Protective effects of rapamycin induced autophagy on CLP septic mice
Introduction
According to the latest guidelines, sepsis is defined as life-threatening organ dysfunction caused by an uncontrolled host immune response to infection [1]. Although a lot of clinical trials have been conducted, the mortality rate of septic patients remains high even in developed countries [2]. It is generally accepted that there is a cytokine storm in acute sepsis. Unbalance of anti-inflammatory and pro-inflammatory reaction is the main pathogenic mechanism of sepsis [3]. Usually, the lung is the first organ damaged by sepsis, and a large number of patients die from acute respiratory distress syndrome (ARDS) in the early stage of sepsis. Recent study has confirmed that intestinal bacteria invaded into the lung during early stage of sepsis causing a disorder of the lung microbiome, mainly characterized by enrichment with the proteobacteria [4,5]. Translocation of proteobacteria in lung was significantly associated with elevated alveolar concentrations of TNF-α, which is an important mediator of pulmonary inflammation in ARDS [6].
Autophagy is a self-protection mechanism responsible for removing misfolded proteins, resisting oxidative stress and degrading microbial pathogens using lysosomal processes [7,8]. Autophagy has been suggested to be involved in the regulation of inflammatory reaction [9,10]. The mechanisms that autophagy can inhibit inflammation, is that autophagy can decrease the cell apoptosis and its secondary necrosis so that inflammation will be constrained [[11], [12], [13], [14]]. Alternatively, autophagy negatively regulates inflammasome activation and inhibits the inflammation caused by pyroptosis [15,16]. Studies also found that the deficiency of autophagy-related protein in macrophages can lead to a high expression of IL-1β and IL-18 after stimulated by lipopolysaccharide (LPS) [10]. Therefore, it is of interesting to study whether autophagy is a valuable target for regulation of acute immunoreaction in early stage of sepsis. Here, mTORC1 inhibitor, rapamycin, is used in our study to activate cell autophagy. Rapamycin is a powerful anticancer agent. Studies have demonstrated that rapamycin protects septic mice through different mechanisms, such as by inhibition of Th17 cell proliferation, blocking the NF-κB pathway, promoting the expansion of Treg cells, and inhibiting the production of inflammatory mediators [[17], [18], [19], [20]]. In the present study, we focused on the effect of rapamycin on regulation of the balance of autophagy and inflammasome during the acute stage of sepsis.
Section snippets
Animals
Male C57BL/6 mice (10 weeks old, SLAC, Shanghai, China) were maintained at animal experimental center of Shanghai Jiaotong University under 12-h day and night cycle with free access to food and water at least 1 week before experiments. All experimental procedures were carried out according to the guidelines of Institutional Animal Care and Use Committee of Shanghai (A2015035).
Cell culture and stimulation
Peritoneal macrophages were obtained by peritoneal lavage in C57BL/6 mice, then approximately 1 × 105 cells/well were
Rapamycin treatment inhibited pyroptosis of macrophages and reduced the release of inflammatory cytokines
Pyroptosis is programmed cell death that can induce very deleterious inflammatory response. We are interested to investigate whether autophagy can inhibit pyroptosis of peritoneal macrophages caused by enteric bacteria infection. LDH release assay showed that cell death was significantly lower in the rapamycin-treated group compared with the control group (Fig. 1A). Protective effect of rapamycin on pyroptosis is dose dependent. Cell death in control group was 30.7 ± 3.03%, while the group
Discussion
There is a storm of inflammatory factors in sepsis, the strong inflammatory reaction in the early stage leads to a long-term immune exhaustion and inhibition state in the late stage, which breaks the balance between the promotion and depression of inflammatory response. ARDS is the leading cause of death in patients with sepsis. Therefore, the control of inflammatory response in lungs during the early stage of sepsis is vital for the treatment of sepsis. Autophagy is an intracellular process of
Conclusion
In summary, our study demonstrated activation of autophagy by rapamycin treatment decreases inflammsome activation and pyroptosis in lung. Treatment either pre- or post CLP sepsis, rapamycin protects mice from septic death significantly. Therefore, regulation of autophagy is a valuable target for effective therapy of sepsis in acute inflammatory phase.
Conflict of interest
The authors declare that they have no conflict of interest.
CRediT authorship contribution statement
Zhenxia Wang: Formal analysis, Validation. Yan Li: Conceptualization, Writing - review & editing. Xiaowei Yang: Formal analysis, Methodology. Lian Zhang: Formal analysis, Methodology. Huiming Shen: Funding acquisition, Resources. Weihong Xu: Funding acquisition, Resources. Congli Yuan: Investigation, Project administration, Supervision.
Acknowledgments
This work was supported by grant of Shanghai Science of Technology Commission (18490712700), SJTU translational medicine cross fund (ZH2018ZDA37), Science and Technology Commission of Sichuan Province (2017JZ0010), the Med-X (YG2016MS69). Open foundation of State Key Laboratory of Dairy Biotechnology (SKLDB2017-005). The authors thank Changcan Li for proofreading the article.
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2020, International Review of Cell and Molecular BiologyCitation Excerpt :Increased autophagic flux upon host cell infection can selectively degrade invading bacteria to prevent cell death altogether (Amer and Swanson, 2005; Gutierrez et al., 2004; Nakagawa et al., 2004). Consistent with this, several reports have concluded that activation of autophagy can prevent pyroptosis (Li et al., 2019; Tu et al., 2019; Wang et al., 2019; Zi et al., 2019) or that the inhibition of autophagy can enhance pyroptotic cell death (Qiu et al., 2018; Yu et al., 2019). It is important to note that in order to prevent pyroptosis, the autophagosome often targets large protein complexes or organelles for degradation, not just individual signaling molecules, and thus creates a rather different feature of pyroptosis regulation by autophagy compared to ferroptosis and necroptosis.
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