Hypercapnia promotes NLRP3 inflammasome activation in microglia by activating P2X7R after lipopolysaccharide-induced activation of the TLR4/NF-κB signaling pathway

Abstract

Background

Sepsis is an uncontrolled inflammatory response to infection and is closely associated with the occurrence of acute respiratory distress syndrome (ARDS). Low tidal volume lung ventilation and permissive hypercapnia is a recognized therapy for ARDS. However, whether permissive hypercapnia aggravates sepsis-associated encephalopathy (SAE) remains unclear. The present study investigated whether hypercapnia contributed to the development of SAE through the purinergic 2X7 receptor (P2X7R) by activating the Nod-like receptor protein 3 (NLRP3) inflammasome in sepsis.

Methods

The SAE model was established by intracranial injection of lipopolysaccharide (LPS) (1 μg/ml, 5 μl) in C57BL/6 mice. Hypercapnia was induced by mechanical ventilation with a high concentration of CO₂ (5 % CO₂, 21 % O₂ and 74 % N₂) in vivo. Toll-like receptor 4 (TLR4) and P2X7R knockout (KO) mice were employed in the study, while in vitro, BV2 microglial cells were treated with LPS or a high concentration of CO₂ (15 % CO₂ + 20 % O₂). Immunofluorescence and western blot analysis were used to assess the expression levels of TLR4, NF-κB, phosphorylated (p)-NF-κB, P2X7R, pro-caspase-1, caspase-1, pro-IL-1β, IL-1β, pro-IL-18 and IL-18. ATP levels in the cell culture medium were detected by fluorometric assay.

Result

The results revealed that, compared with the sham group, the expression levels of TLR4, p-NF-κB, pro-IL-1β, pro-IL-18 and NLRP3 were significantly upregulated in the LPS and LPS + hypercapnia groups, but not in the hypercapnia group. Although the expression levels of caspase-1, IL-1β and IL-18 were increased slightly in the LPS group, their upregulation was more pronounced in the LPS + hypercapnia group, and it was suppressed when TLR4 was knocked out. Furthermore, P2X7R expression and ATP levels in the cell culture medium remained unchanged in the LPS group compared with the sham group but were remarkably increased both in the hypercapnia and LPS + hypercapnia groups. Additionally, P2X7R KO restrained the caspase-1, IL-1β and IL-18 increased induced by LPS injected intracranially and hypercapnia.

Conclusions

In conclusion, LPS induced the priming step of NLRP3 inflammasome activation, but had little effect on the activation step, while hypercapnia played an important role in the activation step through P2X7R, depending on the priming step stimulated by LPS.