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Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools. We thank S. Gregori and G. Amodio for help with neutrophil isolation and culture experiments; F. Di Salvo, F. Porzio and M. Tassara for patient recruitment and data management; the Center for Omics Sciences, the Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory, Centro Risorse Biologiche at Ospedale San Raffaele; and the Centro Universitario di Statistica per le Scienze Biomediche at Vita-Salute San Raffaele University. Figures were created with Adobe Illustrator and BioRender.com. V.B. and F.V.M. conducted this study as partial fulfillment of a PhD in Molecular Medicine (Basic and Applied Immunology and Oncology program) at Vita-Salute San Raffaele University. R.D.M. is a New York Stem Cell Foundation – Robertson Investigator. M.A.C. and P.S. are supported by grants from the Italian Association for Cancer Research (AIRC) (IG 20339) and the Italian Ministry of University and Research (PRIN 20177J4E75_004). A.A. is supported by the Italian Telethon Foundation (SR-Tiget grant award B02). E.M. and N.C. are supported by fellowships from Fondazione Umberto Veronesi. This study was supported by grants from the Italian Telethon Foundation (SR-Tiget grant award F04 to R.O.) and the Italian Ministry of Health (GR-201602362156 to R.O. and S.C.). Research in the R.O. laboratory is supported by the European Research Council (starting grant 759532, X-TAM) and by AIRC (MFAG 20247 and AIRC 5×1000 special program 22737) Sí

BackgroundSARS-CoV-2 occurs in the majority of children as COVID-19, without symptoms or with a paucisymptomatic respiratory syndrome, but a small proportion of children develop the systemic Multi Inflammatory Syndrome (MIS-C), characterized by persistent fever and systemic hyperinflammation, with some clinical features resembling Kawasaki Disease (KD).ObjectiveWith this study we aimed to shed new light on the pathogenesis of these two SARS-CoV-2-related clinical manifestations.MethodsWe investigated lymphocyte and dendritic cells subsets, chemokine/cytokine profiles and evaluated the neutrophil activity mediators, myeloperoxidase (MPO), and reactive oxygen species (ROS), in 10 children with COVID-19 and 9 with MIS-C at the time of hospital admission.ResultsPatients with MIS-C showed higher plasma levels of C reactive protein (CRP), MPO, IL-6, and of the pro-inflammatory chemokines CXCL8 and CCL2 than COVID-19 children. In addition, they displayed higher levels of the chemokines CXCL9 and CXCL10, mainly induced by IFN-γ. By contrast, we detected IFN-α in plasma of children with COVID-19, but not in patients with MIS-C. This observation was consistent with the increase of ISG15 and IFIT1 mRNAs in cells of COVID-19 patients, while ISG15 and IFIT1 mRNA were detected in MIS-C at levels comparable to healthy controls. Moreover, quantification of the number of plasmacytoid dendritic cells (pDCs), which constitute the main source of IFN-α, showed profound depletion of this subset in MIS-C, but not in COVID-19.ConclusionsOur results show a pattern of immune response which is suggestive of type I interferon activation in COVID-19 children, probably related to a recent interaction with the virus, while in MIS-C the immune response is characterized by elevation of the inflammatory cytokines/chemokines IL-6, CCL2, and CXCL8 and of the chemokines CXCL9 and CXL10, which are markers of an active Th1 type immune response. We believe that these immunological events, together with neutrophil activation, might be crucial in inducing the multisystem and cardiovascular damage observed in MIS-C.

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan

Innate lymphoid cells (ILC) are developmentally related cell subsets that play a major role in innate defenses against pathogens, in lymphoid organogenesis and in tissue remodeling. The best characterized ILC are natural killer (NK) cells. They are detectable in decidua in the early phases of pregnancy. During the first trimester, NK cells represent up to 50% of decidua lymphocytes. Differently from peripheral blood (PB) NK cells, decidual NK (dNK) cells are poorly cytolytic, and, instead of IFNγ, they release cytokines/chemokines that induce neo-angiogenesis, tissue remodeling, and placentation. dNK interact with resident myeloid cells and participate in the induction of regulatory T cells that play a pivotal role in maintaining an efficient fetal–maternal tolerance. dNK cells may originate from CD34+ precursor cells present in situ and/or from immature NK cells already present in endometrial tissue and/or from PB NK cells migrated to decidua. In addition to NK cells, also ILC3 are present in human decidua during the first trimester. Decidual ILC3 include both natural cytotoxic receptor (NCR)+ and NCR− cells, producing respectively IL-8/IL-22/GM-CSF and TNF/IL-17. NCR+ILC3 have been shown to establish physical and functional interactions with neutrophils that, in turn, produce factors that are crucial for pregnancy induction/maintenance and for promoting the early inflammatory phase, a fundamental process for a successful pregnancy. While NCR+ILC3 display a stable phenotype, most of NCR−ILC3 may acquire phenotypic and functional features of NCR+ILC3. In conclusion, both NK cells and ILC3 are present in human decidua and may establish functional interactions with immune and myeloid cells playing an important role both in innate defenses and in tissue building/remodeling/placentation during the early pregnancy. It is conceivable that altered numbers or function of these cells may play a role in pregnancy failure.

