Immunological Medicine ISSN: 2578-5826 (Online) Journal homepage: www.tandfonline.com/journals/timm20 Antibodies against Clostridium butyricum in the children of mothers at risk for gestational diabetes Celeste Peterson, Aili Tagoma, Kristi Alnek, Anu Bärenson, Tamara Vorobjova, Ija Talja, Helis Janson, Anne Kirss, Siiri Kõljalg, Aki Sinkkonen, Marja Irmeli Roslund, Raivo Uibo & the HEDIMED Investigator Group To cite this article: Celeste Peterson, Aili Tagoma, Kristi Alnek, Anu Bärenson, Tamara Vorobjova, Ija Talja, Helis Janson, Anne Kirss, Siiri Kõljalg, Aki Sinkkonen, Marja Irmeli Roslund, Raivo Uibo & the HEDIMED Investigator Group (23 May 2025): Antibodies against Clostridium butyricum in the children of mothers at risk for gestational diabetes, Immunological Medicine, DOI: 10.1080/25785826.2025.2504021 To link to this article: https://doi.org/10.1080/25785826.2025.2504021 © 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Japanese Society of Clinical Immunology. View supplementary material Published online: 23 May 2025. Submit your article to this journal Article views: 390 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=timm20 https://www.tandfonline.com/journals/timm20?src=pdf https://www.tandfonline.com/action/showCitFormats?doi=10.1080/25785826.2025.2504021 https://doi.org/10.1080/25785826.2025.2504021 https://www.tandfonline.com/doi/suppl/10.1080/25785826.2025.2504021 https://www.tandfonline.com/doi/suppl/10.1080/25785826.2025.2504021 https://www.tandfonline.com/action/authorSubmission?journalCode=timm20&show=instructions&src=pdf https://www.tandfonline.com/action/authorSubmission?journalCode=timm20&show=instructions&src=pdf https://www.tandfonline.com/doi/mlt/10.1080/25785826.2025.2504021?src=pdf https://www.tandfonline.com/doi/mlt/10.1080/25785826.2025.2504021?src=pdf http://crossmark.crossref.org/dialog/?doi=10.1080/25785826.2025.2504021&domain=pdf&date_stamp=23%20May%202025 http://crossmark.crossref.org/dialog/?doi=10.1080/25785826.2025.2504021&domain=pdf&date_stamp=23%20May%202025 https://www.tandfonline.com/action/journalInformation?journalCode=timm20 ORIGINAL ARTICLE Immunological Medicine Antibodies against Clostridium butyricum in the children of mothers at risk for gestational diabetes Celeste Petersona, Aili Tagomaa , Kristi Alneka, Anu Bärensona,b, Tamara Vorobjovaa, Ija Taljaa, Helis Jansona, Anne Kirssc, Siiri Kõljalgd,e, Aki Sinkkonenf, Marja Irmeli Roslundf, Raivo Uiboa and the HEDIMED Investigator Groupg* aDepartment of Immunology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia; bChildren’s Clinic, Tartu University Hospital, Tartu, Estonia; cWomen’s Clinic, Tartu University Hospital, Tartu, Estonia; dDepartment of Microbiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia; eLaboratory of Clinical Microbiology, United Laboratories, Tartu University Hospital, Tartu, Estonia; fHorticulture Technologies, Natural Resources Institute Finland, Turku, Finland; gHuman Exposomic Determinants of Immune Mediated Diseases (HEDIMED) Investigator group, www. hedimed.eu ABSTRACT Gestational diabetes mellitus (GDM) is linked to an imbalance in gut microbiota composition, which can be transferred to the mother’s offspring. Clostridium butyricum, known for its health benefits in diabetes and allergy, lacks sufficient data regarding its effect on the immune system’s development in the offspring of mothers with GDM. This study assessed antibody responses against C. butyricum T2F3 in children of mothers at risk for GDM, involving 88 children aged 1–6 years. Antibody responses were measured with flow cytometry and immunoblot. Lower IgG median fluorescence intensity (MFI) values and fewer IgA and IgG bands against C. butyricum were detected in children of mothers with GDM. Maternal body mass index was positively associated with children’s IgG MFI and number of IgG bands. Fewer IgA bands were detected in children with higher IgE levels, atopic dermatitis, asthma, and allergic rhinitis. More IgG bands were detected in children with higher anti-β-lactoglobulin IgG levels. Children with autoimmune risk-related HLA-DR3/DQ2.5 had fewer IgA bands, while those with neutral HLA-DR1/DQ5 had higher IgA, but lower IgG MFI. These results indicate that maternal prenatal changes could affect their offspring’s immune response against C. butyricum. Moreover, C. butyricum could have a protective role against allergic sensitization. GRAPHICAL ABSTRACT Abbreviations:  AU: Arbitrary units; BMI: Body mass index; BSA: Bovine serum albumin; CFU: Colony-forming unit; ELISA: Enzyme-linked immunosorbent assay; FAA: Fastidious Anaerobe © 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Japanese Society of Clinical Immunology. CONTACT Aili Tagoma aili.tagoma@ut.ee Department of Immunology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia. *HEDIMED Investigator group: