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. 2021 Jul 5:12:686111.
doi: 10.3389/fimmu.2021.686111. eCollection 2021.

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion

Jean-François Llitjos  1   2   3 , Yacine Bounab  4 , Christophe Rousseau  1   2 , Sophie Dixneuf  4 , Blandine Rimbault  4 , Jean-Daniel Chiche  1   2   3 , Julien Textoris  5   6   7 , Frédéric Pène  1   2   3 , Christophe Védrine  4
Affiliations

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion

Jean-François Llitjos et al. Front Immunol. .

Abstract

Objective: The 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.

Methods: Seven 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.

Results: Consistent 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.

Conclusion: 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.

Keywords: immune suppression; microfluidic; monocyte; septic shock; tolerance.

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Conflict of interest statement

JT is an employee of bioMérieux, S.A. YB and CV are inventors on a patent application based on certain ideas described in this manuscript and may receive financial compensation via their employer’s rewards to inventors’ scheme. The remaining 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
Figure 1
Flow cytometry assessment of monocyte deactivation. Monocytes were stimulated in vitro with 1 μg/mL LPS for 180 minutes. (A) Proportions of TNFα-positive monocytes without or with LPS stimulation in healthy donors (n = 10) and septic shock patients (n=7). (B, C) TNFα intracellular staining (with or without LPS stimulation) and HLA-DR membrane expression. (D) Representative dot-plots of intracellular production of TNFα. Data are expressed as mean and standard deviation. *p < 0.001.
Figure 2
Figure 2
Single-cell dynamic microfluidic analysis of monocyte TNFα secretion under LPS stimulation. (A, B) Heatmaps expressing TNFα secretion rate over time in one healthy donor (988 cells) (A) and one septic shock patient (1021 cells) (B). Cells were clustered based on TNFα secretion rate at 60 min. Secretion rate is expressed in TNFα molecules per second. (C) Means of maximal amount of TNFα molecules in cells. (D) Mean time to reach the maximal concentration of TNFα in each cell. (E) Proportion of monocytes secreting TNFα (molecules/sec) over time after LPS stimulation. (F, G) Proportions of secreting monocytes clustered by TNFα secretion rates over time in healthy donors (F) and septic shock patients (G). *p < 0.05.
Figure 3
Figure 3
Single cell dynamic microfluidic monitoring of monocyte phagocytosis. (A) Kinetics of phagocytosis activity of one monocyte starting at 90 min. Brightfield images (upper panel) illustrate cell migration toward the beadline (from center to the left of the droplet). Fluorescence images (lower panel) show the impact of phagocytosis on the beadline fluorescence signal (fuzzy fluorescence signal at 180 min due to the loss of beadline integrity). (B) Cumulative proportions of monocytes with phagocytosis of magnetic beadline over 180 min after LPS stimulation.
See this image and copyright information in PMC

References

    1. 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(8):801–10. 10.1001/jama.2016.0287 - DOI - PMC - PubMed
    1. Monneret G, Lepape A, Voirin N, Bohé J, Venet F, Debard A-L, et al. Persisting Low Monocyte Human Leukocyte Antigen-DR Expression Predicts Mortality in Septic Shock. Intensive Care Med (2006) 32(8):1175–83. 10.1007/s00134-006-0204-8 - DOI - PubMed
    1. Hotchkiss RS, Monneret G, Payen D. Sepsis-Induced Immunosuppression: From Cellular Dysfunctions to Immunotherapy. Nat Rev Immunol (2013) 13(12):862–74. 10.1038/nri3552 - DOI - PMC - PubMed
    1. Landelle C, Lepape A, Voirin N, Tognet E, Venet F, Bohé J, et al. Low Monocyte Human Leukocyte Antigen-DR Is Independently Associated With Nosocomial Infections After Septic Shock. Intensive Care Med (2010) 36(11):1859–66. 10.1007/s00134-010-1962-x - DOI - PubMed
    1. Avraham R, Haseley N, Brown D, Penaranda C, Jijon HB, Trombetta JJ, et al. Pathogen Cell-To-Cell Variability Drives Heterogeneity in Host Immune Responses. Cell (2015) 162(6):1309–21. 10.1016/j.cell.2015.08.027 - DOI - PMC - PubMed

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