Chemotactic Gradient Research Articles

A compartmentalized microfluidic platform to investigate immune cells cross-talk in rheumatoid arthritis

The dysregulation of the immune system plays a crucial role in the pathogenesis of manyfold diseases, among which we find rheumatoid arthritis (RA), an autoimmune disease characterized by chronic inflammation in synovial joints, leading to pain and disability. Immune cells such as pro-inflammatory macrophages and T helper 1 (Th1) cells drive the inflammatory cascade. Thus, including immune system inin vitromodels is pivotal to recapitulate and better understand the complex interactions between these immune cell subsets and their secreted mediators. Here, a compartmentalized microfluidic platform is presented, for precise confinement of circulating immune cells in organs-on-chip. The integration of innovative normally-closed sieving valves allows, through minimal waste of biological material, to co-culture different immune cell types (e.g. macrophages and Th1). Moreover, the platform allows to stimulate cell subsets separately, and to assess their cross-talk at desired time points. Functional validation of the platform demonstrates its ability to create stable chemotactic gradients, allowing for induction and evaluation of Th1 cells migration. In a proof-of-concept study, the platform allowed to assess Th1 T cells migration towards pro-inflammatory macrophages, thus replicating a characteristic interaction among immune cells triggered during RA onset. These results thus support the suitability of the platform to study immune cells cross-talk and migration phenomena, being potentially applicable to a manyfold immune cell mechanisms, both involved in RA progression and in different immune-mediated pathologies.

Gradient-induced instability in tumour spheroids unveils the impact of microenvironmental nutrient changes

Tumours often display invasive behaviours that induce fingering, branching and fragmentation processes. The phenomenon, known as diffusional instability, is driven by differential cell proliferation, migration, and death due to the presence of metabolite and catabolite concentration gradients. An understanding of the intricate dynamics of this spatially heterogeneous process plays a key role in the investigation of tumour growth and invasion. In this study, we developed an in vitro tumour invasion assay to investigate cell invasiveness in tumour spheroids under a chemotactic stimulus. Our method, employing tumour spheroids seeded in a 3D collagen gel within a microfluidic chemotaxis chamber, focuses on the role of diffusive gradients. Using Time-Lapse Microscopy, the dynamic evolution of tumour spheroids was monitored in real-time, providing a comprehensive view of the morphological changes and cell migration patterns under different chemotactic conditions. Specifically, we explored the impact of fetal bovine serum (FBS) gradients on the behaviour of CT26 mouse colon carcinoma cells and compared the effects of varying FBS concentrations to two isotropic control conditions. Furthermore, a finite element in silico model was developed to quantify the diffusive flow of nutrients in the chemotaxis chamber and obtain a detailed understanding of tumour dynamics. Our findings reveal that the presence of a chemotactic gradient significantly influences tumour invasiveness, with higher concentrations of nutrients associated with increased cancer growth and cell migration.

Towards a More Objective and High-throughput Spheroid Invasion Assay Quantification Method.

Multicellular spheroids embedded in 3D hydrogels are prominent in vitro models for 3D cell invasion. Yet, quantification methods for spheroid cell invasion that are high-throughput, objective and accessible are still lacking. Variations in spheroid sizes and the shapes of the cells within render it difficult to objectively assess invasion extent. The goal of this work is to develop a high-throughput quantification method of cell invasion into 3D matrices that minimizes sensitivity to initial spheroid size and cell spreading and provides precise integrative directionally-dependent metrics of invasion. By analyzing images of fluorescent cell nuclei, invasion metrics are automatically calculated at the pixel level. The initial spheroid boundary is segmented and automated calculations of the nuclear pixel distances from the initial boundary are used to compute common invasion metrics (i.e., the change in invasion area, mean distance) for the same spheroid at a later timepoint. We also introduce the area moment of inertia as an integrative metric of cell invasion that considers the invasion area as well as the pixel distances from the initial spheroid boundary. Further, we show that principal component analysis can be used to quantify the directional influence of a stimuli to invasion (e.g., due to a chemotactic gradient or contact guidance). To demonstrate the power of the analysis for cell types with different invasive potentials and the utility of this method for a variety of biological applications, the method is used to analyze the invasiveness of five different cell types. In all, implementation of this high-throughput quantification method results in consistent and objective analysis of 3D multicellular spheroid invasion. We provide the analysis code in both MATLAB and Python languages as well as a GUI for ease of use for researchers with a range of computer programming skills and for applications in a variety of biological research areas such as wound healing and cancer metastasis.