Anna, Eurén; Heikki, Hyöty; Kalle, Kurppa; Jutta, Laiho; Olli, Laitinen; Jussi, Lehtonen; Katri, Lindfors; Maria, Lönnrot; Johannes, Malkamäki; Henna, Numminen; Noora, Nurminen; Matti, Nykter; Sami, Oikarinen; Leena, Puustinen; Niila, Saarinen; Amirbabak, Sioofy-Khojine; Keijo, Viiri; Daniel, Agardh; Carin, Andrén, Aronsson; Markus, Lundgren; Iida, Mäkelä; Martin, Romantschuk; Laura, Soininen; Nicolai, Lund-Blix; Maria, Magnus; Aino-Kaisa, Rantala; Lars, Stene; Ketil, Størdal; German, Tapia; Laura, Elo; Sini, Junttila; Riitta, Lahesmaa; Johanna, Lempainen; Robert, Moulder; Omid, Rasool; Tomi, Suomi; Jorma, Toppari; Ubaid, Ullah; Riitta, Veijola; Aleksandr, Peet; Kärt, Simre; Vallo, Tillmann; Elena, Bargagli; Francesco, Dotta; Laura, Nigi; Guido, Sebastiani; Leena, Hakola; Hannu, Kiviranta; Panu, Rantakokko; Suvi, Virtanen; Ondrej, Cinek; Eva, Fronkova; Jaroslav, Havlik; Emilia, Barannik; Matthieu, Molinier; Tarja, Nevanen; Juha, Pajula; Eija, Parmes; Juha, Pärkkä; Jukka, Ranta; Jyri, Rökman; Petri, Saviranta; Peter, Ylén; Alar, Aints; Anu, Bärenson; Anne, Kirss; Ivo, Laidmäe; Aili, Tagoma; Raivo, Uibo; Tamara, Vorobjova; Loïc, Burr; Stefano, Cattaneo; Hui, Chai-Gao; Peter, Cristofollini; Silvia, Generelli; Samantha, Paoletti; Edith, Ruth; Gabriele, Berg; Wisnu, Wicaksono; Kristi, Hoffman; Joseph, Petrosino; Daniel, Schmidtmann; Rainer, Thiel; Rosanna, Salo; Lauri, Häme; Alexander, Berler; Apostolia, Karabatea; Korina, Papadopoulou; Hans, Bisgaard; Klaus, Bønnelykke; Sarah, Brandt; Astrid, Sevelsted; Jakob, Stokholm; Jonathan, Thorsen; Mikael, Knip; Marja, Roslund; and Aki, Sinkkonen. Supplemental data for this article can be accessed online at https://doi.org/10.1080/25785826.2025.2504021. https://doi.org/10.1080/25785826.2025.2504021 This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ARTICLE HISTORY Received 18 July 2024 Accepted 6 March 2025 KEYWORDS Clostridium butyricum; gestational diabetes mellitus; atopic dermatitis; asthma; antibody http://orcid.org/0000-0003-0184-9703 http://www.hedimed.eu%ef%bb%bf http://www.hedimed.eu%ef%bb%bf mailto:aili.tagoma@ut.ee https://doi.org/10.1080/25785826.2025.2504021 https://doi.org/10.1080/25785826.2025.2504021 http://creativecommons.org/licenses/by-nc/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1080/25785826.2025.2504021&domain=pdf&date_stamp=2025-5-22 http://www.tandfonline.com htp://www.tandfonline.com 2 C. PETERSON ET AL. Agar; FITC: Fluorescein isothiocyanate; GDM: Gestational diabetes mellitus; GWG: Gestational weight gain; HLA: Human leukocyte antigen; I-FABP: Fatty-acid binding protein; kUA/l: Allergen-specific kilo units per liter; MFI: Median fluorescence intensity; OD: Optical density; OGTT: Oral glucose tolerance test; PBS: Phosphate-buffered saline; PVDF: Polyvinylidene fluoride; T1D: Type 1 diabetes; T2D: Type 2 diabetes 1.  Introduction Gestational diabetes mellitus (GDM) is a metabolic disorder which influences the health of both moth- ers and their children [1,2]. Gestational gut dysbiosis is characteristic of women who develop GDM. Moreover, the imbalance of the maternal microbiota can be transmitted to the offspring, causing abnor- malities in the development of their immune system [3–6]. Microbiota dysbiosis at an early age is associ- ated with various immune-mediated diseases and has been shown to promote allergy development [2,3,6,7]. Maternal dysbiosis during GDM has been associ- ated with lower abundance of beneficial gut com- mensals [7,8]. The vital position of biodiversity in immunoregulatory pathways was described earlier by Nurminen et  al. and Roslund et  al. who showed that an increase in the skin and gut biodiversity pro- moted an anti-inflammatory milieu [9,10]. They also found that the gut microflora is impacted by the soil microbiota and the relative abundance of Clostridiales in the gut is affected by a change in biodiver- sity [9,11]. Early childhood living environment is associated with immune-mediated diseases, such as type 1 diabe- tes (T1D) and allergic diseases [12–14]. Contact with microbially rich soil containing Clostridium sp. pro- motes immune regulation [11–14]. For example, C.  butyricum participates in the activation of regulatory T cells, which helps balance type 1 and type 2 T helper cells, thereby preventing pro-allergic reactions and IgE antibody production [15,16]. Moreover, C.  butyricum is one of the first commensals to colonize the newborn’s gut [16]. It is a Gram-positive butyrate producing bac- terium that can strengthen the gut barrier, reduce gut permeability, and its metabolites can promote the pro- liferation of other important gut commensals, such as bifidobacteria and lactobacilli [17,18]. Most studies of C. butyricum and immune system-associated diseases, such as allergies, have been conducted by using mouse models [15,19]. However, there is a gap in the knowledge regarding the impact of soil-originating strain, such as C. butyricum on humans. Moreover, the presence of C. butyricum in the offspring of mothers with GDM and its association with the immune system of these children during the first years of their lives have not been established. The current study aimed to fill in this gap by assessing differences between antibody reactivity towards C. butyricum in the children of mothers who developed GDM compared to the chil- dren of mothers who did not. Additionally, we aimed to find associations of detected antibody reactivity with the clinical and demographic characteristics of children and mothers, as well as with children’s intestinal permeability. 2.  Materials and methods 2.1.  