Modulation of functional characteristics of resident and thioglycollate-elicited peritoneal murine macrophages by a recombinant banana lectin - Fig 7

<p><b>Impact of TLR2- and CD14-triggered mechanisms in production of IL-10 (A, B), TNFα (C, D) and NO (E, F) upon rBanLec stimulation of peritoneal RMs (A, C, E) and TGMs (B, D, F) from BALB/c.</b> RMs and TGMs were stimulated with rBanLec (1, 5 and 10 μg/ml) in the presence of anti-TLR2 or anti-CD14 blocking monoclonal antibodies (20 μg/ml) for 48h. Cytokines and NO were measured in supernatant by ELISA and colorimetric method using Griess reagent, respectively. Bars presented mean concentration ± SE. Corresponding mean concentrations of IL-10, TNFα and NO measured upon incubation under the same conditions but without blocking antibodies are indicated by black solid line and are considered as referent. The significance of the observed differences, due to incubation with particular blocking antibody, was calculated by one-way repeated ANOVA followed by Bonferroni’s multiple comparison test (p <0.05*, p <0.005**, p <0.0001***). LPS–lipopolysaccharide, PEPG–peptidoglycan.</p

Binding of rBanLec to TLR2, TLR4 and CD14 –the effect of methyl-α-D-mannopyranoside.

<p>TLR2, TLR4 and CD14 were extracted from TGMs lysate with specific monoclonal antibodies adsorbed onto microplate. Biotin-labeled rBanLec (10 μg/ml), pre-incubated with or without 0.5 M methyl-α-D-mannopyranoside (α-D-Man), was added to the wells. Alkaline phosphatase / <i>p-</i>nitrophenylphosphate system was used for the visualization of rBanLec binding. Results are presented as mean A₄₀₅ ± SE. The significance of the observed differences was calculated by one-way repeated ANOVA followed by Bonferroni’s multiple comparison test (p <0.05*, p <0.005**, p <0.0001***). Solid lines indicate compared groups.</p

Anti-PmpC antibody serum levels.

<p>Levels of (A) anti-N-PmpC IgA and (B) anti-N-PmpC IgG and (C) ratio of the levels of N-PmpC-specific IgG1 and IgG2a antibodies in the serum of BALB/c mice immunized via the conjunctiva and age-matched normal controls (nc). All serum samples were collected two weeks after completion of the indicated immunization protocol and were assayed by ELISA. Results for each individual serum sample are presented. The levels of N-PmpC-specific IgG1 and IgG2a were judged according to the A_492/620 values recorded for individual serum samples. The statistical significance of the observed differences was evaluated using Kruskal -Wallis test followed by Dunn's multiple comparisons test to compare between groups (<i>P</i> < 0.05*, <i>P</i><0.005**). Compared groups are indicated by two-head arrow.</p

Anti-CtB antibody levels in serum and on mucosal surfaces.

<p>(A-C) Anti-CtB IgA levels and (C-E) anti-CtB IgG levels in the serum (A and D), tears (B and E) and vaginal washes (C and F) of BALB/c mice immunized via the conjunctiva and age-matched controls (nc). All samples were collected two weeks after the completion of the indicated immunization protocol and were assayed by ELISA. Results for each individual sample are presented. The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (<i>P</i> < 0.05*, <i>P</i><0.005**). Compared groups are indicated by two-head arrow.</p

<i>In vitro</i> analysis of proliferation of primary SMLN cells subjected to N-PmpC and CtB stimulation.

<p>Bar graph showing the proliferation indices (PI) of N-PmpC- and CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva. The proliferation indices of SMLN cells isolated from age-matched control mice (nc) are presented on the graph as a solid line (mean value) and dotted lines (upper and lower standard error values). The number of viable SMLN cells was assessed using the Cell Counting Kit<i>-</i>8 following a 48 h culture period in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10⁶ CFU/ml for CtB). PIs were calculated for each mouse. The results are presented as the mean PIs ± SE for each experimental group (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc <i>P</i> < 0.05*, <i>P</i><0.005**; between immunized groups <i>P</i> < 0.05^#, <i>P</i><0.005^##).</p

Phenotyping of SMLN T lymphocytes by FACS analysis.

