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Role of Vimentin in Modulating Immune Cell Apoptosis And www.nature.com/scientificreports OPEN Role of vimentin in modulating immune cell apoptosis and infammatory responses in sepsis Received: 21 March 2018 Longxiang Su1, Pan Pan2, Peng Yan3, Yun Long1, Xiang Zhou1, Xiaoting Wang1, Ruo Zhou4, Accepted: 27 March 2019 Bo Wen4, Lixin Xie3 & Dawei Liu1 Published: xx xx xxxx New diagnostic biomarkers or therapeutic targets for sepsis have substantial signifcance for critical care medicine. In this study, 192 diferentially expressed proteins were selected through iTRAQ. Based on cluster analysis of protein expression dynamics and protein-protein interactions, hemopexin, vimentin, and heat shock protein 90 were selected for further investigation. It was demonstrated that serum vimentin (VIM) levels were signifcantly increased in patients with sepsis and septic shock compared to controls and that VIM expression was signifcantly increased in lymphocytes isolated from septic shock and sepsis patients compared to controls. Moreover, a nonsurvivor group had higher serum VIM levels and VIM expression in lymphocytes. Caspase-3 was signifcantly upregulated in Jurkat T cells lacking VIM and when exposed to LPS compared to control cells. In contrast, caspase-3 was reduced nearly 40% in cells over-expressing VIM. IL-2, IL-10 and IFN-α levels were signifcantly decreased in cells lacking VIM compared to control cells, whereas they were not signifcantly altered in cells over- expressing VIM. These fndings suggest that VIM modulates lymphocyte apoptosis and infammatory responses and that VIM could be a new target for the diagnosis and prognostic prediction of patients with sepsis or septic shock. Sepsis refers to the presence of a serious infection in combination with organ dysfunction1. Due to sepsis’s sig- nifcant clinical heterogeneity and nonspecifc characteristics, timely and accurate diagnosis of sepsis—as well as prognosis of disease severity—is clinically difcult. Although signifcant advances in the clinical treatment of sep- sis have been made, the mortality associated with sepsis remains high2. It has been reported that nearly 250,000 cases of sepsis result in death in the USA annually3, equivalent to approximately 59.6 deaths per 100,000 individ- uals4. In China, it has been reported that nearly 37 of every 100 ICU admissions were for severe sepsis or septic shock, and the overall ICU and hospital mortality rates were 28.7% and 33.5%, respectively5. Te development of sepsis is an extremely complex and rapid pathophysiological process involving infammation/anti-infammation cascades, humoral/cellular immunity, and hemodynamic abnormalities6. Once a diagnosis of sepsis is made, and standard treatment initiated, it is ofen difcult to diferentiate between patients more likely to achieve positive, rather than negative, outcomes. Biomarkers have the potential to guide clinicians to extend treatment beyond standard regiments and to predict patient outcomes. Two types of biomark- ers can be identifed in clinical practice: diagnostic or prognostic biomarkers used independent of therapy and biomarkers used as an adjunct to guide treatment7. Regardless of classifcation, the demand for new and accurate biomarkers for sepsis or septic shock is high. In 2010, Pierrakos and Vincent estimated that at least 178 diferent sepsis biomarkers have been reported in the scholarly literature8; 4 years later, Sandquist and Wong reported that soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), IL-27, soluble urokinase-type plasminogen activator receptor (suPAR), neutrophil CD64, presepsin, cell-free DNA (cfDNA), and certain miRNAs could be potential biomarkers for the diagnosis, prognosis and treatment of sepsis9. 1Department of Critical Care Medicine, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, 1 Shuaifuyuan, Dongcheng District, Beijing, 100730, China. 2Department of Critical Care Medicine, Beijing Tiantan Hospital, Capital Medical University, Tiantan Xili the 6th, Dongcheng District, Beijing, 100050, China. 3Department of Respiratory and Critical Care Medicine, Chinese PLA General Hospital, 28th Fuxing Rd, Haidian District, Beijing, 100853, China. 