Increasing data from the literature point to a neutrophil-mediated role via cytokine production in several aspects of mammalian biology, including angiogenesis. In such regard, neutrophils have been shown to synthetize and release a number of molecules able to promote, directly or indirectly, the growth and migration of endothelial cells, in turn inducing the formation of new blood vessels from preexisting ones. Interestingly, neutrophil-derived cytokines can be involved either in physiological or in pathological angiogenesis, depending on either the functioning or dysregulation of sophisticated interplays among different cell types, extracellular matrix and soluble mediators within the microenvironment. Our review resumes the most interesting studies elucidating the role of neutrophil-derived cytokines in human physiological and pathological angiogenesis. When appropriate, supporting observations generated in animal models will be also mentioned. Particular emphasis will be given to VEGF and PK2/Bv8, rather than CXCL8/IL-8 and OSM. We will also discuss the potential role of neutrophil-derived cytokines such as FGF2, Ang1 and IL-17, whose roles in angiogenesis - albeit anticipated - remain to be elucidated.

The neuropeptide substance P (SP), a main mediator of neurogenic inflammation, has been shown to have direct and modulatory effects on functional responses of polymorphonuclear leucocytes (PMNL). In this study, we further investigated the effects exerted by SP on human PMNL functions. Pretreatment of PMNL with SP resulted in an increase of superoxide anion (O2-) production in response to formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A (Con A) and opsonized zymosan (STZ). In contrast, the O2- production induced by tumour necrosis factor (TNF) was strongly inhibited by pretreatment with SP. Both enhancement and inhibition of O2- response were exerted by SP in a dose-dependent manner and at concentrations which did not directly stimulate O2- production. These effects were rapid in onset, and occurred after 5 min of preincubation of cells with the neuropeptide. At concentrations that modulated O2- production by PMNL, SP also directly stimulated release of the chemotactic cytokine interleukin-8 (IL-8). Induction of IL-8 release required a longer incubation time (1 hr) with SP and was preceded by an increase of IL-8 mRNA steady-state levels. Furthermore, as well as directly stimulating IL-8 production, SP was also able to enhance the IL-8 release induced by other stimuli such as FMLP and TNF. The results of this study indicate that, in addition to the rapid and differential modulation of O2- production, SP also induces long-term changes such as IL-8 synthesis and release, and can thus amplify the process of PMNL recruitment to the inflammatory site.

In myeloid blasts, the expression and release of the multifunctional chemokine IL-8 could be expected to be differentiation-associated.We investigated the profile of interleukin-8 (IL-8) expression and release by leukemic cells obtained at diagnosis from 42 untreated adult patients with acute myeloid leukemia of various FAB subtypes (2 M0, 7 M1, 6 M2, 6 M3, 10 M4 and 11 M5). IL-8 transcripts were evaluated by Northern blot and densitometric analysis. IL-8 release by myeloid blasts was evaluated by a specific ELISA either in sera at diagnosis or in supernatants (SN) obtained from cultured leukemic cells.In basal conditions, Northern blot analysis revealed detectable IL-8 transcripts in 15/29 cases, eleven of which were classified as M4-M5 and 4 as FAB M0-M3. Densitometric analysis of IL-8 transcript bands showed higher expression in M4-M5 than in M0-M3 cases (mean values +/- SD: 16.5 +/- 21 and 0.77 +/- 1.36 densitometric units, respectively; p = 0.012). Higher IL-8 serum levels were observed in leukemic patients as opposed to normal controls (mean values +/- SD: 0.53 +/- 0.75 vs 0.003 +/- 0.014 ng/mL, respectively; p = 0.006). Furthermore, a trend (though not of statistical significance) towards higher IL-8 serum values was observed in M4-M5 as opposed to M0-M3 subtypes. After 24 hours of culture, the majority of myeloid blasts (95%) spontaneously released detectable amounts of IL-8 into SN. However, M4-M5 released substantially higher amounts of IL-8 than M0-M3 blasts (mean +/- SD: 68 +/- 46 and 8.5 +/- 12 ng/mL, respectively; p0.001). This difference between M0-M3 and M4-M5 blasts was already observed after 6 hours of culture and increased over 72 hours.Our findings confirm and further support the preferential release of high levels of IL-8 by myeloid blasts showing monocytic differentiation.