end using cutadapt (version 1.12) 59. Finally, low-quality reads, with more than 30 percent of the bases with quality below 20, were filtered out using the FASTX package (version 0.0.14). Processed reads were aligned against the mouse genome using TopHat (v2.1.1) 60. The genome version was GRCm38, with annotations from Ensembl release 89. Htseq-count (version 0.6.0) 61 was then used for quantification of raw counts per gene per sample, excluding short or otherwise unwanted gene types, such as rRNA or miRNA. Normalization and differential expression analysis were performed with the DESeq2 package (version 1.12.4) 62. Genes with a sum of counts less than 10 over all samples were filtered out before normalization. Differential expression, comparing control (mIg+) and treated (mIgneg) groups, was calculated with default parameters, except that the independent filtering algorithm was used. The statistical significance threshold was taken as an adjusted p-value (padj) less than 0.1. Exact commands with the full parameters used can be found under GEO accession GSE274186.

Gene set enrichment analysis.
The whole differential expression data were subjected to gene set enrichment analysis using GSEA 63. GSEA uses all differential expression data (cut-off independent) to determine whether a priori–defined sets of genes show statistically significant, concordant differences between two biological states. GSEA was run against the hallmark gene set collection from the molecular signatures database (MSigDB, v6.2, July 2018).

Western blot analysis
Parotid gland or gingival tissues were isolated and homogenized in ice-cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 0.1% SDS, 0.5 mM EDTA) supplemented with a protease inhibitor cocktail (Sigma). Following homogenization, tissue debris was removed by centrifugation (15,000 g for 10 min at 4 °C), and protein concentration was determined using a NanoDrop spectrophotometer. Samples were prepared by adding Tris-Glycine-SDS Sample Buffer followed by 2% β-mercaptoethanol (Sigma-Aldrich), and then heated at 90°C for 10-15 min. Equal amounts of protein (50 µg) were loaded onto 7.5% acrylamide gels and resolved by SDS-PAGE. Proteins were transferred to Trans-Blot Turbo Mini nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo Transfer System at 25 V, 1.3 A for 7 min. Membranes were blocked with PBS containing 0.05% Tween-20 (PBST) and 5% non-fat dried milk for 1 hr at room temperature, and incubated overnight at 4°C on a shaker with HRP-conjugated goat primary antibody (1:5000 dilution in blocking buffer). The membrane was then washed 3 times in PBST before the blots were reacted with ECL substrate (Western blot detection kit, Advansta). Images were captured using a ChemiDocTM MP Image System (Bio-Rad). Bands were normalized to the band intensity of GAPDH.

ELISA and serum analysis
Blood was drawn from mice, and the sera were stored at -80 °C. Maternal microbiota was collected by swabs and enriched on blood agar overnight. Ninety-six-well plates (Nunc) were coated overnight at 4°C with maternal microbiota in 0.1 M bicarbonate buffer (pH 9). The plates were washed twice with PBST and blocked with PBS 10% FCS (2 hr at room temperature). Subsequently, mouse serum samples diluted serially in PBS were added to the wells for 3 hr incubation at RT. This was followed by four washes in PBST and the addition of anti-mouse peroxidases-conjugated IgG1, IgG2a, and IgG2b, IgG3, IgA, and IgM antibodies (Southern Biotech). After incubation for 2 hr at RT, the plates were washed five times, and 100 μL/well of tetramethyl benzidine (TMB) solution (Southern Biotech) was added for 5 min, followed by the addition of 100 μL of TMB stop solution (Southern Biotech). Absorption was read at 450 nm using MULTISKAN SkyHigh (Thermo Scientific). For general characterization of antibody subtypes presented in the neonate's serum, we used the SBA Clonotyping System-HRP kit (Southern Biotech) according to the manufacturer’s instructions. For quantifying IgG concentrations in the parotid glands, saliva, and serum, an IgG competitive inhibition ELISA kit (USCN Life Science Inc.) was used according to the manufacturer’s protocol.

