Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocyte differentiation into macrophages.

Monocytes activated by pro-inflammatory signals adhere to the vascular endothelium and migrate from the bloodstream to the tissue ultimately differentiating into macrophages. Cell mechanics and adhesion play a crucial role in macrophage functions during this inflammatory process. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we used various tools to quantify the morphology, adhesion, and viscoelasticity of monocytes and differentiatted macrophages. Combination of atomic force microscopy (AFM) high resolution viscoelastic mapping with interference contrast microscopy (ICM) at the single-cell level revealed viscoelasticity and adhesion hallmarks during monocyte differentiation into macrophages. Quantitative holographic tomography imaging revealed a dramatic increase in cell volume and surface area during monocyte differentiation and the emergence of round and spread macrophage subpopulations. AFM viscoelastic mapping showed important stiffening (increase of the apparent Young's modulus, E0) and solidification (decrease of cell fluidity, ß) on differentiated cells that correlated with increased adhesion area. These changes were enhanced in macrophages with a spread phenotype. Remarkably, when adhesion was perturbed, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the stiffer and more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activities. Thus, our results revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. METHODS: We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. RESULTS: siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. CONCLUSIONS: The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.

DEN-Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease's progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver's inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.

Hypoxia Drives Dihydropyrimidine Dehydrogenase Expression in Macrophages and Confers Chemoresistance in Colorectal Cancer.

Colorectal adenocarcinoma is a leading cause of death worldwide, and immune infiltration in colorectal tumors has been recognized recently as an important pathophysiologic event. In this context, tumor-associated macrophages (TAM) have been related to chemoresistance to 5-fluorouracil (5-FU), the first-line chemotherapeutic agent used in treating colorectal cancers. Nevertheless, the details of this chemoresistance mechanism are still poorly elucidated. In the current study, we report that macrophages specifically overexpress dihydropyrimidine dehydrogenase (DPD) in hypoxia, leading to macrophage-induced chemoresistance to 5-FU via inactivation of the drug. Hypoxia-induced macrophage DPD expression was controlled by HIF2α. TAMs constituted the main contributors to DPD activity in human colorectal primary or secondary tumors, while cancer cells did not express significant levels of DPD. In addition, contrary to humans, macrophages in mice do not express DPD. Together, these findings shed light on the role of TAMs in promoting chemoresistance in colorectal cancers and identify potential new therapeutic targets. SIGNIFICANCE: Hypoxia induces HIF2α-mediated overexpression of dihydropyrimidine dehydrogenase in TAMs, leading to chemoresistance to 5-FU in colon cancers.

A Universal Model for the Log-Normal Distribution of Elasticity in Polymeric Gels and Its Relevance to Mechanical Signature of Biological Tissues.

The mechanosensitivity of cells has recently been identified as a process that could greatly influence a cell's fate. To understand the interaction between cells and their surrounding extracellular matrix, the characterization of the mechanical properties of natural polymeric gels is needed. Atomic force microscopy (AFM) is one of the leading tools used to characterize mechanically biological tissues. It appears that the elasticity (elastic modulus) values obtained by AFM presents a log-normal distribution. Despite its ubiquity, the log-normal distribution concerning the elastic modulus of biological tissues does not have a clear explanation. In this paper, we propose a physical mechanism based on the weak universality of critical exponents in the percolation process leading to gelation. Following this, we discuss the relevance of this model for mechanical signatures of biological tissues.

Piezoelectric Actuated Glass Plate for Liquid Density and Viscosity Measurement.

This paper reports on a new system for liquid density and viscosity measurement based on a freely suspended rectangular vibrating plate actuated by piezoelectric ceramic (PZT) actuators. The Lamb mode used for these measurements allows us to infer both the density and viscosity in a larger range as compared to the existing gold-standard techniques of MEMS resonators. The combination of the measured resonance frequency and quality factor enables extraction of density and viscosity of the surrounding liquid. The system is calibrated while performing measurements in water glycerol solutions with a density range from 997 to 1264 kg/m3 and viscosity from 1.22 to 985 mPa·s, which is a larger dynamic range compared to existing mechanical resonators showing an upper limit of 700 mPa·s. The out-of-plane vibrating mode exhibits quality factor of 169, obtained in deionized water (1.22 mPa·s viscosity), and 93 for pure glycerol with a viscosity of 985 mPa·s. This Lamb wave resonating sensor can achieve measurement in fairly large viscosity media while keeping a quality factor superior to 90. Measurements performed on oil validate the use of the Lamb system. Oil density is evaluated at 939 kg/m3 and dynamic viscosity at 43 mPa·s which corresponds to our expected values. This shows the possibility of using the sensor outside of the calibration range.

