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The use of mature neutrophil (granulocyte) transfusions for the treatment of neutropenic patients with invasive fungal infections (IFIs) has been the focus of multiple clinical trials. Despite these efforts, the transfusion of mature neutrophils has resulted in limited clinical benefit, likely owing to problems of insufficient numbers and the very short lifespan of these donor cells. In this report, we employed a system of conditionally immortalized murine neutrophil progenitors that are capable of continuous expansion, allowing for the generation of unlimited numbers of homogenous granulocyte-macrophage progenitors (GMPs). These GMPs were assayed in vivo to demonstrate their effect on survival in 2 models of IFI: candidemia and pulmonary aspergillosis. Mature neutrophils derived from GMPs executed all cardinal functions of neutrophils. Transfused GMPs homed to the bone marrow and spleen, where they completed normal differentiation to mature neutrophils. These neutrophils were capable of homing and extravasation in response to inflammatory stimuli using a sterile peritoneal challenge model. Furthermore, conditionally immortalized GMP transfusions significantly improved survival in models of candidemia and pulmonary aspergillosis. These data confirm the therapeutic benefit of prophylactic GMP transfusions in the setting of neutropenia and encourage development of progenitor cellular therapies for the management of fungal disease in high-risk patients., (©2022 Society for Leukocyte Biology.)

We report the results from a multicenter retrospective study of 69 adult patients who underwent haploidentical blood or marrow transplantation (haplo-BMT) with post-transplantation cyclophosphamide (PTCy) for chronic phase myelofibrosis. The median age at BMT was 63 years (range, 41-74). Conditioning regimens were reduced intensity in 54% and nonmyeloablative in 39%. Peripheral blood grafts were used in 86%. The median follow-up was 23.1 months (range, 1.6-75.7). At 3 years, the overall survival, relapse-free survival (RFS), and graft-versus-host-disease (GVHD)-free-RFS were 72% (95% CI 59-81), 44% (95% CI 29-59), and 30% (95% CI 17-43). Cumulative incidences of non-relapse mortality and relapse were 23% (95% CI 14-34) and 31% (95% CI 17-47) at 3 years. Spleen size ≥22 cm or prior splenectomy (HR 6.37, 95% CI 2.02-20.1, P = 0.002), and bone marrow grafts (HR 4.92, 95% CI 1.68-14.4, P = 0.004) were associated with increased incidence of relapse. Cumulative incidence of acute GVHD grade 3-4 was 10% at 3 months and extensive chronic GVHD was 8%. Neutrophil engraftment was reported in 94% patients, at a median of 20 days (range, 14-70). In conclusion, haplo-BMT with PTCy is feasible in patients with myelofibrosis. Splenomegaly ≥22 cm and bone marrow grafts were associated with a higher incidence of relapse in this study., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Neutrophils play an essential role in innate immune responses to bacterial and fungal infections, and loss of neutrophil function can increase the risk of acquiring lethal infections in clinical settings. Here, we show that engineered neutrophil-primed progenitors derived from human induced pluripotent stem cells can produce functional neutrophil-like cells at a clinically applicable scale that can act rapidly in vivo against lethal bacterial infections. Using 5 different mouse models, we systematically demonstrated that these neutrophil-like cells migrate to sites of inflammation and infection and increase survival against bacterial infection. In addition, we found that these human neutrophil-like cells can recruit murine immune cells. This system potentially provides a straight-forward solution for patients with neutrophil deficiency: an off-the-shelf neutrophil transfusion. This platform should facilitate the administration of human neutrophils for a broad spectrum of physiological and pathological conditions., (© 2021 by The American Society of Hematology.)

Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks ( p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks ( p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.

Mutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing cells.

Hemorrhagic shock induces an aberrant immune response characterized by simultaneous induction of a proinflammatory state and impaired host defenses. The objective of this study was to evaluate the impact of conditionally immortalized neutrophil progenitors (NPs) on this aberrant immune response. We employed a mouse model of hemorrhagic shock, followed by the adoptive transfer of NPs and subsequent inoculation of Staphylococcus aureus to induce pneumonia. We observed that transplant of NPs decreases the proportion of host neutrophils that express programmed death ligand 1 and intercellular adhesion molecule 1 in the context of prior hemorrhage. Following hemorrhage, NP transplant decreased proinflammatory cytokines in the lungs, increased neutrophil migration into the airspaces, and enhanced bacterial clearance. Further, hemorrhagic shock improved NP engraftment in the bone marrow. These results suggest that NPs hold the potential for use as a cellular therapy in the treatment and prevention of secondary infection following hemorrhagic shock., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The elderly are susceptible to serious infections by Streptococcus pneumoniae (pneumococcus), which calls for a better understanding of the pathways driving the decline in host defense in aging. We previously found that extracellular adenosine (EAD) shaped polymorphonuclear cell (PMN) responses, which are crucial for controlling infection. EAD is produced by CD39 and CD73, and signals via A1, A2A, A2B, and A3 receptors. The objective of this study was to explore the age-driven changes in the EAD pathway and its impact on PMN function. We found in comparison to young mice, PMNs from old mice expressed significantly less CD73, but similar levels of CD39 and adenosine receptors. PMNs from old mice failed to efficiently kill pneumococci ex vivo; however, supplementation with adenosine rescued this defect. Importantly, transfer of PMNs expressing CD73 from young mice reversed the susceptibility of old mice to pneumococcal infection. To identify which adenosine receptor(s) is involved, we used specific agonists and inhibitors. We found that A1 receptor signaling was crucial for PMN function as inhibition or genetic ablation of A1 impaired the ability of PMNs from young mice to kill pneumococci. Importantly, activation of A1 receptors rescued the age-associated defect in PMN function. In exploring mechanisms, we found that PMNs from old mice failed to efficiently kill engulfed pneumococci and that A1 receptor controlled intracellular killing. In summary, targeting the EAD pathway reverses the age-driven decline in PMN antimicrobial function, which has serious implications in combating infections., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

