Isolation of Human Milk Supernatants
HM was obtained from a cohort of traditional farming lifestyle and urban mothers at 1–3 months postpartum and cryopreserved as part of a study that assessed the effect of farming lifestyle on maternal and infant immunity(11). Traditional farming lifestyle and urban lifestyle mothers in this cohort were recruited from the Old Order Mennonite (OOM) population of western New York and suburban/urban Rochester, NY, respectively. OOM mothers are known to have increased rates and duration of breastfeeding in comparison to their urban lifestyle counterparts(12, 13). All participants were recruited after written informed consent. The study protocol was approved by the University of Rochester Medical Center Research Subjects Review Board. Cryopreserved HM samples were thawed and milk fat and cells were eliminated from human milk samples using differential centrifugation followed by filtration through a 0.22µm filter. Isolated supernatants were utilized in experiments.
HEK-hTLR4 Cell Experiments
The human TLR-transfected cell lines HEK-Blue hTLR4 and HEK-Blue hTLR2 were purchased from Invivogen (San Diego, CA) and maintained in an incubator at 37°C in 5% CO2. The morphology of these cells is epithelial. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Corning, Woodland, CA), 1X penicillin/streptomycin (Gibco, Grand Island, NY), 100ug/mL Normocin (InvivoGen, San Diego, CA), and 2mM L-glutamine (Gibco, Grand Island, NY), as well as the proper selection of reagents as per the manufacturer’s instructions. For cell line assays, HEK-Blue pre-defined detection media (Invivogen, San Diego, CA) absent of FBS was re-suspended as per the manufacturer’s instructions, protected from light, stored at 4°C, and used within 1 week of reconstitution. Assay standards of LPS (Sigma-Aldrich, St. Louis, MO) were thawed, vortexed, and serially diluted 1:3 in phosphate buffered saline (PBS) (Gibco, Grand Island, NY) from a 250 ug/mL stock. Cells were treated with 12.5µg/mL Pam3-Cys-Ser-Lys 4 (Pam3CSK4) (InvivoGen, San Diego, CA) or 333 ng/mL of LPS spike-in along with 10% HM supernatants or high activity fraction (HF) as appropriate. Cells at 50–80% confluence were then detached, counted, and re-suspended in warm HEK-Blue detection media and seeded in 96-well tissue culture treated microplates as per the manufacturer’s instruction. Plates were then incubated at 37°C in 5% CO2 for 16–18 hours and NF-κB-inducible secreted embryonic alkaline phosphatase enzyme (SEAP) was read on a BioTek Epoch spectrophotometer (Winooski, VT) at optical density 620 (OD 620). Data was normalized to LPS.
Fast Protein Liquid Chromatography (FPLC)
Prior to FPLC, prepared HM samples were casein depleted. Pre-cleared HM pH was lowered to 4.5 using 2M HCl. Precipitated casein was then removed by centrifugation at 4400rpm for 30 minutes at room temperature. Supernatants were isolated and pH was returned to 6.6. These supernatants were then fractionated via anion exchange chromatography using a HiTrap Q FF 5x5 mL column (GE Healthcare, Uppsala, Sweden) with a buffer gradient starting with 20mM Tris 35mM NaCl to 20mM Tris 1M NaCl, pH 8.2. Active fractions where further purified via size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare, Uppsala, Sweden). Effluent protein concentration was monitored by absorbance at 280nm. Fractions were tested in HEK-hTLR4 assays, detailed above, to identify fractions containing enhancing effect on LPS-signaling.
Mass Spectrometry
The high activity HM fraction was run in a 4–12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel to separate proteins. The region of interest was excised and dehydrated with acetonitrile. Peptides were extracted the next day by addition of 50% acetonitrile, 0.1% trifluoroacetic acid (TFA), then dried down in a CentriVap concentrator (Labconco, Kansas City, MO). A Fusion Lumos Tribrid mass spectrometer (Thermo Fisher) was used to analyze the peptides. Raw data was searched using the SEQUEST search engine within the Proteome Discoverer software platform, version 2.4 (Thermo Fisher), using the SwissProt Homo sapiens database. Percolator was used as the false discovery rate (FDR) calculator, filtering out peptides which had a q-value greater than 0.01.