Measuring cell displacements in opaque tissues: dynamic light scattering in the multiple scattering regime.

Coherent light scattered by tissues brings structural and dynamic information, at depth, that standard imaging techniques cannot reach. Dynamics of cells or sub-cellular elements can be measured thanks to dynamic light scattering in thin samples (single scattering regime) or thanks to diffusive wave spectroscopy in thick samples (diffusion regime). Here, we address the intermediate regime and provide an analytical relationship between scattered light fluctuations and the distribution of cell displacements as a function of time. We illustrate our method by characterizing cell motility inside half millimeter thick multicellular aggregates.

Toward Microfluidic Label-Free Isolation and Enumeration of Circulating Tumor Cells from Blood Samples.

The isolation, analysis, and enumeration of circulating tumor cells (CTCs) from cancer patient blood samples are a paradigm shift for cancer patient diagnosis, prognosis, and treatment monitoring. Most methods used to isolate and enumerate these target cells rely on the expression of cell surface markers, which varies between patients, cancer types, tumors, and stages. Here, we propose a label-free high-throughput platform to isolate, enumerate, and size CTCs on two coupled microfluidic devices. Cancer cells were purified through a Vortex chip and subsequently flowed in-line to an impedance chip, where a pair of electrodes measured fluctuations of an applied electric field generated by cells passing through. A proof-of-concept of the coupling of those two devices was demonstrated with beads and cells. First, the impedance chip was tested as a stand-alone device: (1) with beads (mean counting error of 1.0%, sizing information clearly separated three clusters for 8, 15, and 20 um beads, respectively) as well as (2) with cancer cells (mean counting error of 3.5%). Second, the combined setup was tested with beads, then with cells in phosphate-buffered saline, and finally with cancer cells spiked in healthy blood. Experiments demonstrated that the Vortex HT chip enriched the cancer cells, which then could be counted and differentiated from smaller blood cells by the impedance chip based on size information. Further discrimination was shown with dual high-frequency measurements using electric opacity, highlighting the potential application of this combined setup for a fully integrated label-free isolation and enumeration of CTCs from cancer patient samples. © 2019 International Society for Advancement of Cytometry.

3D type I collagen environment leads up to a reassessment of the classification of human macrophage polarizations.

Macrophages have multiple roles in development, tissue homeostasis and repair and present a high degree of phenotypic plasticity embodied in the concept of polarization. One goal of macrophage biology field is to characterize these polarizations at the molecular level. To achieve this task, it is necessary to integrate how physical environment signals are interpreted by macrophages under immune stimulation. In this work, we study how a 3D scaffold obtained from polymerized fibrillar rat type I collagen modulates the polarizations of human macrophages and reveal that some traditionally used markers should be reassessed. We demonstrate that integrin ß2 is a regulator of STAT1 phosphorylation in response to IFNγ/LPS as well as responsible for the inhibition of ALOX15 expression in response to IL-4/IL-13 in 3D. Meanwhile, we also find that the CCL19/CCL20 ratio is reverted in 3D under IFNγ/LPS stimulation. 3D also induces the priming of the NLRP3 inflammasome resulting in an increased IL-1ß and IL-6 secretion. These results give the molecular basis for assessing collagen induced immunomodulation of human macrophages in various physiological and pathological contexts such as cancer.

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

Calreticulin Release at an Early Stage of Death Modulates the Clearance by Macrophages of Apoptotic Cells.

