N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in eukaryotic mRNAs. Recent studies have demonstrated that m6A methylation influences all aspects of mRNA metabolism, including mRNA processing, translation and decay. Particularly, research evidence suggests that m6A methylation marks mRNAs for degradation; one important functions of m6A methylation is to sort mRNA transcripts into a fast track for translation and decay. Fast translation followed by fast decay can generate a translation ?pulse? to satisfy the need for a burst of protein synthesis in response to a pathophysiological stress. This theory, however, remains to be tested in vivo. Sepsis is a major clinical problem and leading cause of death in patients within intensive care units. Sepsis is usually caused by Gram-negative bacterial infection that triggers a fast cytokine storm from the host immune response. Cytokines are critical to eliminate the infection, but excessive production can cause tissue and organ damage to the host. Therefore, a proper resolution of the cytokine response is critical for the host's wellbeing. Macrophages are the most predominant producer of cytokines and chemokines during infection. We hypothesize that macrophage pro-inflammatory cytokines are ?fast track? genes whose mRNAs are m6A methylated during infection and m6A methylation is crucial for the resolution of the cytokine storm. METTL14 is a key component of the m6A methyltransferase. Our preliminary data showed that macrophage METTL14 is induced by lipopolysaccharide (LPS); in LPS-induced sepsis model, mice with myeloid cell-specific deletion of METTL14 produced and maintained much higher levels of serum pro- inflammatory cytokines and suffered much higher mortality compared with control mice. These observations support the hypothesis that impaired m6A methylation delays the decay of pro-inflammatory cytokine mRNAs leading to over-sustained cytokine production. We propose two aims to test the hypothesis.
In Aim 1 we will assess the effect of METTL14 deletion on the expression and stability of pro-inflammatory cytokines and chemokines in macrophages at baseline and under LPS stimulation by studying peritoneal macrophages and bone marrow-derived macrophages isolated from METTL14flox/flox and METTL14flox/flox;LysM- Cre mice.
In Aim 2 we will assess the effect of METTL14 deletion on transcriptome-wide mRNA m6A methylation, expression, synthesis and degradation in macrophages at baseline and under LPS stimulation. We will perform m6A RNA-IP-seq and RNA-seq in macrophages from METTL14flox/flox and METTL14flox/flox;LysM-Cre mice, and identify transcripts that are affected by METTL14 deletion in both m6A methylation and expression in opposite directions. We will also perform s4U-seq experiments in METTL14flox/flox and METTL14flox/flox;LysM-Cre macrophages to assess cytokine and chemokine mRNA synthesis and degradation. This is the first study, to our knowledge, to investigate the role of macrophage m6A methylation in the resolution of cytokine storm in sepsis and will advance our understanding of systemic inflammation.
N6-methyladenosine (m6A) is the most abundant post-transcriptional modification of mRNAs. In vitro studies have demonstrated that m6A modification influences all fundamental aspects of mRNA metabolism, particularly mRNA stability, but its in vivo function in mammalian physiology remains unknown. In this application we will investigate the role of macrophage m6A mRNA modification in the regulation of macrophage inflammatory response and in the resolution of cytokine storm in sepsis.