We analyzed data relative to cell content in 88 consecutive patients receiving HLA haploidentical bone marrow (BM) transplants with post-transplantation cyclophosphamide (PT-CY). Median age was 54.5 (range, 17-72); diagnoses were acute leukemia (n = 46), lymphoproliferative disorders (n = 24), myelofibrosis (n = 11) and myelodysplastic syndromes (n = 5). Total nucleated cell (TNC) and CD34+, CD3+, CD4+ and CD8+ cell doses were stratified as higher than first, second and third quartile and the dose effect on various clinical outcomes was assessed. Median time to engraftment was 17 days for neutrophils and 24 days for platelets. To receive a dose of TNC ≥3.2 x 10 6 /kg or CD34+ cells ≥2.7 x 10 6 /kg significantly shortened the time to neutrophil and platelet engraftment and reduced the blood product requirements in the 30-day period after transplantation. Overall, TNC and CD34+ cell doses had no effect on acute graft-versus-host disease (GVHD) incidence, whereas patients receiving higher CD3+ and CD8+ cell doses seemed to have less chronic GVHD. No effect on non-relapse mortality, progression-free survival and overall survival was observed at different cell dose thresholds. These data suggest that in HLA haploidentical BM transplant with PT-CY, appropriate cell doses are relevant to the engraftment. The association between low CD3+/CD8+ cells and chronic GVHD deserves further investigation., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Bacterial and fungal infections are a major cause of morbidity and mortality in neutropenic patients. Donor-derived neutrophil transfusions have been used for prophylaxis or treatment for infection in neutropenic patients. However, the short half-life and the limited availability of large numbers of donor-derived neutrophils for transfusion remain a significant hurdle in the implementation of neutrophil transfusion therapy. Here, we investigate the in vitro and in vivo activity of neutrophils generated from human induced pluripotent stem cells (iPSC), a potentially unlimited resource to produce neutrophils for transfusion. Phenotypic analysis of iPSC-derived neutrophils reveal reactive oxygen species production at similar or slightly higher than normal peripheral blood neutrophils, but have an ∼50%-70% reduced Escherichia coli phagocytosis and phorbol 12-myristate 13-acetate induced formation of neutrophil extracellular traps (NET). Signaling of granulocytic precursors identified impaired AKT activation, but not ERK or STAT3, in agonist-stimulated iPSC-derived neutrophils. Expression of a constitutively activated AKT in iPSC-derived neutrophils restores most phagocytic activity and NET formation. In a model of bacterial induced peritonitis in immunodeficient mice, iPSC-derived neutrophils, with or without corrected AKT activation, migrate similarly to the peritoneal fluid as peripheral blood neutrophils, whereas the expression of activated AKT significantly improves their phagocytic activity in vivo. Stem Cells Translational Medicine 2019;8:557-567., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6C hi CX 3 CR1 lo monocytes/macrophages to pro-resolving Ly6C lo CX 3 CR1 hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2 -/- neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.

Tumor-associated neutrophils (TANs) regulate many processes associated with tumor progression, and depending on the microenvironment, they can exhibit pro- or antitumor functions. However, the molecular mechanisms regulating their tumorigenicity are not clear. Using transplantable tumor models, we showed here that nicotinamide phosphoribosyltransferase (NAMPT), a molecule involved in CSF3R downstream signaling, is essential for tumorigenic conversion of TANs and their pro-angiogenic switch. As a result tumor vascularization and growth are strongly supported by these cells. Inhibition of NAMPT in TANs leads to their antitumor conversion. Adoptive transfer of such TANs into B16F10-tumor bearing mice attenuates tumor angiogenesis and growth. Of note, we observe that the regulation of NAMPT signaling in TANs, and its effect on the neutrophil tumorigenicity, are analogous in mice and human. NAMPT is up-regulated in TANs from melanoma and head-and-neck tumor patients, and its expression positively correlates with tumor stage. Mechanistically, we found that targeting of NAMPT suppresses neutrophil tumorigenicity by inhibiting SIRT1 signaling, thereby blocking transcription of pro-angiogenic genes. Based on these results, we propose that NAMPT regulatory axis is important for neutrophils to activate angiogenic switch during early stages of tumorigenesis. Thus, identification of NAMPT as the critical molecule priming protumor functions of neutrophils provides not only mechanistic insight into the regulation of neutrophil tumorigenicity, but also identifies a potential pathway that may be targeted therapeutically in neutrophils. This, in turn, may be utilized as a novel mode of cancer immunotherapy., (© 2018 UICC.)

Accumulation of neutrophils in the brain is a hallmark of cerebral ischemia and considered central in exacerbating tissue injury. Intercellular adhesion molecule (ICAM)-1 is upregulated on brain endothelial cells after ischemic stroke and considered pivotal in neutrophil recruitment as ICAM-1-deficient mouse lines were found protected from experimental stroke. Translation of therapeutic inhibition of ICAM-1 into the clinic however failed. This prompted us to investigate stroke pathogenesis in Icam1 tm1Alb C57BL/6 mutants, a true ICAM-1 null mouse line. Performing transient middle cerebral artery occlusion, we found that absence of ICAM-1 did not ameliorate stroke pathology at acute time points after reperfusion. Near-infrared imaging showed comparable accumulation of neutrophils in the ischemic hemispheres of ICAM-1 null and wild type C57BL/6 mice. We also isolated equal numbers of neutrophils from the ischemic brains of ICAM-1 null and wild type C57BL/6 mice. Immunostaining of the brains showed neutrophils to equally accumulate in the leptomeninges and brain parenchymal vessels of ICAM-1 null and wild type C57BL/6 mice. In addition, the lesion size was comparable in ICAM-1 null and wild type mice. Our study demonstrates that absence of ICAM-1 neither inhibits cerebral ischemia-induced accumulation of neutrophils in the brain nor provides protection from ischemic stroke.