Study population The study comprised 88 children, with median age of 1.95 (ranged 1–6 years), born to mothers who were at risk for GDM during their pregnancy according to the Estonian Gynaecologists’ Society guidelines and who participated in the GDM study of the University of Tartu during 2014–2019 [20]. The study was approved by the University of Tartu Research Ethics Committee (Estonia; protocols 298/M-21 and 315/M-18) and complied with documents of the Declaration of Helsinki. Written informed consent from the children’s parents or guardians was obtained before participating in the study. Among the 88 chil- dren included in the study, the mothers of 38 had been diagnosed with GDM and the remaining 50 children acted as the reference. GDM was diagnosed based on the oral glucose tolerance test (OGTT) according to the International Association of Diabetes and Pregnancy Study Groups Consensus Panel [21]. Dietary counseling was provided to all mothers diag- nosed with GDM, which was sufficient to maintain their blood glucose levels within the normal range. Venous blood samples were collected from the chil- dren at the Women’s Clinic of Tartu University Hospital, Estonia (2020–2021), and the separated serum and plasma were stored at −40 and −80 °C, respectively, until further analysis. 2.2.  Background characteristics Background information about the mothers and children was collected from questionnaires. Maternal abnormal gestational weight gain (GWG) was calcu- lated according to the Institute of Medicine and National Research Council Committee guidelines Immunological Medicine 3 based on the mother’s pre-pregnancy body mass index (BMI) [22]. The GWG values were also con- sidered as abnormal or normal variables. Data about the children’s diagnoses of interest [atopic dermatitis (L20) and respiratory-related: allergic rhinitis (J30) and asthma (J45.0 and J45.1) according to ICD-10] were obtained from Electronic Health Records. Neonatal hypoglycemia was diagnosed in accordance with the established monitoring guidelines for Estonia. Because only one child in our study experi- enced a postnatal infection, we excluded this vari- able from our analysis due to its low frequency. Blood glucose, C-peptide and vitamin D levels were measured at the United Laboratories of Tartu University Hospital according to their routine proto- col. Serum total IgA was detected using enzyme-linked immunosorbent assay (ELISA; Nordic BioSite, Sweden) according to the manufacturer’s instructions. 2.3.  Intestinal fatty acid-binding protein and anti-β-lactoglobulin antibody detection To evaluate intestinal epithelial damage, intestinal fatty-acid binding protein (I-FABP) levels were detected from serum samples (diluted 1/10) using the ELISA test kit (HK406-02, Hycult Biotech, the Netherlands). The obtained results were calculated according to the manufacturer’s directions and expressed in pg/ml. IgA and IgG antibodies against β-lactoglobulin were measured using ELISA as described by Savilahti et  al. with some modifications [23]. Microtiter plates (Nunc PolySorp, Denmark) were coated with bovine lactoglobulin (2 µg/ml; Sigma-Aldrich, USA) in a carbonate-bicarbonate buf- fer and incubated overnight +4 °C. The plates were washed with phosphate-buffered saline (PBS)-Tween 20 (0.05%) and blocked with 1% normal horse serum in PBS for 1 h at +37 °C. Diluted serum (1/20 in 1% horse serum-PBS) was applied in triplicate (2 coated with and 1 without the antigen) and the plates were incubated for 1 h at +37 °C. After the plates were washed, 100 µl of horseradish peroxidase-conjugated rabbit anti-human IgA (diluted 1/4000; Dako, Glostrup, Denmark) or IgG (diluted 1/4000; Dako) was added and incubated for 1 h at +37 °C. 100 µl of o-phenylenediamine dihydrochloride (Thermo Fisher Scientific, Sweden) in citrate buffer and H2O2 (Sigma-Aldrich, USA) was added after washing. The reaction was stopped after 10 min (at room tempera- ture) with 1 N H2SO4, and optical density (OD) was measured at 492 nm. Four serum samples positive for IgE antibodies against milk protein (f2, Thermo Fisher Scientific/Phadia, Sweden) were used as a pos- itive reference pool. The detected OD values were used to calculate arbitrary units (AU) using the for- mula: studied sera (mean OD of two wells − OD without the antigen)/reference pool (mean OD of two wells − OD without antigen). 2.4.  IgE sensitization IgE antibody sensitization to allergens was analyzed with ImmunoCap 100 (Thermo Fisher Scientific/ Phadia, Sweden) with the Phadiatop Infant test kit (Thermo Fisher Scientific/Phadia) according to the manufacturer’s instructions, with a cut-off value of ≥ 0.35 kUA/L (allergen-specific kilo units per liter) for positivity. In addition, a cut-off of ≥ 0.7 kUA/L was used to identify participants with definite aller- gic sensitization [24]. The IgE sensitization test included chicken egg, cow’s milk, peanut, shrimp, Dermatophagoides pteronyssinus, cat, dog, birch, thyme, ragweed and wall pellitory (Parietaria juda- ica) allergens. 2.5.  HLA genotyping PCR-based lanthanide labelled oligonucleotide hybrid- ization with time-resolved fluorometry was used for HLA-DR/DQ genotyping as described previously [25,26]. Based on the data, the DR1/DQ5, DR3/ DQ2.5, DR4/DQ8 and DR15/DQ6.2 haplotypes were coded as yes or no variables. The risk for developing autoimmune T1D has been associated with the DR3/ DQ2.5 and DR4/DQ8 haplotypes, and protective risk and neutral risk have been regarded as haplotypes DR15/DQ6.2 and DR1/DQ5, respectively. 2.6.  