4Shenzhen Proteome Engineering Laboratory, BGI Shenzhen, Shenzhen, 518083, Guangdong Province, China. Longxiang Su and Pan Pan contributed equally. Correspondence and requests for materials should be addressed to L.X. (email: [email protected]) or D.L. (email: dwliu98@163. com) SCIENTIFIC REPORTS | (2019) 9:5747 | https://doi.org/10.1038/s41598-019-42287-7 1 www.nature.com/scientificreports/ www.nature.com/scientificreports With recent developments and increased use of basic biotechnological methods, including genomics, tran- scriptomics, proteomics and metabolomics platform technologies in biomedical and clinical research, newly discovered potential biomarkers for sepsis diagnosis and prognosis have increased. In this context, we have focused on the application of clinical proteomics and metabolomics for the diagnosis and prognosis of sepsis. Specifcally, we have assessed various urinary biomarkers at diferent stages of sepsis development and identi- fed lysosome-associated membrane protein-1 (LAMP-1) as being closely related to a poorer prognosis, which revealed the important role of autophagy during the progression of sepsis10–12. Additionally, we have also previ- ously reported the important role of sulfur-containing amino acids and vitamin D in sepsis11,13. We have further performed proteomics analysis of blood samples collected from patients with sepsis or septic shock, and we found that serum vimentin levels were signifcantly increased in these patients. Vimentin is an intermediate flament expressed in variety of cells, including lymphocytes and neutrophils. Recent studies have suggested that vimentin plays a role in many cell and tissue functions, including apoptosis of neutrophils and lymphocytes14–18. Since apoptosis of innate immune cells—particularly lymphocyte apoptosis— has been recognized as an important step in the pathogenesis of sepsis, and as vimentin plays a role in lympho- cyte apoptosis, we hypothesized that disruption of vimentin in lymphocytes results in cell death and that soluble vimentin released by apoptotic cells into blood circulation could serve as a potential biomarker for the prognosis of sepsis. To test this hypothesis, the current study was carried out in the following three stages: screening target proteins from patient blood samples and selecting vimentin as a target protein, validation of the vimentin in the clinical setting of sepsis, and in vitro experimental studies to explore the role of vimentin in regulating apoptosis of lymphocytes in response to endotoxin exposure. Materials and Methods Patients. Patients who were admitted to the Respiratory Intensive Care Unit (ICU), Surgical ICU, and Emergency ICU at Chinese People’s Liberation Army (PLA) General Hospital from May 2010 to Oct 2011 were enrolled in the current study. Systemic Infammatory Response Syndrome (SIRS) and sepsis were diagnosed based on the 1991 ACCP/SCCM Sepsis Directory19 and the revised criteria in the 2001 and 2008 updates20,21. Patients with two or more of the following signs were eligible for SIRS diagnosis: (1) a temperature >38 °C or <36 °C, (2) a pulse rate >90 beats/min, (3) a respiratory rate >20 breaths/min or hyperventilation with a partial −1 pressure of arterial carbon dioxide (PaCO2) <32 mmHg, or (4) a white blood cell (WBC) count >12,000 µL or <4,000 µL−1, or >10% immature cells. Severe sepsis was defned as sepsis combined with organ dysfunction. Once a vasoactive drug had to be used afer fuid resuscitation, the case was defned as septic shock. In this study, 100 participants were randomly enrolled and assigned to a SIRS (n = 20), sepsis (n = 20), severe sepsis (consisting of severe sepsis, n = 10; and septic shock, n = 10), or death groups (patients with septic shock who died during treatment, n = 20). For the control group, 20 healthy volunteers were enrolled. Te study protocol of this stage was approved by the Ethics Committee of the Chinese PLA General Hospital (project No. 20111013-007) and was registered with the Clinical Trials Register (NCT01493466). Te clinical validation stage utilized a prospective study conducted from July 2016 to December 2016 in the Department of Critical Care Medicine, Peking Union Medical College Hospital. Sepsis 3.0 was adopted to select patients for validation of potential proteins1. Sepsis was then defned as life-threatening organ dysfunction (an acute change in total SOFA score ≥2 points) caused by a dysregulated host response to infection. Patients with septic shock were identifed based on a clinical presentation of sepsis with persistent hypotension requiring vas- opressors to maintain a MAP ≥65 mmHg and a serum lactate level >2 mmol/L despite adequate volume resus- citation. A total of 31 patients were randomly selected and enrolled in this stage of study, and these patients were divided into sepsis (n = 12) and septic shock (n = 19) groups. On day 28, these patients were further classifed as survivors (n = 21) and nonsurvivors (n = 10). Additionally, 10 surgical postoperative, noninfected patients were assigned to the control group. Te study protocol of this stage was approved by the Ethics Committee of the Peking Union Medical College Hospital (project No. ZS1080) and was registered with the Clinical Trials Register (NCT 03253146). Patients were excluded if they were (1) younger than 18 years; (2) had acquired immunodefciency
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