Man scripClick here to ie linked References SummaryClassically considered short-lived, purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that in the lungs occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands. circulation (Hidalgo et al., 2019) and reduced transcriptional activity preclude genetic adaptation to tissue environments (Silvestre-Roig et al., 2016). Existing evidence has shown, however, that cancer can instruct different transcriptional profiles, resulting in functions that can either promote, or counteract, tumoral growth and metastasis (Coffelt et al., 2016). Similar heterogeneous behavior has been reported in the context of stroke,

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A novel bacterium-free approach for rapid assembly of flavivirus infectious cDNAs using circular polymerase extension reaction was applied to generate infectious cDNA for the virulent New South Wales isolate of the Kunjin strain of West Nile virus (KUNV) that recently emerged in Australia. Recovered virus recapitulated the genetic heterogeneity present in the original isolate. The approach was utilized to generate viral mutants with designed phenotypic properties and to identify E protein glycosylation as one of the virulence determinants. Generation of flavivirus infectious clones has been traditionally hindered by the toxicity of their full-length cDNAs in bacteria. Various approaches have been employed to overcome this problem, including the use of very-low-copy-number plasmids (1, 2), bacterial artificial chromosomes (3), cosmid vectors (4), specific Escherichia coli strains (5, 6), mutation of cryptic bacterial promoter sites in the flavivirus genome (7), separation of the genome into two plasmids followed by in vitro ligation and RNA transcription (8)(9)(10)(11)(12), and use of a yeast recombination system to assemble full-length clones (13,14). All of these approaches require substantial efforts, are time-consuming, and are prone to introducing undesired mutations during amplification of plasmid DNA in bacteria and during in vitro RNA transcription by bacteriophage DNA-dependent RNA polymerases (T7 or SP6). A bacterium-free approach involving either in vitro ligation of two large overlapping cDNA fragments generated by reverse transcription and PCR (RT-PCR) or linking these two large cDNA fragments by fusion PCR was developed for tick-borne encephalitis virus (TBEV) (15). The resulting product containing the SP6 RNA polymerase promoter upstream of TBEV sequence was then used to produce RNA by in vitro transcription and virus was recovered by injecting in vitro-transcribed RNA into the brain of suckling mice. This approach allows rapid generation of infectious cDNA without a need for plasmid DNA amplification in bacteria; however, it still requires an in vitro RNA transcription step as well as the highly sensitive suckling mouse model to recover infectious virus. The former is prone to introduction of undesired mutations, and the latter is not applicable for flaviviruses that do not replicate well in mice (e.g., dengue viruses). We and others have previously developed infectious cDNA clones for West Nile virus (WNV) and Japanese encephalitis virus (JEV) in which the in vitro RNA transcription step is eliminated by replacing SP6 or T7 polymerase promoters with the cytomegalovirus (CMV) promoter, thus allowing transcription of viral RNA in cells directly from plasmid DNAs by the cellular RNA polymerase II (16-18). Although an infectious cDNA clone for the Kunjin strain of West Nile virus (KUNV) has been demonstrated to be relatively stable, further manipulations, including mutagenesis could render it less stable (A. Khromykh, unpublished results). To solve the problem of stability for CMV-based JEV and WNV i...

Summary Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We show active chromatin remodelling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses, and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Our study therefore represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b + CD15 + CD10 lo/– CD64 – band neutrophils and CD66b hi CD15 + CD10 lo/– CD64 +/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

COVID-19 is characterised by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterised by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2 specific T cells to elucidate markers which may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that whilst some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation.

Acute inflammation recruits neutrophils with a band-shaped nucleus to the circulation. This neutrophil population was recently shown to have superior antibacterial capacity. Early recruitment of banded neutrophils to an infection site will likely improve the outcome of the immune response, yet it critically depends on efficient migration. However, the current dogma states that the segmentation of the mature neutrophil nucleus has evolved to favor migration through narrow pores as found between endothelial cells and in the interstitium. Therefore, we hypothesized that banded neutrophils migrate less efficiently than neutrophils with segmented nuclei, whereas recently described neutrophils with hypersegmented nuclei would in turn migrate more efficiently. Acute inflammation was evoked in a human model of experimental endotoxemia to recruit neutrophil subsets with different nuclear segmentation to the circulation. To simulate migration toward an infection site, migration of the subsets was studied in in vitro models of transendothelial migration or interstitial chemokinesis and chemotaxis. In both models, nuclear segmentation did not increase migration speed. In dense collagen matrices, the speed of the hypersegmented neutrophils was even reduced compared with the banded neutrophils. Fluorescence microscopy suggested that the hypersegmented neutrophils displayed reduced rear release and deposited more membrane vesicles. Vice versa, migration through narrow pores did not induce nuclear segmentation in the neutrophils. In conclusion, like neutrophils with a segmented nucleus, the banded subset exhibited efficient migration through narrow pores. These findings suggest that the nucleus does not preclude the banded subset from reaching an infection site.

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