Preconception allergen sensitization can induce B10 cells in offspring: a potential main role for maternal IgG

C57BL/6 inbred wild-type (WT) or IL-10-genetically-deficient male and female mice were used at 8–10 weeks of age. The animals were purchased through the Central Animal Facility of the School of Medicine and Institute of Biomedical Sciences—USP. The offspring (F1) of both sexes were evaluated during the neonatal period, and samples from at least three independent experiments were studied.

Peripheral blood mononuclear cells (PBMCs) and sera were collected from volunteers who were previously classified as atopic or non-atopic individuals according to their clinical status and who voluntarily submitted to a skin prick test (SPT) to confirm their atopic state. These individuals were classified into two groups: atopic individuals [clinically allergic and reactive to at least two allergens, n = 14, age (mean ± SE) = 31.1 ± 2.86] and non-atopic individuals [without any clinical allergy symptoms and unreactive to any tested allergen, n = 14, age (mean ± SE) = 28.2 ± 3.11].

Each PBMC sample was provided by a different donor and analyzed in three independent experiments.

The subjects voluntarily submitted to a SPT according to European standards [12] with an adapted panel of allergens that included the Brazil pattern (Blomia tropicalis, Canis familiaris, Periplaneta americana, Aspergillus fumigatus, Penicillium notatum, Alternaria alternata, Cladosporium herbarum, Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Felis domesticus).

In brief, a drop of each allergen extract, histamine (positive control) or allergen diluent (negative control—IPI-Asac, Brazil) was applied to the volar aspect of the forearm. A superficial skin puncture was made through each allergen or control drop with a hypodermic lancet (Alko, Brazil) without inducing bleeding. Fifteen minutes after puncture, the transverse diameter of each wheal reaction was measured. We considered a reaction to be positive when the wheal was 3 mm greater than the wheal diameter of the negative control. As exclusion criteria, we adopted the use of antihistamines, glucocorticosteroids and some other systemic drugs that can influence the results 15 days before the test. We also excluded volunteers with severe eczema or dermographism.

Both purified IgGs were sterilized using 0.20-micron filters (Corning, Germany), and their IgG concentrations were determined with the Coomassie Protein Assay Reagent (Pierce, USA) according to the manufacturer’s instructions.

Female WT mice were immunized subcutaneously with 6 mg of Alum (FURP, Sao Paulo) only or supplemented with 1500 μg of OVA (EndoFit™—endotoxin levels <1 EU/mg; InvivoGen, San Diego, CA, USA), 100 μg of myelin oligodendrocyte glycoprotein (MOG) peptide fragment 35–55 (Sigma, USA) or 100 μg of Dp (LoTox—Indoor Biotechnologies, USA). These animals where boosted intraperitoneally (i.p.) after 10 and 20 days with the same immunization antigen at the following doses: 1000 μg of OVA, 100 μg of MOG or 100 μg of Dp in saline. Thee females that were immunized with Alum only were boosted with saline alone. All females were mated 21 days post-immunization. The pups were maintained with their respective mothers during the breast-feeding period. Some groups of offspring from the immunized and non-immunized mothers were immunized with the same antigen used for maternal immunization. Specifically, 3-day-old offspring were treated i.p. with 100 μg of OVA or 10 μg of Dp in 0.6 mg of Alum and boosted after 10 days with the same antigen/dose in saline. Experimental analyses of the offspring were performed at 20 days of age. The OVA immunization protocols were also performed using IL-10^−/− or IL-10^−/+ mice.

The OVA-specific IgG1, IgM and total IgE antibodies were measured by ELISA as previously described [13]. To measure the total IgE level, a standard curve was used (Pharmingen, USA). The anti-OVA and anti-Dp Ab levels are expressed as optical densities. The anaphylactic anti-OVA IgE titer was measured through passive cutaneous anaphylaxis (PCA) as previously described [7].