Microbiota analysis
The oral cavity of anesthetized B6 mice was swabbed for 30 seconds. The swabs were then processed for DNA isolation using the Zymo Quick-DNA Fungal\Bacterial DNA Extraction Kit according to the manufacturer’s instructions. Library preparation, sequencing, and analysis were performed by Hay Laboratories (Israel) as follows: Libraries were prepared using a two-step PCR protocol. Primers 515F–806R, which target the V4 region of the 16S SSU rRNA, were used for PCR amplification. 515F (ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGT) and 806R (TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCT) (Earth Microbiome Project). The PCR reaction products were cleaned using AMPure beads and then subjected to a second PCR using the Fluidigm Access Array Primers for the addition of Illumina adaptor and index sequences. DNA was purified using Kapa Pure Beads at a ratio of 0.65X and quantified with Qubit using Denovix dsDNA high sensitivity assay. DNA size and integrity were quantified by Tapestation using Agilent DNA screen tape and reagents. The libraries were then sequenced on a dedicated MiSeq (Illumina) machine with 5-30% PhiX using MiSeq Reagent Kit v2 500PE. Demultiplexing was performed using bcl2fastq with default parameters, allowing for 0 mismatches. Data was then mapped to PhiX using bowtie2 to remove PhiX control, and unmapped reads were quantified, collected, and examined using fastQC. Bioinformatics analysis was performed with QIIME 2. Briefly, demultiplexed fastq files were processed for adapter sequence removal using the Cutadapt trim-paired plugin, followed by denoising with DADA2 (via q2-dada2) to identify all observed amplicon sequence variants (ASVs). ASVs were aligned with Mafft (via q2-alignment) and used to construct a phylogeny with Fasttree2 (via q2-phylogeny). Taxonomy was assigned to ASVs using the q2-feature-classifier classify-sklearn naïve Bayes taxonomy classifier, which was pretrained against sequences extracted from Greengenes 13_S8 99% OTUs sequences using primers set 515F-806R. Qiime2 output files were used as inputs to ‘MicrobiomeAnalyst—comprehensive statistical, visual and meta-analysis of microbiome data’ online tool (https://www.microbiomeanalyst.ca/MicrobiomeAnalyst/home.xhtml), after minor manual format editing.
To examine the absolute abundance of certain bacteria, DNA was prepared from oral swabs using the Zymo Quick-DNA Fungal\Bacterial DNA Extraction Kit according to the manufacturer’s instructions, and then directly subjected to RT-PCR analysis using designated primers. qPCR reaction was performed using Power SYBR Green PCR Master Mix (Quanta-BioSciences Inc.). The following reaction conditions were used: 10 min at 95°C, 40 cycles of 15 sec at 95°C and 60 sec at 60°C. The samples were normalized to the 18S as control mRNA by the change in cycling threshold (ΔCT) method, and calculated based on 2-ΔCT.

Permeability assay
20 mg of FITC (Sigma) was dissolved in 100µl DMSO (Sigma) and then diluted 1:10 with acetone. The FITC solution was applied using a pipette on the buccal mucosa of anesthetized 8-week-old mice (40 μL per mouse). After 30 min, the mice were euthanasied with isoflurane in a desiccator , the oral cavity was washed with DDW, and the buccal tissues were collected, embedded in OCT, and cryosectioned into 10-μm-thick sections. Signals were visualized, and digital images were obtained using an Olympus BX51 fluorescence microscope. For toluidine blue experiments, 40 mg of toluidine blue was dissolved in 250 μL DMSO (Sigma) and then diluted 1:1 with acetone. The toluidine blue solution was applied using a pipette on the buccal mucosa of anesthetized 1-week-old mice (20 μL per mouse) or 8-week-old mice (50 μL per mouse). After 30 min, the mice were euthanasied and the buccal tissues were collected, embedded in OCT, and cryosectioned into 10-μm-thick sections. Signals were visualized, and digital images were obtained using a Nikon spinning disk confocal microscope.

Epithelium permeability to IgG antibodies
One-week-old and eight-week-old mice were anesthetized, and 5 or 15 μL (adjusted based on body weight ratio) of Pacific Blue anti-mouse CD45.2 antibody (0.5 mg/mL) was carefully applied to the gingiva. After 30 min, the mice were euthanasied with isoflurane in a desiccator , and the oral epithelium was collected, processed to obtain a single-cell suspension, and analyzed using an Aurora flow cytometer (Cytek).

FITC application
20mg of FITC (Sigma) was dissolved in 100 μL DMSO (Sigma), and the solution was diluted in acetone (1:1). Mice were anesthetized, and 40 μL of the solution was carefully applied to the gingiva. The parotid glands were excised 2 days after the application for flow cytometry analysis.

