Real-time Deformability Cytometry Research Articles

Membrane localization of actin filaments stabilizes giant unilamellar vesicles against external deforming forces

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape. Using giant unilamellar vesicles (GUVs) as models for cell membranes, we show that actin filaments can arrange along the membrane to form cortex-like structures. Notably, this organization is achieved using only actin and crowders as a minimal set of components. We utilize surface micropatterning to examine actin filament organization in deformed GUVs adhered to various pattern shapes. Our findings indicate that at the periphery of spherical GUVs, actin bundles align along the membrane. However, in highly curved regions of adhered GUVs, actin bundles avoid crossing the highly curved edges perpendicular to the adhesion site and instead remain in the lower curved regions by aligning parallel to the micropatterned surface. Furthermore, the actin bundles increase the stiffness of the GUVs, effectively counteracting strong deformations when GUVs adhere to micropatterns. This finding is corroborated by real-time deformability cytometry on GUVs with synthetic actin cortices. By precisely manipulating the shape of GUVs, our study provides a minimal system to investigate the interplay between actin structures and the membrane. Our findings provide insights into the spatial organization of actin structures within crowded environments, specifically inside GUVs that resemble the size and shape of cells. This study advances our understanding of actin network organization and functionality within cell-sized compartments.

Pilot study on the influence of acute alcohol exposure on biophysical parameters of leukocytes.

Objective: This pilot study explores the influence of acute alcohol exposure on cell mechanical properties of steady-state and activated leukocytes conducted with real-time deformability cytometry. Methods: Nineteen healthy male volunteers were enrolled to investigate the effect of binge drinking on biophysical properties and cell counts of peripheral blood leukocytes. Each participant consumed an individualized amount of alcohol to achieve a blood alcohol concentration of 1.2 ‰ as a mean peak. In addition, we also incubated whole blood samples from healthy donors with various ethanol concentrations and performed stimulation experiments using lipopolysaccharide and CytoStim™ in the presence of ethanol. Results: Our findings indicate that the biophysical properties of steady-state leukocytes are not significantly affected by a single episode of binge drinking within the first two hours. However, we observed significant alterations in relative cell counts and a shift toward a memory T cell phenotype. Moreover, exposure to ethanol during stimulation appears to inhibit the cytoskeleton reorganization of monocytes, as evidenced by a hindered increase in cell deformability. Conclusion: Our observations indicate the promising potential of cell mechanical analysis in understanding the influence of ethanol on immune cell functions. Nevertheless, additional investigations in this field are warranted to validate biophysical properties as biomarkers or prognostic indicators for alcohol-related changes in the immune system.

IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression...

The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

Increased red blood cell deformation in children and adolescents after SARS-CoV-2 infection

Severe coronavirus disease 2019 (COVID-19) is associated with hyperinflammation, hypercoagulability and hypoxia. Red blood cells (RBCs) play a key role in microcirculation and hypoxemia and are therefore of special interest in COVID-19 pathophysiology. While this novel disease has claimed the lives of many older patients, it often goes unnoticed or with mild symptoms in children. This study aimed to investigate morphological and mechanical characteristics of RBCs after SARS-CoV-2 infection in children and adolescents by real-time deformability-cytometry (RT-DC), to investigate the relationship between alterations of RBCs and clinical course of COVID-19. Full blood of 121 students from secondary schools in Saxony, Germany, was analyzed. SARS-CoV-2-serostatus was acquired at the same time. Median RBC deformation was significantly increased in SARS-CoV-2-seropositive compared to seronegative children and adolescents, but no difference could be detected when the infection dated back more than 6 months. Median RBC area was the same in seropositive and seronegative adolescents. Our findings of increased median RBC deformation in SARS-CoV-2 seropositive children and adolescents until 6 months post COVID-19 could potentially serve as a progression parameter in the clinical course of the disease with an increased RBC deformation pointing towards a mild course of COVID-19.

