Calcineurin/NFAT1/Smad2 signaling regulates microglial autophagy that contributes to neuroinflammation and cognitive deficit in mice with sepsis-associated encephalopathy.

OBJECTIVE: Sepsis-induced encephalopathy is a critical determinant of mortality, driven by microglial activation and excessive autophagy. However, the underlying mechanisms remain unclear.

METHODS: Sepsis was induced in wild-type and nuclear factor of activated T cells (NFAT) 1-deficient mice via cecal ligation and puncture. Hippocampal morphology, microglial autophagy, polarization, and inflammatory cytokine expressions were analyzed. Cognitive function was evaluated using the Morris water maze and fear conditioning tests. In vitro, BV2 microglia were stimulated with lipopolysaccharide (LPS), followed by genetic manipulation of calcineurin, NFAT1 or Smad2 to investigate underlying mechanisms.

RESULTS: In wild-type mice, sepsis induced microglial autophagy, M1 polarization and neuroinflammation, resulting in cognitive impairment. These changes were accompanied by upregulated NFAT1 and elevated phosphorylation of Smad2 in hippocampal microglia. Notably, the sepsis-induced effects were attenuated by either pharmacological inhibition of autophagy (using 3-methyladenine) or genetic NFAT1 deficiency. Smad2 overexpression in NFAT1-deficient mice reversed sepsis-induced pathological phenotype, suggesting a functional dependency on Smad2 downstream of NFAT1. Corroborating the in vivo findings, in vitro experiments demonstrated that knockdown of calcineurin, NFAT1 or Smad2 suppressed LPS-induced autophagy and inflammatory responses in microglial cells. Furthermore, Smad2 overexpression rescued the effects of NFAT1 knockdown on LPS-exposure cells.

CONCLUSION: The calcineurin/NFAT1 pathway may interact with Smad2 signaling promotes microglial autophagy during sepsis, exacerbating neuroinflammation and cognitive impairment. These findings support targeting this pathway for treating or even preventing sepsis-induced encephalopathy.