Fostering experiments
Pregnant J H T+/+ and J H T-/- dams were timed-mated to deliver simultaneously, and within 24 hours of birth, the dams and offspring were exchanged to generate four groups: (1) mIg+ pups nursed by J H T+/+ dams, (2) mIgneg pups nursed by J H T-/- dams, (3) mIg+ pups nursed by J H T-/- dams, and (4) mIgneg pups nursed by J H T+/+ dams. Pups were fostered with litters of matching size and sex distribution to ensure equal nutritional access and maternal care. Cross-fostering was confirmed by daily monitoring of maternal behavior and pup survival. Tissues were collected at the indicated timepoints for downstream analysis.

Perinatal broad-spectrum antibiotic treatment
Pregnant dams were provided with a combination of amoxicillin (80 mg/L) and tylosin (40 mg/L) in their drinking water starting from embryonic day 15 until their offspring reached 4 weeks of age. The antibiotic-containing water was replaced every three days to maintain potency. Control dams received regular drinking water. All treatments were administered ad libitum.

IgG-Seq analysis
To assess the binding specificity of maternal antibodies to oral bacteria, we performed IgG-Seq using serum from 2-week-old mIg⁺ and mIgneg offspring. Due to the limited bacterial biomass obtained from direct oral swabbing, oral bacteria were isolated from swabs collected from the respective dams and cultured on blood agar under aerobic and anaerobic conditions to generate sufficient material (termed input microbiota) for downstream analysis. Bacterial colonies were harvested by centrifugation, washed in PBS, and resuspended to a standardized optical density (OD600). Aliquots of the cultured bacteria were incubated with heat-inactivated serum (1:100 dilution) from 2-week-old mIg⁺ or mIgneg offspring for 30 min at 4°C. After washing to remove unbound antibodies, IgG-coated bacteria were labeled with PE-conjugated anti-mouse IgG antibody, followed by magnetic separation using an Anti-PE MicroBeads UltraPure system (Miltenyi Biotec). The labeled suspension was loaded onto a pre-equilibrated magnetic column and washed multiple times with PBS to remove unbound material. The IgG-bound ("positive") fraction was then eluted by removing the column from the magnetic field.
DNA was extracted using 270 μL of GT lysis buffer and 30 μL of proteinase K (both from the MagCore Genomic DNA Tissue Kit), combined with 200 μL of the sample in bead beating tubes (Type C, GeneAid). Bead beating was carried out for 2 min using a Biospec homogenizer. Samples were then incubated at 60°C for 2 hr and subsequently processed on the MagCore extraction system (RBC Bioscience) using the MagCore Genomic DNA Tissue Kit cartridges and standard protocol.
For DNA amplificationan initial PCR was performed using 2 μL of DNA as template in a 25 μL reaction volume with PCRBio Hot Start Ready Mix and custom primers targeting the V4 region of the 16S rRNA gene (based on the Earth Microbiome Project). These primers included CS1/CS2 adapter sequences. Amplification was carried out for 25 cycles. Primer sequences: CS1_515F (V4_F): ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGT and CS2_806R (V4_R): TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCT. 2 μL of the PCR product (containing CS1/CS2 adaptors) was used as template in a 10 μL reaction for 10 additional cycles using the Fluidigm Access Array Barcode Library protocol (2 μL barcode per reaction). Following amplification, DNA was purified using KAPA Pure Beads at a 0.65× ratio and quantified using Qubit with the DeNovix dsDNA High Sensitivity Assay. DNA fragment size and integrity were assessed using the Agilent TapeStation system with DNA ScreenTape and reagents.
Samples were then sequenced on a dedicated Illumina MiSeq instrument using the MiSeq Reagent Kit v2 (500-cycle, paired-end) with 30% PhiX spike-in. Demultiplexing was performed using bcl2fastq with default settings, allowing zero mismatches in the index reads. Reads were then aligned to the PhiX genome using Bowtie2, and unmapped reads were retained for downstream analysis. Read quality was assessed using FastQC.
For sequencing analysis, demultiplexed reads were imported into the CLC Genomics Workbench (Qiagen) and analyzed using the built-in 16S Microbiome pipeline. The workflow included quality filtering, adapter trimming, and OTU clustering under default parameters. Reads with a Phred quality score below 25 or length <150 bp were discarded, and a maximum of two ambiguous nucleotides per read was allowed. Only reads between 200-500 bp were retained. Chimeric sequences and singletons were identified and removed. Remaining high-quality sequences were clustered into operational taxonomic units (OTUs) by alignment to the SILVA database at 97% sequence similarity.