Isolation and preparation of C. butyricum The C. butyricum strain T2F3 was isolated from the commercial gardening soil sample no. RG166 T2N5_2019 collected from Finland in spring 2019. The soil “Nurmikkomulta” contained peat and min- eral soil, and composted sludge as fertilizer, which was enhanced with composted coniferous bark and composted dung, including chicken dung. The soil samples were cultivated on Fastidious Anaerobe Agar (FAA, Lab M, Heywood, UK) plates at +36.5 °C in an anaerobic glove chamber (5% CO2, 5% H2, and 90% N2; Concept 400, Biotrace, Bridgend, UK) for one week. After isolation, the strain T2F3 was incubated on FAA agar for six hours at the same temperature and anaerobic glove chamber condi- tions. Depending on the experiment, the C. butyri- cum strain T2F3 was harvested in 20% glycerin solution or PBS at a cell density of 108–109 CFU/ml and stored at −80 °C until use. 4 C. PETERSON ET AL. 2.7.  Detection of C. butyricum surface-bound antibodies by flow cytometry The method for detecting anti-bacterial antibodies using flow cytometry was adapted from Moor et  al. [27] Briefly, C. butyricum T2F3 samples stored in a 20% glycerin solution were thawed and washed twice (6000 g, +4 °C for 10 min) in a sterile filtered 1xPBS solution. The bacterial solution was brought to a concentration of 5 × 106 cells per sample using a PBS-BSA buffer [filtered PBS with 2% bovine serum albumin (BSA, Thermo Fisher Scientific, USA) and 0.02% NaN3 (Serva, Germany)]. For complement inactivation, thawed serum samples were heated at +56 °C for 30 min and centrifuged at 16,000g at +4 °C for 5 min. The supernatant was passed through a 0.22 μm spin filter column (VWR Centrifugal Filters, US) and diluted with a PBS-BSA buffer to serial dilutions of 1/81, 1/243, 1/729 and 1/2187. The diluted serum samples (25 μl) were incubated with 25 μl of bacteria for 15 min at room tempera- ture and washed twice with PBS-BSA (3320 g, +4 °C for 10 min). Next, 50 μl of 1/1 pooled fluorophore-conjugated goat anti-human IgA [diluted 1/200; fluorescein isothiocyanate (FITC) conjugated, Jackson ImmunoResearch, USA] and IgG (diluted 1/214; Alexa Fluor 647 conjugated, Jackson ImmunoResearch, USA) antibodies were added and incubated for 15 min. The washing step was repeated twice. To visualize bacteria, 1/1000 diluted SYTOX™ orange nucleic acid stain (Invitrogen™) was added together with 1/1000 diluted Triton X-100 (Sigma-Aldrich, USA) and incubated for 20 min. All incubation steps were performed in the dark at room temperature. The samples were acquired using LSRFortessa™ (BD Biosciences, USA) with 10,000 target events recorded for each sample. Flow cytom- etry data was analyzed using the FACSDiva™ version 6.2 (BD Biosciences, USA). For each sample, median fluorescence intensity (MFI) for IgA and IgG was recorded, the obtained values reflecting antibody amount against C. butyricum’s surface epitopes. To ensure that all MFI values from the analysis were positive, the scale’s zero point was shifted to the lowest MFI value and all MFI values were subse- quently adjusted. In addition, the proportions of bacteria bound with only IgA antibodies, with only IgG antibodies, or with both IgA and IgG antibodies per 10,000 bacteria were recorded. 2.8.  Detection of C. butyricum specific antibodies by immunoblot assay For the immunoblot experiment, the bacterial cells stored in PBS were washed three times with the PBS buffer and disrupted with 0.1 mm glass beads (30 sec of cell disruption, 10 min on ice and repeated approx- imately 10 times; Biospec Products, USA). The pro- tein assay solution (Bio-Rad, USA) was used to detect protein concentration in samples, and 100 μg protein was loaded on a gradient gel with the SDS-PAGE sample buffer [62.5 mM Tris (pH 6.8), 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerin, a few grains of bro- mophenol blue]. Antigens were separated for the immunoblot according to Nilsson et  al. using the ver- tical electrophoresis system SE-600 (Hoefer, San Fransisco, CA, USA) and transferred to a polyvi- nylidene fluoride (PVDF) membrane (0.45 μm pore size) using a semi-dry electroblotter (Hoefer) [28]. All blue 10–250 kDa molecular weight markers (Bio-Rad, USA) were used. Plasma was diluted in the incubating buffer [1.25 g/L gelatin hydrolysate, 0.25 g/L Tween-20, 6.1 g/L NaCl (Merck, USA), 0.06 g/L Tris Base] 1/50 for IgA and 1/100 for IgG detection, and incubated overnight on a shaker at +4 °C. Horseradish peroxidase-conjugated rabbit polyclonal anti-human IgA or IgG antibodies (Invitrogen, USA) were applied at 1/2000 dilution and incubated for two hours at constant shaking at room temperature. The reaction was stopped using distilled water, and the number of bands on the PVDF membrane was counted. The higher the band count, the more IgA or IgG antibody reactions were found in the sample. 2.9.  Statistical analysis Categorical characteristics were compared using the χ2 test or Fisher’s exact test, and continuous charac- teristics were compared using the Welch two-sample t-test for a normal distribution and the Wilcoxon rank sum test for a non-normal distribution. Correlations between children’s and mother’s clinical data and bacterial antibody response values were found using Spearman’s correlation. Linear regres- sion with antibody parameters on the log2 scale was employed to ensure a better linear fit. In addition, Poisson regression was used. Regression models were adjusted for the child’s age and sex, if not stated oth- erwise. p-value < 0.05 was considered statistically significant. Statistical analysis was done using R (version 4.3.1, The R Foundation for Statistical Computing, Vienna, Austria). 