The Concentration of Large Extracellular Vesicles Differentiates Early Septic Shock From Infection


Posted: 2021-10-04 19:00:00
Front Med (Lausanne) . 2021 Sep 16;8:724371. doi: 10.3389/fmed.2021.724371. eCollection 2021. Affiliations Expand Affiliations 1 Faculty of Medicine, Department of Biomedical Sciences and Biomedical Engineering, Prince of Songkla University, Songkhla, Thailand. 2 Faculty of Medicine, Department of Internal Medicine, Prince of Songkla University, Songkhla, Thailand. 3 Faculty of Medicine, Department of Family and Preventive Medicine, Prince of Songkla University, Songkhla, Thailand. 4 Faculty of Medicine Siriraj Hospital, Department of Research and Development, Mahidol University, Bangkok, Thailand. Item in Clipboard Latthawan Monnamorn et al. Front Med (Lausanne). 2021. Show details Display options Display options Format Front Med (Lausanne) . 2021 Sep 16;8:724371. doi: 10.3389/fmed.2021.724371. eCollection 2021. Affiliations 1 Faculty of Medicine, Department of Biomedical Sciences and Biomedical Engineering, Prince of Songkla University, Songkhla, Thailand. 2 Faculty of Medicine, Department of Internal Medicine, Prince of Songkla University, Songkhla, Thailand. 3 Faculty of Medicine, Department of Family and Preventive Medicine, Prince of Songkla University, Songkhla, Thailand. 4 Faculty of Medicine Siriraj Hospital, Department of Research and Development, Mahidol University, Bangkok, Thailand. Item in Clipboard CiteDisplay options Display options Format Abstract Septic shock represents a subset of sepsis with severe physiological aberrations and a higher mortality rate than sepsis alone. Currently, the laboratory tools which can be used to identify the state of septic shock are limited. In pre-clinical studies, extracellular vesicles (EVs), especially large EVs (lEVs), have been demonstrated a role as functional inflammatory mediators of sepsis. However, its longitudinal trend during the disease course has not been explored. In this study, the quantities and subtypes of plasma-derived lEVs were longitudinally compared between patients with septic shock (n = 21) and non-sepsis infection (n = 9), who presented within 48 h of their symptom onset. Blood specimens were collected for seven consecutive days after hospital admission. lEVs quantification and subtyping were performed using an imaging flow cytometer. The experiments revealed a higher lEVs concentration in septic shock patients than infected patients at the onset of the disease. In septic shock patients, lEVs concentration decreased over time as opposed to infected patients whose lEVs concentration is relatively static throughout the study period. The major contributors of lEVs in both septic shock and infected patients were of non-leukocyte origins; platelets, erythrocytes, and endothelial cells released approximately 40, 25, and 15% of lEVs, respectively. Among lEVs of leukocyte origins, neutrophils produced the highest number of EVs. Nevertheless, the proportion of each subtype of lEVs among the given amount of lEVs produced was similar between septic shock and infected patients. These findings raise the possibility of employing lEVs enumeration as a septic shock identifying tool, although larger studies with a more diverse group of participants are warranted to extrapolate the findings to a general population. Keywords: extracellular vesicles; imaging flow cytometry; microparticles; microvesicles; sepsis; septic shock. Copyright © 2021 Monnamorn, Seree-aphinan, Molika, Vichitkunakorn, Pattanapanyasat, Khwannimit and Navakanitworakul. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figures Figure 1 Imaging flow cytometry analysis for… Figure 1 Imaging flow cytometry analysis for the identification of EVs' subtypes. Image Data Exploration… Figure 1 Imaging flow cytometry analysis for the identification of EVs' subtypes. Image Data Exploration and Analysis Software (IDEAS®) version 6.2 was used for the analysis. Doublets were excluded using “Spot count” on SSC channel (A). This function is an image-based propriety software function that is suitable for counting events that appear as spots (e.g., parasite, phagocytosed particles) rather than a ring of fluorescent cell membrane. Events with spot counts of more than one was categorized as doublets and excluded from the analysis. Among singlets, large EVs were gated based on their intrinsically low SSC intensity (B). Extracellular