Limits...
Toll-like receptor 4 confers inflammatory response to Suilysin.

Bi L, Pian Y, Chen S, Ren Z, Liu P, Lv Q, Zheng Y, Zhang S, Hao H, Yuan Y, Jiang Y - Front Microbiol (2015)

Bottom Line: In this study, we found that suilysin (SLY) is the main protein inflammatory stimulus of SS2 and that native SLY (nSLY) stimulated cytokines independently of its haemolytic ability.We demonstrated for the first time that SLY stimulates immune cells through TLR4.In addition, the Myd88 adaptor-p38-MAPK pathway was involved in this process.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Beijing, China ; Beijing Key Laboratory of Immunology Regulatory and Organ Transplantation, Basic Research Lab of Organ Transplant Institute, 309th Hospital of the People's Liberation Army Beijing, China.

ABSTRACT
Streptococcus suis serotype 2 (SS2) is an emerging human pathogen worldwide. A large outbreak occurred in the summer of 2005 in China. Serum samples from this outbreak revealed that levels of the main proinflammatory cytokines were significantly higher in patients with streptococcal toxic-shock-like syndrome (STSLS) than in patients with meningitis only. However, the mechanism underlying the cytokine storm in STSLS caused by SS2 remained unclear. In this study, we found that suilysin (SLY) is the main protein inflammatory stimulus of SS2 and that native SLY (nSLY) stimulated cytokines independently of its haemolytic ability. Interestingly, a small amount of SLY (Å Mol/L) induced strong, long-term TNF-α release from human PBMCs. We also found that nSLY stimulated TNF-α in wild-type macrophages but not in macrophages from mice that carried a spontaneous mutation in TLR4 (P712H). We demonstrated for the first time that SLY stimulates immune cells through TLR4. In addition, the Myd88 adaptor-p38-MAPK pathway was involved in this process. The present study suggested that the TLR4-dependent inflammatory responses induced by SLY in host might contribute to the STSLS caused by SS2 and that p38-MAPK could be used as a target to control the release of excess TNF-α induced by SS2.

No MeSH data available.


Related in: MedlinePlus

SLY induces strong, long-term TNF-α release from human PBMCs. (A) Heated, inactivated nSLY could not induce TNF-α release from PBMCs. The nSLY was heated at 100°C for 30 min and then incubated with PBMCs at a concentration of 100 ng/ml. Culture supernatants were harvested and assayed for TNF-α production by ELISA. Each symbol represents the level of TNF-α (pg/ml) released from the PBMCs from one donor in one independent experiment. Horizontal lines indicate the mean for each group, and the data in were analyzed by Student's unpaired t-test. Dose (B) and time-course (C) of production of TNF-α in PBMCs from different donors treated with nSLY (100 ng/ml). Dose response (D) of TNF-α production in THP-1 macrophage cells treated with nSLY or LPS. Culture supernatants were harvested and assayed for cytokine production by ELISA. Data are expressed as the mean ± SD for three independent experiments. nSLY, native SLY; nSLY-heat, the native SLY was heated at 100°C for 30 min. *P < 0.05; **P < 0.01; ***P < 0.001. The comparisons between test groups and the control in (D) were analyzed by a One-Way ANOVA test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4481166&req=5

Figure 2: SLY induces strong, long-term TNF-α release from human PBMCs. (A) Heated, inactivated nSLY could not induce TNF-α release from PBMCs. The nSLY was heated at 100°C for 30 min and then incubated with PBMCs at a concentration of 100 ng/ml. Culture supernatants were harvested and assayed for TNF-α production by ELISA. Each symbol represents the level of TNF-α (pg/ml) released from the PBMCs from one donor in one independent experiment. Horizontal lines indicate the mean for each group, and the data in were analyzed by Student's unpaired t-test. Dose (B) and time-course (C) of production of TNF-α in PBMCs from different donors treated with nSLY (100 ng/ml). Dose response (D) of TNF-α production in THP-1 macrophage cells treated with nSLY or LPS. Culture supernatants were harvested and assayed for cytokine production by ELISA. Data are expressed as the mean ± SD for three independent experiments. nSLY, native SLY; nSLY-heat, the native SLY was heated at 100°C for 30 min. *P < 0.05; **P < 0.01; ***P < 0.001. The comparisons between test groups and the control in (D) were analyzed by a One-Way ANOVA test.

Mentions: Unlike most other cytokines, TNF-α administered in vivo produces dramatic physiological changes and large doses of TNF-α can result in tissue damage and metabolic changes that clearly mimic endotoxic shock (Tracey et al., 1987; Hesse et al., 1988; Whicher and Evans, 1990). TNF-α was chosen as the main marker of inflammatory activity in the present study. We purified the nSLY from the supernatant of a bacterial culture as in our previous study (Ren et al., 2012) and detected its ability to induce the TNF-α release from immune cells. To exclude the possibility that the observed inflammatory activity of nSLY is attributable to contaminating LPS or other endotoxins, removal of endotoxin from the proteins was carried out. Subsequently, the final endotoxin contamination for nSLY, rSLY, and SLYP353V used in this study is less than 0.03 EU/ml, a concentration below that which is known to cause cell activation (Martin and Dorf, 1991). Moreover, we used heat to abolish the activity of nSLY to induce TNF-α release from PBMCs. As shown in Figure 2A, the inflammatory response was lost when nSLY was heated (at 100°C for 30 min), indicating that this activity of nSLY was due to the protein, not to endotoxin contamination.