Ex vivo expansion in a clinical grade medium, containing growth factors from human platelets, enhances migration capacity of adipose stem cells.

Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated Supernatant Rich in Growth Factors (SRGF) can replace fetal bovine serum (FBS) to expand ASC by a clinical grade compliant protocol. The therapeutic potential of ASC is based also on their homing capacity toward inflammatory/cancer sites: oriented cell migration is a fundamental process in this scenario. We investigated the impact of SRGF on ASC migration properties. The motility/migration potential of ASC expanded in 5% SRGF was analyzed, in comparison to 10% FBS, by standard wound healing, bidimensional chemotaxis and transwell assays, and by millifluidic transwell tests. Mechanisms involved in the migration process were investigated by transient protein overexpression. In comparison to standard 10% FBS, supplementation of the cell culture medium with 5% SRGF, strongly increased migration properties of ASC along the chemotactic gradient and toward cancer cell derived soluble factors, both in static and millifluidic conditions. We showed that, independently from applied migratory stimulus, SRGF expanded ASC were characterized by far lower expression of α-smooth muscle actin (αSMA), a protein involved in the cell migration machinery. Overexpression of αSMA induced a significant and marked decrease in migration capacity of SRGF expanded ASC. In conclusion, 5% SRGF addition in the cell culture medium increases the migration potential of ASC, reasonably through appropriate downregulation of αSMA. Thus, SRGF could potentially improve the therapeutic impact of ASC, both as modulators of the immune microenviroment or as targeted drug delivery vehicles in oncology.

Chemokine‐Based Injectable Navigation System for Enhancing CAR‐T Cell Therapy Against Solid Tumors

AbstractChimeric antigen receptor (CAR)‐T cell therapy has demonstrated poor efficacy for solid tumors. The low number and poor functional persistence of CAR‐T cells in tumors are the main limiting factors. Here a Chemokine‐based Injectable platform for enhancing adoptive T cells’ Efficacy (CITE) is presented. CITE consists of peritumorally‐injected immunogel co‐loading chemotactic CXCL9 particles with PD‐1 antibody (aPD1) and intravenously‐infused tumor‐penetrating peptide iRGD. Immunogel formed drug depots and allowed sustained release of cargo peritumorally while iRGD facilitated tumor infiltration of gel‐released drugs, thus remarkably increasing the amount of CXCL9 and aPD1 in the tumor. CITE induced an effective chemotactic gradient to promote the tumor‐specific migration of CAR‐T cells and boosted the function of infiltrated T cells by blocking immunosuppression via a two‐in‐one approach. Consequently, CITE yielded a 218‐fold increase in the intratumoral number of transferred T cells and substantially improved the efficacies of transferred T cell receptor (TCR)‐T cells and CAR‐T cells in multiple syngeneic tumor models, including immunologically “cold” tumors, in immunocompetent mice. More importantly, CITE enhanced systemic antitumor immunity and elicited immune memory, enabling CITE to treat multimodal, metastatic, and recurrent tumors. CITE is broadly applicable to adoptive cell therapies involving CAR‐T or TCR‐T cells.

Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.

The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.

Systemic cellular migration: The forces driving the directed locomotion movement of cells.

Directional motility is an essential property of cells. Despite its enormous relevance in many fundamental physiological and pathological processes, how cells control their locomotion movements remains an unresolved question. Here, we have addressed the systemic processes driving the directed locomotion of cells. Specifically, we have performed an exhaustive study analyzing the trajectories of 700 individual cells belonging to three different species (Amoeba proteus, Metamoeba leningradensis, and Amoeba borokensis) in four different scenarios: in absence of stimuli, under an electric field (galvanotaxis), in a chemotactic gradient (chemotaxis), and under simultaneous galvanotactic and chemotactic stimuli. All movements were analyzed using advanced quantitative tools. The results show that the trajectories are mainly characterized by coherent integrative responses that operate at the global cellular scale. These systemic migratory movements depend on the cooperative nonlinear interaction of most, if not all, molecular components of cells.

Disulfiram Inhibits Opsonin-Independent Phagocytosis and Migration of Human Long-Lived In Vitro Cultured Phagocytes from Multiple Inflammatory Diseases.

Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads® M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.

An agent-based modeling approach for lung fibrosis in response to COVID-19.

The severity of the COVID-19 pandemic has created an emerging need to investigate the long-term effects of infection on patients. Many individuals are at risk of suffering pulmonary fibrosis due to the pathogenesis of lung injury and impairment in the healing mechanism. Fibroblasts are the central mediators of extracellular matrix (ECM) deposition during tissue regeneration, regulated by anti-inflammatory cytokines including transforming growth factor beta (TGF-β). The TGF-β-dependent accumulation of fibroblasts at the damaged site and excess fibrillar collagen deposition lead to fibrosis. We developed an open-source, multiscale tissue simulator to investigate the role of TGF-β sources in the progression of lung fibrosis after SARS-CoV-2 exposure, intracellular viral replication, infection of epithelial cells, and host immune response. Using the model, we predicted the dynamics of fibroblasts, TGF-β, and collagen deposition for 15 days post-infection in virtual lung tissue. Our results showed variation in collagen area fractions between 2% and 40% depending on the spatial behavior of the sources (stationary or mobile), the rate of activation of TGF-β, and the duration of TGF-β sources. We identified M2 macrophages as primary contributors to higher collagen area fraction. Our simulation results also predicted fibrotic outcomes even with lower collagen area fraction when spatially-localized latent TGF-β sources were active for longer times. We validated our model by comparing simulated dynamics for TGF-β, collagen area fraction, and macrophage cell population with independent experimental data from mouse models. Our results showed that partial removal of TGF-β sources changed the fibrotic patterns; in the presence of persistent TGF-β sources, partial removal of TGF-β from the ECM significantly increased collagen area fraction due to maintenance of chemotactic gradients driving fibroblast movement. The computational findings are consistent with independent experimental and clinical observations of collagen area fractions and cell population dynamics not used in developing the model. These critical insights into the activity of TGF-β sources may find applications in the current clinical trials targeting TGF-β for the resolution of lung fibrosis.

CCR7 acts as both a sensor and a sink for CCL19 to coordinate collective leukocyte migration.

Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein-coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization.

Sensitivity Analysis of a 2D Stochastic Agent-Based and PDE Diffusion Model for Cancer-on-Chip Experiments

The present work extends a previous paper where an agent-based and two-dimensional partial differential diffusion model was introduced for describing immune cell dynamics (leukocytes) in cancer-on-chip experiments. In the present work, new features are introduced for the dynamics of leukocytes and for their interactions with tumor cells, improving the adherence of the model to what is observed in laboratory experiments. Each system’s solution realization is a family of biased random walk trajectories, affected by the chemotactic gradients and in turn affecting them. A sensitivity analysis with respect to the model parameters is performed in order to assess the effect of their variation on both tumor cells and on leukocyte dynamics.

A Mathematical Modelling Study of Chemotactic Dynamics in Cell Cultures: The Impact of Spatio-temporal Heterogeneity

As motivated by studies of cellular motility driven by spatiotemporal chemotactic gradients in microdevices, we develop a framework for constructing approximate analytical solutions for the location, speed and cellular densities for cell chemotaxis waves in heterogeneous fields of chemoattractant from the underlying partial differential equation models. In particular, such chemotactic waves are not in general translationally invariant travelling waves, but possess a spatial variation that evolves in time, and may even oscillate back and forth in time, according to the details of the chemotactic gradients. The analytical framework exploits the observation that unbiased cellular diffusive flux is typically small compared to chemotactic fluxes and is first developed and validated for a range of exemplar scenarios. The framework is subsequently applied to more complex models considering the chemoattractant dynamics under more general settings, potentially including those of relevance for representing pathophysiology scenarios in microdevice studies. In particular, even though solutions cannot be constructed in all cases, a wide variety of scenarios can be considered analytically, firstly providing global insight into the important mechanisms and features of cell motility in complex spatiotemporal fields of chemoattractant. Such analytical solutions also provide a means of rapid evaluation of model predictions, with the prospect of application in computationally demanding investigations relating theoretical models and experimental observation, such as Bayesian parameter estimation.