3.  Results 3.1.  Children’s clinical characteristics and antibody parameters The background characteristics were similar for chil- dren born to mothers diagnosed with GDM and for children born to the mothers without the diagnosis, except for a few differences (Table 1). The HLA-DR3/ Immunological Medicine 5 DQ2.5 haplotype was significantly more common in children of the non-GDM group (p = 0.026). Additionally, in the GDM group children had higher levels of anti-β-lactoglobulin IgA antibodies (p = 0.015). Although the IgE test results at ≥ 0.35 kUA/L were similar between the compared groups, there was a trend for a higher proportion of children with positive IgE at ≥ 0.7 kUA/L in the GDM group (p = 0.053). As expected, maternal OGTT values (p < 0.001), as well as pre-pregnancy and antepartum BMI (p = 0.014 and p = 0.035, respectively) were higher in the GDM group. Comparison of the data about children’s antibody reaction between children born to the mothers with GDM and those without GDM is shown in ESI Table S1. The proportion of bacteria with antibodies bound to the bacterial surface is shown at the serum dilution of 1/243, which best distinguished both IgA and IgG antibody responses. Serum dilution of 1/2187 was excluded from statistical analysis for it being too dilute. Overall, the antibody reactivity detected with flow cytometry was similar between the two groups. At the same time, bacteria bound with both IgA and IgG antibodies were detectable in only a few children (median values for both groups were 0%; Table S1). Immunoblot analysis revealed a trend for higher number of IgA bands in children belonging to the non-GDM group (median 12 vs 16, p = 0.056; Table S1). 3.2.  Correlations Figure 1 shows Spearman’s correlation between anti- body values and children’s clinical characteristics. With children’s increasing age, percentage of bacteria bound with IgA (r = 0.22, p = 0.044) and values of bacteria-binding IgA and IgG MFI (at dilution 1/81, r = 0.21, p = 0.049 and r = 0.38, p < 0.001, respectively) increased. On the other hand, number of IgA and IgG bands correlated negatively with age (r = −0.53, p < 0.001 and r = −0.67, p < 0.001, respectively). Vitamin D levels correlated with IgA and IgG MFI values (at dilution 1/81, r = −0.22, p = 0.044 and r = −0.25, p = 0.019, respectively). Total IgA levels had inverse correlation with number of IgA and IgG bands (r = −0.35, p = 0.002 and r = −0.32, p = 0.004, respectively), but positive correlation with IgG MFI values (at dilution 1/81, r = 0.22, p = 0.043). Additionally, significant correlation was found between anti-β-lactoglobulin IgG antibodies and number of IgG bands (r = 0.31, p = 0.005). 3.3.  Regression models In regression analysis, only the data from the serum dilution 1/243 was used, as it best distinguished both IgA and IgG antibody responses. Crude models (data not shown) showed that living in urban or rural areas did not affect children’s antibody reactiv- ity against C. butyricum T2F3. However, children’s daycare attendance was associated with higher IgG MFI values (β = 0.77, p = 0.003), but with lower num- ber of IgA and IgG bands (β = −0.23, p < 0.001 and β = −0.53, p < 0.001, respectively), compared to chil- dren staying at home. Additionally, inverse associa- tions with number of IgA and IgG bands were found for children born via scheduled cesarean section compared to emergency cesarean section (β = −0.36, p = 0.008 and β = −0.48, p = 0.002, respectively). Also, more IgA and IgG bands were detected in children who were breastfed during the study period (β = 0.31, p < 0.001 and β = 0.36, p < 0.001, respectively). I-FABP values were positively associated with number of IgA bands (β = 0.0002, p = 0.010). In adjusted models, we found that after adjust- ment for pre-pregnancy BMI, in addition to child’s age and sex, the children of mothers with GDM showed lower IgG MFI values (β = −0.52, p = 0.028) and fewer IgA and IgG bands (β = −0.26, p < 0.001 and β = −0.30, p < 0.001, respectively). Similarly, higher maternal pregnancy glucose levels after 1h of the OGTT were associated with fewer number of IgG bands in their children (model adjusted for same covariates, β = −0.04, p = 0.049). On the other hand, in a model adjusted for maternal GDM diag- nosis and child’s age and sex, the children of moth- ers with higher pre-pregnancy BMI showed a higher IgG MFI value (β = 0.05, p = 0.025) and more IgG bands (β = 0.02, p = 0.002). More specifically, the chil- dren of obese mothers had more IgG bands com- pared to the children of mothers with normal weight (β = 0.22, p = 0.012). However, children whose moth- ers gained more weight during gestation had fewer IgA and IgG bands (β = −0.01, p = 0.020 and β = −0.02, p = 0.003, respectively). Additionally, adjusted models revealed fewer IgA bands in children diagnosed with either atopic der- matitis (β = −0.22, p = 0.003) or respiratory-related diagnoses (β = −0.23, p = 0.032). An inverse associa- tion was also found between number of detected IgA bands and the IgE levels (β = −0.06, p = 0.029), as well as between number of detected IgA bands and the level of total IgA (β = −0.09, p = 0.005). Children who had hypoglycemia after birth had lower per- centages of bacteria bound with IgA (β = −1.13, p = 0.033), in a model adjusted for maternal GDM diagnosis and child’s age and sex. Children with the