Chronic granulomatous disease (CGD) is a congenital disorder characterized by recurrent life-threatening bacterial and fungal infections and development of severe inflammation secondary to a congenital defect in 1 of the 5 phagocyte oxidase (phox) subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Hematopoietic cell transplant (HCT) is a curative treatment for patients with CGD that provides donor neutrophils with functional NADPH and superoxide anion production. Many characteristics of CGD, including preexisting infection and inflammation and the potential for cure with mixed-donor chimerism, influence the transplant approach and patient outcome. Because of the dangers of short-term death, graft-versus-host disease, and late effects from chemotherapy, HCT historically has been reserved for patients with high-risk disease and a matched donor. However, as advances in CGD and HCT treatments have evolved, recommendations on transplant eligibility also must be amended, but the development of modern guidelines has proven difficult. In this review, we provide an overview of HCT in patients with CGD, including the debate over HCT indications in them, the unique aspects of CGD that can complicate HCT, and a summary of transplant outcomes.

Severe influenza virus infection can lead to life-threatening pathology through immune-mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza-mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage-1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell-mediated pathology after influenza infection. Wild-type (WT) and CD11b -/- mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b -/- mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b -/- mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b -/- mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune-mediated rather than caused by virus-induced damage. In contrast to adoptive transfer of CD11b -/- neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza-induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza-induced, T cell-mediated disease.

Objective: Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure., Approach and Results: Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of β2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis., Conclusions: Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury., (© 2018 American Heart Association, Inc.)

Objective: Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1β in the formation of murine experimental AAAs. Here, the hypothesis that IL-1β-induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested., Approach and Results: NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1β. In vitro, IL-1RA attenuated IL-1β-induced NETosis in human neutrophils. Mechanistically, IL-1β treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-β-induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage-day 3 of aneurysm induction. IL-1β-knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1β-knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation., Conclusions: Altogether, the results suggest a dominant role of IL-1β-induced NETosis in AAA formation., (© 2018 American Heart Association, Inc.)

Ischemia/reperfusion is a common cause of acute kidney injury (AKI). However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we investigated the role of peptidyl arginine deiminase-4 (PAD4), which converts arginine to citrulline and plays a role in epigenetic regulation and inflammation, in renal ischemia/reperfusion injury. PAD4 expression was highly induced in infiltrating leukocytes 24 hours following renal ischemia and reperfusion. This induction was accompanied by citrullination of histone H3 and formation of neutrophil extracellular traps in kidneys of wild-type mice. By contrast, PAD4-deficient mice did not form neutrophil extracellular traps, expressed lower levels of pro-inflammatory cytokines and were partially protected from renal ischemia/reperfusion-induced AKI. Furthermore, PAD4-deficient mice recovered kidney function 48 hours after ischemia/reperfusion, whereas kidney function in the wild-type mice progressively worsened. Administration of DNase I, which degrades neutrophil extracellular traps or the PAD-specific inhibitor YW3-56 before ischemia, partially prevented renal ischemia/reperfusion-induced AKI. Notably, transfer of neutrophils from wild-type, but not from PAD4-deficient mice, was sufficient to restore renal neutrophil extracellular trap formation and impair kidney function following renal ischemia/reperfusion. Thus, neutrophil PAD4 plays a pivotal role in renal ischemia/reperfusion-induced AKI., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Granulocyte transfusions (GTs) are seldom used as a life-saving therapy for neutropenic patients with severe infections. Despite compelling evidence of GT efficacy in retrospective and prospective case series, no study has been successful in demonstrating a definite advantage for recipients in controlled clinical trials. This review critically revises some aspects emerging from past experience that might have weakened the evidence of GT benefits. Some specific issues relevant to the efficacy of this therapeutic approach, such as primary infection, delivered doses and schedules, and immunologic effects of GTs, are discussed. Importantly, the awareness of biologic effects accompanying the transfusion of neutrophils might support their use at standardized doses and may definitely convey significant advantages to the recipient patients., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Clinically, systemic inflammatory response syndrome (SIRS) occurs after serious trauma or sepsis. In sepsis, neutrophils are the major effector cells responsible for eliminating pathogens. However, the role of neutrophils in development of SIRS, especially in local inflammatory area, is controversial. In this study, we established a SIRS mouse model characterized with cytokine-mediated lethal shock by intraperitoneal injection of oligodexynucleotides containing CpG motifs (CpG ODN) in D-galactosamine (D-GalN) sensitized mice based on our previous work and found that abundant neutrophils were rapidly recruited into the peritoneal cavity, where some neutrophils expressed surface TLR9 (sTLR9), defined as sTLR9 + neutrophils. Along with the progression of SIRS, the expression of sTLR9 in sTLR9 + neutrophils isolated from peritoneal lavage cells (PLCs) was declined in accompany with an increase in the level of the inflammatory cytokine TNFα and a decrease in the level of the anti-inflammatory cytokine IL-10 in Ly6G + PLCs. When using CCT ODN, an oligodeoxynucleotide with CCT repeats, which we have previously shown to be capable of rescuing mice from lethal shock, the expression of sTLR9 on neutrophils was significantly elevated. Adoptive therapy using early recruited neutrophil-rich PLCs containing sTLR9 + neutrophils that express high levels of sTLR9, could rescue mice from SIRS. Overall, the data reveal that the early recruited sTLR9 + neutrophils may, at least in the area of local inflammation, play a protective role during SIRS development and provide a method to rescue the patients with severe SIRS via the up-regulation of sTLR9 levels on the surface of neutrophils or via adoptive therapy with protective sub-populations of neutrophils., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Obesity is associated with chronic, low-grade inflammation, which can disrupt homeostasis within tissue microenvironments. Given the correlation between obesity and relative risk of death from cancer, we investigated whether obesity-associated inflammation promotes metastatic progression. We demonstrate that obesity causes lung neutrophilia in otherwise normal mice, which is further exacerbated by the presence of a primary tumour. The increase in lung neutrophils translates to increased breast cancer metastasis to this site, in a GM-CSF- and IL5-dependent manner. Importantly, weight loss is sufficient to reverse this effect, and reduce serum levels of GM-CSF and IL5 in both mouse models and humans. Our data indicate that special consideration of the obese patient population is critical for effective management of cancer progression.