Human Serum Albumin Depletion
HSA was depleted from HM supernatants using PureProteome Albumin Magnetic Beads (Millipore, Burlington, MA) according to the manufacturer’s protocol. Isolated albumin-free (AF) HM supernatants were then concentrated using Amicon Ultra 0.5ml 3K centrifugal filters (Millipore, Burlington, MA) and centrifuged down at 14,000xg for 5 minutes. Concentrated samples were then sterile filtered through a 0.22µm filter.
Endotoxin Measurement
Endotoxin content of HSA was determined from a 50 µg/ml solution of HSA using Pierce Chromogenic Endotoxin Quant Kit (Thermo Scientific, Rockford, IL) using kit instructions.
Caco-2 Cell Experiments
Caco-2 cells were cultivated in DMEM, 10% FBS, 4mM L-glutamine, 1X penicillin/streptomycin or DMEM, 1X insulin-transferrin-selenium (Gibco, Grand Island, NY), 4mM L-glutamine, 1X penicillin/streptomycin for HSA depletion experiments at 37°C 5% CO2. 1×105 cells were seeded in the upper chamber of 24-well plate transwells on a polycarbonate membrane (0.4µm diameter pores) (Corning Costar, Corning, NY). Cells were cultured for 7–21 days until the trans-epithelium electrical resistance (TEER), as measured using a chopstick electrode ohmmeter, reached at least 300 Ω×cm2. 10% PBS, 1 µg/ml LPS, 10% HM supernatants, 10% HM supernatants plus 1 µg/ml LPS, 10% albumin-free (AF) HM supernatants, 10% AF HM supernatants plus 1 µg/ml LPS, 10% AF HM supernatants plus 10 µg/ml Fraction V HSA (EMD Millipore, Billerica, MA), or 10% AF HM supernatants plus 1µg/ml LPS plus 10 µg/ml Fraction V HSA were added in the upper chamber and incubated for one hour at 37°C 5% CO2. Cells were lysed using RLT Lysis Buffer and RNA was extracted using Qiagen RNeasy Plus Mini Kit (Qiagen, Germantown, MD). For quantitative real-time (qRT) PCR gene expression studies, RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).
RNAseq and Analysis
RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). 1 ng of total RNA was pre-amplified with the SMARTer Ultra Low Input kit v4 (Clontech, Mountain View, CA) per manufacturer’s recommendations. 150 pg of cDNA was used to generate Illumina compatible sequencing libraries with the NexteraXT library preparation kit (Illumina, San Diego, CA) per manufacturer’s protocols. The amplified libraries were hybridized to the Illumina flow cell and sequenced using the NextSeq 550 sequencer (Illumina, San Diego, CA). Single end reads of 75 nucleotides (nt) were generated for each sample.
Raw reads generated from the Illumina basecalls were demultiplexed using bcl2fastq version 2.19.0. Quality filtering and adapter removal are performed using FastP version 0.20.1. Processed/cleaned reads were then mapped to the human reference genome (GRCh38.p13) using STAR_2.7.6a. Gene level read quantification was derived using the subread-2.0.1 package (featureCounts) with a GTF annotation file (Genocode-31). Differential expression analysis was performed using DESeq2-1.28.1 with a P-value threshold of 0.05 within R version 4.0.2 (https://www.R-project.org/). Z-score heatmaps of normalized counts were generated using the pheatmap package.
Quantitative Real-Time PCR (qRT-PCR)
Expression of IL8, CXCL1, CXCL2, CCL20, TNFAIP3 (A20), NFKBIA, and ZFP36 was compared to reference gene β2 microglobulin using a TaqMan probe-based qPCR gene expression assay (Applied Biosystems, Warrington, United Kingdom) performed on a BioRad CFX96 Real-Time system. The assay was carried out using the manufacturer’s TaqMan qRT-PCR protocol using pre-made primer/probe sets (IDT, Morrisville, NC) for each gene of interest (Supplemental Table 1). Assays were conducted in technical triplicate. The relative expression of genes of interest was calculated using the 2−ΔΔCT method and analyzed using the CFX Maestro software (BioRad). Data was normalized to the LPS control across each plate of biological replicates.
Statistics
GraphPad Prism was used to graph and perform a one-way analysis of variance (ANOVA) to statistically analyze all data. Patterns were deemed synergistic if the average relative gene expression of Caco-2 cells treated with both HM and LPS was greater than the sum of the average relative gene expression of Caco-2 cells treated with HM or LPS alone.