DR1/DQ5 HLA haplotype showed higher values of IgA but lower IgG MFI (β = 0.52, p = 0.019 and β = −0.58, p = 0.033, respectively), in a model adjusted for maternal GDM diagnosis and child’s age and sex. https://doi.org/10.1080/25785826.2025.2504021 https://doi.org/10.1080/25785826.2025.2504021 https://doi.org/10.1080/25785826.2025.2504021 6 C. PETERSON ET AL. Additionally, children with the DR3/DQ2.5 haplo- type had fewer IgA bands (model adjusted for the same covariates, β = −0.22, p = 0.011). Furthermore, the child’s vitamin D and random blood glucose lev- els were inversely associated with IgG MFI values (β = −0.007, p = 0.041) and with number of detected IgG bands (β = −0.17, p = 0.007), respectively. However, there occurred positive association with children’s anti-β-lactoglobulin IgG antibody levels and IgG MFI values and number of IgG bands (β = 0.76, p = 0.049 and β = 0.37, p = 0.001, respectively). 4.  Discussion GDM has been shown to be associated with micro- biota dysbiosis [2,7]. When transferred to the moth- er’s offspring, these changes in the gut can cause abnormalities in the development of the immune system with several consequences, including allergy development in these children [3–6]. However, the knowledge of the effect of C. butyricum on the immune system in the offspring of GDM mothers is inadequate. This study assessed associations between antibody reactions against soil-isolated C. butyricum T2F3 in children born to mothers at risk for GDM during pregnancy. In addition, we aimed to find associations of C. butyricum’s antibody reactivity with children’s and mothers’ background characteris- tics and children’s intestinal permeability. We found that children of mothers with GDM showed lower IgG MFI values and fewer IgA and IgG bands reacting with C. butyricum, which sug- gests that children in the GDM group may have a diminished anti-C. butyricum antibody response. Table 1. C omparison of the clinical data of children and their mothers in the GDM and non-GDM groups. Clinical characteristics GDM group (n = 38) Non-GDM group (n = 50) p-value Children  A ge (years) 2.3 (1–5.8) 1.9 (1–6.3) 0.686   Sex (male) 22 (57.9%) 26 (52%) 0.738  M ode of birth    Vaginal 31 (81.6%) 39 (78%) 0.584    Scheduled cesarean section 2 (5.3%) 6 (12%)   E mergency cesarean section 5 (13.2%) 5 (10%)   Hypoglycemia 9 (23.7%) 8 (16%) 0.528   Residence   U rban area 17 (44.7%) 29 (58%) 0.309    Rural area 21 (55.3%) 21 (42%)  D aycare attendance 19 (50%) 22 (44%) 0.798   Breast milk consumption 7 (18.4%) 15 (30%)  0.320  D uration of breastfeeding (months) 10 (5–16.3) 13 (11–17) 0.088  L aboratory analysis      Total IgA (mg/mL) 1.2 (1–1.4) 1.3 (0.9–2.2) 0.494   G lucose (mmol/L) 4.5 (4.2–5) 4.6 (4.2–4.9) 0.858   C -peptide (nmol/L) 0.62 (0.38–0.99) 0.60 (0.38–0.81) 0.139    Vitamin D (nmol/L) 73.4 (58.3–94.6) 65.3 (56.1–77.9) 0.086    I-FABP (pg/ml) 427 (293.8–712.8) 522.8 (298.2–872.2) 0.667   A nti-β-lactoglobulin IgA (AU) 0.25 (0.14–0.56) 0.12 (0.05–0.27) 0.015   A nti-β-lactoglobulin IgG (AU) 0.72 (0.51–0.9) 0.59 (0.38–0.9) 0.207   IgE sensitization          IgE (kUA/L) 0.13 (0.05–0.56) 0.12 (0.05–0.29) 0.435    IgE test ≥ 0.35 kUA/L 13 (34.2%) 11 (22%) 0.348    IgE ≥ 0.7 kUA/L 8 (21.1%) 3 (6%) 0.053  D iagnosis  of interest   A topic dermatitis 12 (31.6%) 10 (20%) 0.320    Respiratory-related 4 (10.5%) 8 (16%) 0.542   HLA haplotype   D R1/DQ5 8 (21.1%) 11 (22%) 1   D R3/DQ2.5 3 (7.9%) 13 (26%) 0.026   D R4/DQ8 4 (10.5%) 6 (12%) 1   D R15/DQ6.2 12 (31.6%) 14 (28%) 1 Mothers during pregnancy  A ge (years) 31.6 (21–40) 32 (19–44) 0.608  O ral glucose tolerance test (mmol/L)    Fasting glucose 5 (4.6–5.3) 4.6 (4.4–4.9) < 0.001    1h glucose 10.1 (8.6–10.5) 7.1 (5.9–7.9) < 0.001    2h glucose 7.5 (6.6–8.5) 5.8 (4.9–6.5) < 0.001   Body mass index (BMI; kg/m2)    Pre-pregnancy 26.1 (23.7–29.6) 23.6 (21.2–27.9) 0.014   A ntepartum 29.7 (27.6–36.1) 29 (26–31.7) 0.035   Pre-pregnancy BMI factor (kg/m2)   U nderweight (BMI < 18.5) 1 (2.6%) 2 (4%) 0.354   N ormal weight (BMI 18.5–24.9) 15 (39.5%) 28 (56%)   O verweight (BMI 25–29.9) 13 (34.2%) 12 (24%)   O bese (BMI ≥ 30) 9 (23.7%) 7 (14%)  G estational weight gain (kg) 11 (8–15) 13.1 (10.2–17) 0.357  A bnormal gestational weight gain 20 (52.6%) 28 (56%) 0.862 Statistically significant differences (p < 0.05) are highlighted in bold. Immunological Medicine 7 Paun et  al. demonstrated that a lower antibody response directed towards gut commensals was asso- ciated with their lower abundance in the gut and vice versa [29]. This association is likely driven by the ability of commensal gut microbes to induce serum IgA and IgG responses, which in turn facili- tate colonization and functioning [27]. In women with GDM, reduced presence of species annotated to Clostridium (sensu stricto) has been reported [7,8]. However, to our knowledge, no studies have previ- ously reported C. butyricum’s abundance in the off- spring of these women. Therefore, we do not know whether the vertical transmission of Clostridium spe- cies from non-GDM mothers to their offspring is greater than it is from mothers with GDM, which could then cause increased exposure to Clostridium and hence stronger immune response. This hypoth- esis needs to be confirmed by future studies. Interestingly, both maternal and child’s blood glu- cose levels were inversely associated with number of detected IgG bands reacting with