Bacterial adhesion assay
Oral swabs were obtained from dams via swabbing and cultured on blood agar plates under aerobic and anaerobic conditions to generate sufficient bacterial biomass. Colonies were harvested, resuspended in sterile PBS, and adjusted to a standardized optical density. Bacterial suspensions were incubated with heat-inactivated serum (1:10 dilution) from 2-week-old offspring for 1 hr at 4°C with gentle rotation to allow antibody binding. For the adhesion assay, immortalized oral epithelial cells (4NQO-L) were seeded in 24-well plates and cultured overnight in DMEM (Sigma-Aldrich, Rehovot, Israel) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 µg/mL; Biological Industries, Beit Haemek, Israel). The cells were cultured at 37 °C and 5% CO2 until they reached confluence. Before infection, cells were washed with pre-warmed DMEM 10% FCS 1% L-glutamine 1% medium, washed, and then incubated with the serum-opsonized bacteria at a multiplicity of infection of 1:100 for 1 hr at 37°C in 5% CO₂. Following incubation, wells were washed extensively with PBS to remove non-adherent bacteria. Cells were then lysed, and serial dilutions of the lysates were plated on blood agar to quantify adherent bacteria by CFU enumeration.

Phagocytosis assays
Oral bacteria were collected from dams via swabbing and cultured on blood agar plates under aerobic and anaerobic conditions. Bacterial colonies were harvested, washed in sterile PBS, resuspended, and incubated with heat-inactivated serum (1:10 dilution) from 2-week-old offspring for 1 hr at 4°C with gentle rotation to allow opsonization. RAW264.7 cells were seeded in 24-well plates and cultured overnight in DMEM (Sigma-Aldrich, Rehovot, Israel) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 µg/mL; Biological Industries, Beit Haemek, Israel). The cells were cultured at 37 °C and 5% CO2. Cells were then exposed to opsonized bacteria at a multiplicity of infection (MOI) of [1:100] for 1 hr at 37°C and 5% CO₂. After incubation, cells were washed thoroughly with sterile PBS to remove extracellular bacteria. Extracellular bacteria were killed by treatment with 6 mg/mL Metronidazole (Sigma-Aldrich) and 0.3 mg/mL Gentamycin (Biological Industries) for 1 hr. The medium was then replaced with complete DMEM, cells were incubated for 1 hr, and then lysed in sterile distilled water for 20 min. Lysates were plated on blood agar to quantify internalized bacteria by CFU enumeration. In a separate experiment, oral bacteria collected from dams were labeled with 10 µM CFSE (carboxyfluorescein succinimidyl ester; Invitrogen) in PBS for 1 hr at room temperature, with shaking in the dark. Excess dye was removed by three washes in 15 mL PBS. The labeled bacteria were then incubated with heat-inactivated serum from 2-week-old offspring (1:20 dilution) for 1 hr at 4 °C with gentle rotation to allow for antibody opsonization. Freshly isolated neutrophils (0.5 million), obtained from the femurs of adult WT mice using a Histopaque density gradient, were then added to the opsonized CFSE-labeled bacteria at a multiplicity of infection (MOI) of 10:1 and incubated at 37 °C for 30 min. After incubation, cells were thoroughly washed with cold PBS and stained with anti-Ly6G antibody to identify neutrophils. Phagocytosis was assessed by flow cytometry.

Experimental periodontitis
Mice were anesthetized, and silk ligatures (5/0, SMI, Belgium) were tied around both second maxillary molars as previously described (Abe and Hajishengallis 2013). Seven to nine days after ligature placement, the mice were euthanasied with isoflurane in a desiccator and analyzed.

Quantification of saliva flow
Mice were held at a 45° angle (head down), and the oral cavity was filled with a cone-shaped sponge for 15 min. The sponge was then inserted into a 0.5 mL tube with a hole in the bottom that was placed in a 2 mL tube and centrifuged for 3 min at 5000 g.

