Survival of Streptococcus suis in Porcine Blood Is Limited by the Antibody- and Complement-Dependent Oxidative Burst Response of Granulocytes

Growth of S. suis is impaired in the presence of hydrogen peroxide. S. suis strains 10, A3286/94, 16085/3b, and 13-00283-02, representing three different serotypes (cps2, cps9, and cps7), as well as a capsule-deficient S. suis mutant (10cpsΔEF) were grown in THB medium. After 2 h, hydrogen peroxide was added at the indicated concentrations (percent, weight per volume). The OD₆₀₀ was measured every hour and is depicted on the y axis. Data from one representative experiment out of four are shown.

Antibody-mediated induction of the oxidative burst in porcine granulocytes in response to S. suis. (A) S. suis strain 10 was incubated with three sera, serum from colostrum-deprived piglets (CDS) (free of IgG), serum containing moderate levels of IgM and IgG antibodies against S. suis strain 10 (moderate Ab serum), and anti-S. suis strain 10 hyperimmune serum. Subsequently, the whole-blood cell pellet of healthy donor piglets was added, and ROS production in blood granulocytes was measured by flow cytometry (n = 3). Bars and error bars represent means and standard deviations, and significant differences are indicated (**, P < 0.01; ***, P < 0.001). (B) Anti-S. suis IgM and IgG antibody levels in the chosen experimental sera, listed as ELISA units. The prime-booster hyperimmune serum was defined to include 100 ELISA units of IgG and 100 ELISA units of IgM. Note that ELISA units for the different Ig classes are therefore not comparable.

Inhibition of NADPH oxidase by apocynin leads to a decreased oxidative burst in porcine granulocytes and increased survival of S. suis in blood reconstituted with hyperimmune sera, without affecting granulocyte viability. (A) Oxidative burst of granulocytes in porcine blood in response to in vitro infection with different S. suis serotypes with or without the NADPH oxidase inhibitor apocynin. The oxidative burst induced by S. suis and hyperimmune sera was set at 100% (control [ctr]). The reduction in the oxidative burst by the addition of apocynin (n = 5 to 6) is depicted as a percentage of the oxidative burst without the inhibitor. (B) Survival factors of S. suis serotypes 2, 9, and 7 in the presence of hyperimmune sera with or without the addition of the NADPH oxidase inhibitor apocynin as the quotients of CFU per milliliter after 1 h and CFU per milliliter directly after in vitro infection with S. suis (n = 5 to 6). (C) The viability of granulocytes after the bactericidal assay (B) was measured by flow cytometry by staining with the fixable viability dye eF506 (n = 3). Bars and error bars represent means and standard deviations. Statistical analyses between controls and the respective apocynin-treated samples were done by an unpaired t test (n = 5 to 6/group) (*, P < 0.05; **, P < 0.01; n.s., not significant).