C. butyricum. Furthermore, children with postnatal hypoglyce- mia—potentially influenced by maternal hyperglyce- mia—had lower percentage of bacteria coated with IgA, suggesting a potential link between glucose reg- ulation and immune interactions with C. butyricum. Several studies have described the link between C.  butyricum and diabetes. An anti-diabetic effect of C. butyricum CGMCC0313.1 was reported in a type 2 diabetic (T2D) mouse model where C. butyricum reduced glucose levels and improved insulin resis- tance [30]. In addition, Zhou et al. reported decreased abundance of C. butyricum in the gut of diabetic mice [31]. In humans, negative association was found between Clostridium cluster IV and 2h plasma glucose levels in women with GDM, as well as between certain Clostridium species and fasting glu- cose levels in subjects with T2D [7,32]. Therefore, C. butyricum could protect against diabetes develop- ment by reducing blood glucose levels in persons at risk for diabetes, including the offspring of mothers with GDM. In addition to hyperglycemia, several GDM risk factors are also associated with changes in the moth- er’s and child’s gut microbiota. Su et  al. showed that maternal higher pre-pregnancy and antepartum BMI were associated with higher abundance of the Clostridium genus in the newborn’s gut [33]. Therefore, we aimed to investigate whether maternal BMI and GWG could also contribute to children’s antibody reactivity against C. butyricum. We found that the children of mothers with higher pre-pregnancy Figure 1.  Spearman’s rank correlation between children’s clinical data and parameters of C. butyricum’s antibody reactivity. Bacteria with antibodies (%) represent the proportion of bacteria with either surface-bound IgA, IgG, or both IgA and IgG (at dilution 1/243). MFI indicates the intensity of antibody fluorescence against bacterial surface proteins. The parameters obtained from immunoblot experiment are presented as the number of IgA and IgG bands. Only the data indicating significant correla- tions with parameters of antibody reactivity are shown. Red color indicates positive correlation and blue color indicates neg- ative correlations (p < 0.05). MFI, median fluorescence intensity. 8 C. PETERSON ET AL. BMI had higher C. butyricum IgG MFI and higher number of detected IgG bands. Furthermore, mater- nal pre-pregnancy obesity was associated with an increased number of IgG bands. These associations might indicate increased exposure to this bacterium in these children. Notably, higher maternal GWG was associated with a reduced number of detected IgA and IgG bands reacting with C. butyricum. However, considering that women with lower pre-pregnancy BMI typically experience greater weight gain during pregnancy compared to those with higher BMI [34], our findings align with the above observation of a positive correlation between maternal pre-pregnancy BMI and increased IgG reactivity. Another important finding was that we detected fewer IgA bands in children diagnosed with either atopic dermatitis or respiratory-related diagnoses. Decreased seroreactivity against commensals is observed in allergy-prone children [29,35]. Therefore, lower IgA in children with atopic dermatitis might be linked to reduced Clostridium abundance, as pre- viously reported [36]. This may also reflect dimin- ished mucosal immune stimulation and barrier dysfunction, consistent with Dzidic et  al.’s finding of weaker IgA responses to gut commensals in allergic children, indicating impaired mucosal barrier func- tion [37]. Also, C. butyricum has demonstrated allergy-preventive potential [19,38,39], modulating regulatory T cells via IL-10 production [16,19,39] which is further supported by our observation of a negative association between the number of C.  butyricum-reactive IgA bands and IgE level. Increased intestinal permeability can also be indi- cated by increased levels of IgA and IgG antibodies to the food allergen β-lactoglobulin [40,41]. While children of mothers with GDM exhibited higher anti-β-lactoglobulin IgA levels, we surprisingly found no corresponding increase in I-FABP, a marker of intestinal barrier dysfunction [42]. Similarly, despite C. butyricum’s known gut-strenghtening properties [16], I-FABP levels were not associated with anti-C. butyricum’s antibody responses. Only increased anti-β-lactoglobulin IgG antibody levels were accom- panied by higher anti-C. butyricum’s IgG MFI values and number of IgG bands. However, further research, including the assessment of other intestinal permea- bility markers like zonulin [41], is needed to clarify the interplay between β-lactoglobulin sensitization, C. butyricum, and intestinal barrier function. Previous research has demonstrated that autoimmune-associated HLA haplotypes influence the colonization of gut bacteria and play a crucial role in regulating the immune response to these microbes [29,43,44]. Consistent with our findings, which revealed a decrease in C. butyricum-specific IgA bands among children carrying the HLA-DR3/ DQ2.5 haplotype, Paun et  al. also noted diminished anti-commensal antibody responses in individuals with T1D who carried either the DR3 or DR4 hap- lotype [29]. These haplotypes have previously been linked to the decreased abundance of specific com- mensal bacteria, such as bifidobacteria. Furthermore, Berryman et  al. reported a dose-dependent relation- ship, where the