<p>Bar graph showing the contribution (expressed in %) of specific T cell populations to the overall T cell pool within SMLNs isolated from BALB/c mice two weeks after the completion of the indicated immunization protocol. The mean percentages of (A) CD4+ and CD8+, (B) CD25+, and (C) CD4-CD25+ and CD4+CD25+ cells within T lymphocytes pool are presented. Lymphocytes were gated according to their position within the forward scatter (FSC) vs side scatter (SSC) and T cells were marked according to the expression of CD3+ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157875#pone.0157875.s001" target="_blank">S1 Fig</a>). Assessment of Foxp3 and CD25 co-expression was performed for CD4+ lymphocytes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157875#pone.0157875.s002" target="_blank">S2 Fig</a>) and mean percentages of Foxp3+CD25+ within CD4+ lymphocytes (D) are presented. The mean percentage of corresponding T cell population recorded in SMLN isolated from age-matched control mice (nc) is presented on each graph as a solid line (mean value) and dotted lines (upper and lower standard error values). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc <i>P</i> < 0.05*, <i>P</i><0.005**; between immunized groups <i>P</i> < 0.05^#, <i>P</i><0.005^##).</p

Effect of N-PmpC and CtB stimulation on the <i>in vitro</i> cytokine pattern in SMLN cells isolated after ocular mucosal immunization with N-PmpC and N-PmpC/LB.

<p>Bar graphs representing the levels of IFNγ, IL-4, IL-10 and IL-17A in the supernatants of non-stimulated, N-PmpC- or CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva (bars). The corresponding measurements in SMLN cells isolated from age-matched controls (nc) are presented on the graphs as a solid line (mean value) and dotted lines (upper and lower standard error values). SMLN cells were cultured at 37°C under a 5% CO₂ atmosphere for 48 h in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10⁶ CFU/ml for CtB). The results are presented as the mean concentrations ± SE (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc stimulated in a same way <i>P</i> < 0.05*, <i>P</i><0.005**; vs non-stimulated samples of the same group <i>P</i> < 0.05^#, <i>P</i><0.005^##).</p

Anti-PmpC antibody levels from mucosal surfaces.

<p>(A-B) Anti-N-PmpC IgA levels and (C-D) anti-N-PmpC IgG levels in the tears (A and C) and vaginal washes (B and D) of BALB/c mice immunized via the conjunctiva and age-matched controls (nc). All samples were collected two weeks after the completion of the indicated immunization protocol and were assayed by ELISA. Results for each individual sample are presented. The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (<i>P</i> < 0.05*, <i>P</i><0.005**). Compared groups are indicated by two-head arrow.</p

The abundance of neutrophils in the conjunctiva and CALT of guinea pigs infected with a single ocular instillation of three different <i>C</i>. <i>caviae</i> doses.

<p>The neutrophils infiltration was evaluated in paraffin-embedded conjunctival sections by immunohistochemical staining for myeloperoxidase, a neutrophil-specific enzyme. Conjunctival sections were prepared from samples collected at screening time points within the post-infection period (day 4 –dpi4, day 7 –dpi7, day 14 –dpi14, day 21 –dpi21). ExtrAvidin^®−Peroxidase/DAB system was used for visualisation of myeloperoxidase presence.</p

Experimental design.

<p>Guinea pigs were infected on day 0 via inoculation of 1×10², 1×10⁴ or 1×10⁶ IFUs of <i>C</i>. <i>caviae</i> directly into the conjunctival sac (arrow). Pathological signs were followed daily for 21 days post-infection (dashed line). Days 4, 7, 14 and 21 post-infection are chosen as time points when sampling of mucosal washes, sera and swabs were performed, and <i>ex vivo</i> analyses were performed (asterisks).</p