Rationale: Mitochondrial oxidative stress (mitoOS) has been shown to be increased in various cell types in human atherosclerosis and with aging. However, the role of cell type-specific mitoOS in atherosclerosis in the setting of advanced age and the molecular mechanisms remains to be determined in vivo., Objective: The aim of this study was to examine the role of myeloid cell mitoOS in atherosclerosis in aged mice., Approach and Results: Lethally irradiated low-density lipoprotein receptor-deficient mice ( Ldlr -/- ) were reconstituted with bone marrow from either wild-type or mitochondrial catalase (mCAT) mice. mCAT transgenic mice contain ectopically expressed human catalase gene in mitochondria, which reduces mitoOS. Starting at the age of 36 weeks, mice were fed the Western-type diet for 16 weeks. We found that mitoOS in lesional myeloid cells was suppressed in aged mCAT→ Ldlr -/- chimeric mice compared with aged controls, and this led to a significant reduction in aortic root atherosclerotic lesion area despite higher plasma cholesterol levels. Neutrophil extracellular traps (NETs), a proinflammatory extracellular structure that contributes to atherosclerosis progression, were significantly increased in the lesions of aged mice compared with lesions of younger mice. Aged mCAT→ Ldlr -/- mice had less lesional neutrophils and decreased NETs compared with age-matched wild-type→ Ldlr -/- mice, whereas young mCAT→ and wild-type→ Ldlr -/- mice had comparable numbers of neutrophils and similar low levels of lesional NETs. Using cultured neutrophils, we showed that suppression of mitoOS reduced 7-ketocholesterol-induced NET release from neutrophils of aged but not younger mice., Conclusions: MitoOS in lesional myeloid cells enhanced atherosclerosis development in aged mice, and this enhancement was associated with increased lesional NETs. Thus, mitoOS-induced NET formation is a potentially new therapeutic target to prevent atherosclerosis progression during aging., (© 2017 American Heart Association, Inc.)

Angiotensin-converting enzyme (ACE) inhibitors are widely used to reduce blood pressure. Here, we examined if an ACE is important for the antibacterial effectiveness of neutrophils. ACE knockout mice or mice treated with an ACE inhibitor were more susceptible to bacterial infection by methicillin-resistant Staphylococcus aureus (MRSA). In contrast, mice overexpressing ACE in neutrophils (Neu ACE mice) have increased resistance to MRSA and better in vitro killing of MRSA, Pseudomonas aeruginosa , and Klebsiella pneumoniae ACE overexpression increased neutrophil production of reactive oxygen species (ROS) following MRSA challenge, an effect independent of the angiotensin II AT1 receptor. Specifically, as compared with wild-type (WT) mice, there was a marked increase of superoxide generation (>twofold, P < .0005) in Neu ACE neutrophils following infection, whereas ACE knockout neutrophils decreased superoxide production. Analysis of membrane p47-phox and p67-phox indicates that ACE increases reduced NAD phosphate oxidase activity but does not increase expression of these subunits. Increased ROS generation mediates the enhanced bacterial resistance of Neu ACE mice because the enhanced resistance is lost with DPI (an inhibitor of ROS production by flavoenzymes) inhibition. Neu ACE granulocytes also have increased neutrophil extracellular trap formation and interleukin-1β release in response to MRSA. In a mouse model of chemotherapy-induced neutrophil depletion, transfusion of ACE-overexpressing neutrophils was superior to WT neutrophils in treating MRSA infection. These data indicate a previously unknown function of ACE in neutrophil antibacterial defenses and suggest caution in the treatment of certain individuals with ACE inhibitors. ACE overexpression in neutrophils may be useful in boosting the immune response to antibiotic-resistant bacterial infection., (© 2017 by The American Society of Hematology.)