relative abundance of Bifidobacterium was the lowest in individuals homozygous for the DR4/DQ8 haplotype, but the highest in those homo- zygous for the haplotype DR1/DQ5 [43]. They sug- gested that HLA variants associated with increased risk for autoimmune diseases may act as gatekeepers in the gut, influencing the balance of beneficial microbes and contributing to dysbiosis [43]. Our observation of higher IgA MFI values in children with the HLA-DR1/DQ5 haplotype, further elabo- rates these findings. On the other hand, Vitamin D is known to modulate the adaptive and innate response of the immune system [45]. Some studies have shown that as vitamin D can also affect anti-microbial response, it could suppress the immu- noglobulin production suppressing these bacteria [46,47]. Our results are consistent with this hypoth- esis, as we detected higher vitamin D levels in chil- dren with lower IgG MFI against C. butyricum. Since C. butyricum T2F3 was isolated from gar- dening soil we expected that children living in urban areas would be more exposed to the studied strain. However, there were no differences in antibody reac- tivity towards C. butyricum between children living in rural and urban areas, which might indicate that children in the compared groups were equally exposed to the bacterium. To assess children’s antibody response to C. butyr- icum, we used flow cytometry and immunoblot assays, revealing somewhat divergent results. We found a negative correlation between children’s age and the number of IgA and IgG bands detected via immunoblot but a positive correlation between age and IgA and IgG MFI values measured by flow cytometry. This difference may indicate that anti- bodies targeting conformational epitopes on intact bacterial cell surfaces, detected via flow cytometry, become more prevalent with age. On the other hand, immunoblot assay uses bacterial cell homogenate and allows the detection of antibodies bound to lysed bacterial components, rather than intact cell surfaces [28]. Since C. butyricum can exist as spores in the gut in vivo [16,48], potentially altering exposed epitopes, we ensured that the flow cytometry proto- col was completed within a short time frame (~ 3 h) before acquiring samples. This precaution helped prevent C. butyricum from reaching a significant Immunological Medicine 9 level of spore formation. Consequently, the obtained results only describe immune reactivity against the vegetative form of C. butyricum. The use of two different methods for analyzing antibody reactivity against C. butyricum can be seen as a strength of the study, enabling to detect both bacterial surface-specific and intracellular epitopes. Furthermore, the children of the study groups belonged to various age groups, facilitating assess- ment of immunological changes against C. butyri- cum across different age ranges. However, the small sample size poses a limitation the study. With a lim- ited number of participants, we were only able to categorize children based on the presence of the diagnoses of interest. Categorizing them according to symptoms would have likely led to an underpowered evaluation. Larger sample sizes in future studies should be used to explore immune responses based on allergic symptoms. Additionally, there were no samples from children younger than one year, which would have enabled us to better study the maternal effect on the offspring’s microbiota and immune sys- tems. Furthermore, we did not account for maternal nutrition during pregnancy, a factor known to sig- nificantly impact the offspring’s microbiota and immune system. These limitations may have restricted our ability to fully assess maternal contri- butions to these developmental processes. 5.  Conclusions The children of mothers who were diagnosed with GDM had lower IgG MFI and fewer IgA and IgG bands against C. butyricum. These results indicate that maternal health during pregnancy influences the child, subsequently affecting the response of off- spring’s immune system to C. butyricum. Factors such as maternal glucose levels, BMI and child’s HLA hap- lotypes, glucose, vitamin D and anti-β-lactoglobulin IgG antibody levels were also found to affect antibody reactivity. In addition, the results suggest a possible protective role of C. butyricum against developing atopic dermatitis and respiratory-related diagnoses in children. Together, these findings highlight the com- plex interplay between maternal health, microbial exposure and offspring immunity and underline the importance of further research to understand and potentially mitigate the risk of immune-related condi- tions in children born to mothers at risk for GDM. Acknowledgements We are grateful to Mrs. Laura Lauren from the Women’s Clinic, Tartu University Hospital, for blood sample collec- tion, and to Mrs. Kaja Metsküla from the Department of Immunology, University of Tartu, for laboratory assistance. We also thank Prof. Jorma Ilonen from the Immunogenetic Laboratory of the University of Turku, Finland, for per- forming HLA genotyping. Authors’ contributions C.P.: Writing – original draft, formal analysis, visualiza- tion. C.P., A.T., K.A., A.B., T.V., I.T. and H.J.: Investigation. A.K. and A.B.: Participant recruitment. A.S., M.I.R. and S.K.: Resources. R.U. and A.T.: Project administration, supervision, writing – review and editing. 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