Lps from escherichia coli serotype 0111 b4

LPS from Escherichia coli serotype 0111:B4 and cFN from skin fibroblasts were from Sigma-Aldrich. TAK-242, TLR1/2 agonist Pam₃Csk₄, and TLR9 agonist CpG ODNs were purchased from Invivogen. pFN was from BD Bioscience. The following antibodies were used: anti-FN was from Chemicon (Merck-Millipore), and anti–EDA FN (FN-3E2), anti-FN (FN IST-4), and anti–β-actin were all from Sigma-Aldrich. Anti–EDA FN (IST-9) and anti–type III collagen were from Abcam. Anti–β3 integrin, anti-TLR4 (clone 25 and M-300), and anti-α4 (clone C19) were from Santa Cruz Biotechnology. Anti-mouse CD49d (α4 integrin) with blocking function was from BioLegend (clone 9C10). Anti–phospho-ERK 1/2, anti-ERK 1/2, anti–phospho–STAT-5, anti–STAT-5, anti–phospho p65 (NF-κB), and p65 (NF-κB) were purchased from Cell Signaling Technologies. EDA⁺ and EDA⁻ recombinant peptides were produced as previously described (Losino et al., 2013 (link)). All the experiments were performed with the same batch for each peptide.

As a model for monocytes, the THP-1 cell line (number TIB-202l; American Type Culture Collection, Manassas, VA) was used, realizing the cell culture within RPMI 1640 culture media, which was supplemented with 10% inactivated FBS and 1% antibiotic-antimycotic (Life Technologies, Grand Island, NY).
In order to obtain activated monocytes, THP-1 cells were exposed to 1 µg/ml LPS from Escherichia coli serotype 0111:B4 (Sigma–Aldrich) for 48 h. The THP-1 cells (resting monocytes) and the activated monocytes that were obtained were seeded at a density of 7 × 105 cells/ml on a six-well plate, either without hormonal stimulus (W/S) or with hormonal stimulus (i.e. LLH (low) and HLH (high) hormone concentrations in the first trimester or third trimester). To the obtain macrophages, 3 × 106 THP-1 cells were differentiated with 200 nM phorbol myristate acetate for 3 d, followed by resting culture with fresh RPMI for 5 d.^{17 (link)} Thereafter, the macrophages that were obtained were exposed to the same treatments as previously described for monocytes. All cell cultures were carried out in a humidified atmosphere at 37°C and CO₂ 5%, in triplicate for each condition.
Next, we evaluated the effect of the pregnancy hormone mixture on cytokine production and surface marker expression in resting and activated monocytes and macrophages derived from THP-1 cells with measurements detailed below.

DMEM, heat inactivated FBS, Hank's balanced salt solution (HBSS) and PBS were purchased from Bio-Rad Laboratories, Inc. Penicillin/streptomycin sulfate, amphotericin B and L-glutamine were purchased from Mediatech, Inc. Cocaine hydrochloride (Ecgonine methyl ester benzoate; molecular weight, 339.8), trypan blue, sulfanilamide, LPS from Escherichia coli serotype 0111:B4, N-(1-naphthyl)-ethylenediamine (NED), phosphoric acid, and EDTA were supplied by Sigma-Aldrich; Merck KGaA. IFNγ protein was obtained from Novus Biologicals Ltd. All other routinely used agents were analytical grade.

RC (batch number, 1711028050) was purchased from Anhui Huizhongzhou TCM Pieces Co., Ltd. (Anhui, China) and authenticated by Prof. Qi-nan Wu (College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China). Voucher specimens were deposited at the Museum of Materia Medica, Nanjing University of Chinese Medicine. Diazepam (DZP) was purchased from Huazhong Pharmaceutical Co., Ltd. (Hubei, China). LPS (from Escherichia coli, serotype 0111: B4) and pentobarbital sodium were from Sigma-Aldrich (St. Louis, MO, USA). Radioimmunoassay (RIA) kits for 5-HT (20210202), DA (20210129), IL-1β (20210201), TNF-α (20200130), IL-10 (20200201), and IL-4 (20200203) were purchased from Beijing Sino-UK Institute of Biological Technology (Beijing, China). Two primary antibodies were used in the study: rabbit anti-NeuN (Abcam, ab236870, 1:40000) and rabbit anti-Syn I (Abcam, ab254349, 1:10000).

To determine the function of HAECs-cocultured neutrophils, the amount of the phagocytosis of them after activation was assessed; briefly, an aliquot of the washed neutrophils (after coculture) were stimulated with 50 ng/ml endotoxin (LPS from Escherichia coli, serotype 0111: B4, Sigma) at 37℃, 5% CO2, 90% humidity for 30 min in 96-well v-bottom plate. Afterwards, fluorescent latex beads (1.0 μm mean size, Sigma) were added (the final concentration of beads were about 2×10⁵ beads/ml) and neutrophils were incubated for another 30 min (37℃, 5% CO2, 90% humidity). After incubation, the cells were washed twice with a large volume of cold PBS and resuspended in cold FACS solution contained 0.5% formaldehyde, then were run on the flow cytometer. Negative controls were treated as mentioned except LPS stimulation.
Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson). The data were acquired using CellQuest (Becton Dickson) and then analyzed by FlowJo software version X.