Immune cell extravasation in an organ-on-chip to model lung inflammation

Acute respiratory distress syndrome (ARDS) is a severe lung condition with high mortality and various causes, including lung infection. No specific treatment is currently available and more research aimed at better understanding the pathophysiology of ARDS is needed. Most lung-on-chip models that aim at mimicking the air-blood barrier are designed with a horizontal barrier through which immune cells can migrate vertically, making it challenging to visualize and investigate their migration. In addition, these models often lack a barrier of natural protein-derived extracellular matrix (ECM) suitable for live cell imaging to investigate ECM-dependent migration of immune cells as seen in ARDS. This study reports a novel inflammation-on-chip model with live cell imaging of immune cell extravasation and migration during lung inflammation. The three-channel perfusable inflammation-on-chip system mimics the lung endothelial barrier, the ECM environment and the (inflamed) lung epithelial barrier. A chemotactic gradient was established across the ECM hydrogel, leading to the migration of immune cells through the endothelial barrier. We found that immune cell extravasation depends on the presence of an endothelial barrier, on the ECM density and stiffness, and on the flow profile. In particular, bidirectional flow, broadly used in association with rocking platforms, was found to significantly delay extravasation of immune cells in contrast to unidirectional flow. Extravasation was increased in the presence of lung epithelial tissue. This model is currently used to study inflammation-induced immune cell migration but can be used to study infection-induced immune cell migration under different conditions, such as ECM composition, density and stiffness, type of infectious agents used, and the presence of organ-specific cell types.

Integration of immune cells in organs-on-chips: a tutorial.

Viral and bacterial infections continue to pose significant challenges for numerous individuals globally. To develop novel therapies to combat infections, more insight into the actions of the human innate and adaptive immune system during infection is necessary. Human in vitro models, such as organs-on-chip (OOC) models, have proven to be a valuable addition to the tissue modeling toolbox. The incorporation of an immune component is needed to bring OOC models to the next level and enable them to mimic complex biological responses. The immune system affects many (patho)physiological processes in the human body, such as those taking place during an infection. This tutorial review introduces the reader to the building blocks of an OOC model of acute infection to investigate recruitment of circulating immune cells into the infected tissue. The multi-step extravasation cascade in vivo is described, followed by an in-depth guide on how to model this process on a chip. Next to chip design, creation of a chemotactic gradient and incorporation of endothelial, epithelial, and immune cells, the review focuses on the hydrogel extracellular matrix (ECM) to accurately model the interstitial space through which extravasated immune cells migrate towards the site of infection. Overall, this tutorial review is a practical guide for developing an OOC model of immune cell migration from the blood into the interstitial space during infection.

Ackr1-deficient mice are protected from lethal SARS-CoV-2 challenge

Abstract To date, there have been more than 613 million confirmed cases of COVID-19, with over 6 million deaths worldwide. In severe cases, the causative agent of the COVID-19, SARS-CoV-2, induces acute respiratory distress syndrome. To study the roles of chemokine pathways during COVID-19 pathogenesis, we performed an infection screen in mice deficient in chemokine ligands and receptors using the mouse-adapted strain of the virus. Weight loss and death were recorded for 2 weeks after infection of wild type mice, and mice genetically deficient for Ccr2, Ccr5, Ccr6, Cxcr3, Cxcr6, Cxcl10 or the atypical chemokine receptor Ackr1. Acute weight loss was observed in all infected mouse strains; however, Ackr1−/−mice uniquely displayed markedly increased survival compared to wild type mice. ACKR1 (also known as the Duffy antigen receptor for chemokines) is a non-signaling receptor expressed on endothelial cells, erythrocytes and a subset of neurons that binds to a wide range of chemokines thereby controlling their availability as a scavenger and shaping chemotactic gradients. Comprehensive expression analysis revealed Ackr1 as the most highly induced chemokine receptor in infected lung post-infection. Of note, COVID-19 mortality is disproportionately low in West Africa, overlapping with the geographic distribution of genetic deficiency of erythrocyte ACKR1 due to a single nucleotide polymorphism in the ACKR1 promoter that interrupts binding of GATA-1. Further studies are required to understand the modulation of SARS-CoV-2 pathogenesis by Ackr1 and its potential to explain the trajectory and limited mortality of Covid-19 in Africa and to serve as a potential therapeutic target.