vesicles, subtyped by their cellular origin, were identified with a double positivity of CD9 and their corresponding markers; CD235 for erythrocytes, CD41A for platelets, CD146 for endothelial cells, CD66b for neutrophils, CD14 for monocytes, CD19 for B lymphocytes, and CD3 for T lymphocytes (C). CD, cluster of differentiation; SSC, side scatter; EV, extracellular vesicles. Figure 2 Plasma concentration and size distribution… Figure 2 Plasma concentration and size distribution of extracellular particles in PFP samples of patients… Figure 2 Plasma concentration and size distribution of extracellular particles in PFP samples of patients with septic shock and infection. PFP samples were collected daily for seven consecutive days after hospital admission for analysis. Nanoparticle tracking analysis was employed to measure the concentration of extracellular particles of varying sizes within PFP samples. The daily concentrations of extracellular particles were presented in boxplot graphs using median and interquartile range (A). The particles varied in sizes, with the majority of them had a diameter of approximately 100 nm (B). Wilcoxon rank-sum tests were performed to compare the concentration of extracellular particles between septic shock and infected patients. *p-value < 0.05 for Wilcoxon rank-sum tests. PFP, platelet-free plasma. Figure 3 Assessing extracellular vesicle isolates by… Figure 3 Assessing extracellular vesicle isolates by western blot and electron microscopy. Using the differential… Figure 3 Assessing extracellular vesicle isolates by western blot and electron microscopy. Using the differential centrifugation technique, EVs were concentrated and separated from PFP samples of septic shock and infected patients. The isolates were examined with western blot using the standard SDS-PAGE method and JEM-2101 transmission electron microscope (TEM). Western blot confirmed the presence of extracellular vesicles (i.e., CD9, CD63 positivity) and the absence of co-isolated contaminants of cell debris (i.e., CYC1 negativity) (A). As demonstrated by ApoA1 positivity, there was some degree of lipoprotein contamination within the isolates from both septic shock and infected patients. TEM illustrated numerous single EVs and the absence of protein aggregates or other amorphous substances within the isolates (B). Morphologically, EVs appeared as a cup-shaped structure with an approximate size of around 100–200 nm. ApoA1, Apolipoprotein A1; CD, cluster of differentiation; CYC1, cytochrome C1; EV, extracellular vesicle; PFP, platelet-free plasma. Figure 4 The longitudinal trends of large… Figure 4 The longitudinal trends of large EVs concentration and the proportion of each subtype… Figure 4 The longitudinal trends of large EVs concentration and the proportion of each subtype in patients with septic shock and non-sepsis infection. EVs were isolated from PFP samples collected daily for seven consecutive days after hospital admission. Large EVs concentration (A) and their proportion (B) sorted by subtypes were quantified by the imaging flow cytometry analysis. The results were illustrated in line graphs with error bars using median and IQR. The comparison of the results between septic shock and non-sepsis patients were performed with Wilcoxon rank sum tests. *p-value from Wilcoxon rank-sum test <0.05. EVs, extracellular vesicles; IQR, interquartile range; PFP, platelet-free plasma. References Reinhart K, Daniels R, Kissoon N, Machado FR, Schachter RD, Finfer S. Recognizing sepsis as a global health priority - a WHO resolution. N Engl J Med. (2017) 377:414–7. 10.1056/NEJMp1707170 - DOI - PubMed Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. . The Third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. (2016) 315:801–10. 10.1001/jama.2016.0287 - DOI - PMC - PubMed Delano MJ, Ward PA. The immune system's role in sepsis progression, resolution, and long-term outcome. Immunol Rev. (2016) 274:330–53. 10.1111/imr.12499 - DOI - PMC - PubMed Hotchkiss RS, Moldawer LL, Opal SM, Reinhart K, Turnbull IR, Vincent JL. Sepsis and septic shock. Nat Rev Dis Primers. (2016) 2:16045. 10.1038/nrdp.2016.45 - DOI - PMC - PubMed van der Poll T, van de Veerdonk FL, Scicluna BP, Netea MG. The immunopathology of sepsis and potential therapeutic targets. Nat Rev Immunol. (2017) 17:407–20. 10.1038/nri.2017.36 - DOI - PubMed Show all 43 references

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