National screening programs use dried blood specimens to detect abnormal metabolism or aberrant protein function in infants shortly after birth, thus identifying disorders that are not clinically evident in the newborn period. Gut microbiota metabolites and immunological acute phase proteins are capable of revealing potential immune aberrations. Microbial metabolites interact with xenobiotic receptors (i.e., aryl hydrocarbon and pregnane-X) and maintain gastrointestinal tissue health, supported by acute-phase proteins, functioning as sensors of microbial immunomodulation and homeostasis. The delivery mode (vaginal or cesarean section) shapes the microbial colonization, which substantially modulates both the immune system's response and mucosal homeostasis. This study profiled microbial metabolites of the kynurenine and tryptophan pathway and acute phase proteins in 134 neonatal dried blood specimens. We newly established neonatal blood levels of the aryl hydrocarbon receptor microbial ligands (indole-3-aldehyde, indole-3-butyric acid, and indole-3-acetamide) on the second day of life. Furthermore, we observed divergent microbial metabolic profiles in neonates born vaginally or via cesarean section, hypothesizing potential microbial immunomodulatory influence. In summary, these findings suggest the supportive role of human gut microbiota in developing and maintaining immune system homeostasis.

DBS proteins were extracted, processed, and analyzed by UHPLC-MS as described previously (Vidova et al., 2019). In brief, the DBS extract's total protein content was determined using BCA (cat. #23227, Thermo Fisher, Waltham, MA) in extracts diluted 100-fold with 50 mM ammonium bicarbonate buffer. A dilution series (31.25 – 2000 µg/ml) of bovine serum albumin standard in 50 mM ammonium bicarbonate buffer was used to generate a 7-point calibration curve. Spectrophotometric absorbance was measured at 562 nm. Mass spectrometry protein assays performed in 30 µl of DBS extract mixed with 10 µl of the internal standard solution in 5% of acetonitrile, containing isotopically labeled standard peptides (Table S-3) and with 3 µl of trypsin (1 µg/µl). Samples were incubated (17 h at 37°C, orbital shaking), and the enzymatic proteolysis was quenched by adding 200 µl of 2% formic acid in water (pH < 3). Tryptic peptides were purified and desalted, applying solid phase extraction (Oasis PRIME HLB 96-well plate, 30 mg, Waters, Milford, MA). The solid-phase extraction protocol: the sample loaded onto the cartridge, washed with 300 µl of 2% formic acid in water (pH <3), eluted with 50% acetonitrile with 2% formic acid (pH <3), and the eluate dried in the SpeedVac. Before UHPLC-MS analysis, peptides were reconstituted in 50 µl of 5% acetonitrile with 0.1% formic acid. Processed DBS samples were injected (5 µL) on the UHPLC-QQQ system (Infinity 1260 and 6495B from Agilent Technologies, USA). We utilized a reversed-phase analytical column (C18 Peptide CSH; 1.7 μm, 2.1 mm i.d. x 100 mm; cat. #186006937, Waters, Milford, MA) and the previously described method (Vidova et al., 2019). Extracted DBS were analyzed in triplicates. Samples were injected (2 µl) on the UHPLC-QQQ system equipped with a reverse-phase analytical column (Acquity UHPLC CSHTM C18 Column; 1.7 μm, 2.1 mm x 100 mm; cat. #186005297, Waters, Milford, MA) thermostated to 40°C. The mobile phase consisted of buffer A (water with 0.1% formic acid) and buffer B (acetonitrile: water; 95:5 with 0.1% formic acid). The gradient elution program (0-14 min) was: 0.0 min 5% B, 5 min 10% B, 10 min 95% B, 11.99 95% B, 12.0 5% B, 14 min 5% B. The mobile phase flow was 0.3 mL/min. A standard-flow Jet Stream electrospray source operated in positive SRM ion mode with a capillary voltage of 3.5 kV. Additional parameters were: gas flow rate 15 L/min at 160°C, sheath gas pressure 25 PSI at 250°C, and nozzle voltage 500 V. SRM libraries were generated using Optimizer software (Agilent Technologies) on standard solutions of individual metabolites. For the metabolite identification, 2-4 SRM qualifier transitions were monitored per metabolite (Supplementary Table S-5), and a best-performing SRM transition was used for the quantification (Supplementary Figure S-1). Peak integration and visual inspection performed in Skyline software (Version 20.1.0.155; MacCoss Lab, Univ. of Washington).

DBS samples from 134 neonates (20 delivered via cesarean section and 114 delivered vaginally) collected under IRB approval were part of the CELSPAC-TNG study at Faculty Hospital Brno (Ethical Committee CELSPAC/EK/4/2016, in 2016-2017). Characteristics of individual neonates, including their gestational age, delivery mode, sex, birth weight, birth length, Apgar score, DBS sampling, and anamnesis of mothers and neonates, are shown in Supplementary Table S-1. The study subjects were female (n=56) and male (n=78), with an average birth weight of 3494 g and an average birth length of 50.5 cm (Supplementary Tables S-1 and S-2). We show the average, minimal and maximal values for birth length, weight, gestation age, Apgar score, and the delay from the birth to DBS sampling for VD and CD neonates separately in Supplementary Tables S-2. For DBS sampling, a small amount of capillary blood from the heel prick was soaked into Whatman 903 filter paper and allowed to dry at room temperature for 3 hours. DBS punches (1/8" or 3 mm) were stored in the freezer at -80°C until analysis.