Passive redirection filters minimize red blood cell contamination during neutrophil chemotaxis assays using whole blood.

Neutrophils are the most numerous white blood cells and are the first to arrive at sites of inflammation and infection. Thus, neutrophil behavior provides a comprehensive biomarker for antimicrobial defenses. Several microfluidic tools have been developed to test neutrophil chemotaxis, phagocytosis, extrusion of extracellular traps, etc. Traditional tools rely on purified neutrophil samples, which require lengthy and expensive isolation procedures from large volumes of blood. In the absence of such isolation, visualizing neutrophils in blood is complicated by the overwhelming number of red blood cells (RBCs), which outnumber neutrophils by 1000 : 1. Recently, several microfluidic technologies have been designed to analyze neutrophils directly in blood, by separating neutrophils on selectin coated surfaces before the migration assay or blocking the advance of RBCs with the moving neutrophils. However, RBC contamination remains an issue, albeit with a reduced ratio, down to 1 : 1. Here, we present an RBC-debulking strategy for neutrophil assays based on microscale passive redirection filters (PRFs) that reduce RBC contamination down to as few as a 1 : 17 RBC to neutrophil ratio. We compare the performance of different PRF designs and measure changes in neutrophil chemotaxis velocity and directionality following immune stimulation of whole blood.

Inflammatory Glycoprotein YKL-40 Is Elevated after Coronary Artery Bypass Surgery and Correlates with Leukocyte Chemotaxis and Myocardial Injury, a Pilot Study.

The aim of the present study was to investigate the levels of YKL-40 during and after coronary artery bypass grafting surgery (CABG) and to establish possible connections between YKL-40 and markers of oxidative stress, inflammation, and myocardial injury. Patients undergoing elective CABG utilizing cardiopulmonary bypass (CPB) were recruited into the study. Blood samples were collected at the onset of anesthesia, during surgery and post-operatively. Levels of YKL-40, 8-isoprostane, interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and troponin T (TnT) were measured by immunoassay. YKL-40 levels increased significantly 24 h after CPB. Positive correlation was seen between post-operative TnT and YKL-40 levels (r = 0.457, p = 0.016) and, interestingly, baseline YKL-40 predicted post-operative TnT increase (r = 0.374, p = 0.050). There was also a clear association between YKL-40 and the chemotactic factors MCP-1 (r = 0.440, p = 0.028) and IL-8 (r = 0.484, p = 0.011) linking YKL-40 to cardiac inflammation and fibrosis following CABG. The present results show, for the first time, that YKL-40 is associated with myocardial injury and leukocyte-activating factors following coronary artery bypass surgery. YKL-40 may be a factor and/or biomarker of myocardial inflammation and injury and subsequent fibrosis following heart surgery.

Optimization of the Transwell® assay for the analysis of neutrophil chemotaxis using flow cytometry to refine the clinical investigation of immunodeficient patients.

Chemotaxis is the directed movement of neutrophils towards an infected site. This physiological process can be reproduced using a modified Boyden chamber, such as the Transwell® support. Different techniques can be used to count neutrophils after migration to the lower chamber of the holder. The present study supports the use of an optimized Transwell® assay coupled with a flow cytometry-based method (Sysmex XN-9000) to detect chemotaxis abnormalities. A reference interval of neutrophil's chemotaxis was determined as part of this work. A first step involves the extraction of neutrophils from whole blood. The migration of neutrophils from the upper to the lower support chamber is subsequently directed by a chemoattractant gradient using N-formyl-l-Methionyl-l-Leucyl-l-Phenylalanine (fMLP). Neutrophils collected in the lower chamber are finally counted by flow cytometry. The original protocol was optimized through the comparison of different parameters. The use of Polymorphprep®, in the extraction of neutrophils, showed an improvement of the neutrophils yield of 1.65 times (57.5% of recovery) compared to the extraction using the Ficoll-Hypaque® gradient. A solution containing 5% of Bovin Serum Albumin (BSA) was used to suspend the extracted neutrophils, stabilize their viability and preserve their integrity. The mechanical agitation of the Transwell® permeable supports during migration did not show an increase in neutrophil yield. A migration time of 1 h 30 was identified as the best time for collecting the largest number of neutrophils after migration. Finally, we demonstrated that scraping the bottom of the well after migration improved neutrophil collection from the lower chamber by 1.9-fold compared to a non-scraping method. In conclusion, our results support the use of Polymorphprep® and a 5% BSA solution in the suspension, without agitation of the medium. An incubation time of 1 h 30 was identified as optimal for neutrophil migration through the chamber. Scraping the bottom after neutrophil migration improved neutrophil collection yield. Normal adult values were obtained with directed migration equal to 32.4% ±13.41% on 15 men and 18 women.