Calreticulin (CRT) is a well-known "eat-me" signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study, we focus on the role of the newly exposed CRT following cell death induction. We show that if CRT increases at the outer face of the plasma membrane and is well recognized by C1q even when phosphatidylserine is not yet detected, CRT is also released in the surrounding milieu and is able to interact with phagocytes. We observed that exogenous CRT is endocytosed by THP1 macrophages through macropinocytosis and that internalization is associated with a particular phenotype characterized by an increase of cell spreading and migration, an upregulation of CD14, an increase of interleukin-8 release, and a decrease of early apoptotic cell uptake. Importantly, CRT-induced pro-inflammatory phenotype was confirmed on human monocytes-derived macrophages by the overexpression of CD40 and CD274, and we found that monocyte-derived macrophages exposed to CRT display a peculiar polarization notably associated with a downregulation of the histocompatibility complex of class II molecules hampering its description through the classical M1/M2 dichotomy. Altogether our results highlight the role of soluble CRT with strong possible consequences on the macrophage-mediated immune response to dying cell.

Proteomic Signature Reveals Modulation of Human Macrophage Polarization and Functions Under Differing Environmental Oxygen Conditions.

Macrophages are innate immune cells which can react to a large number of environmental stimuli thanks to a high degree of plasticity. These cells are involved in a variety of tissue functions in homeostasis, and they play essential roles in pathological contexts. Macrophages' activation state, which determines their functional orientation, is strongly influenced by the cellular environment. A large body of macrophage literature is devoted to better defining polarizations from a molecular viewpoint. It is now accepted that a multidimensional model of polarization is needed to grasp the broad phenotype repertoire controlled by environmental signals. The study presented here aimed, among other goals, to provide a molecular signature of various polarizations in human macrophages at the protein level to better define the different macrophage activation states. To study the proteome in human monocyte-derived macrophages as a function of their polarization state, we used a label-free quantification approach on in-gel fractionated and LysC/Trypsin digested proteins. In total, 5102 proteins were identified and quantified for all polarization states. New polarization-specific markers were identified and validated. Because oxygen tension is an important environmental parameter in tissues, we explored how environmental oxygen tension, at either atmospheric composition (18.6% O2) or "tissue normoxia" (3% O2), affected our classification of macrophage polarization. The comparative results revealed new polarization-specific makers which suggest that environmental oxygen levels should be taken into account when characterizing macrophage activation states. The proteomic screen revealed various polarization-specific proteins and oxygen sensors in human macrophages. One example is arachidonate 15-lipoxygenase (ALOX15), an IL4/IL13 polarization-specific protein, which was upregulated under low oxygen conditions and is associated with an increase in the rate of phagocytosis of apoptotic cells. These results illustrate the need to consider physicochemical parameters like oxygen level when studying macrophage polarization, so as to correctly assess their functions in tissue.

Identifying exposition to low oxygen environment in human macrophages using secondary ion mass spectrometry and multivariate analysis.

RATIONALE: Macrophages are innate immune cells presenting a strong phenotypic plasticity and deeply involved in tissue homeostasis. Oxygen environmental tension is a physical parameter that could influence their polarizations. In this study we use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to describe how various polarizations are modified by a low oxygen exposure. METHODS: TOF-SIMS experiments were performed using an IONTOF ToF-SIMS 5-100 (ION-TOF GmbH, Munster, Germany). Analysis was performed using a pulsed 25 keV Bi3+ beam, sputtering was performed using a 250 eV Cs beam. Cells were fixed by paraformaldehyde before TOF-SIMS analysis. RESULTS: Multivariate analysis of the TOF-SIMS spectra provided ion species associated with the exposure of macrophages to low oxygen concentration. We were able to obtain some species, specific of a particular polarization, advocating for the use of macrophages as reporter cells of oxygen tension in tissues. CONCLUSIONS: Our study demonstrates that macrophage molecular signature to low oxygen environment is dependent on their polarization. TOF-SIMS shows the clear capability to produce species revealing this exposition. This result opens the way to the use of TOF-SIMS as a tool to explore hypoxia in human tissues.

Extracellular Vesicles: Isolation Methods.

Extracellular vesicles (EVs) have recently been at the center of attention of cellular biologists and physicians as their role in intercellular communications has become progressively revealed. EVs display a huge diversity concerning their biogenesis and functions, leading to a still evolving classification comprising exosomes, microvesicles and apoptotic bodies. One of the main technical challenges to studying EVs is to isolate them without interfering with their structure, in order to be able to reveal their functions and to use them as biomarkers. Moreover, the new area of therapeutically using EVs needs clinical grade methods of isolation. In this review, different methods disposable to researchers and clinicians to isolate EVs are described, focusing on the physical principles that allow understanding the advantages and limitations of each technique. The new growing field of microfluidic systems which offers the opportunity to associate isolation and characterization on a single chip will be presented highlighting its potential in the field of EV studies.