Complement partially mediates oxidative burst induction but is not crucial for the killing of S. suis strain 10 in blood reconstituted with hyperimmune serum raised against cps2 strain 10. Hyperimmune serum raised against S. suis strain 10 was used to reconstitute porcine blood subsequently infected with S. suis strain 10 to analyze the oxidative burst (shown as percentages of Rho123⁺ granulocytes) (A) and bacterial survival (shown as survival factors) (B) in the untreated control sample (ctr) or in the presence of the complement inhibitor VCP (100 μg/ml), the NADPH oxidase inhibitor apocynin (Apo) (1.5 mM), or a combination of both. Survival factors were determined after 2 h at 37°C after in vitro infection with 2 × 10⁶ CFU/ml S. suis strain 10. To investigate the S. suis-induced oxidative burst, the same sample treatments were conducted in the absence of S. suis (n = 3). PMA (0.1 μg/ml) was used as a positive control in oxidative burst experiments. Bars and error bars represent means and standard deviations. For statistical analysis, the Kruskal-Wallis test with Dunn’s multiple-comparison test was used (n = 6). All S. suis in vitro-infected groups (black brackets) or only the subgroups of controls versus VCP and VCP plus Apo (gray brackets) were included in statistical analyses (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Influence of complement on the oxidative burst (A) and the survival of cps2 S. suis strain 10 (B) in porcine blood reconstituted with IgG-depleted hyperimmune serum. IgG-depleted hyperimmune serum raised against cps2 strain 10, supplemented with 10% CDS, was used to reconstitute porcine blood samples subsequently infected with S. suis. The oxidative burst (shown as percentages of Rho123⁺ granulocytes) (A) and bacterial survival (shown as survival factors) (B) were analyzed in the presence of the complement inhibitor VCP (100 μg/ml), the NADPH oxidase inhibitor apocynin (Apo) (1.5 mM), or a combination of both as well as in an untreated sample (ctr). Bacterial survival factors were determined after 2 h at 37°C after in vitro infection with 2 × 10⁶ CFU/ml S. suis strain 10. To determine S. suis-specific mechanisms of oxidative burst induction, the same sample treatments were conducted in the absence of S. suis (n = 3). PMA (0.1 μg/ml) was used as a positive control in oxidative burst experiments. Bars and error bars represent means and standard deviations. For statistical analysis, the Kruskal-Wallis test with Dunn’s multiple-comparison test was used (n = 6). All S. suis in vitro-infected groups (black brackets) or only the subgroups of controls versus VCP and VCP plus Apo (gray brackets) were included in statistical analyses (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Impact of IgM on killing of S. suis (A) and induction of the oxidative burst in granulocytes (B) in porcine blood reconstituted with IgG-depleted hyperimmune serum. Shown are bacterial survival factors (A) and the oxidative burst responses of granulocytes (B) in S. suis strain 10-infected porcine blood reconstituted with IgG-depleted hyperimmune serum (raised against cps2 strain 10) pretreated with PBS as a control (ctr) or with the IgM protease Ide_Ssuis_h or its nonfunctional control rIde_Ssuis_h_95°C (heat inactivated) or rIde_Ssuis_h_C195S. Particular serum treatments were used without further treatment (left), were heat inactivated (56°C for 30 min) (middle and left) for complement inactivation, or were additionally treated with the NADPH oxidase inhibitor apocynin (1.5 mM) (right). The oxidative burst was measured by flow cytometry and is depicted as a percentage of Rho123⁺ cells of all granulocytes. Bars and error bars represent means and standard deviations, and significant differences are indicated. Statistical significances were calculated using the Kruskal-Wallis test with Dunn’s multiple-comparison test (n = 3 to 6) (*, P < 0.05; **, P < 0.01).

Influence of complement and NADPH oxidase inhibition on the rate of phagocytosis of porcine granulocytes in blood reconstituted with hyperimmune serum. (A) Blood reconstituted with hyperimmune serum raised against cps2 strain 10 was infected with CellTrace far-red (FR)-labeled S. suis strain 10, and samples were analyzed by flow cytometry to estimate the oxidative burst response (Rho123 signal) within phagocytic granulocytes. (B) Complete phagocytosis rate depicted as a percentage of S. suis FR-positive granulocytes. Serum was untreated, heat inactivated (56°C for 30 min) to inhibit complement, and/or treated with the NADPH oxidase inhibitor apocynin (1.5 mM). Bars and error bars represent means and standard deviations, and significant differences are indicated. Statistical significances were calculated using the Kruskal-Wallis test with Dunn’s multiple-comparison test (n = 7) (*, P < 0.05; ***, P < 0.001).

Impact of IgM and complement on phagocytosis and ROS production of porcine granulocytes in blood reconstituted with IgG-depleted hyperimmune serum. Fluorescently labeled S. suis strain 10 bacteria were used to infect porcine blood reconstituted with IgG-depleted hyperimmune serum (raised against cps2 strain 10) pretreated with PBS as a control (PBS) or with the IgM protease Ide_Ssuis_h to analyze the phagocytosis rate and oxidative burst within phagocytic granulocytes (A) and bacterial survival (B). Serum was left without inhibitor (ctr), treated with VCP (100 μg/ml) for complement inactivation, and/or treated with the NADPH oxidase inhibitor apocynin (1.5 mM). The phagocytosis rate and oxidative burst response of granulocytes were measured by flow cytometry and are depicted as percentages of S. suis FR-positive and Rho123⁺ cells of all granulocytes, respectively. Bars and error bars represent means and standard deviations, and significant differences are indicated. Statistical significances were calculated using the Kruskal-Wallis test with Dunn’s multiple-comparison test (n = 6) (marked with brackets) and the Wilcoxon test for each group of samples versus the Ide_Ssuis-treated equivalent (without brackets) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). n.d., not determined.