Dynamics of cell rounding during detachment

Animal cells undergo repeated shape changes, for example by rounding up and respreading as they divide. Cell rounding can be also observed in interphase cells, for example when cancer cells switch from a mesenchymal to an ameboid mode of cell migration. Nevertheless, it remains unclear how interphase cells round up. In this article, we demonstrate that a partial loss of substrate adhesion triggers actomyosin-dependent cortical remodeling and ERM activation, which facilitates further adhesion loss causing cells to round. Although the path of rounding in this case superficially resembles mitotic rounding in involving ERM phosphorylation, retraction fiber formation, and cortical remodeling downstream of ROCK, it does not require Ect2. This work provides insights into the way partial loss of adhesion actives cortical remodeling to drive cell detachment from the substrate. This is important to consider when studying the mechanics of cells in suspension, for example using methods like real-time deformability cytometry (RT-DC).

Rapid single-cell physical phenotyping of mechanically dissociated tissue biopsies

During surgery, rapid and accurate histopathological diagnosis is essential for clinical decision making. Yet the prevalent method of intra-operative consultation pathology is intensive in time, labour and costs, and requires the expertise of trained pathologists. Here we show that biopsy samples can be analysed within 30 min by sequentially assessing the physical phenotypes of singularized suspended cells dissociated from the tissues. The diagnostic method combines the enzyme-free mechanical dissociation of tissues, real-time deformability cytometry at rates of 100–1,000 cells s−1 and data analysis by unsupervised dimensionality reduction and logistic regression. Physical phenotype parameters extracted from brightfield images of single cells distinguished cell subpopulations in various tissues, enhancing or even substituting measurements of molecular markers. We used the method to quantify the degree of colon inflammation and to accurately discriminate healthy and tumorous tissue in biopsy samples of mouse and human colons. This fast and label-free approach may aid the intra-operative detection of pathological changes in solid biopsies.

Identification of a Distinct Monocyte-Driven Signature in Systemic Sclerosis Using Biophysical Phenotyping of Circulating Immune Cells.

Pathologically activated circulating immune cells, including monocytes, play major roles in systemic sclerosis (SSc). Their functional characterization can provide crucial information with direct clinical relevance. However, tools for the evaluation of pathologic immune cell activation and, in general, of clinical outcomes in SSc are scarce. Biophysical phenotyping (including characterization of cell mechanics and morphology) provides access to a novel, mostly unexplored layer of information regarding pathophysiologic immune cell activation. We hypothesized that the biophysical phenotyping of circulating immune cells, reflecting their pathologic activation, can be used as a clinical tool for the evaluation and risk stratification of patients with SSc. We performed biophysical phenotyping of circulating immune cells by real-time fluorescence and deformability cytometry (RT-FDC) in 63 SSc patients, 59 rheumatoid arthritis (RA) patients, 28 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) patients, and 22 age- and sex-matched healthy donors. We identified a specific signature of biophysical properties of circulating immune cells in SSc patients that was mainly driven by monocytes. Since it is absent in RA and AAV, this signature reflects an SSc-specific monocyte activation rather than general inflammation. The biophysical properties of monocytes indicate current disease activity, the extent of skin or lung fibrosis, and the severity of manifestations of microvascular damage, as well as the risk of disease progression in SSc patients. Changes in the biophysical properties of circulating immune cells reflect their pathologic activation in SSc patients and are associated with clinical outcomes. As a high-throughput approach that requires minimal preparations, RT-FDC-based biophysical phenotyping of monocytes can serve as a tool for the evaluation and risk stratification of patients with SSc.

