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

Expression of CD4 and CD8 on SMLN lymphocytes.

<p>Cell suspension for BD FACSVerse^™ analyses were prepared from SMLN and splenic lymphocytes collected at defined time points (post-infection: day 4 –dpi4, day 7 –dpi7, day 14 –dpi14, and day 21 –dpi21) from guinea pigs infected with <i>C</i>. <i>caviae</i> (infectious doses per eye, expressed in IFU, assigned on the x-axis) and age-matched non-infected guinea pigs (nc; used negative controls). The start of infection is considered as day 0. The expression of CD4 and CD8 are presented as percentages of CD4⁺ and CD8⁺ cells within lymphocyte gates, respectively. Lymphocytes were gated according to their position within the forward scatter vs. side scatter plot. The statistical significance of the observed differences was evaluated using the two-way ANOVA test followed by Tukey’s multiple comparisons test. The statistical significance of the differences in parameter values between specific time points (compared groups indicated with arrows) for guinea pigs infected with an equal amount of <i>C</i>. <i>caviae</i> is indicated as follows: * P<0.05, ** P<0.005, and *** P<0.001.</p

The levels of <i>C</i>. <i>caviae</i>-specific IgA (A) and IgG (B) in the sera of guinea pigs infected with a single ocular instillation of three different <i>C</i>. <i>caviae</i> doses.

<p>The amount of <i>C</i>. <i>caviae</i> applied per guinea pig eye (expressed in IFU) and screening time points within post-infection period (day 4 –dpi4, day 7 –dpi7, day 14 –dpi14, and day 21 –dpi21) are indicated on the x-axis. The start of infection is considered as day 0. Age-matched non-infected guinea pigs (nc) were used as negative controls. The level of specific antibodies is expressed as a <i>relative concentration</i>, calculated as the A_450/650 for a sample divided by the lowest A_450/650 recorded within the same assay for the corresponding nc sample. The statistical significance of the observed differences was evaluated using the two-way ANOVA test followed by Tukey’s multiple comparisons test. The statistical significance of differences in parameter values between specific time points (compared groups indicated with arrows) for guinea pigs infected with an equal amount of <i>C</i>. <i>caviae</i> is indicated as follows: * P<0.05, ** P<0.005, and *** P<0.001.</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

Proliferation of SMLN cells after stimulation with live <i>C</i>. <i>caviae</i>.

<p>Cell cultures were prepared from SMLN and spleen cells collected at defined time points (post-infection: day 4 –dpi4, day 7 –dpi7, day 14 –dpi14, and day 21 –dpi21) from guinea pigs infected with <i>C</i>. <i>caviae</i> (infectious doses per eye, expressed in IFU, assigned on x-axes) and age-matched non-infected guinea pigs (nc; used as negative controls). The start of infection is considered as day 0. The PI is calculated per individual sample and defined as a ratio of number of viable cells per well present in a stimulated culture to the number of viable cells per well present in the corresponding non-stimulated culture. The statistical significance of the observed differences was evaluated using the two-way ANOVA test followed by Tukey’s multiple comparisons test. The statistical significance of the differences in parameter values between specific time points (compared groups indicated with arrows) for guinea pigs infected with an equal amount of <i>C</i>. <i>caviae</i> is indicated as follows: * P<0.05, ** P<0.005, and *** P<0.001.</p

Ocular pathology scores (A) and <i>C</i>. <i>caviae</i> load (B) in guinea pigs infected with a single ocular instillation of three different <i>C</i>. <i>caviae</i> doses.

<p>The amount of <i>C</i>. <i>caviae</i> applied per guinea pig eye (expressed as IFU) and screening time points within the post-infection period (day 4 –dpi4, day 7 –dpi7, day 14 –dpi14, and day 21 –dpi21) are indicated on the x-axis. The start of infection is considered as day 0. Age-matched non-infected guinea pigs (nc) were used as negative controls. Statistical significance of the observed differences was evaluated using two-way ANOVA test followed by Tukey’s multiple comparisons test. The statistical significance of differences in parameter values between specific time points (compared groups indicated with arrows) for guinea pigs infected with an equal amount of <i>C</i>. <i>caviae</i> is indicated as follows: * P<0.05, ** P<0.005, and *** P<0.001.</p