Table 1.
Bacterial strains used.
Fig 1.
Variable sensitivity of lactobacilli to lysozyme digestion and NOD2 activation.
Live or UV-killed lactobacilli were digested with lysozyme for 2 h and OD600 assessed at t = 0 and 2h. Duplicate 2 h lysozyme digests were separated into soluble or insoluble portions via centrifugation and transfected into HEK hNOD2 cells. Positive control, L18-MDP, and negative control, Pam2CSK4, were treated similarly. (a) Data (mean with SEM) is displayed as % sensitivity to lysozyme digestion for each bacterial variant. Means with the same letter are not significantly different (P > 0.05) from each other. (b) Data (mean with SEM) is reported as the fold increase of NFκB/AP-1 activation over negative control as determined by chemiluminescent detection of culture supernatant SEAP after 44–48 h co-culture. N = 3–8 samples per group.
Fig 2.
BMDM phenotype after 24 h co-culture with lactobacilli.
BMDM derived from wild type C57BL/6 or Tlr2-/-, Nod2-/-, or Casp1-/- mice, were co-cultured with either negative control (no bacteria), TLR2/6 agonist (Pam2CSK4), or Cell Trace Violet stained lactobacilli bacteria (10 CFU bacteria per 1 cell) as indicated for 2 or 24 h prior to harvest for flow cytometric analysis. (a) Cells were gated on 7-AAD-F4/80+cells to assess purity and then 7-AAD- BMDM subdivided into Bacteria+ (Bacteria +) or Bacteria- (Exposed) populations, and the percentage of CD11b+, and subsequent MHCII+ cells, determined. Data (mean with SEM) represented as the (b) percentage of Bacteria+ BMDM cells or (c) MFI (median fluorescence intensity) of respective bacteria within Bacteria+ BMDM cells across treatments and BMDM genotypes. Asterisk (*) indicates statistically significant difference (P < 0.05) between 24 h data and corresponding 2 h treatment; bracket denotes significance between respective treatments within the same group and timepoint. 24 hr BMDM phenotype data displayed as (d) the percentage (mean with SEM) of CD11b+MHCII+, (e) CD11b+, and (f) 7-AAD+ BMDM cells across bacterial treatments and BMDM genotypes. Asterisk (*) denotes a significant difference (P < 0.05) between respective treatments and no bacteria control. Bracket alone denotes significant difference (P < 0.05) between treatments within a BMDM genotype. N = 5–6 separate experiments.
Fig 3.
BMDM from (a) wild type C57BL/6, (b) Tlr2-/-, (c) Nod2-/-, and (d) Casp1-/- mice were co-cultured with CFSE-labeled lactobacilli (10 CFU per 1 cell; green). After 24 h, media was removed, cells were washed and stained with actin-specific phalloidin (red) and DNA-specific DAPI (blue), and subsequently evaluated using fluorescence microscopy. Representative images are shown. N = 6 separate experiments.
Fig 4.
Cytokine and chemokine production by BMDM after 24 h co-culture with lactobacilli.
BMDM from wild type C57BL/6, Tlr2-/-, Nod2-/-, or Casp1-/- mice were co-cultured with NCK1785, NCFM, or NCFM mutants (NCK2025 or NCK2031) (10 CFU bacteria per 1 cell) for 24 h and culture supernatants harvested for cytokine and chemokine quantification using a 12-plex Milliplex MAP mouse assay. Box (percentiles) and whisker (minimum and maximum) data for (a) IL-6, IL-1β, and TNF-α and (b) MIP-1α, KC, IL-12 (p40), and IL-10 displayed for lactobacilli across BMDM genotype. Asterisk (*) denotes a significant difference (P < 0.05) in cytokine concentration between the respective knock-out BMDM and wild type BMDM treatment. Bracket denotes significant difference (P < 0.05) between treatments within a BMDM genotype. N = 5–6 separate experiments.
Fig 5.
Differential IgG response of immunized C57BL/6 mice based upon Nod2 expression and IL-1β.
