Abstract
ObjectiveThe development of advanced single-cell technologies to decipher inter-cellular heterogeneity has enabled the dynamic assessment of individual cells behavior over time, overcoming the limitation of traditional assays. Here, we evaluated the feasibility of an advanced microfluidic assay combined to fluorescence microscopy to address the behavior of circulating monocytes from septic shock patients.MethodsSeven septic shock patients and ten healthy volunteers were enrolled in the study. Using the proposed microfluidic assay we investigated the production over time of LPS-elicited TNFα by single monocytes encapsulated within droplets. Cellular endocytic activity was assessed by internalization of magnetic nanoparticles. Besides, we assessed HLA-DR membrane expression and LPS-induced TNFα production in monocytes through classical flow cytometry assays.ResultsConsistent with the flow cytometry results, the total number of TNFα molecules secreted by encapsulated single monocytes was significantly decreased in septic shock patients compared to healthy donors. TNFα production was dampened as soon as 30 and 60 minutes after LPS stimulation in monocytes from septic patients. Furthermore, the microfluidic assay revealed heterogeneous individual behavior of monocytes from septic shock patients. Of note, monocytes from both healthy donors and patients exhibited similar phagocytic activities over time.ConclusionThe microfluidic assay highlights the functional heterogeneity of monocytes, and provides in-depth resolution in assessing the hallmark monocyte deactivation encountered in post-septic immunosuppression.
Highlights
Sepsis is a complex life-threatening syndrome caused by a dysregulated host response to infection [1]
Consistent with the flow cytometry results, the total number of TNFa molecules secreted by encapsulated single monocytes was significantly decreased in septic shock patients compared to healthy donors
The microfluidic assay highlights the functional heterogeneity of monocytes, and provides in-depth resolution in assessing the hallmark monocyte deactivation encountered in post-septic immunosuppression
Summary
Sepsis is a complex life-threatening syndrome caused by a dysregulated host response to infection [1]. The hallmark features of sepsis-induced immunosuppression includes monocyte deactivation defined by reduced expression of HLADR, dampened pro-inflammatory cytokine production towards ex vivo lipopolysaccharide (LPS) stimulation, lymphocyte apoptosis, expansion of regulatory T-cells, apoptosis and defective antigen presentation of dendritic cells [2, 3]. We have developed an ultrasensitive in-droplet immunoassay, in which an advanced microfluidic assay combined to fluorescence microscopy allows addressing the time course of LPS-elicited TNFa production by single monocytes encapsulated within droplets [7]. Thousands of single in-droplet cells can thereby be monitored at the same time This miniaturized assay enables simultaneous and dynamic measurements of protein secretion rate, cell surface marker, viability and phagocytic activity. In order to better characterize monocyte deactivation in sepsis, we applied this technique to blood samples obtained from septic shock patients as well as from healthy volunteers as controls
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