The spleens were collected, and their cells were isolated for culture or flow cytometry analyses. Single-cell suspensions were prepared with cell strainers (BD Biosciences, MA, USA) and placed in Petri dishes containing RPMI 1640 culture medium (Sigma, USA). The cell suspension was treated with lysis buffer (Biosource—ACK Lysis Buffer, Rockville, MD, USA) for 2 min, and the cell suspension was washed twice with RPMI medium. The cells were subsequently resuspended in 1 mL of RPMI medium with 10% FBS (III HyClone, Logan, UT, USA), and the cellular viability was quantified with 0.5% Trypan blue in a Neubauer chamber.

For surface staining, single-cell suspensions were prepared in flow cytometry buffer (PBS, 1% BSA). Anti-CD19 (1D3), anti-B220 (RA3-6B2), anti-CD1d (1B1), anti-IgM (R6-60.2) or anti-CD16/32 (2.4G2) antibodies directly conjugated to Cy-Chrome, PE, APC, Horizon V450, PE-Texas Red, PerCP, PerCP-Cy5 or FITC (BD Biosciences) were used at their optimal concentrations, as determined through titration experiments. Cell gating was based on the specific isotype control values as well as the fluorochrome minus 1 (FMO) setting when needed. The gating strategy for B cells that produce IL-10 and B10 cells is illustrated in Additional file 1: Figure S1. All analyses were performed using FlowJo software (Tree Star Inc.). For intracellular staining, the cells were first surface-stained, fixed (PBS, 1% formaldehyde—Sigma), permeabilized (PBS, 0.5% Saponin—Sigma), and subjected to intracellular staining with IL-10 (JES5-16E3) or a matching isotype. For the evaluation of intracellular cytokines, the cells were cultured for 24 h at 3 × 10⁶ cells/mL in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (HyClone) without stimulus in the presence of 10 mg/mL brefeldin A during the last 12 h (Sigma-Aldrich). After the staining of surface markers, the cells were fixed with 4% formaldehyde in PBS (Merck) and then subjected to intracellular staining. Instrument compensation was performed using micro beads adsorbed with anti-rat/anti-hamster antibodies (CompBeads, BD Biosciences) and their conjugated antibodies. The acquisition of 50,000 events per sample was performed in the lymphocyte quadrant (as determined by the ratio of size to granularity) on an LSRFortessa cytometer (BD Biosciences, USA), and data analysis was performed using FlowJo software 7.6.5 (Tree Star, USA).

For the measurement of serum cytokines, the Th1/Th2/Th17A mouse CBA kit (Cytometric Bead Assay, BD Biosciences) was used according to the manufacturer’s instructions. In brief, microspheres of different fluorochrome intensities were sensitized with anti-IL-6 and anti-IL-10 and incubated either with the serum or supernatant for the generation of a standard curve or in the presence of capture antibodies for the same cytokine conjugated to PE for 2 h at room temperature. After washing with buffer supplied by the manufacturer, the microspheres were analyzed with an LSRFortessa cytometer (BD Biosciences, USA), and the levels were determined using CBA Analysis Software.

The BAL was prepared in PBS, and red blood cells were lysed using ammonium chloride lysis buffer. The BAL cells were stained as described by van Rijt et al. [14] with the optimal concentrations of the following monoclonal antibodies provided by BD Biosciences (San Diego, CA, USA): MHCII (2G9), CCR3 (101.111), CD11c (HL3), CD3 (145-2C11) and B220 (RA3-6B2). To prevent nonspecific binding to Fc receptors, a 2.4 G2-blocking reagent (6 µg/mL) was added to the monoclonal antibody mix. Lymphocytes were identified as FSClo/SSClo cells expressing CD3 or B220, and B cells were distinguished from T cells through MHCII expression in the (B220/CD3)⁺ gate. Granulocytes were recognized as non-autofluorescent highly granular (SSChi) cells. Within this gate, eosinophils were defined as cells that expressed the eotaxin receptor CCR3, intermediate levels of CD11c, and very low to undetectable levels of MHCII, B220 and CD3. Neutrophils presented a similar scatter profile to eosinophils but lacked CCR3 expression. Dendritic cells were identified as (CD3/B220)⁺ cells and expressed high levels of MHCII and CD11c. Alveolar macrophages were identified as large autofluorescent cells.

