The protective role of YTHDF1-knock down macrophages on the immune paralysis of severe sepsis rats with ECMO

Abstract

Objective: To examine the role of YTHDF1 knock-down macrophages on the immunity of severe sepsis rats with ECMO.

Methods: 15 SD rats were randomly allocated into 3 groups: mild sepsis (I), severe sepsis with ECMO (II), and YTHDF1 knock-down macrophages treatment groups (III). Blood biochemical indexes, different immune factors and brain changes were detected by RT-PCR, ELISA, ELISPOT and HE staining. Isolated macrophages subtypes and signal proteins were detected by flow cytometry, western blot and m6A RNA methylation test.

Results: The levels of HMGB1, RAGE, YTHDF1 and IL-17 in peripheral blood were significantly higher (p < 0.01), while the level of CXCL9 and TNF-α, and LPS-specific CD8⁺CTL function were significantly decreased in group II compared with group I (p < 0.01). The ratio of CD63⁺ macrophages (p < 0.05) and CD64⁺ macrophages (p< 0.05) decreased and the level of elastase (p < 0.01) and CCR2^highCX3CR1^low/CCR2^lowCX3CR1^high (p < 0.01) of macrophages increased in group II. The above were consistent with the severity of biochemical indicators, the increasing endothelial injury factor (Ang2/Ang1), lower endothelial protective factor (sTie2), severer brain injury in group II. After YTHDF1 knock-down macrophages treatment, the above indexes' changes were opposite when Group III versus Group II through the down-regulation of m6A RNA methylation of JAK2/STAT3 (p < 0.01) and protein expression of ^PJAK2/^PSTAT3 (p < 0.05) in isolated macrophages.

Conclusions: YTHDF1 knock-down macrophages improved the immune paralysis of macrophages, Th1/Th17 and CTL and reduced the entry of macrophages into the brain to cause endothelial damage of severe sepsis rats with ECMO through the inhibition of HMGB1/RAGE and YTHDF1, m6A RNA methylation of JAK2/STAT3 and ^PJAK2/^PSTAT3 proteins expression in macrophages.

Keywords: Endothelial injury; Immunity; Macrophages; Severe sepsis with ECMO; YTHDF1 knock-down.