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Neutrophil migration is essential for inflammatory responses to kill pathogens; however, excessive neutrophilic inflammation also leads to tissue injury and adverse effects. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified 8 miRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (Cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Our results therefore reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide directions in alleviating systemic inflammation.

Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) provide a genetically tractable system to study host defense responses of human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus . GATA1-knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and have improved neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal β-glucan to the pentose phosphate pathway (PPP), similar to primary human neutrophils. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the metabolic shift . Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response .

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility 1 . Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases 2 . However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.

Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D.

Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against . GATA1 knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal β-glucan, including an upregulation of the pentose phosphate pathway (PPP), similar to primary human neutrophils . Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill but is not necessary for the upregulation of PPP. Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Neutropenic patients are at high risk for fungal and bacterial infections and can benefit from granulocyte transfusion therapy. Human induced pluripotent stem cells (iPSCs) could provide a robust source of neutrophil-like cells for infusion as they can be generated in large quantities and do not require a donor. However, dampened intracellular signaling limits their cellular activation and response. Here, we show that we can engineer iPSC-derived neutrophils (iNeutrophils) for enhanced motility and anti-microbial functions. Deletion of the PTP1B phosphatase increased iNeutrophil PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration and phagocytosis. PTP1B deletion also increased production of inflammatory cytokines, including the neutrophil chemoattractant IL-8. Furthermore, PTP1B-KO iNeutrophils displayed a highly activated morphology and were more responsive to the fungal pathogen Aspergillus fumigatus. KO iNeutrophils efficiently migrated to and swarmed hyphae resulting in inhibition of fungal growth. Taken together, deletion of the PTP1B phosphatase removes the “brakes” on iPSC-derived intracellular signaling and neutrophil function. Deletion of PTP1B increases iPSC-derived neutrophil intracellular signaling to improve motility and phagocytosis. Deletion of PTP1B enhances iPSC-derived neutrophil swarming response and ability to inhibit fungal growth.

Calcium signaling is an important early step in wound healing, yet how these early signals promote regeneration remains unclear. Peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes, catalyze citrullination, a post-translational modification that alters protein function and has been implicated in autoimmune diseases. We generated a mutation in the single zebrafish ancestral pad gene, padi2, resulting in a loss of detectable calcium-dependent citrullination. The padi2 mutants exhibit impaired resolution of inflammation and regeneration after caudal fin transection. Further, we identified a new subpopulation of cells displaying citrullinated histones within the notochord bead following tissue injury. Citrullination of histones in this region was absent and wound-induced proliferation was perturbed in Padi2-deficient larvae. Taken together, our results show that Padi2 is required for the citrullination of histones within a group of cells in the notochord bead, and for promoting wound-induced proliferation required for efficient regeneration. These findings identify Padi2 as a potential intermediary between early calcium signaling and subsequent tissue regeneration. Golenberg et al. developed a citrullination-deficient zebrafish and demonstrated a role for Padi2 in fin wound responses and regeneration. This work identified a distinct population of cells within the regenerative notochord bead that exhibited wound-induced histone citrullination.

Neutrophil migration is essential for inflammatory responses to kill pathogens and cause tissue injury. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA overexpression screen in zebrafish, and identified eight microRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting CDK2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils. Chemotaxis of primary human neutrophils are also reduced by CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Together, our results reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide new directions in alleviating systemic inflammation. Footnotes * https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127174

Leukocytes forge paths through interstitial spaces by exerting forces to overcome confining mechanical pressures provided by surrounding cells. While such mechanical cues regulate leukocyte motility, engineering an in vitro system that models the deformable cellular environment encountered in vivo has been challenging. Here, microchannels are constructed with a liquid‐liquid interface that exerts confining pressures similar to cells in tissues, and thus, is deformable by cell‐generated forces. Consequently, the balance between migratory cell‐generated and interfacial pressures determines the degree of confinement. Pioneer cells that first contact the interfacial barrier require greater deformation forces to forge a path for migration, and as a result migrate slower than trailing cells. Critically, resistive pressures are tunable by controlling the curvature of the liquid interface, which regulates motility. By granting cells autonomy in determining their confinement, and tuning environmental resistance, interfacial deformations match those of surrounding cells in vivo during interstitial neutrophil migration in a larval zebrafish model. It is discovered that neutrophils employ a bleb‐based mechanism of force generation to deform a soft barrier exerting cell‐scale confining pressures. In all, this work introduces a tunable in vitro material interface that replicates confining pressures applied by soft tissue environments. Leukocytes sense mechanical cues provided by surrounding cells to regulate motility. Replicating the confining pressures applied by soft, elastic tissues in vitro is challenging. Here, the use of liquid‐liquid interfaces is introduced to model forces exerted by surrounding cells. Confining pressures are tunable by controlling interfacial curvature, and replicate confinement observed during neutrophil interstitial motility in vivo. Interaction with the interface induces a transition from actin‐rich pseudopodial migration to a bleb‐based mechanism of force‐generation to deform an interface exerting physiologically relevant mechanical resistance.

Surgical site infection (SSI) remains the most common complication of surgery around the world. WHO does not make recommendations for changing gloves and instruments before wound closure owing to a lack of evidence. This study aimed to test whether a routine change of gloves and instruments before wound closure reduced abdominal SSI. ChEETAh was a multicentre, cluster randomised trial in seven low-income and middle-income countries (Benin, Ghana, India, Mexico, Nigeria, Rwanda, South Africa). Any hospitals (clusters) doing abdominal surgery in participating countries were eligible. Clusters were randomly assigned to current practice (42) versus intervention (39; routine change of gloves and instruments before wound closure for the whole scrub team). Consecutive adults and children undergoing emergency or elective abdominal surgery (excluding caesarean section) for a clean–contaminated, contaminated, or dirty operation within each cluster were identified and included. It was not possible to mask the site investigators, nor the outcome assessors, but patients were masked to the treatment allocation. The primary outcome was SSI within 30 days after surgery (participant-level), assessed by US Centers for Disease Control and Prevention criteria and on the basis of the intention-to-treat principle. The trial has 90% power to detect a minimum reduction in the primary outcome from 16% to 12%, requiring 12 800 participants from at least 64 clusters. The trial was registered with ClinicalTrials.gov, NCT03700749. Between June 24, 2020 and March 31, 2022, 81 clusters were randomly assigned, which included a total of 13 301 consecutive patients (7157 to current practice and 6144 to intervention group). Overall, 11 825 (88·9%) of 13 301 patients were adults, 6125 (46·0%) of 13 301 underwent elective surgery, and 8086 (60·8%) of 13 301 underwent surgery that was clean–contaminated or 5215 (39·2%) of 13 301 underwent surgery that was contaminated–dirty. Glove and instrument change took place in 58 (0·8%) of 7157 patients in the current practice group and 6044 (98·3%) of 6144 patients in the intervention group. The SSI rate was 1280 (18·9%) of 6768 in the current practice group versus 931 (16·0%) of 5789 in the intervention group (adjusted risk ratio: 0·87, 95% CI 0·79–0·95; p=0·0032). There was no evidence to suggest heterogeneity of effect across any of the prespecified subgroup analyses. We did not anticipate or collect any specific data on serious adverse events. This trial showed a robust benefit to routinely changing gloves and instruments before abdominal wound closure. We suggest that it should be widely implemented into surgical practice around the world. National Institute for Health Research (NIHR) Clinician Scientist Award, NIHR Global Health Research Unit Grant, Mölnlycke Healthcare.