Multifactorial assessment of neutrophil chemotaxis efficiency from a drop of blood.

Following injury and infection, neutrophils are guided to the affected site by chemoattractants released from injured tissues and invading microbes. During this process (chemotaxis), neutrophils must integrate multiple chemical signals, while also responding to physical constraints and prioritizing their directional decisions to generate an efficient immune response. In some clinical conditions, human neutrophils appear to lose the ability to chemotax efficiently, which may contribute both directly and indirectly to disease pathology. Here, a range of microfluidic designs is utilized to test the sensitivity of chemotaxing neutrophils to various perturbations, including binary decision-making in the context of channels with different chemoattractant gradients, hydraulic resistance, and angle of approach. Neutrophil migration in long narrow channels and planar environments is measured. Conditions in which neutrophils are significantly more likely to choose paths with the steepest chemoattractant gradient and the most direct approach angle, and find that migration efficiency across planar chambers is inversely correlated with chamber diameter. By sequential measurement of neutrophil binary decision-making to different chemoattractant gradients, or chemotactic index in sequential planar environments, data supporting a model of biased random walk for neutrophil chemotaxis are presented.

Infiltrating T lymphocytes and programmed cell death protein-1/programmed death-ligand 1 expression in endometriosis-associated ovarian cancer.

OBJECTIVE: To characterize T lymphocyte infiltration and programmed cell death protein-1 (PD-1)/programmed death-ligand 1 (PD-L1) expression in early-stage endometriosis-associated ovarian cancer (EAOC), ovarian endometriosis (OE), atypical endometriosis (AE), and deep endometriosis (DE). DESIGN: Case-control, retrospective study. SETTING: Research University Hospital. PATIENT(S): A total of 362 patients with a histologic diagnosis of EAOC, OE, AE, or DE were identified between 2000 and 2019 from Fondazione Policlinico Universitario Agostino Gemelli IRCCS and Gemelli Molise SpA tissue data banks. A 1:1 propensity score-matched method yielded matched pairs of 55 subjects with EAOC, 55 patients with OE, 12 patients with AE, and 42 patients with DE, resulting in no differences in family history of cancer, parity, and use of oral contraceptives. INTERVENTION(S): Immunohistochemistry assays using the following primary antibodies: CD3+; CD4+; CD8+; PD-1; and PD-L1. MAIN OUTCOME MEASURE(S): To characterize T lymphocyte infiltration and PD-1/PD-L1 expression in 4 different endometriosis-related diseases. RESULT(S): Endometriosis-associated ovarian cancer cases displayed significantly higher levels of PD-1/PD-L1 expression compared with all other endometriosis-related diseases (vs. OE vs. AE vs. DE). Moreover, a significantly lower count of infiltrating T lymphocytes was observed in EAOC cases compared with OE ones. Finally, one-third of OE cases showed a cancer-like PD-1/PD-L1 expression profile. CONCLUSION(S): Endometriosis-associated ovarian cancer is characterized by higher levels of PD-1/PD-L1 expression compared with benign endometriosis-related diseases. This profile was found in one-third of clinically benign cases, suggesting that it develops early in the carcinogenesis process.

