HISA big data in biomedicine and healthcare 2013 conference

Additional file 5. Biofilm formation by the S. suis serotype 2 (S2) and serotype 9 (S9) wild-type and agI/II -deficient mutant strains in the absence of porcine fibrinogen. Biofilm formation capacity was quantified after 24 h of incubation at 37 °C in the absence of porcine fibrinogen. Data represent the mean ± SEM from at least three independent experiments

Additional file 1: Table S1. of Preferential production of G-CSF by a protein-like Lactobacillus rhamnosus GR-1 secretory factor through activating TLR2-dependent signaling events without activation of JNKs

Expression of cytokines and chemokines in immortalized BMDMs treated with GR-1 and TLR ligands. (DOC 105 kb

<i>S</i>. <i>suis</i> induces NET release which is favored by the presence of the capsular polysaccharide (CPS).

(A, B) Non-primed purified porcine neutrophils were stimulated with S. suis (at a multiplicity of infection (MOI) of 1 or 10), or positive controls phorbol-12-myristate-13-acetate (PMA; 1 μM) and lipopolysaccharide (LPS; 2 μg/mL). C- designate unstimulated control cells. After stimulation, released double-stranded DNA was measured in the supernatant using Picogreen. (A) Kinetics of DNA release by porcine neutrophils stimulated with S. suis. * represents a significant difference when compared to C- (P (B) Effect of S. suis virulence factors on its ability to induce NET release by neutrophils. Neutrophils were stimulated with S. suis, its non-encapsulated mutant (S. suis ΔcpsF) or its suilysin (sly)-negative mutant (S. suis Δsly) at a MOI of 10 for 180 min. * represents a significant difference when compared to C- (P S. suis wild-type strain (P (C) Neutrophiles stimulated with S. suis release long DNA fibers called NETs. Nucleus of living cells was marked with Hoechst 33342 before seeding in coverslips. Neutrophils (non-primed) were then stimulated with S. suis wild-type strain (MOI of 10) for 2 h in a medium containing SytoxGreen, a marker of DNA. Images were obtained by confocal microscopy.</p

<i>S</i>. <i>suis</i> induces a significant production of IL-8 by porcine neutrophils after 12 h of stimulation but present cytotoxic effects.

Purified porcine neutrophils were stimulated for different times with S. suis wild type at various multiplicity of infection (MOI) or with positive control lipopolysaccharide (LPS; 100 ng/mL). C- correspond to unstimulated control cells. (A) The IL-8 in the supernatant was quantified by ELISA. (B) Cytotoxicity was determined by measuring the amounts of lactate dehydrogenase (LDH) in fresh supernatant using a colorimetric reaction. The dotted line represents the threshold of 25% under which the condition is considered non-cytotoxic. * represents a significant difference compared to C- (P (PDF)</p

Proposed model for <i>S</i>. <i>suis</i> activation of porcine neutrophils.

The figure depicts how S. suis activates the five main functions of porcine neutrophils (blue squares), the role of its capsular polysaccharide (CPS) (green squares) and the influence of the priming by the granulocyte colony-stimulating factor (G-CSF) and the granulocyte-macrophages colony-stimulating factor (GM-CSF) (yellow squares). ① Neutrophils weakly kill S. suis due to its CPS and priming with G-CSF and GM-CSF do not enhance killing. ② S. suis induces a slight production of IL-8 by neutrophils although CPS prevents full activation. GM-CSF activates and primes neutrophils for IL-8 production. ③ S. suis induced neutrophil extracellular trap (NET) release by neutrophils which depends on the expression of the CPS. ④ S. suis do not stimulates neutrophil degranulation since CPS protect bacteria. ⑤ S. suis do not induce reactive oxygen species (ROS) production by neutrophils but no role can be attributable to the CPS.</p

G-CSF and GM-CSF do not promote <i>S</i>. <i>suis</i> killing mediated by neutrophils.

Purified neutrophils were infected with S. suis wild-type strain or its non-encapsulated mutant (S. suis ΔcpsF) at a multiplicity of infection (MOI) of 1. Before contact, neutrophils were primed with 50 ng/mL of G-CSF or GM-CSF, or left unprimed (non-treated). After 90 min of incubation, the remaining live bacteria were plated on agar plates and colony-forming units were counted. Percentage of killing was calculated in comparison to bacterial growth without neutrophils. * indicates a significant difference compared to S. suis wild-type (P < 0.001), as determined using the t-test or the Mann-Whitney rank sum test.</p

The cytotoxicity induced by <i>S</i>. <i>suis</i> on porcine neutrophils after 12h depends on the expression of the capsular polysaccharide (CPS) and the suilysin.

Purified porcine neutrophils were stimulated for 12 h with S. suis wild type, S. suis ΔcpsF, S. suis Δsly at a multiplicity of infection (MOI) of 1. After incubation, the supernatant was collected and the lactate dehydrogenase (LDH) measured by a colorimetric reaction. The LDH is released by lysed cells and its amount reflects the percentage of cytotoxicity. The dotted line represents the threshold of 25% under which the condition is considered non-cytotoxic. * represents a significant difference (P (PDF)</p

Neutrophils do not produce reactive oxygen species (ROS) when stimulated by <i>S</i>. <i>suis</i>.

Purified porcine neutrophils (non-primed) were plated and stimulated with S. suis at a multiplicity of infection (MOI) of 1 or 10, or positive control lipopolysaccharide (LPS; 100 ng/mL). C- represents unstimulated control cells. ROS were measured using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) reactive which turns fluorescent when oxidized. Fluorescence was read after stimulation at an Ex/Em of 495/520 nm. (A) ROS production kinetics by stimulated porcine neutrophils. (B) Role of S. suis virulence factors in ROS production by neutrophils. Cells were stimulated with S. suis, its non-encapsulated mutant (S. suis ΔcpsF) or its suilysin (sly)-negative mutant (S. suis Δsly) at multiplicity of infection (MOI) of 1; or LPS for 120 min. * represents a significant increase compared to C- cells (*P < 0.05), as determined using the t-test or the Mann-Whitney rank sum test.</p

<i>S</i>. <i>suis</i> serotype 2 strains used in this study.

<p>¹ All strains from healthy carrier pigs were isolated from tonsils.</p><p><i>S</i>. <i>suis</i> serotype 2 strains used in this study.</p

<i>S</i>. <i>suis</i> capsular polysaccharide (CPS) prevents degranulation of neutrophils exposed to bacteria.

Porcine neutrophils (non-primed) were stimulated with S. suis or controls, and myeloperoxidase (MPO) release evaluated by a colorimetric reaction with 3,3’,5,5’-tetramethylbenzidine hydrochloride (TMB) and oxygen peroxide (H2O2). (A) Degranulation kinetic of S. suis-stimulated neutrophils. Cells were stimulated with S. suis wild-type strain at a multiplicity of infection (MOI) of 1 or the positive control phorbol-12-myristate-13-acetate (PMA; 1 μM) for different incubation times. (B) Degranulation induced by isogenic mutants of S. suis. Cells were then stimulated with S. suis, its non-encapsulated mutant (S. suis ΔcpsF) or its suilysine (sly)-negative mutant (S. suis Δsly); or positive control PMA (1 μM) for 180 min. * represents a significant difference compare to C- cells stimulated with PBS (P S. suis wild-type strain (P < 0.05), as determined using the t-test or the Mann-Whitney rank sum test.</p