Wild type Nod2+/+ or Nod2-/- C57BL/6 mice were immunized as described in methods with buffer only, NCK1895, or NCK2166. At sacrifice, lymphocytes were isolated from Peyer’s patches (PP), large intestine (LI), female reproductive tract (FRT), and spleen; and analyzed by flow cytometry. (a) Data represented as mean percentage (and SEM) of 7-AAD-CD45+CD19+CD38- B cells across tissue type from NCK1895 and NCK2166 immunized mice. (b) Lymphocytes were additionally applied to total IgA ELISpot assay for 20 h of incubation without stimulation. Data represented as spot forming units (SFU) per 1 × 106 live (7AAD-) B cells (CD45+CD19+ and/or CD45+CD38+). At sacrifice, serum and cecal contents were harvested, serially diluted, and applied to antigen-specific colorimetric ELISA, using (c) HIV IIIB lysate (d) or lactobacilli SlpA as antigen, with α-IgG or -IgA detection antibody. Data are represented as absorbance (mean with SEM) across serial dilutions along with the standard curve value (open circle or square) derived from control. Seroconversion of immunized animals was defined as 2-fold absorbance over the standard curve at 2 or more dilutions. The number of NCK2166 or NCK1895 immunized animals that met this criterion is shown in parentheses. (e) Small intestine ex vivo cellular supernatants were utilized in a customized 14-plex Milliplex MAP mouse TH17 cytokine magnetic bead assay and results reported as pg/mL of cytokine per g of tissue cultured. Only IL-1β cytokine data (mean with SEM) is shown with assay lower limit of detection (LLOD) indicated by horizontal dashed line. (f) Lymphocytes isolated at sacrifice from PP, MLN, and spleen were stained for flow cytometry or incubated in duplicate with or without AT-2 inactivated HIV IIIB lysate for IFN-γ ELISpot assay. Spots were counted after 2 nights of incubation and data (mean with SEM) represented as the difference of spot forming units (SFU) from HIV IIIB stimulated and unstimulated cells per 1 × 106 live, CD45+ cells. Asterisk (*) indicates significance (P < 0.05) of difference between treatment group and buffer-only group of the same Nod2 genotype status; (**), between treatment group and NCK1895-treated group of the same Nod2 genotype status; (***) between treatment group and Nod2-/- mice receiving same treatment. N = 4–6 animals per group.
Fig 6.
Differential IgG and IgA response of BALB/c immunized mice based upon Nod2 expression.
Wild type Nod2+/+ or Nod2-/- BALB/c mice were immunized with buffer only, NCK1895, or GAD31. (a) At sacrifice, lymphocytes were isolated from Peyer’s patches (PP), large intestine (LI), female reproductive tract (FRT), and spleen; applied to total IgA ELISpot assay for 20 h of incubation without stimulation. Data represented as spot forming units (SFU) per 1 × 106 live B cells (CD45+CD19+ and/or CD45+CD38+) across tissue per immunization group. N = 4–6 animals per group. (b) Fecal extract and vaginal wash were harvested from GAD31 immunized mice at week 0 and week 12/sacrifice and applied to a quantitative colorimetric ELISA to evaluate total IgA. Data (mean plus SEM) are represented as μg/mL of total IgA. Asterisk (*) indicates significance (P < 0.05) of difference between indicated treatment groups. N = 12 animals per group. At 0, 2, 4, 6, 8, 10, and 12 weeks/sacrifice serum, feces, and vaginal wash were harvested, serially diluted, and applied to antigen-specific colorimetric ELISA, using HIV MPER (c and d) or lactobacilli SlpA as antigen (e), with α-IgG or -IgA detection antibody. Data are represented as absorbance (mean with SEM) across time at 1:100 dilution for MPER-specific and 1:1000 for SlpA-specific serum and 1:10 for vaginal wash and feces. Average control values at the same dilution are represented as open circle or square. Seroconversion of immunized animals was defined as absorbance 3-fold greater than the average absorbance from control animals, and the number of GAD31 immunized animals that met this criterion is shown in parentheses; N = 6–12 animals per group (c). The same criterion was used for endpoint titer data, with values under detection limit assigned 0; N = 12 animals per group (d and e). (f) Lymphocytes isolated at sacrifice from PP, LI, FRT, and spleen were stained for flow cytometry or incubated in duplicate with or without HIV MPER for IFN-γ ELISpot assay. Spots were counted after 2 nights of incubation and data (mean with SEM) represented as the difference of SFU from HIV MPER stimulated and unstimulated cells per 1 × 106 live, CD45+ cells; N = 4–6 animals per group. Asterisk (*) indicates significance (P < 0.05) of difference between treatment group and buffer-only group of the same Nod2 genotype status; (***), between treatment group and Nod2-/- mice receiving same treatment.