Mesenchymal Stem Cells Enhance Chemotaxis of Activated T Cells through the CCL2-CCR2 Axis In Vitro.

Activation and migration of donor T cells to the host target organs are critical mechanisms in the pathogenesis of graft-versus-host disease (GVHD). The role of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor CCR2 in the recruitment of T cells during immune or inflammatory response is also well known. For elucidation of the mechanism of the therapeutic effect of human bone marrow derived-mesenchymal stem cells (MSC) in GVHD, we studied the effect of these cells on migration of activated donor T cells through the CCL2-CCR2 axis in vitro. MSC were expanded from donors' bone marrow mononuclear cells. After co-culturing of IL-2-activated T cells with allogeneic MSC at different ratios, the levels of CCL2 in supernatants were measured by ELISA, and CCR2 expression in CD4+/CD8+ T cells subsets were detected by flow cytometry. The effect of MSC on the migration of activated T cells in the Transwell system was studied in the absence or presence of CCL2. Our results show that CCL2 levels in supernatants of co-cultures were significantly higher than in MSC monoculture and this increase depended on the number of MSC. MSC inhibited proliferation of T cells, but did not change the percentages of CD4+ and CD8+ T cells subsets. MSC can up-regulate the CCR2 expression in CD8+ subsets rather than in CD4+ subsets; MSC enhanced migration of IL-2-activated T cells to CCL2 by increasing the expression of CCR2. The data demonstrate that MSC can enhance chemotaxis of cytokine-activated T cells through the CCL2-CCR2 axis in vitro.

B Cell-Activating Factor Antagonism Attenuates the Growth of Experimental Abdominal Aortic Aneurysm.

B cell-activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

Characterizing Intra-Tumor and Inter-Tumor Variability of Immune Cell Infiltrates in Murine Syngeneic Tumors.

Murine tumors are indispensable model systems in preclinical immuno-oncology research. While immunologic heterogeneity is well-known to be an important factor that can influence treatment outcome, there is a severe paucity of data concerning the nature of this heterogeneity in murine tumor models. Using serial sectioning methodology combined with IHC analysis and whole-slide image analysis, the depth-dependent variation in immune-cell abundance in tumor specimens was investigated at single-cell resolution. Specifically, intra- and intertumor variability in cell density of nine immune-cell biomarkers was quantified in multiple murine tumor models. The analysis showed that intertumor variability was typically the dominant source of variation in measurements of immune-cell densities. Statistical power analysis revealed the effect of group size and variance in immune-cell density on the predictive power of detecting a statistically meaningful fold-change in immune-cell density. Intertumor variability in the ratio of immune-cell densities showed distinct patterns in select tumor models and revealed the existence of strong correlations between select biomarker pairs. Furthermore, the relative proportion of immune cells at different depths across tumor samples was preserved in some but not all tumor models, thereby revealing the existence of compositional heterogeneity. Taken together, these results reveal novel insights into the nature of immunologic heterogeneity, which is not accessible through typical omics approaches.

Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation.

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.

Exosomes mediate LTB4 release during neutrophil chemotaxis.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.

Sequential expression of neutrophil chemoattractants in cerebrospinal fluid after subarachnoid hemorrhage.

OBJECTIVE: Neutrophils induce inflammation through the exocytosis of cytotoxic granule proteins. Recently, neutrophils have been reported to be an independent parameter associated with unfavorable outcomes after subarachnoid hemorrhage (SAH). However, the mechanism by which neutrophils accumulate within the CSF after SAH remains undetermined. METHODS: Concentrations of C5a, epithelial neutrophil activating peptide 78 (ENA-78), interleukin-8 (IL-8), growth-regulated oncogene-α (GRO-α), neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase (MPO) were measured serially until day 14 in the CSF of 10 patients with SAH. CSF samples obtained from patients suffering from unruptured aneurysms were used as controls. RESULTS: The concentrations of C5a and ENA-78 were significantly increased on day 1, while those of IL-8 and GRO-α significantly increased during days 3-7 compared with those of the control samples. The levels of NGAL and MPO, components of neutrophil granules, significantly increased during days 1-5 and days 1-3, respectively, after SAH and gradually decreased thereafter. The correlations between ENA-78 and C5a on day 1, IL-8 and GRO-α on days 3-7, and NGAL and MPO on days 1-3 were significant. CONCLUSION: These neutrophil chemoattractants might be serially involved in the infiltration of neutrophils into the CSF after SAH. Migrated neutrophils play an important role in inflammatory reactions in the central nervous system after SAH.