Cisplatin is an effective chemotherapeutic agent and widely used in treatment of various solid organ malignancies, including head and neck, ovarian, and testicular cancers. However, the induction of acute kidney injury (AKI) is one of its main side effects. Leukotriene B 4 receptor 1 (BLT1) mediates the majority of physiological effects of leukotriene B 4 (LTB 4 ), a potent lipid chemoattractant generated at inflammation sites, but the role of the LTB 4 -BLT1 axis in cisplatin-induced AKI remains unknown. Here we found upregulated LTB 4 synthesis and BLT1 expression in the kidney after cisplatin administration. Cisplatin was found to directly upregulate gene expression of leukotriene A 4 hydrolase and stimulate LTB 4 production in renal tubular epithelial cells. Reduced kidney structural/functional damage, inflammation, and apoptosis were observed in BLT1 -/- mice, as well as in wild-type mice treated with the LTA4H inhibitor SC-57461A and the BLT1 antagonist U-75302. Neutrophils were likely the target of this pathway, as BLT1 absence induced a significant decrease in infiltrating neutrophils in the kidney. Adoptive transfer of neutrophils from wild-type mice restored kidney injury in BLT1 -/- mice following cisplatin challenge. Thus, the LTB 4 -BLT1 axis contributes to cisplatin-induced AKI by mediating kidney recruitment of neutrophils, which induce inflammation and apoptosis in the kidney. Hence, the LTB 4 -BLT1 axis could be a potential therapeutic target in cisplatin-induced AKI., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Intestinal ischemia/reperfusion (I/R) causes barrier impairment and bacterial influx. Protection against I/R injury in sterile organs by hypoxic preconditioning (HPC) had been attributed to erythropoietic and angiogenic responses. Our previous study showed attenuation of intestinal I/R injury by HPC for 21 days in a neutrophil-dependent manner., Aim: To investigate the underlying mechanisms of neutrophil priming by HPC, and explore whether adoptive transfer of primed neutrophils is sufficient to ameliorate intestinal I/R injury., Methods: Rats raised in normoxia (NM) and HPC for 3 or 7 days were subjected to sham operation or superior mesenteric artery occlusion for I/R challenge. Neutrophils isolated from rats raised in NM or HPC for 21 days were intravenously injected into naïve controls prior to I/R., Results: Similar to the protective effect of HPC-21d, I/R-induced mucosal damage was attenuated by HPC-7d but not by HPC-3d. Naïve rats reconstituted with neutrophils of HPC-21d rats showed increase in intestinal phagocytic infiltration and myeloperoxidase activity, and barrier protection against I/R insult. Elevated free radical production, and higher bactericidal and phagocytic activity were observed in HPC neutrophils compared to NM controls. Moreover, increased serum levels of tumor necrosis factor α (TNFα) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were seen in HPC rats. Naïve neutrophils incubated with HPC serum or recombinant TNFα, but not CINC-1, exhibited heightened respiratory burst and bactericidal activity. Lastly, neutrophil priming effect was abolished by neutralization of TNFα in HPC serum., Conclusions: TNFα-primed neutrophils by HPC act as effectors cells for enhancing barrier integrity under gut ischemia.

Background: Nosocomial pneumonias are common in trauma patients and so interventions to prevent and treat nosocomial pneumonia may improve outcomes. Our prior work strongly suggests that tissue injury predisposes to infections like nosocomial pneumonia because mitochondrial debris originating from injured cells contains damage-associated molecular patterns that can reduce neutrophil (PMN) migration into the airway and diminish PMN function in response to bacterial inoculation of the airway. This suggested that putting exogenous "normal" PMN into the airway might be beneficial., Methods: Postinjury pneumonia (PNA) commonly arises in two groups, early, community-acquired PNA (CAP) and later hospital-acquired PNA (HAP). Posttraumatic early-onset CAP and late-onset HAP were modeled in CD-1 mice using Staphylococcus aureus or Pseudomonas aeruginosa instilled intratracheal (i.t.) at clinically relevant times with or without extrapulmonary injuries mimicked by an intraperitoneal application of mitochondrial damage-associated molecular patterns. We applied bone marrow-derived PMN (BM-PMN) intratracheally to assess their effect on bacterial clearance in the lung., Results: BM-PMN instillation i.t. had no untoward clinical effects on recipient animals. In both the early/CAP and late/HAP models, clearance of the bacterial inoculum from the lung was suppressed by mitochondrial debris and restored to uninjured levels by i.t. instillation of exogenous BM-PMN. Furthermore, PMN instillation cleared the inoculum of P. aeruginosa that could not be cleared by uninjured mice. Instillation of PMN into the lung, even across strains (CD-1 vs. C57BL/6) had no injurious effect., Conclusion: These initial studies suggest PMN instillation (i.t.) is worthy of further study as a potential adjunctive therapy aimed at decreasing the morbidity of lung infections in trauma patients. Moreover, PMN instillation (i.t.) may represent a unique means of preventing or treating pneumonia after serious injury that is completely independent of the need for antibiotic use.

Limited cell numbers in umbilical cord blood (UCB) grafts present a major impediment to favorable outcomes in adult transplantation, largely related to delayed or failed engraftment. The advent of UCB transplantation (UCBT) using two grafts successfully circumvents this obstacle, despite the engraftment of only one unit. Preclinical models suggested that the addition of UCB T cells at the time of transplant can enhance engraftment. We tested whether ex vivo activation by CD3/CD28 costimulation and expansion of T cells from a single UCB graft would be safe and feasible in adults with advanced hematologic malignancies, with an overall objective of optimizing engraftment in single unit UCBT. In this phase 1 study, recipients of single UCB units were eligible if the unit was stored in two adequate fractions. Dose limiting toxicity was defined as grade 3 or grade 4 GVHD within 90 days of UCBT. Four patients underwent UCBT; all were treated at the first dose level (10(5) cells/kg). At the 10(5) cells/kg dose level two subjects experienced grade 3 intestinal GVHD, thus meeting stopping criteria. For three subjects, neutrophil engraftment was early (12, 17, and 20 days), while one subject experienced primary graft failure. We observed early donor T cell trafficking and found that expanded T cells produced supraphysiologic levels of cytokines relevant to engraftment and to lymphoid differentiation and function. Taken together, these preliminary data suggest rapid engraftment in recipients of a single UCBT combined with relatively low doses of activated T cells, though potentially complicated by severe GVHD., (© 2016 Wiley Periodicals, Inc.)