Quantification and statistical analysis
Data are expressed as means ± SEM. Statistical tests were performed using a two-sided, unpaired t-test comparing two groups, and a one-way ANOVA comparing more than two groups. A p-value of <0.05 was considered significant. Detailed information on the number (n) of biological samples and animals used is mentioned in the figure legends. * P < 0.05 **, P < 0.01, and *** P < 0.00.1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study explores the influence of maternal antibodies, specifically IgG transferred both in utero and through breastfeeding, on the development of oral and salivary mucosal immunity in offspring. Using an experimental mouse model with crossbreeding, researchers conducted immunological, microbiological, and transcriptomic analyses to assess how maternal IgG shapes oral immune homeostasis in neonates and adults. The findings reveal that maternal IgG reaches neonatal salivary glands and saliva, where it interacts with maternal oral microbiota. It plays a crucial role in regulating microbial colonization, immune system activation, and epithelial development. Notably, the absence of in utero-derived IgG led to increased activation of T- and B-cells, an altered IgG/IgA balance, higher microbial loads, and greater vulnerability to experimental periodontitis in adulthood. These results highlight the distinct contributions of prenatal and postnatal antibodies in establishing oral immunity and promoting long-term oral health.\nFollowing review of the manuscript, a few areas for improvement are identified:\n1.The data indicate that maternal IgG suppresses early adaptive immunity, but the discussion could be enhanced by incorporating potential molecular pathways, such as FcγRIIB signaling and Treg induction, and connecting these mechanisms to the transcriptomic changes observed.\n2.While the manuscript addresses the complementary roles of antibodies derived in utero and from breastmilk, these insights are dispersed across various sections. A clear visual or tabular summary would help consolidate these findings and improve overall clarity.\n3.The IgG-seq results highlight bacterial families targeted by maternal IgG. To underscore the significance of this discovery, the discussion could delve deeper into the immunological relevance of these taxa, such as their pathogenic potential or links to oral dysbiosis, to better illustrate their impact on long-term oral health.\n4.It is briefly mentioned that maternal immunization has the potential to enhance offspring oral immunity. Expanding on this idea by discussing its feasibility, possible targets, and limitations, such as species-specific differences and the timing of antibody transfer, would add depth and strengthen the manuscript’s overall impact.", "reviewer_2": "Naamneh et al have investigated the role of maternal-derived (both prenatal and postnatal) IgG on early-life and long-term immune homeostasis. This is a crucial question in the field and needs to be interrogated. However, the following needs to be addressed to make the manuscript is conclusive.\n1. The authors need to make the introduction focused, highlighting the central questions. The introduction currently has a lot of information without a clearly defined direction.\n2. Line 92 and experiments performed in Fig 1: It is unclear how conclusions from this experiment are different from those conducted in a previous paper by the same group (Cell Reports, 2023). In the Cell Reports paper, the authors show that maternal IgG is transferred to saliva. It is not clear how Fig 1 of this current paper is novel. If this is only to establish that this phenomenon is also observed in mIg+/neg mice, then all of Fig 1 needs to be in supplemental.\n3. Please elaborate the discussion section on why CD4+ T cells, CD8+ T cells and B cells were lower in mIg+ mice and what benefit does that provide to early life immunity.\n4. Please include a discussion section for the transcriptomic alteration observed among mIg+ and mIg-neg mice and what advantage does that provide to early life immunity.\n5. It is important to show how the mucosal immune environment of mIg+ and mIg-neg mice impacts their susceptibility to oral infections or any oral diseases."}, "author_response": "We would first like to thank the reviewers for the careful and critical evaluation of our manuscript and for the constructive suggestions to improve its quality. We have addressed each comment in detail below.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis study explores the influence of maternal antibodies, specifically IgG transferred both in utero and through breastfeeding, on the development of oral and salivary mucosal immunity in offspring. Using an experimental mouse model with crossbreeding, researchers conducted immunological, microbiological, and transcriptomic analyses to assess how maternal IgG shapes oral immune homeostasis in neonates and adults. The findings reveal that maternal IgG reaches neonatal salivary glands and saliva, where it interacts with maternal oral microbiota. It plays a crucial role in regulating microbial colonization, immune system activation, and epithelial development. Notably, the absence of in utero-derived IgG led to increased activation of T- and B-cells, an altered IgG/IgA balance, higher microbial loads, and greater vulnerability to experimental periodontitis in adulthood. These results highlight the distinct contributions of prenatal and postnatal antibodies in establishing oral immunity and promoting long-term oral health.