Methamphetamine facilitates pulmonary and splenic tissue injury and reduces T cell infiltration in C57BL/6 mice after antigenic challenge.

Methamphetamine (METH) is a strong addictive central nervous system stimulant. METH abuse can alter biological processes and immune functions necessary for host defense. The acquisition and transmission of HIV, hepatitis, and other communicable diseases are possible serious infectious consequences of METH use. METH also accumulates extensively in major organs. Despite METH being a major public health and safety problem globally, there are limited studies addressing the impact of this popular recreational psychostimulant on tissue adaptive immune responses after exposure to T cell dependent [ovalbumin (OVA)] and independent [lipopolysaccharide (LPS)] antigens. We hypothesized that METH administration causes pulmonary and splenic tissue alterations and reduces T cell responses to OVA and LPS in vivo, suggesting the increased susceptibility of users to infection. Using a murine model of METH administration, we showed that METH causes tissue injury, apoptosis, and alters helper and cytotoxic T cell recruitment in antigen challenged mice. METH also reduces the expression and distribution of CD3 and CD28 molecules on the surface of human Jurkat T cells. In addition, METH decreases the production of IL-2 in these T-like cells, suggesting a negative impact on T lymphocyte activation and proliferation. Our findings demonstrate the pleotropic effects of METH on cell-mediated immunity. These alterations have notable implications on tissue homeostasis and the capacity of the host to respond to infection.

Controlling T cells spreading, mechanics and activation by micropatterning.

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.

Arhgef6 (alpha-PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence.

Immunity is governed by successful T cell migration, optimized to enable a T cell to fully scan its environment without wasted movement by balancing speed and turning. Here we report that the Arhgef6 RhoGEF (aka alpha-PIX; αPIX; Cool-2), an activator of small GTPases, is required to restrain cell migration speed and cell turning during spontaneous migration on 2D surfaces. In Arhgef6-/- T cells, expression of Arhgef7 (beta-PIX; ßPIX; Cool-1), a homolog of Arhgef6, was increased and correlated with defective activation and localization of Rac1 and CDC42 GTPases, respectively. Downstream of Arhgef6, PAK2 (p21-activated kinase 2) and LIMK1 phosphorylation was reduced, leading to increased activation of Cofilin, the actin-severing factor. Consistent with defects in these signaling pathways, Arhgef6-/- T cells displayed abnormal bilobed lamellipodia and migrated faster, turned more, and arrested less than wild-type (WT) T cells. Using pharmacologic inhibition of LIMK1 (LIM domain kinase 1) to induce Cofilin activation in WT T cells, we observed increased migration speed but not increased cell turning. In contrast, inhibition of Cdc42 increased cell turning but not speed. These results suggested that the increased speed of the Arhgef6-/- T cells is due to hyperactive Cofilin while the increased turning may be due to abnormal GTPase activation and recruitment. Together, these findings reveal that Arhgef6 acts as a repressor of T cell speed and turning by limiting actin polymerization and lamellipodia formation.

Pannexin Channel Regulation of Cell Migration: Focus on Immune Cells.

The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.

High Endothelial Venules Accelerate Naive T Cell Recruitment by Tumor Necrosis Factor-Mediated R-Ras Upregulation.

