Upregulation of TLR4-Dependent ATP Production Is Critical for Glaesserella parasuis LPS-Mediated Inflammation

Abstract

Glaesserella parasuis (G. parasuis), an important pathogenic bacterium, cause Glässer's disease, and has resulted in tremendous economic losses to the global swine industry. G. parasuis infection causes typical acute systemic inflammation. However, the molecular details of how the host modulates the acute inflammatory response induced by G. parasuis are largely unknown. In this study, we found that G. parasuis LZ and LPS both enhanced the mortality of PAM cells, and at the same time, the level of ATP was enhanced. LPS treatment significantly increased the expressions of IL-1β, P2X7R, NLRP3, NF-κB, p-NF-κB, and GSDMD, leading to pyroptosis. Furthermore, these proteins' expression was enhanced following extracellular ATP further stimulation. When reduced the production of P2X7R, NF-κB-NLRP3-GSDMS inflammasome signaling pathway was inhibited, and the mortality of cells was reduced. MCC950 treatment repressed the formation of inflammasome and reduced mortality. Further exploration found that the knockdown of TLR4 significantly reduced ATP content and cell mortality, and inhibited the expression of p-NF-κB and NLRP3. These findings suggested upregulation of TLR4-dependent ATP production is critical for G. parasuis LPS-mediated inflammation, provided new insights into the molecular pathways underlying the inflammatory response induced by G. parasuis, and offered a fresh perspective on therapeutic strategies.

Keywords: ATP; G. parasuis LPS; P2X7R; TLR4; acute inflammatory response; inflammation; pharmacological target.

Figure 1

G. parasuis LPS enhanced the mortality and the ATP level of PAM cells. (A,B) Quantification of mortality. PAM cells were treated with G. parasuis LZ (A) and 50 μg/mL LPS (B) for 8 h, and cell viability was measured by CCK-8. (C,D) Representative images of cell proliferation were determined by EdU cell proliferation assay, and quantification of EdU⁺ cell. n = 10. (E,F) ATP levels in PAM cells. After PAM cells were affected with G. parasuis at MOI = 10 for 8 h, PAM cells were lysed and the cell lysates were analyzed for ATP levels. Data represent mean ± SEM, n = 3, ** p < 0.01.

Figure 2

ATP induces inflammation and increases P2X7 expression. (A,B) ELISA analysis of IL-1β normalized to the control. PAM cells were treated with G. parasuis LZ (A) /50 μg/mL LPS (B) in the presence and absence of ATP, apyrase, and nigericin. (C) mRNA expression measured by qRT-PCR for IL-1β level normalized to the control. (D) Western blot analysis of P2X7R, NLRP3, NF-κB, p-NF-κB, and GSDMD expression in PAM cells. Cells were treated with or without LPS in the presence and absence of nigericin. All proteins were normalized to the level of β-actin. Data represent mean ± SEM, n = 3, * p < 0.05, ** p < 0.01.

Figure 3

A740003 regulates P2X7 function and inhibits inflammation. (A) Western blot analysis of P2X7R, NLRP3 expression in PAM cells. All proteins were normalized to the level of β-actin. Cells were treated with or without 50 μg/mL LPS in the presence and absence of A-740003 (10 μM) (B) Western blot analysis of NF-κB, p- NF-κB, and GSDMD expression in PAM cells. All proteins were normalized to the level of β-actin. (C) ELISA analysis of IL-1β normalized to the control. (D) Quantification of mortality. After PAM cells were treated, cell viability was measured by CCK-8. (E) Representative images of immunofluorescence staining. Differentiated PAM cells were treated with or without LPS in the presence and absence of 0.1 μM A-740003. Nuclei were stained by DAPI in Blue and NF-kB p65 were stained in green, then observed using an inverted fluorescence microscope, 100×. Data represent mean ± SEM, n = 3, * p < 0.05, ** p < 0.01.

Figure 4

MCC950 reduced NLRP3 expression and inhibited inflammation. (A) Western blot analysis of NLRP3, IL-1β p17, and cleaved caspase1 expression in PAM cells. PAM cells were treated with 50 μg/mL LPS in the presence of a different concentration of MCC950 (0, 0.01, 0.1, 1 μM). All proteins were normalized to the level of β-actin. (B) Western blot analysis of GSDMD expression in PAM cells. PAM cells were treated with LPS in the presence of 0.1 μM MCC950. (C) ELISA analysis of IL-1β normalized to the control. (D) Quantification of mortality. After PAM cells being treated, cell viability was measured by CCK-8. Data represent mean ± SEM, n = 3, ** p < 0.01.

Figure 5

Inhibition of TLR4 reduced inflammation and increased cell viability. (A) mRNA expression measured by qRT-PCR for Tlr4 level normalized to the negative control. (B) ATP levels in PAM cells. After cell transfection, and cells were treated with LPS. PAM cells were lysed and the cell lysates were analyzed for ATP levels. (C) Quantification of mortality. After cell transfection, all PAM cells were treated with 50 μg/mL LPS, and test the cell survival rate by CCK8. (D) Representative Western blot analysis of NLRP3 andIL-1β p17 were normalized based on the internal control β-actin. After cell transfection, all PAM cells were treated with LPS, and tested the expression of NLRP3 and IL-1β p17. Data represent mean ± SEM, n = 3, ** p < 0.01.