Toll-like receptor 4 confers inflammatory response to Suilysin.

Bi L, Pian Y, Chen S, Ren Z, Liu P, Lv Q, Zheng Y, Zhang S, Hao H, Yuan Y, Jiang Y - Front Microbiol (2015)

SLY induces strong, long-term TNF-α release from human PBMCs. (A) Heated, inactivated nSLY could not induce TNF-α release from PBMCs. The nSLY was heated at 100°C for 30 min and then incubated with PBMCs at a concentration of 100 ng/ml. Culture supernatants were harvested and assayed for TNF-α production by ELISA. Each symbol represents the level of TNF-α (pg/ml) released from the PBMCs from one donor in one independent experiment. Horizontal lines indicate the mean for each group, and the data in were analyzed by Student's unpaired t-test. Dose (B) and time-course (C) of production of TNF-α in PBMCs from different donors treated with nSLY (100 ng/ml). Dose response (D) of TNF-α production in THP-1 macrophage cells treated with nSLY or LPS. Culture supernatants were harvested and assayed for cytokine production by ELISA. Data are expressed as the mean ± SD for three independent experiments. nSLY, native SLY; nSLY-heat, the native SLY was heated at 100°C for 30 min. *P < 0.05; **P < 0.01; ***P < 0.001. The comparisons between test groups and the control in (D) were analyzed by a One-Way ANOVA test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481166&req=5

Figure 2: SLY induces strong, long-term TNF-α release from human PBMCs. (A) Heated, inactivated nSLY could not induce TNF-α release from PBMCs. The nSLY was heated at 100°C for 30 min and then incubated with PBMCs at a concentration of 100 ng/ml. Culture supernatants were harvested and assayed for TNF-α production by ELISA. Each symbol represents the level of TNF-α (pg/ml) released from the PBMCs from one donor in one independent experiment. Horizontal lines indicate the mean for each group, and the data in were analyzed by Student's unpaired t-test. Dose (B) and time-course (C) of production of TNF-α in PBMCs from different donors treated with nSLY (100 ng/ml). Dose response (D) of TNF-α production in THP-1 macrophage cells treated with nSLY or LPS. Culture supernatants were harvested and assayed for cytokine production by ELISA. Data are expressed as the mean ± SD for three independent experiments. nSLY, native SLY; nSLY-heat, the native SLY was heated at 100°C for 30 min. *P < 0.05; **P < 0.01; ***P < 0.001. The comparisons between test groups and the control in (D) were analyzed by a One-Way ANOVA test.
Mentions: Unlike most other cytokines, TNF-α administered in vivo produces dramatic physiological changes and large doses of TNF-α can result in tissue damage and metabolic changes that clearly mimic endotoxic shock (Tracey et al., 1987; Hesse et al., 1988; Whicher and Evans, 1990). TNF-α was chosen as the main marker of inflammatory activity in the present study. We purified the nSLY from the supernatant of a bacterial culture as in our previous study (Ren et al., 2012) and detected its ability to induce the TNF-α release from immune cells. To exclude the possibility that the observed inflammatory activity of nSLY is attributable to contaminating LPS or other endotoxins, removal of endotoxin from the proteins was carried out. Subsequently, the final endotoxin contamination for nSLY, rSLY, and SLYP353V used in this study is less than 0.03 EU/ml, a concentration below that which is known to cause cell activation (Martin and Dorf, 1991). Moreover, we used heat to abolish the activity of nSLY to induce TNF-α release from PBMCs. As shown in Figure 2A, the inflammatory response was lost when nSLY was heated (at 100°C for 30 min), indicating that this activity of nSLY was due to the protein, not to endotoxin contamination.

Bottom Line: In this study, we found that suilysin (SLY) is the main protein inflammatory stimulus of SS2 and that native SLY (nSLY) stimulated cytokines independently of its haemolytic ability.We demonstrated for the first time that SLY stimulates immune cells through TLR4.In addition, the Myd88 adaptor-p38-MAPK pathway was involved in this process.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Beijing, China ; Beijing Key Laboratory of Immunology Regulatory and Organ Transplantation, Basic Research Lab of Organ Transplant Institute, 309th Hospital of the People's Liberation Army Beijing, China.

ABSTRACT
Streptococcus suis serotype 2 (SS2) is an emerging human pathogen worldwide. A large outbreak occurred in the summer of 2005 in China. Serum samples from this outbreak revealed that levels of the main proinflammatory cytokines were significantly higher in patients with streptococcal toxic-shock-like syndrome (STSLS) than in patients with meningitis only. However, the mechanism underlying the cytokine storm in STSLS caused by SS2 remained unclear. In this study, we found that suilysin (SLY) is the main protein inflammatory stimulus of SS2 and that native SLY (nSLY) stimulated cytokines independently of its haemolytic ability. Interestingly, a small amount of SLY (Å Mol/L) induced strong, long-term TNF-α release from human PBMCs. We also found that nSLY stimulated TNF-α in wild-type macrophages but not in macrophages from mice that carried a spontaneous mutation in TLR4 (P712H). We demonstrated for the first time that SLY stimulates immune cells through TLR4. In addition, the Myd88 adaptor-p38-MAPK pathway was involved in this process. The present study suggested that the TLR4-dependent inflammatory responses induced by SLY in host might contribute to the STSLS caused by SS2 and that p38-MAPK could be used as a target to control the release of excess TNF-α induced by SS2.

No MeSH data available.


Related in: MedlinePlus