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 ROS production were reduced. PTP1B-deficient neutrophils were more responsive to A. fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine IL-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Cell Type-Specific Transcriptome Profiling Reveals a Role for Thioredoxin During Tumor Initiation.

Neutrophils in the tumor microenvironment exhibit altered functions. However, the changes in neutrophil behavior during tumor initiation remain unclear. Here we used Translating Ribosomal Affinity Purification (TRAP) and RNA sequencing to identify neutrophil, macrophage and transformed epithelial cell transcriptional changes induced by oncogenic RasG12V in larval zebrafish. We found that transformed epithelial cells and neutrophils, but not macrophages, had significant changes in gene expression in larval zebrafish. Interestingly, neutrophils had more significantly down-regulated genes, whereas gene expression was primarily upregulated in transformed epithelial cells. The antioxidant, thioredoxin (txn), a small thiol that regulates reduction-oxidation (redox) balance, was upregulated in transformed keratinocytes and neutrophils in response to oncogenic Ras. To determine the role of thioredoxin during tumor initiation, we generated a zebrafish thioredoxin mutant. We observed an increase in wound-induced reactive oxygen species signaling and neutrophil recruitment in thioredoxin-deficient zebrafish. Transformed keratinocytes also showed increased proliferation and reduced apoptosis in thioredoxin-deficient larvae. Using live imaging, we visualized neutrophil behavior near transformed cells and found increased neutrophil recruitment and altered motility dynamics. Finally, in the absence of neutrophils, transformed keratinocytes no longer exhibited increased proliferation in thioredoxin mutants. Taken together, our findings demonstrate that tumor initiation induces changes in neutrophil gene expression and behavior that can impact proliferation of transformed cells in the early tumor microenvironment.

Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.

Neutrophils are cells at the frontline of innate immunity that can quickly activate effector functions to eliminate pathogens upon stimulation. However, little is known about the metabolic adaptations that power these functions. Here we show rapid metabolic alterations in neutrophils upon activation, particularly drastic reconfiguration around the pentose phosphate pathway, which is specifically and quantitatively coupled to an oxidative burst. During this oxidative burst, neutrophils switch from glycolysis-dominant metabolism to a unique metabolic mode termed 'pentose cycle', where all glucose-6-phosphate is diverted into oxidative pentose phosphate pathway and net flux through upper glycolysis is reversed to allow substantial recycling of pentose phosphates. This reconfiguration maximizes NADPH yield to fuel superoxide production via NADPH oxidase. Disruptions of pentose cycle greatly suppress oxidative burst, the release of neutrophil extracellular traps and pathogen killing by neutrophils. Together, these results demonstrate the remarkable metabolic flexibility of neutrophils, which is essential for their functions as the first responders in innate immunity.

Immune Cell Paracrine Signaling Drives the Neutrophil Response to A. fumigatus in an Infection-on-a-Chip Model.

INTRODUCTION: Neutrophils act as first responders during an infection, following signals from the pathogen as well as other host cells to migrate from blood vessels to the site of infection. This tightly regulated process is critical for pathogen clearance and, in many cases, eliminates the pathogen without the need for an additional immune response. It is, therefore, critical to understand what signals drive neutrophil migration to infection in a physiologically relevant environment. METHODS: In this study, we used an infection-on-a-chip model to recapitulate many important aspects of the infectious microenvironment including an endothelial blood vessel, an extracellular matrix, and the environmental fungal pathogen Aspergillus fumigatus. We then used this model to visualize the innate immune response to fungal infection. RESULTS: We found that A. fumigatus germination dynamics are influenced by the presence of an endothelial lumen. Furthermore, we demonstrated that neutrophils are recruited to and swarm around A. fumigatus hyphae and that the presence of monocytes significantly increases the neutrophil response to A. fumigatus. Using secreted protein analysis and blocking antibodies, we found that this increased migration is likely due to signaling by MIP-1 family proteins. Finally, we demonstrated that signal relay between neutrophils, mediated by LTB4 signaling, is also important for sustained neutrophil migration and swarming in response to A. fumigatus infection in our system. CONCLUSIONS: Taken together, these results suggest that paracrine signaling from both monocytes and neutrophils plays an important role in driving the neutrophil response to A. fumigatus.

Neutrophil plasticity in the tumor microenvironment.

Neutrophils act as the body's first line of defense against infection and respond to diverse inflammatory cues, including cancer. Neutrophils display plasticity, with the ability to adapt their function in different inflammatory contexts. In the tumor microenvironment, neutrophils have varied functions and have been classified using different terms, including N1/N2 neutrophils, tumor-associated neutrophils, and polymorphonuclear neutrophil myeloid-derived suppressor cells (PMN-MDSCs). These populations of neutrophils are primarily defined by their functional phenotype, because few specific cell surface markers have been identified. In this review, we will discuss neutrophil polarization and plasticity and the function of proinflammatory/anti-inflammatory and protumor/antitumor neutrophils in the tumor microenvironment. We will also discuss how neutrophils with the ability to suppress T-cell activation, referred to by some as PMN-MDSCs, fit into this paradigm.

Long-Term Persistence of Anti-HIV Broadly Neutralizing Antibody-Secreting Hematopoietic Cells in Humanized Mice.

Broadly neutralizing antibodies (bNAbs) are among the most promising strategies to achieve long-term control of HIV-1 in the absence of combination antiretroviral therapy. Passive administration of such antibodies in patients efficiently decreases HIV-1 viremia, but is limited by the serum half-life of the protein. Here, we investigated whether antibody-secreting hematopoietic cells could overcome this problem. We genetically modified human CD34+ hematopoietic stem and progenitor cells (HSPCs) to secrete bNAbs and transplanted them into immunodeficient mice. We found that the gene-modified cells engraft and stably secrete antibodies in the peripheral blood of the animals for the 9 months of the study. Antibodies were predominantly expressed by human HSPC-derived T- and B cells. Importantly, we found that secreted PGT128 was able to delay HIV-1 viremia in vivo and also prevent a decline in CD4+ cells. Gene-modified cells were maintained in bone marrow and were also detected in spleen, thymus, lymph nodes, and gut-associated lymphoid tissue. These data indicate that the bNAb secretion from HSPC-derived cells in mice is functional and can affect viral infection and CD4+ cell maintenance. This study paves the way for potential applications to other diseases requiring long-lasting protein or antibody delivery.

A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.

Hematopoietic reconstitution after bone marrow transplantation is thought to be driven by committed multipotent progenitor cells followed by long-term engrafting hematopoietic stem cells (HSCs). We observed a population of early-engrafting cells displaying HSC-like behavior, which persisted long-term in vivo in an autologous myeloablative transplant model in nonhuman primates. To identify this population, we characterized the phenotype and function of defined nonhuman primate hematopoietic stem and progenitor cell (HSPC) subsets and compared these to human HSPCs. We demonstrated that the CD34+CD45RA-CD90+ cell phenotype is highly enriched for HSCs. This population fully supported rapid short-term recovery and robust multilineage hematopoiesis in the nonhuman primate transplant model and quantitatively predicted transplant success and time to neutrophil and platelet recovery. Application of this cell population has potential in the setting of HSC transplantation and gene therapy/editing approaches.