Bacillus subtilis Provides Long-Term Protection in a Murine Model of Allergic Lung Disease by Influencing Bacterial Composition

C57B1/6 mice, age 6–10 weeks old, were treated through oral gavage with 10^9 wild-typespores, 10^9spores unable to produce exopolysaccharide (EPS), or sterile PBS as a negative control.andspores were generated via exhaustion, as previously described [ 22 ]. After 24 h, mice were sensitized intranasally with 100 µg of house-dust mites (HDM,XPB82S3A2.5, Stallergenes Greer, Lenoir, NC, USA) in 100 µL of sterile PBS. After 5 days of initial treatment, mice were once again treated orally withspores as described above. On days 6–9, mice that were in the “week 1” group were sensitized intranasally with 25 µg HDM in 100 µL sterile PBS. These mice were euthanized on day 12. Bronchial alveolar lavage (BAL) was collected by flushing lungs with 0.8 mL BAL fluid (10% FCS, 1 mM EDTA, 1X PBS). Blood was collected through cardiac puncture and spun down at 8000 rpm for 8 min, and serum was collected and frozen for ELISA assays. Lungs were excised and fixed in formalin for 24 h. BALF was counted using a hemocytometer to determine total cells/mL, cytospun onto slides, and assessed for percent eosinophils, neutrophils, lymphocytes, and macrophages using Diff-Quick staining and counting 100 cells per slide (Dade Behring, Deerfield, IL, USA). The total cells/mL of each cell type was determined by calculating the number of cells/mL based on the percent of each cell type. On days 13–16, mice that were in the “week 2” group were sensitized intranasally with 25 µg HDM in 100 µL sterile PBS. On day 19, these mice were euthanized, and tissues were collected as described above. Intranasal sensitizations were repeated for groups “week 3” through “week 8,” mice were euthanized, and tissues were collected.

2.3. ELISA

CCL24:

96-well plates were coated with 100 µL of biotinylated anti-mouse CCL24 in carbonate buffer at 4 °C overnight (Peprotech, Cranbury, NJ, USA). The plates were washed three times with PBST buffer (1xPBS, 0.05% Tween 20) and blocked with 3% BSA in PBS for 2 h at room temperature. The plates were washed again and BALF was added to the wells along with antibody standard recombinant murine CCL24 (Peprotech). After three washes, the plates were treated with secondary antibody Biotin anti-murine CCL24 (Peprotech) and incubated at room temperature for 1 h. The plates were washed and stained with HRP-avidin (Biolegend, San Diego, CA, USA) in the dark at room temperature for 1 h. After washing the plates, TMB substrate solution (Biolegend) was added to the wells, and once there was a color change the plates were measured on a MultiSkan™ plate reader (Thermo Fisher, Waltham, MA, USA) at OD 405 nm.

HDM-specific IgE and IgG1:

96-well plates were coated with 100 µL of HDM (5 µg/µL) in carbonate buffer at 4 °C overnight. The plates were washed three times with PBST buffer (1xPBS, 0.05% Tween 20) and blocked with 3% BSA in PBS for 2 h at room temperature. The plates were washed again and serum was added to the wells. After subsequent washes, the plates were treated with secondary antibody Biotin anti-mouse IgE or IgG1 and incubated at room temperature for 1 h. The plates were washed and stained with HRP-avidin (Biolegend) in the dark at room temperature for 1 h. After washing the plates, TMB substrate solution (Biolegend) was added to the wells and once there was a color change, the plates were measured on a MultiSkan™ plate reader (Thermo Fisher, Waltham, MA, USA) at OD 405 nm.

Total IgE and IgG1:

96-well plates were coated with 100 µL of rat anti-mouse IgE or anti-mouse IgG1 (BD Biosciences, San Jose, CA) in carbonate buffer at 4 °C overnight. The plates were washed three times with PBST buffer (1xPBS, 0.05% Tween 20) and blocked with 3% BSA in PBS for 2 h at room temperature. The plates were washed again and serum was added to the wells along with antibody standards (BD Biosciences). The plates were incubated at 4 °C overnight. The plates were washed and stained with HRP-avidin (Biolegend) in the dark at room temperature for 1 h. After washing the plates, TMB substrate solution (Biolegend) was added to the wells and once there was a color change, the plates were measured on a MultiSkan™ plate reader (Thermo Fisher, Waltham, MA, USA) at OD 405 nm.