Microscopic DTI accurately identifies early glioma cell migration: correlation with multimodal imaging in a new glioma stem cell model.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion. In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n = 6), sham (n = 3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood-brain barrier leakage and tumor angiogenesis were detected later, at three months. To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D-DTI (80 µm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n = 6), sham (n = 3)). DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D⊥ ) changes derived from ex vivo microscopic 3D-DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p < 0.001) was observed, while in the CC a - 28% decrease in FA (p < 0.005) and a + 95% increase in D⊥ (p < 0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two-photon microscopy in the same brains were correlated (p < 0.001, r = 0.69), validating FA and D⊥ as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization. During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D-DTI and to validate DTI parameters FA and D⊥ as biomarkers for glioma cell invasion.

Neutrophil-Expressed p21/waf1 Favors Inflammation Resolution in Pseudomonas aeruginosa Infection.

Neutrophil-associated inflammation during Pseudomonas aeruginosa lung infection is a determinant of morbidity in cystic fibrosis (CF). Neutrophil apoptosis is a key factor in inflammation resolution and is controlled by cytosolic proliferating cell nuclear antigen (PCNA). p21/Waf1, a cyclin-dependent kinase inhibitor, is a partner of PCNA, and its mRNA is up-regulated in human neutrophils during LPS challenge. We show here that, after 7 days of persistent infection with P. aeruginosa, neutrophilic inflammation was more prominent in p21(-/-) compared with wild-type (WT) mice. Notably, no intrinsic defect in the phagocytosis of apoptotic cells by macrophages was found in p21(-/-) compared with WT mice. Inflammatory cell analysis in peritoneal lavages after zymosan-induced peritonitis showed a significantly increased number of neutrophils at 48 hours in p21(-/-) compared with WT mice. In vitro analysis was consistent with delayed neutrophil apoptosis in p21(-/-) compared with WT mice. Ectopic expression of p21/waf1 in neutrophil-differentiated PLB985 cells potentiated apoptosis and reversed the prosurvival effect of PCNA. In human neutrophils, p21 messenger RNA was induced by TNF-α, granulocyte colony-stimulating factor, and LPS. Neutrophils isolated from patients with CF showed enhanced survival, which was reduced after treatment with a carboxy-peptide derived from the sequence of p21/waf1. Notably, p21/waf1 was detected by immunohistochemistry in neutrophils within lungs from patients with CF. Our data reveal a novel role for p21/waf1 in the resolution of inflammation via its ability to control neutrophil apoptosis. This mechanism may be relevant in the neutrophil-dominated inflammation observed in CF and other chronic inflammatory lung conditions.

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Republished: Antineutrophil cytoplasmic antibody-associated vasculitides: is it time to split up the group?

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a heterogeneous group of diseases corresponding to necrotising inflammation of small vessels with a wide range of clinical presentations. At least two of the diseases are believed to exhibit a common ground of pathophysiological mechanisms. These are granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis) and microscopic polyangiitis (MPA). ANCA directed against proteinase 3 (PR3) are preferentially associated with GPA, and anti-myeloperoxidase (MPO) ANCA are associated mainly with MPA and eosinophilic GPA (formerly known as Churg-Strauss syndrome). Anti-MPO and anti-PR3 antibodies can activate neutrophils in vitro. In vivo data are available for humans and mice on the pathogenicity of anti-MPO but it is more controversial for PR3-ANCA. A recent genome-wide association study of patients with ANCA-associated vasculitides confirmed the genetic contribution to the pathogenesis of these conditions, with significant association of PR3-ANCA and human leukocyte antigen-DP and the genes encoding α1-antitrypsin and PR3. MPO-ANCA were significantly associated with human leukocyte antigen-DQ. Thus, recent results from epidemiological studies, genome-wide association study and therapeutic trials have suggested that these entities are, in fact, distinct. We have summarised these results and discuss the idea that these two entities should be studied separately as the nature of the two auto-antigens suggests at a molecular level despite shared ANCA involvement.