Purpose of Review: The purpose of this review is to report a recently completed multicenter randomized controlled trial of neutrophil/granulocyte transfusions collected from G-CSF + dexamethasone donors to treat neutropenic infections in oncology and transplant patients, within the context of other historic and current clinical trials.The multicenter trial (RING Study) was funded by the NHLBI transfusion medicine/hemostasis clinical trials network., Recent Findings: There was no significant benefit of therapeutic neutrophil/granulocyte transfusions versus antibiotics per intention to treat analysis, but 32% of patients received substandard neutrophil doses. Separate analysis suggested patients given a higher neutrophil doses had better outcomes., Summary: Efficacy of 'high-dose' therapeutic neutrophil/granulocyte transfusions remains unproven, but promising.

In myelofibrosis, the introduction of reduced-intensity conditioning (RIC) preceding allogeneic stem cell transplantation (SCT) resulted in lower transplant-related mortality rates compared with myeloablative conditioning. However, lowering the intensity of conditioning may increase the risk of graft failure in myelofibrosis, although hitherto this has not been indisputably proven. We here report the outcome of 53 patients who underwent allogeneic SCT with different conditioning regimens (RIC and non-myeloablative (NMA)) in three transplantation centers in the Netherlands. The cumulative incidence of graft failure within 60 days after SCT was high (28%), and this was primarily associated with the intensity of the conditioning regimen. Cumulative neutrophil engraftment at 60 days was lower in patients who received NMA conditioning compared with those who received RIC (56% vs 84%, P=0.03). Furthermore, of six patients who received a second transplantation after graft failure, the three patients with RIC regimens subsequently engrafted, whereas the three patients who received a second NMA regimen did not. This study indicates that in myelofibrosis, NMA regimens result in high engraftment failure rates. We propose the use of more intensive conditioning regimens, incorporating busulfan or melphalan.

In haploidentical (haplo)-cord blood (CB) transplantations, early haplo donor engraftment serves as a myeloid bridge to sustainable CB engraftment and is associated with early neutrophil recovery. The conditioning regimens as published for haplo-cord protocols usually contain serotherapy, such as rabbit antithymocyte globulin (ATG) (Thymoglobulin, Genzyme, Cambridge, MA). However, reducing or omitting serotherapy is an important strategy to improve early immune reconstitution after transplantation. The need for serotherapy in successful haplo-cord transplantation, defined as having a haplo-derived myeloid bridge to CB engraftment, has not been investigated before. Two consecutive cohorts of patients underwent transplantation with haplo-CB. The first group underwent transplantation with haplo-CB for active infection and/or an underlying condition with expected difficult engraftment without a conventional donor available. They received a single unit (s) CB and haplo donor cells (CD34(+) selected, 5 × 10(6) CD34(+)/kg). The second cohort included patients with poor-risk malignancies, not eligible for other treatment protocols. They received a sCB and haplo donor cells (CD19/αβTCR-depleted; 5 × 10(6) CD34(+)/kg). Retrospectively in both cohorts, active ATG (Thymoglobulin) levels were measured and post-hematopoietic cell transplantation area under the curve (AUC) was calculated. The influence of ATG exposure for having a successful haplo-myeloid bridge (early haplo donor engraftment before CB engraftment and no secondary neutropenia) and transplantation-related mortality (TRM) were analyzed as primary endpoints. Twenty patients were included (16 in the first cohort and 4 in the second cohort). In 58% of evaluable patients, there was no successful haplo-derived myeloid bridge to CB engraftment, for which a low post-transplantation ATG exposure appeared to be a predictor (P <.001). TRM in the unsuccessful haplo-bridge group was 70% ± 16% versus 12% ± 12% in the successful haplo-bridge group (P = .012). In conclusion, sufficient in vivo T depletion with ATG is required for a successful haplo-myeloid bridge to CB engraftment., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Blood-derived monocytes remove apoptotic cells and terminate inflammation in settings as diverse as atherosclerosis and Alzheimer's disease. They express high levels of the proresolving receptor ALX/FPR2, which is activated by the protein annexin A1 (ANXA1), found in high abundance in inflammatory exudates. Using primary human blood monocytes from healthy donors, we identified ANXA1 as a potent CD14(+)CD16(-) monocyte chemoattractant, acting via ALX/FPR2. Downstream signaling pathway analysis revealed the p38 MAPK-mediated activation of a calcium independent phospholipase A2 with resultant synthesis of lysophosphatidic acid (LPA) driving chemotaxis through LPA receptor 2 and actin cytoskeletal mobilization. In vivo experiments confirmed ANXA1 as an independent phospholipase A2-dependent monocyte recruiter; congruently, monocyte recruitment was significantly impaired during ongoing zymosan-induced inflammation in AnxA1(-/-) or alx/fpr2/3(-/-) mice. Using a dorsal air-pouch model, passive transfer of apoptotic neutrophils between AnxA1(-/-) and wild-type mice identified effete neutrophils as the primary source of soluble ANXA1 in inflammatory resolution. Together, these data elucidate a novel proresolving network centered on ANXA1 and LPA generation and identify previously unappreciated determinants of ANXA1 and ALX/FPR2 signaling in monocytes., (Copyright © 2015 The Authors.)

The recent WHO report on antibiotic resistances shows a dramatic increase of microbial resistance against antibiotics. With only a few new antibiotics in the pipeline, a different drug delivery approach is urgently needed. We have obtained evidence demonstrating the effectiveness of a cell based drug delivery system that utilizes the innate immune system as targeting carrier for antibacterial drugs. In this study we show the efficient loading of neutrophil granulocytes with chlorhexidine and the complete killing of E. coli as well as Fusobacterium necrophorum in in-vitro studies. Fusobacterium necrophorum causes hepatic abscesses in cattle fed high grain diets. We also show in a mouse model that this delivery system targets infections of F. necrophorum in the liver and reduces the bacterial burden by an order of magnitude from approximately 2•106 to 1•105.