\nFollowing review of the manuscript, a few areas for improvement are identified:\n1.The data indicate that maternal IgG suppresses early adaptive immunity, but the discussion could be enhanced by incorporating potential molecular pathways, such as FcγRIIB signaling and Treg induction, and connecting these mechanisms to the transcriptomic changes observed.\nResponse: We thank the reviewer for this insightful suggestion. We have now expanded the Discussion to incorporate potential molecular mechanisms by which maternal IgG may suppress early adaptive immunity. Specifically, we discuss the possible involvement of inhibitory signaling, as well as the induction of Treg cells, both of which could contribute to dampened adaptive immune activation in early life. Importantly, we now link these pathways to the transcriptomic changes observed in our dataset, including the altered expression of genes associated with immune regulation and T cell differentiation. These additions help provide a mechanistic framework that integrates our functional and transcriptomic findings (Page 18 line 22– page 19-20).\n2.While the manuscript addresses the complementary roles of antibodies derived in utero and from breastmilk, these insights are dispersed across various sections. A clear visual or tabular summary would help consolidate these findings and improve overall clarity.\nResponse: We thank the reviewer for this helpful suggestion. To improve clarity and consolidate these findings, we have added a visual summary figure (Fig. 8 in the revised manuscript) that integrates the complementary roles of antibodies acquired in utero and through breastmilk. This schematic highlights their temporal contribution, sites of action, and distinct immunological effects, allowing the key concept to be presented in a unified and accessible manner. We believe this addition substantially improves the overall clarity of the manuscript.\n3. The IgG-seq results highlight bacterial families targeted by maternal IgG. To underscore the significance of this discovery, the discussion could delve deeper into the immunological relevance of these taxa, such as their pathogenic potential or links to oral dysbiosis, to better illustrate their impact on long-term oral health.\nResponse- We thank the reviewer for this important point. We have now expanded the Discussion to further elaborate on the immunological relevance of the bacterial families identified by IgG-seq. Specifically, we discuss their reported pathogenic potential and documented associations with oral dysbiosis and inflammatory oral diseases, thereby situating our findings within the context of long-term oral health (from page 21, line 8 to page 22, line 8).\n4.It is briefly mentioned that maternal immunization has the potential to enhance offspring oral immunity. Expanding on this idea by discussing its feasibility, possible targets, and limitations, such as species-specific differences and the timing of antibody transfer, would add depth and strengthen the manuscript’s overall impact.\nResponse- We thank the reviewer for raising this important point. We have now expanded the Discussion to further elaborate on the relevance of our murine findings to humans and also to the concept of maternal immunization as a strategy to enhance offspring oral immunity (from page 22, line 9 to page 23, line 2).\nReviewer #2 (Remarks to the Author):\nNaamneh et al have investigated the role of maternal-derived (both prenatal and postnatal) IgG on early-life and long-term immune homeostasis. This is a crucial question in the field and needs to be interrogated. However, the following needs to be addressed to make the manuscript is conclusive.\n1. The authors need to make the introduction focused, highlighting the central questions. The introduction currently has a lot of information without a clearly defined direction.\nResponse- We thank the reviewer for this important comment. We have substantially revised the Introduction to improve focus and clarity by streamlining background information and explicitly highlighting the central questions and hypotheses addressed in this study. Redundant or peripheral content was removed or condensed, and the revised Introduction now provides a clearer narrative that guides the reader toward the specific aims of the work (Pages 3-4).\n2. Line 92 and experiments performed in Fig 1: It is unclear how conclusions from this experiment are different from those conducted in a previous paper by the same group (Cell Reports, 2023). In the Cell Reports paper, the authors show that maternal IgG is transferred to saliva. It is not clear how Fig 1 of this current paper is novel. If this is only to establish that this phenomenon is also observed in mIg+/neg mice, then all of Fig 1 needs to be in supplemental.\nResponse-We thank the reviewer for raising this point. We agree that the title of the Results section may not fully convey the scope of novel data in Fig. 1, which could give the impression that these results merely replicate our previous findings. While part of Fig. 1 confirms our earlier work published in Cell Reports (2023), it also presents substantial new information and demonstrates the main claim within the experimental systems used throughout the current study.