P009 IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) is still incompletely understood. However, despite having been described back in the 1980s and reported to be involved in chronic inflammatory arthritis, the role of interleukin 3 (IL-3) signaling via the IL-3 receptor (IL-3R) in the development and perpetuation of chronic intestinal inflammation remains largely unexplored. In this study, we therefore aimed to explore the role of IL-3 signaling via IL-3R in experimental colitis as well as human IBD. Methods We analysed IL-3 expression in lamina propria mononuclear cells (LPMCs) from patients with IBD and from mice with experimental colitis. We compared the development of T cell transfer colitis in Rag1-/- mice after transfer of Il3r-/- and Il3r+/+ naïve T cells and investigated the underlying mechanisms by immunofluorescence and functional cell trafficking assays. The mechanical properties of Il3r-/- and Il3r+/+ T cells were determined by real-time deformability cytometry (RT-DC) and atomic force microscopy (AFM) and cytoskeleton structure was analysed by scanning electron microscopy (SEM) and fluorescence recovery after photobleaching (FRAP). Results The expression of IL-3 and IL3+ T cells were increased in the gut of patients with IBD and IL-3 expression was associated to shorter flare-free survival. In vivo, experimental chronic colitis upon T cell transfer was exacerbated in the absence of IL-3 or IL-3R signalling. This was attributable to IL-3-induced changes in kinase signalling and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of regulatory T (Treg) cells from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Treg cells. (A) IL3 mRNA expression in colon tissue from patients.(B) Flare-free survival of IBD patients.(C) Colitis in Rag1–/– mice after transfer of naïve CD4+ T cells from Il3r+/+ and Il3r-/- mice. Mini-endoscopy (left) and IVIS (right).(D) RT-DC plot (left) and quantification (right) of thymic lymphocytes from Il3r-/- and Il3r+/+ mice.(E) Lightsheet microscopy (left) and quantification of TReg index (right, TReg fraction in Ozanimod/DATK32 per fraction in placebo) in mLNs from Rag1-/- mice with transfer colitis induced by Il3r-/- or Il3r+/+ T cells. Conclusion We uncover a crucial role of IL-3R signalling in regulating TReg mechanobiology and tissue egress. Together, our data demonstrate a central – probably counter-regulatory – role for IL-3 receptor signalling in Treg cells in the pathogenesis of murine and human chronic intestinal inflammation and therefore suggest that IL-3 might be a novel target for future therapeutic approaches in IBD.

A new hyperelastic lookup table for RT-DC.

Real-time deformability cytometry (RT-DC) is an established method that quantifies features like size, shape, and stiffness for whole cell populations on a single-cell level in real-time. A lookup table (LUT) disentangles the experimentally derived steady-state cell deformation and the projected area to extract the cell stiffness in the form of the Young's modulus. So far, two lookup tables exist but are limited to simple linear material models and cylindrical channel geometries. Here, we present two new lookup tables for RT-DC based on a neo-Hookean hyperelastic material numerically derived by simulations based on the finite element method in square and cylindrical channel geometries. At the same time, we quantify the influence of the shear-thinning behavior of the surrounding medium on the stationary deformation of cells in RT-DC and discuss the applicability and impact of the proposed LUTs regarding past and future RT-DC data analysis. Additionally, we provide insights about the cell strain and stresses, as well as the influence resulting from the rotational symmetric assumption on the cell deformation and volume estimation. The new lookup tables and the numerical cell shapes are made freely available.

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements.

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Mechanical characterization of isolated mitochondria under conditions of oxidative stress.

Mechanical properties have been proven to be a pivotal parameter to enhance our understanding of living systems. While research during the last decades focused on cells and tissues, little is known about the role of organelle mechanics in cell function. Here, mitochondria are of specific interest due to their involvement in numerous physiological and pathological processes, e.g., in the production and homeostasis of reactive oxygen species (ROS). Using real-time fluorescence and deformability cytometry, we present a microfluidic technology that is capable to determine the mechanical properties of individual mitochondria at a throughput exceeding 100 organelles per second. Our data on several thousands of viable mitochondria isolated from rat C6 glial cells yield a homogenous population with a median deformation that scales with the applied hydrodynamic stress. In two proof-of-principle studies, we investigated the impact of exogenously and endogenously produced ROS on mitochondria mechanics. Exposing C6 cells to hydrogen peroxide (H2O2) triggers superoxide production and leads to a reduction in mitochondria size while deformation is increased. In a second study, we focused on the knockout of tafazzin, which has been associated with impaired remodeling of the mitochondrial membrane and elevated levels of ROS. Interestingly, our results reveal the same mechanical alterations as observed after the exposure to H2O2, which points to a unified biophysical mechanism of how mitochondria respond to the presence of oxidative stress. In summary, we introduce high-throughput mechanical phenotyping into the field of organelle biology with potential applications for understanding sub-cellular dynamics that have not been accessible before.

The impact of cell culture media on the interaction of biopolymer-functionalized gold nanoparticles with cells: mechanical and toxicological properties

Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.

Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.