Delayed engraftment is a major limitation of cord blood transplantation (CBT), due in part to a defect in the cord blood (CB) cells' ability to home to the bone marrow. Because this defect appears related to low levels of fucosylation of cell surface molecules that are responsible for binding to P- and E-selectins constitutively expressed by the marrow microvasculature, and thus for marrow homing, we conducted a first-in-humans clinical trial to correct this deficiency. Patients with high-risk hematologic malignancies received myeloablative therapy followed by transplantation with 2 CB units, one of which was treated ex vivo for 30 minutes with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose to enhance the interaction of CD34(+) stem and early progenitor cells with microvessels. The results of enforced fucosylation for 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. The median time to neutrophil engraftment was 17 days (range, 12-34 days) compared with 26 days (range, 11-48 days) for controls (P = .0023). Platelet engraftment was also improved: median was 35 days (range, 18-100 days) compared with 45 days (range, 27-120 days) for controls (P = .0520). These findings support ex vivo fucosylation of multipotent CD34(+) CB cells as a clinically feasible means to improve engraftment efficiency in the double CBT setting. The trial is registered to www.clinicaltrials.gov as #NCT01471067., (© 2015 by The American Society of Hematology.)

In this issue of Blood, Popat et al report the results of a clinical trial designed to test the safety and feasibility of a novel strategy, enforced fucosylation, intended to improve engraftment after donor unrelated cord blood (UCB) transplantation in adults. The data show that the strategy is safe, and that engraftment of platelets and neutrophils is faster in recipients of the modified cord blood (CB) unit when compared with historical controls

Rationale: In-hospital outcomes are generally acceptable in patients with type B dissection; however, some patients present with undesirable complications, such as aortic expansion and rupture. Excessive inflammation is an independent predictor of adverse clinical outcomes., Objective: We have investigated the underlying mechanisms of catastrophic complications after acute aortic dissection (AAD) in mice., Methods and Results: When angiotensin II was administered in lysyl oxidase inhibitor-preconditioned mice, AAD emerged within 24 hours. The dissection was initiated at the proximal site of the descending thoracic aorta and propagated distally into an abdominal site. Dissection of the aorta caused dilatation, and ≈70% of the mice died of aortic rupture. AAD triggered CXCL1 and granulocyte-colony stimulating factor expression in the tunica adventitia of the dissected aorta, leading to elevation of circulating CXCL1/granulocyte-colony stimulating factor levels. Bone marrow CXCL12 was reduced. These chemokine changes facilitated neutrophil egress from bone marrow and infiltration into the aortic adventitia. Interference of CXCL1 function using an anti-CXCR2 antibody reduced neutrophil accumulation and limited aortic rupture post AAD. The tunica adventitia of the expanded dissected aorta demonstrated high levels of interleukin-6 (IL-6) expression. Neutrophils were the major sources of IL-6, and CXCR2 neutralization significantly reduced local and systemic levels of IL-6. Furthermore, disruption of IL-6 effectively suppressed dilatation and rupture of the dissected aorta without any influence on the incidence of AAD and neutrophil mobilization., Conclusions: Adventitial CXCL1/granulocyte-colony stimulating factor expression in response to AAD triggers local neutrophil recruitment and activation. This leads to adventitial inflammation via IL-6 and results in aortic expansion and rupture., (© 2015 American Heart Association, Inc.)

We investigated the unit characteristics associated with engraftment after double-unit cord blood (CB) transplantation (dCBT) and whether these could be reliably identified during unit selection. Cumulative incidence of neutrophil engraftment in 129 myeloablative dCBT recipients was 95% (95% confidence interval: 90-98%). When precryopreservation characteristics were analyzed, the dominant unit CD34(+) cell dose was the only characteristic independently associated with engraftment (hazard ratio, 1.43; P = .002). When postthaw characteristics were also included, only dominant unit infused viable CD34(+) cell dose independently predicted engraftment (hazard ratio, 1.95; P < .001). We then examined the determinants of infused viable CD34(+) cell dose (precryopreservation count, postthaw recovery, and postthaw viability) in 402 units thawed at our center. This revealed close correlation between precryopreservation and postthaw CD34(+) cell counts (r(2) = 0.73). Median CD34(+) cell recovery was 101%, although it ranged from 12% to 1480%. Notably, units from non-Netcord Foundation for the Accreditation of Cellular Therapy (Netcord-FACT)-accredited banks were more likely to have low recovery (P < .001). Furthermore, although median postthaw CD34(+) cell viability was 92%, 33 (8%) units had <75% viable CD34(+) cells. Units from non-Netcord-FACT-accredited banks and units with cryovolumes other than 24.5 to 26.0 mL were more likely to have poor postthaw viability. Precryopreservation CD34(+) cell dose and banking practices should be incorporated into CB unit selection., (© 2014 by The American Society of Hematology.)

