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

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.

Trained immunity — basic concepts and contributions to immunopathology

“…Monocytes can also be enriched using magnetic beads 182 ; this approach is fast and very gentle on myeloid cells but does not isolate monocyte populations with the same degree of purity as flow cytometry approaches such as fluorescence-activated cell sorting (FACS). Compared with FACS, microfluidics-based isolation methods are even gentler on myeloid cells and therefore less prone to induce non-specific cell activation, which would have a confounding effect when measuring the cellular response to subsequent challenges with an activating stimulus such as lipopolysaccharide (LPS) 183 . Flow cytometry not only enables the detection of human monocyte populations using CD11b and CD64 184 , 185 as lineage markers, but can also be used to detect cell-specific expression of pro-inflammatory cytokines through intracellular cytokine staining 167 .…”

Single-cell deep phenotyping of cytokine release unmasks stimulation-specific biological signatures and distinct secretion dynamics

“…Previous applications of the DropMap platform focused on measuring a single protein species from purified cell populations, such as antibodies from splenic and bone marrow B cells or tumor necrosis factor α (TNF-α) from enriched human monocytes. 29 , 30 To assess cytokine release comparable with classical CRAs, we adapted the platform to measure up to three cytokines in parallel for every individual cell in a heterogeneous mixture of PBMCs. First, sandwich immunoassays were established for TNF-α, interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-8, and macrophage inflammatory protein 1α (MIP-1α), allowing the detection of said cytokines through measurement of fluorescence relocation on the single-cell level ( Figure 1 ).…”

“…Given that in the previous application of DropMap the stimulation time was fixed to 3 h, 30 we next wanted to investigate the influence of stimulation time. Therefore, we stimulated PBMCs for various amounts of time with LPS in bulk, namely 1, 6, 16, and 24 h, and examined how cytokine secretion changed ( Figures 2 D–2F and S4 A for unstimulated PBMCs).…”

Single-Cell Deep Phenotyping of Cytokine Release Unmasks Stimulation-Specific Biological Signatures and Distinct Secretion Dynamics

“…Previous applications of the DropMap platform focused on measuring a single protein species from purified cell popula-tions, such as antibodies from splenic and bone marrow B cells or tumor necrosis factor a (TNF-a) from enriched human monocytes. 29,30 To assess cytokine release comparable with classical CRAs, we adapted the platform to measure up to three cytokines in parallel for every individual cell in a heterogeneous mixture of PBMCs. First, sandwich immunoassays were established for TNF-a, interferon-g (IFN-g), interleukin-2 (IL-2), IL-6, IL-8, and macrophage inflammatory protein 1a (MIP-1a), allowing the detection of said cytokines through measurement of fluorescence relocation on the single-cell level (Figure 1).…”

“…Different stimulation times alter the frequency of CSCs and their secretion rates in a cytokine-specific manner Given that in the previous application of DropMap the stimulation time was fixed to 3 h, 30 we next wanted to investigate the influence of stimulation time. Therefore, we stimulated PBMCs for various amounts of time with LPS in bulk, namely 1, 6, 16, and 24 h, and examined how cytokine secretion changed (Figures 2D-2F and S4A for unstimulated PBMCs).…”