Abstract 60: An emerging paradigm of Cxcl12 & Cxcr4 involvement in breast cancer metastasis

Abstract The Cxcl12/Cxcr4 signaling axis has been shown to promote metastasis in multiple mouse models of breast carcinoma, and has been linked to breast cancer cell seeding, homing, survival, and proliferation at future metastatic sites. The precise mechanism by which Cxcr4+ breast cancer cells escape the primary tumors (which also highly express Cxcl12), remains poorly understood. By using a novel multichannel immunofluorescence (mIF) based methodology for quantifying chemotactic gradients in fixed tissue, we here demonstrate that Cxcl12 gradients in mouse primary breast tumors are concentrically expressed around sites of cancer cell intravasation known as Tumor Microenvironment of Metastasis (TMEM) doorways. Via distance analysis algorithms, we additionally demonstrate that TMEM doorway-mediated Cxcl12 gradients associate with Cxcr4+ breast cancer cells migrating towards the underlying TMEM doorways. Consistent with this observation, pharmacological inhibition of the Cxcl12/Cxcr4 pathway significantly abrogates the translocation of Cxcr4+ cancer cells to TMEM doorways, suppressing TMEM doorway-mediated metastatic dissemination. However, targeted elimination of the Cxcr4 gene specifically from breast cancer cells, paradoxically results in a suboptimal response, thus suggesting the existence of a bypass or compensatory mechanism. Previously, it was shown that Cxcr4+ tumor-associated macrophages (TAMs) support the invasive and migratory properties of tumor cells that utilize TMEM doorways. We thus hypothesized that, absent Cxcr4 expression in tumor cells, accompanying Cxcr4+ TAMs may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, clodronate-mediated TAM depletion results in the significant suppression of Cxcr4+ cancer cell translocation to TMEM doorways and their subsequent dissemination to the peripheral circulation and future metastatic sites. Finally, we used a variety of stromal and immune cell lineage markers to identify the precise source of TMEM doorway-generated Cxcl12 gradients in mouse primary breast cancers. Despite the fact that blood vessels (irrespective of presence of TMEM doorways) were primarily lined by Pdgfrb+ stromal cells with basal Cxcl12 expression, TMEM-generated Cxcl12 gradients were specifically linked to a subset of Cxcl12+Iba1+ perivascular TAMs. Pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not significantly affect Cxcl12/Cxcr4- mediated translocation of Cxcr4+ tumor cells to TMEM doorways. Overall, our data support a new paradigm implicating the Cxcl12/Cxcr4 axis during the early stages of the metastatic cascade, and point to a new avenue for rationalized antimetastatic treatments for breast cancer. Citation Format: Dimitra P. Anastasiadou, Luis R. Sanchez, Camille L. Duran, Joseph Burt, Xiaoming Chen, Yu Lin, Robert Eddy, Allison S. Harney, David Entenberg, John S. Condeelis, Maja H. Oktay, George S. Karagiannis. An emerging paradigm of Cxcl12 & Cxcr4 involvement in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 60.

The Regulation of Neutrophil Migration in Patients with Sepsis: The Complexity of the Molecular Mechanisms and Their Modulation in Sepsis and the Heterogeneity of Sepsis Patients.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Common causes include gram-negative and gram-positive bacteria as well as fungi. Neutrophils are among the first cells to arrive at an infection site where they function as important effector cells of the innate immune system and as regulators of the host immune response. The regulation of neutrophil migration is therefore important both for the infection-directed host response and for the development of organ dysfunctions in sepsis. Downregulation of CXCR4/CXCL12 stimulates neutrophil migration from the bone marrow. This is followed by transmigration/extravasation across the endothelial cell barrier at the infection site; this process is directed by adhesion molecules and various chemotactic gradients created by chemotactic cytokines, lipid mediators, bacterial peptides, and peptides from damaged cells. These mechanisms of neutrophil migration are modulated by sepsis, leading to reduced neutrophil migration and even reversed migration that contributes to distant organ failure. The sepsis-induced modulation seems to differ between neutrophil subsets. Furthermore, sepsis patients should be regarded as heterogeneous because neutrophil migration will possibly be further modulated by the infecting microorganisms, antimicrobial treatment, patient age/frailty/sex, other diseases (e.g., hematological malignancies and stem cell transplantation), and the metabolic status. The present review describes molecular mechanisms involved in the regulation of neutrophil migration; how these mechanisms are altered during sepsis; and how bacteria/fungi, antimicrobial treatment, and aging/frailty/comorbidity influence the regulation of neutrophil migration.

