Julianne Jorgensen

Neutrophil Interactions Stimulate Evasive Hyphal Branching by Aspergillus fumigatus

Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching.

Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay

Current methods for the diagnosis of sepsis have insufficient precision, causing regular misdiagnoses. Microbiological tests can help to diagnose sepsis, but are usually too slow to have an impact on timely clinical decision-making. Neutrophils have a high sensitivity to infections, yet measurements of neutrophil surface markers, genomic changes and phenotype alterations have had only a marginal effect on sepsis diagnosis. Here, we report a microfluidic assay that measures, from one droplet of diluted blood, the spontaneous motility of neutrophils in the presence of plasma. We measured the performance of the assay in two independent cohorts of critically ill patients suspected of sepsis. Using data from a first cohort, we developed a machine-learning-based scoring system (sepsis score) that segregated patients with sepsis from those without sepsis. We then validated the sepsis score in a double-blind, prospective case–control study. For the 42 patients across the two cohorts, the assay identified sepsis patients with 97% sensitivity and 98% specificity. The neutrophil assay could potentially be used to accurately diagnose and monitor sepsis in larger populations of at-risk patients.A microfluidic assay that identifies sepsis from a single droplet of diluted blood by measuring the spontaneous motility of neutrophils showed 97% sensitivity and 98% specificity in two independent patient cohorts.

Neutrophil chemotaxis and transcriptomics in term and preterm neonates

&NA; Neutrophils play a crucial role in combating life‐threatening bacterial infections in neonates. Previous studies investigating neonatal cell function have been limited because of restricted volume sampling. Here, using novel microfluidic approaches, we provide the first description of neutrophil chemotaxis and transcriptomics from whole blood of human term and preterm neonates, as well as young adults. Ex vivo percent cell migration, neutrophil velocity, and directionality to N‐formylmethionyl‐leucyl‐phenylalanine were measured from whole blood using time‐lapse imaging of microfluidic chemotaxis. Genome‐wide expression was also evaluated in CD66b+ cells using microfluidic capture devices. Neutrophils from preterm neonates migrated in fewer numbers compared to term neonates (preterm 12.3%, term 30.5%, P = 0.008) and at a reduced velocity compared to young adults (preterm 10.1 &mgr;m/min, adult 12.7 &mgr;m/min, P = 0.003). Despite fewer neutrophils migrating at slower velocities, neutrophil directionality from preterm neonates was comparable to adults and term neonates. 3607 genes were differentially expressed among the 3 groups (P < 0.001). Differences in gene expression between neutrophils from preterm and term neonates were consistent with reduced pathogen recognition and antimicrobial activity but not neutrophil migration, by preterm neonates. In summary, preterm neonates have significant disturbances in neutrophil chemotaxis compared to term neonates and adults, and these differences in phenotype appear at the transcriptional level to target inflammatory pathways in general, rather than in neutrophil migration and chemotaxis.

Temporal gradients limit the accumulation of neutrophils toward sources of chemoattractant

Neutrophil trafficking during inflammation is a highly orchestrated process, coordinating neutrophil recruitment, sterilization of the wound, and inflammation resolution. Although the chemotactic signals guiding neutrophil recruitment to sites of inflammation are relatively well understood, our knowledge of mechanisms controlling cessation of neutrophil recruitment and return to normal tissue physiology remains incomplete. To gain insights into these processes, we designed a microfluidic device with an array of chemoattractant reservoirs, which mimics the microenvironment in infected tissues, when multiple clusters of microbes are present. We monitored the temporal dynamics of neutrophil recruitment toward the chemoattractant reservoirs at single cell resolution, for 3 h. We observed robust neutrophil recruitment that reached a plateau after 1.5 h, despite the continuous presence of strong chemoattractant gradients around the reservoirs. The timing of the plateau was dependent on the geometry of the devices and was independent from the number of neutrophils. On the basis of these observations, we ruled out sub-population sensitivity, chemoattractant scavenging, and production of a self-limiting stop signal as potential mechanisms underpinning the plateau in neutrophil recruitment. We found a strong correlation between the temporal stabilization of concentration changes and the plateau in neutrophils recruitment. These results suggest that dynamic aspects of chemoattractant gradients are key for maximizing recruitment during the acute phase of infections and limiting the accumulation of neutrophils as soon as the infection is contained.

Convergent and Divergent Migratory Patterns of Human Neutrophils inside Microfluidic Mazes

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.

Trapped Chromatin Fibers Damage Flowing Red Blood Cells

Neutrophils are the most abundant white blood cells in the circulation and serve antimicrobial functions. One of their antimicrobial mechanisms involves the release of neutrophil extracellular traps (NETs), long chromatin fibers decorated with antimicrobial granular proteins that contribute to the elimination of pathogens. However, the release of NETs has also been associated with disease processes. While recent research has focused on biochemical reactions catalyzed by NETs, significantly less is known about the mechanical effect of NETs in circulation. Here, microfluidic devices and biophysical models are employed to study the consequences of the interactions between NETs trapped in channels and red blood cells (RBCs) flowing in blood over the NETs. It has been found that the RBCs can be deformed and ruptured after interactions with NETs, generating RBC fragments. Significant increases in the number of RBC fragments have also been found in the circulation of patients with conditions in which NETs have been demonstrated to be present in circulation, including sepsis and kidney transplant. Further studies will probe the potential utility of RBC fragments in the diagnostic, monitoring, and treatment of diseases associated with the presence of NETs in circulation.