The axillary lymphoid organ - an external, experimentally accessible immune organ in the zebrafish.

Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs make it challenging to image and study them in living animals. Here, we describe a previously uncharacterized external immune organ in the zebrafish ideally suited for studying immune cell dynamics in vivo, the axillary lymphoid organ (ALO). This small, translucent organ has an outer cortex teeming with immune cells, an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the ALO is readily accessible to external treatment. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.

Single-cell Transcriptional Landscape of Temporal Neutrophil Response to Burn Wound in Larval Zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.

Advancing chemokine research: the molecular function of CXCL8.

CXCL8 and other chemokines have been implicated in tissue inflammation and are attractive candidates for therapeutic targeting to treat human disease.

Infection induced inflammation impairs wound healing through IL-1ß signaling.

Wound healing is impaired by infection; however, how microbe-induced inflammation modulates tissue repair remains unclear. We took advantage of the optical transparency of zebrafish and a genetically tractable microbe, Listeria monocytogenes, to probe the role of infection and inflammation in wound healing. Infection with bacteria engineered to activate the inflammasome, Lm-Pyro, induced persistent inflammation and impaired healing despite low bacterial burden. Inflammatory infections induced il1b expression and blocking IL-1R signaling partially rescued wound healing in the presence of persistent infection. We found a critical window of microbial clearance necessary to limit persistent inflammation and enable efficient wound repair. Taken together, our findings suggest that the dynamics of microbe-induced tissue inflammation impacts repair in complex tissue damage independent of bacterial load, with a critical early window for efficient tissue repair.

Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. Here, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-day time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds the molecular foundation and a comparative single-cell genomic framework to identify neutrophil markers of tissue damage using model organisms.

PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited, as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable. However, current work has indicated that dampened intracellular signaling limits iPSC-derived neutrophil (iNeutrophil) cellular activation and antimicrobial response. Here, we show that protein tyrosine phosphatase 1B (PTP1B) inhibits intracellular signaling and dampens iNeutrophil effector function. Deletion of the PTP1B phosphatase increased PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration, and phagocytosis. In contrast, other effector functions like NETosis and reactive oxygen species production were reduced. PTP1B-deficient neutrophils were more responsive to Aspergillus fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine interleukin-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Neutrophil motility is regulated by both cell intrinsic and endothelial cell ARPC1B.

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.

The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo.

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: (i) Short-term storage (< 8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; (ii) Neutrophils from long-term storage (> 24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. (iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.

Liquid-liquid interfaces enable tunable cell confinement to recapitulate surrounding tissue deformations during neutrophil interstitial migration in vivo.

Cell migration is regulated by an interplay between both chemical and mechanical cues. Immune cells navigate through interstitial spaces and generate forces to deform surrounding cells, which in turn exert opposing pressures that regulate cell morphology and motility mechanisms. Current in vitro systems to study confined cell migration largely utilize rigid materials orders of magnitude stiffer than surrounding cells, limiting insights into how these local physical interactions regulate interstitial cell motility. Here, we first characterize mechanical interactions between neutrophils and surrounding cells in larval zebrafish and subsequently engineer in vitro migration channels bound by a deformable liquid-liquid interface that responds to cell generated pressures yielding a gradient of confinement across the length of a single cell. Tuning confining pressure gradients replicates mechanical interactions with surrounding cells during interstitial migration in vivo . We find that neutrophils favor a bleb-based mechanism of force generation to deform a barrier applying cell-scale confining forces. This work introduces a biomimetic material interface that enables new avenues of exploring the influence of mechanical forces on cell migration.