AdipoRon accelerates bone repair of calvarial defect in diet-induced obesity mice

ObjectivesTo investigate the role of AdipoRon in bone wound healing of calvaria critical-sized defects (CSD) in diet-induced obesity (DIO) mice. Materials and methodsAfter establishing the calvaria CSD in normal-chow (NC), DIO and Adiponectin knockout (APNKO) mice, AdipoRon or vehicle was orally gavaged for 3 weeks. The bone defects were analyzed by micro-CT and H&E staining. The expression of osteogenesis-related factor in the defect area, and the chemotactic gradient of SDF-1 between bone marrow and bone defect area were further analyzed. ResultsAdipoRon downregulated body weight and alleviated fasting blood glucose level of DIO mice after treatment with AdipoRon in 14 and 21 days. Newly formed bone was significantly increased in the defect area of DIO and APNKO mice after treatment with AdipoRon compared with vehicle treatment. No significant difference was shown in NC mice. Furthermore, compared with NC mice, a significant decrease of BV/TV%, Tb.N value and formed bone percentage were shown in DIO and APNKO mice. The treatment with AdipoRon could reverse of decreased value and increase the newly formed bone in those mice. AdipoRon promoted col-1α expression in wound sites in DIO and APNKO mice. AdipoRon nearly quadrupled the chemotactic gradient of SDF-1 by decreasing SDF-1 expression in bone marrow and increasing it in the bone defect area in APNKO and DIO treated mice. ConclusionAdipoRon alleviates the obesity status in DIO mice with calvarial defect and increase new bone formation in calvarial defects in DIO and APNKO mice by modulating chemotactic gradient of SDF-1.

Photolithographic microfabrication of hydrogel clefts for cell invasion studies.

Invasion of migrating cells into surrounding tissue plays a key role in cancer metastasis and immune response. In order to assess invasiveness, most in vitro invasion assays measure the degree to which cells migrate between microchambers that provide a chemoattractant gradient across a polymeric membrane with defined pores. However, in real tissue cells experience soft, mechanically deformable microenvironments. Here we introduce RGD-functionalized hydrogel structures that present pressurized clefts for invasive migration of cells between reservoirs maintaining a chemotactic gradient. Using UV-photolithography, equally spaced blocks of polyethylene glycol-norbornene (PEG-NB) hydrogels are formed, which subsequently swell and close the interjacent gaps. The swelling ratio and final contours of the hydrogel blocks were determined using confocal microscopy confirming a swelling induced closure of the structures. The velocity profile of cancer cells transmigrating through the clefts, which we name 'sponge clamp', is found to depend on the elastic modulus as well as the gap size between the swollen blocks. The 'sponge clamp' discriminates the invasiveness of two distinct cell lines, MDA-MB-231 and HT-1080. The approach provides soft 3D-microstructures mimicking invasion conditions in extracellular matrix.

Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas.

Ciliary ectosomes are vesicles that bud from the ciliary membrane. Isolation and analysis of these structures can shed light on their bioactive cargoes and identify proteins and biomolecules involved in intercellular communication and various physiological processes. Most published methods to isolate ciliary ectosomes are based on their size (100nm to 1μm) to separate cilia-derived vesicles from isolated cilia and/or intact cells. However, it is often difficult to determine the origin of extracellular vesicles and to distinguish ciliary ectosomes from ectosomes budded from the plasma membrane or from exosomes that derive from multivesicular bodies. Here, we describe procedures to isolate and purify ciliary ectosomes from the unicellular green alga, Chlamydomonas reinhardtii, through differential and iodixanol density gradient ultracentrifugation; in this organism, the ciliary membrane is the only membrane directly exposed to the environment and thus ectosomes are of known origin. Ciliary ectosomes contain enzymes and α-amidated peptide products required to mediate peptidergic-signaling cascades; one identified amidated peptide acts as a chemotactic modulator for C. reinhardtii gametes. Classical methods used to assess chemotaxis do not provide quantitative measurements of the chemotactic gradient or the real-time effects on the migration of fast moving cells. Consequently, we developed a chemotaxis assay protocol using microfluidic channel slides that provides quantitative and qualitative measurements of the chemotactic gradient and cell migration. Here, we describe how to establish a stable gradient of a bioactive substance in microfluidic channel slides and perform quantitative assays to assess chemotaxis of both individual cells and populations of C. reinhardtii.