Jeremy Point Virus-ORF6: a novel viral chemokine induces neutrophil trafficking in a microfluidic device

Introduction: Neutrophils are critical to the body's defense mechanisms, primarily acting as first responders to infection and inflammation. These cells exhibit both chemotaxis and retrotaxis. Chemotaxis is the directed movement towards infection sites, in response to chemoattractant agents or chemokines such as IL-8, which binds to CXCR1 and CXCR2 receptors on neutrophils through its ELR-CXC motif (Figure1.A). Conversely, retrotaxis is the process by which neutrophils move away from the inflammatory site, a mechanism that regulates the resolution of inflammation and prevents excessive tissue damage. The recent discovery of Jeremy-Point virus (JPTV) unveiled a unique aspect of viral evolution: the virus encodes JPTV-ORF6, a protein mimicking the human chemokine structure, specifically the ELR-CXC sequence analogous to IL-8. This study explores the chemotactic capability of JPTV-ORF6 in directing neutrophil migration, employing microfluidic devices to accurately measure and compare the migration induced by IL-8 and JPTV-ORF6. Our microfluidic setup allows precise control and observation of the chemotactic and retrotactic responses of neutrophils, providing a clear insight into the potential of JPTV-ORF6 to mimic or interfere with natural immune responses. This comparative analysis not only enhances our understanding of viral manipulation of host immune pathways but also underscores the importance of advanced techniques like microfluidics in studying cellular responses under highly controlled conditions.

Materials and

Methods: We tested the ability of JPTV-ORF6 to induce migration in both a neutrophil cell line (dHL-60) and human primary neutrophils. dHL-60 cells were cultured in IMDM-FBS media followed by a five days differentiation using DMSO. Human primary neutrophils were isolated from freshly drawn blood from healthy donors. The isolation was done using STEM CELL kits. In both conditions, cells were used at a concentration of 5x10^7 cells/mL. We performed experiments using a microfluidic device designed to simulate physiological conditions of cell migration (Figure1B). This device consists of three main chambers, one in the middle and two on the sides, and interconnected by a set of migration channels. We screened different concentrations of JPTV-ORF6 ranging from 1 nM to 1000 nM. IL-8 was tested in the same range for comparison. We used fMLP as a chemoattractant positive control. To monitor cell migration, we used time-lapse imaging microscopy recording images every 2 minutes for 4 hours. Data were collected as video .ND2 files and were analyzed using NIS software to determine cell migration percentage, cell velocity and cell directionality. We performed statistical tests for comparison, including a Two-way ANOVA for parametric data and a Kruskal Wallis test for non-parametric data using R.

Results, Conclusions, and Discussions: dHL-60 (Figure1.C) and human primary neutrophil (Figure1.D) exposed to JPTV-ORF6 exhibited a significantly similar migratory pattern to IL-8 (p > 0.05). However, their migration towards the positive control, fMLP, was significantly stronger (p < 0.00001). Migration towards JPTV-ORF6 (chemotaxis, velocity) and migration away JPTV-ORF6 (velocity) were recorded. No significant difference was observed across all tested concentrations (p > 0.05) suggesting a non-dose-dependent behavior, which has been previously reported for IL-8 (Nat Commun 5, 4787 (2014)). At 10 nM, although dHL-60 cells migrated slower (8.14 ± 2.11 µm/min) (Figure 1E) and human primary neutrophils migrated faster (20.31 ± 4.14 µm/min) (Figure 1F), they migrated at a significantly same velocity across all concentrations (p > 0.05). Both dHL-60 and human primary neutrophils displayed the same directionality (average 0.33 ± 0.07 & 0.33 ± 0.09, p > 0.5). These findings confirm that JPTV-ORF6 is a chemoattractant like IL-8. With this ability to modulate neutrophil trafficking, JPTV-ORF6 expression could be a viral evasion strategy the JPTV uses to take control of its hosts by either infecting its cells or hiding from its immune defense. Thus, developing novel microfluidic tools to decipher immune cell-viral interactions will enable us to design and screen new therapeutic drugs.

Acknowledgements (Optional): National Institute of Health (NIH): 5R21AI156546-02
Jones Lab, UT Dallas / UTSW Medical Center
Allison Lab, UF