To investigate the FcγRIIb expression kinetics in B cells, the phenotypes of 3 × 10⁶ cells/mL normal adult splenic B cells were evaluated by flow cytometry ex vivo or after culture for 24, 48, 72 or 96 h in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (HyClone) in the presence of 50 μg/mL anti-IgM F(ab’)2 (Southern Biotech, USA).

To investigate the in vitro effect of purified maternal antibodies on offspring B cells, splenocytes from 3-day-old offspring of immunized or non-immunized mothers were cultured for 120 h at a density of 3 × 10⁶ cells/mL in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (HyClone) in the presence of 20 μg/mL OVA (Grade V—Sigma, St. Louis, MO, USA) or 100 μg/mL purified IgG from immunized or non-immunized mothers. Some culture wells were previously blocked with 10 μg/mL purified anti-CD16/32 (2.4G2). For the evaluation of intracellular cytokine production, all of the culture wells were given 10 mg/mL brefeldin A (Sigma-Aldrich) 24 h before flow cytometric evaluation.

Thawed, freshly separated PBMCs were washed twice with 10 mL of RPMI medium at 37 °C, centrifuged at 800g for 10 min, and resuspended in 1 mL of RPMI 1640 medium containing 10% fetal bovine serum. A sample of this suspension was diluted 1:2 in Trypan blue (Sigma, USA) for evaluation of the cell viability and count under an optical microscope using a Neubauer chamber (Laboroptik, Germany). After dilution, 1 × 10⁶ viable PBMCs were placed in each well of a culture plate and cultured with 100 μg/mL IgG, which was purified from pools of atopic or non-atopic individuals in RPMI 1640 medium containing 10% fetal bovine serum in a total volume of 200 μL. The culture plate was incubated at 37 °C with 5% CO₂ for 6 days, which was the period determined based on the kinetic assessment. After incubation, 1 mg/mL brefeldin A was added (Sigma, Israel) to each well of the culture plate, and flow cytometry labeling was performed after 24 h.

For extracellular staining, PBMCs at a concentration of 0.5 × 10⁶ were transferred to test tubes, and 1 μL of each antibody was added to the cells (except the unlabeled tubes) and incubated for 30 min at 4 °C while protected from light. After 500 μL of 1X PBS solution was added, the tubes were centrifuged at 800g for 5 min. The supernatant was discarded by inversion, and 300 μL of 1X PBS was added. For fixation, 200 μL of 1% formaldehyde was added for at least 10 min. The PBMCs were stained with mouse anti-human-CD4 conjugated to FITC or APC, anti-human-CD8 conjugated to PE and anti-human-CD19 PE-Cy7 antibodies (BD Pharmingen, NJ, USA) for the identification of B cells (CD4⁻CD8⁻CD19⁺).

For intracellular labeling, the tubes were centrifuged at 800g for 5 min, the supernatant was discarded, and 1 μL of each antibody was added to the cells (except the unlabeled tubes). A 100-µL volume of 1X PBS containing 0.05% saponin was added, and the tubes were stored at 4 °C for 30 min while protected from light. After centrifugation at 800g for 5 min, the supernatant was discarded by inversion, and the cells were resuspended in 300 μL of 1X PBS solution. The PBMCs were stained with mouse anti-human IL-10 conjugated to Horizon V450 (BD Pharmingen, NJ, USA).

The acquisition of 50,000 events per sample was performed in the lymphocyte quadrant (as determined by the ratio of size to granularity) using an LSRFortessa cytometer (BD Biosciences, USA). Compensation was performed using the adsorbed microspheres with anti-mouse antibodies (CompBeads, BD Biosciences, USA), and the same antibodies were used for extra and intracellular staining. The cell gating protocol was based on the specific isotype control values as well as the fluorochrome minus 1 (FMO) setting when needed. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).