\nSpecifically, in this study:\n1. We used a different mouse model (JHT⁻/⁻) to validate maternal IgG transfer to salivary glands and saliva, whereas the previous report employed NSG mice lacking T and B lymphocytes. The JHT⁻/⁻ model is more suitable for these experiments because it specifically lacks B cells while retaining other components of the immune system, allowing us to assess maternal IgG transfer under physiologically relevant conditions. In contrast, NSG mice are highly immunodeficient, which can alter antibody distribution and immune interactions. Using JHT⁻/⁻ mice therefore provides a clearer and more accurate demonstration of passive maternal IgG reaching neonatal mucosal sites.\n2. We included quantitative ELISA measurements of IgG in saliva and neonatal serum, complementing and extending beyond the Western blot analyses previously shown.\n3. Germ-free mice were incorporated to assess the influence of the microbiota on maternal antibody transfer.\n4. We examined the duration of maternal antibody persistence in offspring by crossing heterozygous parents and analyzing JHT⁻/⁻ pups.\n5. Supplementary Fig. S1 already provides additional controls and supporting data that complement Fig. 1, making relocation of Fig. 1 to the Supplementary section unnecessary.\nGiven the substantial novel data, the expanded experimental context, and the relevance of these findings for the rest of the manuscript, we believe Fig. 1 should remain a main figure rather than being moved to the Supplementary Materials.\n3. Please elaborate the discussion section on why CD4+ T cells, CD8+ T cells and B cells were lower in mIg+ mice and what benefit does that provide to early life immunity.\nResponse- We thank the reviewer for this insightful comment. We have now expanded the Discussion to elaborate on the mechanisms underlying the reduced frequencies of CD4⁺ T cells, CD8⁺ T cells, and B cells observed in mIg⁺ mice. Specifically, we discuss how maternal IgG can limit early antigen exposure and antigen presentation through immune complex–mediated mechanisms, thereby dampening adaptive immune activation during early life. We further propose that this transient suppression may be beneficial by preventing premature or excessive adaptive responses during a critical developmental window, promoting immune tolerance, limiting inflammation, and allowing proper maturation of the mucosal immune system. These additions place our findings within the broader context of neonatal immune education and homeostasis (from page 18 line 22 to page 20 line 16).\n4. Please include a discussion section for the transcriptomic alteration observed among mIg+ and mIg-neg mice and what advantage does that provide to early life immunity.\nResponse- We thank the reviewer for this important suggestion. We have now expanded the Discussion to specifically address the transcriptomic differences observed between mIg⁺ and mIg⁻ mice. In this new section, we interpret the altered gene expression patterns in the context of early-life immune regulation, highlighting pathways related to antigen presentation, inflammatory signaling, and immune tolerance. We further discuss how these transcriptional programs likely reflect a maternally imprinted immune state that favors controlled immune activation, limits excessive inflammation, and supports proper immune maturation during early life. This expanded discussion helps clarify the functional significance of the transcriptomic alterations and their potential advantage for early-life immunity (Page 19, lines 18-20, and Page 20, line 9 – Page 21, line 7).\n5. It is important to show how the mucosal immune environment of mIg+ and mIg-neg mice impacts their susceptibility to oral infections or any oral diseases.\nResponse - We thank the reviewer for this important comment. To show how the mucosal immune environment of mIg⁺ and mIg⁻ mice influences the susceptibility to oral infection/disease, we now emphasize our analysis of steady-state adult gingival immunity. As shown in Fig. 6 and 7, the oral mucosa of adult mIg-neg mice exhibits (i) impaired epithelial integrity with reduced barrier function, (ii) elevated oral microbial load with increased colonization by dysbiotic bacteria, and (iii) enhanced steady-state immune activation. These changes reflect oral mucosal dysbiosis, which is known to increase susceptibility of the gingiva to oral infection diseases such as periodontitis. To functionally test this, we employed the ligature-induced periodontitis model, a well-established experimental model of periodontal disease. Using this approach, we demonstrate that offspring lacking maternal antibodies mount an exaggerated inflammatory response to ligature placement and develop significantly greater alveolar bone loss compared with mIg⁺ offspring. Importantly, our data further reveal that antibodies acquired in utero, rather than those derived from breast milk, have a dominant role in determining susceptibility to ligature-induced periodontitis. Together, these findings provide direct functional evidence that maternal antibodies shape the early oral immune environment in a manner that restrains excessive inflammation and protects against periodontal tissue destruction in adulthood. We have highlighted these results more prominently in the Discussion section (Page 21, lines 3-7) and added a schematic summary (Fig. 8) to clearly link the immunological differences between mIg⁺ and mIg⁻ mice to oral disease susceptibility."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed the comments sufficiently.", "reviewer_2": "Thank you for addressing and clarifying the questions that I had. I do not have any more questions."}, "author_response": ""}]

Decision letter