Depressive disorders are associated with increased peripheral blood cell deformability: a cross-sectional case-control study (Mood-Morph)

Pathophysiological landmarks of depressive disorders are chronic low-grade inflammation and elevated glucocorticoid output. Both can potentially interfere with cytoskeleton organization, cell membrane bending and cell function, suggesting altered cell morpho-rheological properties like cell deformability and other cell mechanical features in depressive disorders. We performed a cross-sectional case-control study using the image-based morpho-rheological characterization of unmanipulated blood samples facilitating real-time deformability cytometry (RT-DC). Sixty-nine pre-screened individuals at high risk for depressive disorders and 70 matched healthy controls were included and clinically evaluated by Composite International Diagnostic Interview leading to lifetime and 12-month diagnoses. Facilitating deep learning on blood cell images, major blood cell types were classified and morpho-rheological parameters such as cell size and cell deformability of every individual cell was quantified. We found peripheral blood cells to be more deformable in patients with depressive disorders compared to controls, while cell size was not affected. Lifetime persistent depressive disorder was associated with increased cell deformability in monocytes and neutrophils, while in 12-month persistent depressive disorder erythrocytes deformed more. Lymphocytes were more deformable in 12-month major depressive disorder, while for lifetime major depressive disorder no differences could be identified. After correction for multiple testing, only associations for lifetime persistent depressive disorder remained significant. This is the first study analyzing morpho-rheological properties of entire blood cells and highlighting depressive disorders and in particular persistent depressive disorders to be associated with increased blood cell deformability. While all major blood cells tend to be more deformable, lymphocytes, monocytes, and neutrophils are mostly affected. This indicates that immune cell mechanical changes occur in depressive disorders, which might be predictive of persistent immune response.

Interpretation of cell mechanical experiments in microfluidic systems depend on the choice of cellular shape descriptors.

The capability to parameterize shapes is of essential importance in biomechanics to identify cells, to track their motion, and to quantify deformation. While various shape descriptors have already been investigated to study the morphology and migration of adherent cells, little is known of how the mathematical definition of a contour impacts the outcome of rheological experiments on cells in suspension. In microfluidic systems, hydrodynamic stress distributions induce time-dependent cell deformation that needs to be quantified to determine viscoelastic properties. Here, we compared nine different shape descriptors to characterize the deformation of suspended cells in an extensional as well as shear flow using dynamic real-time deformability cytometry. While stress relaxation depends on the amplitude and duration of stress, our results demonstrate that steady-state deformation can be predicted from single cell traces even for translocation times shorter than their characteristic time. Implementing an analytical simulation, performing experiments, and testing various data analysis strategies, we compared single cell and ensemble studies to address the question of computational costs vs experimental accuracy. Results indicate that high-throughput viscoelastic measurements of cells in suspension can be performed on an ensemble scale as long as the characteristic time matches the dimensions of the microfluidic system. Finally, we introduced a score to evaluate the shape descriptor-dependent effect size for cell deformation after cytoskeletal modifications. We provide evidence that single cell analysis in an extensional flow provides the highest sensitivity independent of shape parametrization, while inverse Haralick's circularity is mostly applicable to study cells in shear flow.

Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping

Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues

Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells’ properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.

Machine learning assisted real-time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.

Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy.

BackgroundSeptic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin‐1β (IL‐1β), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL‐1β in sepsis‐induced cardiomyopathy and cardiac atrophy.Methods Male Nlrp3 knockout (KO) and wild‐type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham‐treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL‐1β effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real‐time deformability cytometry (RT‐DC) analysis were used to investigate functional and mechanical effects of IL‐1β on cardiomyocytes.ResultsHeart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 μL vs. 24.6 ± 8.7 μL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 μL vs. 29.9 ± 9.9 μL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight–tibia length ratio: WT CLP vs. WT sham: −26.6%, P < 0.05; KO CLP vs. KO sham: −3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL‐1β decreased contractility (cell shortening: IL‐1β: −15.4 ± 2.3%, P < 0.001 vs. vehicle, IL‐1RA: −6.1 ± 3.3%, P < 0.05 vs. IL‐1β) and relaxation of adult rat ventricular cardiomyocytes (time‐to‐50% relengthening: IL‐1β: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL‐1RA: 564 ± 247 ms, P < 0.001 vs. IL‐1β), which was attenuated by an IL‐1 receptor antagonist (IL‐1RA). RT‐DC analysis indicated that IL‐1β reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF‐κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL‐1β activated NF‐κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL‐1RA.ConclusionsIL‐1β causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL‐1β pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.