Recruitment of naive T cells to lymph nodes is essential for the development of adaptive immunity. Upon pathogen infection, lymph nodes promptly increase the influx of naive T cells from the circulation in order to screen and prime the T cells. The precise contribution of the lymph node vasculature to the regulation of this process remains unclear. Here we show a role for the Ras GTPase, R-Ras, in the functional adaptation of high endothelial venules to increase naive T cell trafficking to the lymph nodes. R-Ras is transiently up-regulated in the endothelium of high endothelial venules by the inflammatory cytokine tumor necrosis factor (TNF) within 24 hours of pathogen inoculation. TNF induces R-Ras upregulation in endothelial cells via JNK and p38 mitogen-activated protein kinase but not NF-κB. Studies of T cell trafficking found that the loss of function of endothelial R-Ras impairs the rapid acceleration of naive T cell recruitment to the lymph nodes upon inflammation. This defect diminished the ability of naive OT-1 T cells to develop antitumor activity against ovalbumin-expressing melanoma. Proteomic analyses suggest that endothelial R-Ras facilitates TNF-dependent transendothelial migration (diapedesis) of naive T cells by modulating molecular assembly the at T cell-endothelial cell interface. These findings give new mechanistic insights into the functional adaptation of high endothelial venules to accelerate naive T cell recruitment to the lymph nodes.

Glucocorticoids Regulate Circadian Rhythm of Innate and Adaptive Immunity.

Animals have evolved circadian rhythms to adapt to the 24-h day-night cycle. Circadian rhythms are controlled by molecular clocks in the brain and periphery, which is driven by clock genes. The circadian rhythm is propagated from the brain to the periphery by nerves and hormones. Glucocorticoids (GCs) are a class of steroid hormones produced by the adrenal cortex under the control of the circadian rhythm and the stress. GCs have both positive and negative effects on the immune system. Indeed, they are well known for their strong anti-inflammatory and immunosuppressive effects. Endogenous GCs inhibit the expression of inflammatory cytokines and chemokines at the active phase of mice, regulating the circadian rhythm of tissue inflammation. In addition, GCs induce the rhythmic expression of IL-7R and CXCR4 on T cells, which supports T cell maintenance and homing to lymphoid tissues. Clock genes and adrenergic neural activity control the T cell migration and immune response. Taken together, circadian factors shape the diurnal oscillation of innate and adaptive immunity. Among them, GCs participate in the circadian rhythm of innate and adaptive immunity by positive and negative effects.

Tumour-associated neutrophils orchestrate intratumoural IL-8-driven immune evasion through Jagged2 activation in ovarian cancer.

BACKGROUND: Tumour associated neutrophils (TANs) play a controversial role in regulating immune surveillance and immune evasion in various malignancies. Here, we investigated the relevance of TANs with the prognosis and immune microenvironment of epithelial ovarian cancer (EOC). METHODS: We characterised TANs using flow cytometric analysis and immunofluorescence analysis. The prognostic merit of TANs in EOC was evaluated using cox regression analysis. Furthermore, we explored the therapeutic merit of targeting Notch signalling in EOC and determined its involvement in the immune microenvironment. RESULTS: High level of TANs is associated with a dismal prognosis and immune tolerance in EOC. TANs impaired cytotoxic effects of CD8+ T cells partly through Jagged2 (JAG2). Notch pathway blocked using γ-secretase inhibitor LY3039478 and anti-JAG2 antibody led to retarded tumour growth and augmented cytotoxic effects of CD8+ T cells. IL-8 contributes to the recruitment of TANs and the induction of JAG2 expression in TANs. Blockade of CXCR2 signalling reduces tumour growth rate, accompanied by a decreasing amount of TANs and increasing activity of CD8+ T cells. JAG2+TANs is an independent predictor of clinical outcomes. CONCLUSION: JAG2+TANs are closely linked to IL-8-driven immune evasion microenvironment and may serve as a promising therapeutic target for the reinvigoration of anti-tumour immunity.