Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyze four qualitative migration patterns: chemo-attraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence, and fraction of cells responding. We find that established chemokines C5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T-lymphocytes migrate away than towards SDF-1 and their directional migration patterns are not persistent. Overall, our platform characterizes migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

No abstract

Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed in multiple tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes, and its in-depth effects on leukocyte migration is poorly understood. Here, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4+ T-cells. Neogenin, UNC5A and UNC5B were expressed at low levels in unstimulated cells, but increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A-B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bi-directional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4+ T-cells, but it had no demonstrable effects on the migration of purified CD4+CD25+CD127dim T regulatory cells. Furthermore, using a shRNA knockdown approach, we observed that the pro-migratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3+ T cell infiltrates. Neogenin was also observed on CD3+ T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4+ T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.

Capillary plexuses are commonly regarded as reliable networks for blood flow and robust oxygen delivery to hypoxia sensitive tissues. They have high levels of redundancy to assure adequate blood supply after when one or more of the capillaries in the network are blocked by a clot. However, despite having extensive capillary plexuses, many vital organs are often subject to secondary organ injury in patients with severe inflammation. Recent studies have suggested that neutrophils play a role in this pathology, even though their precise contribution remains elusive. Here we investigate the effect of chromatin fibres released from overly-activated neutrophils (neutrophil extracellular traps, NETs) on the flow of blood through microfluidic networks of channels replicating geometrical features of capillary plexuses. In an in vitro setting, we show that NETs can decouple the traffic of red blood cells from that of plasma in microfluidic networks. The effect is astonishingly disproportionate, with NETs from less than 200 neutrophils resulting in more than half of a 0.6 mm2 microfluidic network to become void of red blood cell traffic. Importantly, the NETs are able to perturb the blood flow in capillary networks despite the presence of anti-coagulants. If verified to occur in vivo, this finding could represent a novel mechanism for tissue hypoxia and secondary organ injury during severe inflammation in patients already receiving antithrombotic and anticoagulant therapies.

The migration of T-cell subsets within peripheral tissues is characteristic of inflammation and immunoregulation. In general, the lymphocyte migratory response is assumed directional and guided by local gradients of chemoattractants and/or chemorepellents. However, little is known about how cells explore their tissue environment, and whether lymphocyte activation may influence speed and exploratory patterns of migration. To probe migration patterns by T-cells we designed a microfluidic maze device that replicates critical features of a tissue-like microenvironment. We quantified the migration patterns of unstimulated and mitogen-activated human T-cells at single cell resolution and found significant differences in exploration within microfluidic mazes. While unstimulated lymphocytes migrated in a directed manner, activated T-cells migrated through large areas of the mazes in an exploratory pattern in response to the chemoattractants RANTES (CCL5) and IP-10 (CXCL10). The analysis of migration enabled by the microfluidic devices help develop new methods for determining how human circulating T-cells function in vivo to seek out antigens in health and disease states.

Purpose of review New developments suggest that the graft itself, and molecules expressed within the graft microenvironment dictate the phenotype and evolution of chronic rejection. Recent findings Once ischemia-reperfusion injury, cellular and humoral immune responses target the microvasculature, the associated local tissue hypoxia results in HIF-1α-dependent expression of pro-inflammatory and pro-angiogenic growth factors including vascular endothelial growth factor (VEGF) as a physiological response to injury. However, these genes, and notably VEGF, can promote the recruitment of alloimune T effectors and T regulatory cells into the graft. Semaphorin family molecules may also bind to neuropilin-1 on T cell regulatory cell subsets to stabilize functional responses. mTOR/Akt signaling within endothelial cells regulates cytokineand alloantibody-induced activation and proliferation and their pro-inflammatory phenotype. Inhibition of mTOR and/or Akt results in an anti-inflammatory phenotype and enables the expression of coinhibitory molecules that limit local T cell reactivation and promote immunoregulation. Ligation of neuropilin-1 on T regulatory cells also inhibits Akt-induced responses suggesting common theme for enhancing local immunoregulation and long-term graft survival. Summary Events within the graft initiated by changes within the microvasculature and mTOR/Akt-induced signaling promotes the development of chronic rejection. Semaphorin-neuropilin biology represents a novel avenue for targeting this biology and warrents further investigation.

Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways.Neutrophils are guided towards their target location within tissues by a broad range of chemoattractants, including bacterial products such as N-Formyl-Met-Leu-Phe (fMLF), complement factors such as component 5a (C5a), tissue derived cytokines such as interleukin-8 (IL-8), and leukocyte-released lipid mediators such as Leukotriene B4 (LTB4) [1][2][3][4] . Several of these chemoattractants can be present in a tissue simultaneously. Thus, neutrophils must integrate all signals to migrate effectively. For example, fMLF gradients around bacterial targets are assumed to elicit rapid and directional chemotaxis of neutrophils in the close vicinity of the source 1 . C5a, which is formed when complement component 5 is cleaved by infectious pathogens 5 , is similarly assumed to elicit a rapid, short range chemotaxis response. fMLF and C5a have thus been named end-target chemoattractants. Meanwhile, LTB4 and IL-8 released by multiple cell types during inflammation 4,6,7 are thought to elicit neutrophil recruitment from larger distances, towards the areas of the tissue under stress. They are often called intermediary chemoattractants. However, it is still not completely understood how neutrophils integrate responses to multiple chemoattractants that they encounter during their migration from blood into sites of inflammation [8][9][10] . The current paradigm for neutrophil responses to multiple chemoattractants has evolved from observations of their response to opposing gradients and indicate that neutrophils prioritize between different groups of c...

Advances in therapeutics have dramatically improved short-term graft survival, while the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection. Key changes include EC injury, activation and uncontrolled leukocyte-induced angiogenesis. We propose that all of these changes may lead to abnormal blood flow patterns, local tissue hypoxia and an associated overexpression of HIF-1α-inducible genes including Vascular Endothelial Growth Factor. We also discuss how regulators of mTOR-mediated signaling within EC are of critical importance in microvascular stability and the inhibition of chronic rejection. Finally, we discuss recent findings indicating that miRNAs regulate EC stability, and their potential as novel non-invasive biomarkers of allograft rejection. Altogether, this review provides insights into molecular events, genes and signals that promote chronic rejection and their potential as biomarkers for the future development of interruption therapeutics.