We evaluated the role of mismatched third-party hematopoietic stem cells (TPC) in shortening the neutropenia after umbilical cord blood transplantation (UCBT). A TPC graft was given to 7/37 children with UCBT to support engraftment due to anticipated increased risk of nonengraftment (N=6) or active infection (N=1). TPC grafts were collected with apheresis from haploidentical family members. The median UCB and CD34 cell counts were 5.10 (range, 4.13 to 9.98)×10/kg and 5.98 (range, 4.40 to 14.00)×10/kg, respectively. The median time to neutrophil engraftment was shorter in the patients with TPC (12 d; range, 9 to 24 d) than those without (23 d; range, 12 to 44 d) (P=0.010). TPC chimerism was lost in median at 28 (range, 24 to 103) days posttransplant. TPC grafts from mothers engrafted similarly as the grafts from other family members. UCB graft cell count and the use of methotrexate posttransplant strongly contributed to engraftment. TPC may form a valuable transient bridging graft over the neutropenia after UCBT in patients with anticipated high-risk of nonengraftment or toxicity due to pretransplant infections.

Engraftment outcomes following autologous transplantation correlate poorly to infused stem cell number. We evaluated 446 consecutive patients who underwent autologous transplantation at our centre between 2001 and 2012. The impact of pre-transplant and collection factors together with CD34(+) dosing ranges on engraftment, hospital length of stay (LOS) and survival endpoints were assessed in order to identify factors which might be optimized to improve outcomes for patients undergoing autologous transplantation using haemopoietic progenitor cells-apheresis (HPC-A). Infused CD34(+) cell dose correlated to platelet but not neutrophil recovery. Time to platelet engraftment was significantly delayed in those receiving low versus medium or high CD34(+) doses. Non-remission status was associated with slower neutrophil and platelet recovery. Increasing neutrophil contamination of HPC-A was strongly associated with slower neutrophil recovery with infused neutrophil dose/kg recipient body weight ≥3 × 10(8)/kg having a significant impact on time to neutrophil engraftment (p = 0.001). Higher neutrophil doses/kg in HPC-A were associated with days of granulocyte colony stimulation factor (G-CSF) use, HPC-A volumes >500 ml and higher NCC in HPC-A. High infused neutrophil dose/kg and age >65 years were associated with longer hospital LOS (p = 0.002 and 0.011 respectively). Only age, disease and disease status predicted disease-free survival (DFS) and overall survival (OS) in our cohort (p < 0.005). Non-relapse mortality was not affected by low dose of CD34(+) (<2 × 10(6)/kg). In conclusion, our study shows that CD34(+) remains a useful and convenient marker for assessing haemotopoietic stem cell content and overall engraftment capacity post-transplant. Neutrophil contamination of HPC-A appears to be a key factor delaying neutrophil recovery. Steps to minimize the degree of neutrophil contamination in HPC-A product may be associated with more rapid neutrophil engraftment and reduced hospital LOS.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: The use of hematopoietic progenitor cell (HPC) transplant has risen over the past two decades. A variety of adverse events (AEs) of varying severity have been noted during HPC infusions. These AEs have been associated with several factors such as the amount of dimethyl sulfoxide and white blood cells in the HPC product. We performed a single-institution retrospective analysis to determine the effect of two different HPC infusion techniques, manual push with syringes versus infusion from bags with the aid of gravity, on the occurrence of infusion-related AEs., Study Design and Methods: Infusions between December 2008 and November 2010 involving peripheral blood HPCs were reviewed. Pertinent clinical and HPC product-related information was recorded. Data were analyzed to determine the incidence of infusion-related AEs and its association with patient and product-related variables., Results: We found 461 AEs in 645 patients during the study period. A total of 325 (50%) experienced at least one AE. Flushing was the most common type of AE followed by nausea and hypertension. The use of syringe infusion was more commonly associated with AEs (odds ratio, 1.82 [95% confidence interval, 1.32-2.50]; p=0.002). Other independent risk factors were cryopreserved products and the amount of polymorphonuclear leukocytes in the product., Conclusion: To our knowledge, this is the first study examining the effect of two different infusion techniques on infusion-related AEs. Our findings suggest that the use of bags for infusion protected the patients from AEs., (© 2014 AABB.)

The aim of this study was to investigate predictive factors for rapid engraftment after allogeneic peripheral blood stem cell transplantation (alloPBSCT) in patients with acute leukemia. Two hundred sixty-two patients receiving alloPBSCT were analyzed. Subset analyses of donor stem cells were conducted using a flow cytometric method. The correlation between rapid engraftment of neutrophils, platelets, and donor stem cells doses, as well as other recipient and donor clinical factors, was analyzed. In univariate analysis, factors correlated with neutrophil engraftment (≥0.5 × 10(9)/L) by day 12 were achievement of complete remission (CR) after induction chemotherapy (CR1) before hematopoietic cell transplantation (HCT) and high numbers of CD34+ cells, CD3+ T cells, and CD3+/CD4+ T cells. Factors correlated with platelet engraftment (≥20 × 10(9)/L) by day 12 were achievement of CR1 before HCT, donor and recipient sex mismatch, and high numbers of mononuclear cells, CD34+ cells, CD3+ T cells, CD3+/CD4+ T cells, CD3+/CD8+ T cells, and CD56+ NK cells. In multivariate analysis, independent predictive factors for rapid neutrophil and platelet engraftment were CR1 before HCT (p < 0.001 and p = 0.002, respectively), high number of donor CD34+ cells (p = 0.005 and p < 0.001, respectively), and high number of CD3+ T cells (p = 0.005 and p = 0.001, respectively). In conclusion, achieving CR1 before HCT, as well as larger quantities of donor CD34+ and CD3+ T cells, may predict rapid neutrophil and platelet engraftment after PBSCT.

Biofilm infections often lead to significant morbidity due to their chronicity and recalcitrance to antibiotics. We have demonstrated that methicillin-resistant Staphylococcus aureus (MRSA) biofilms can evade macrophage (MΦ) antibacterial effector mechanisms by skewing MΦs toward an alternatively activated M2 phenotype. To overcome this immune evasion, we have used two complementary approaches. In the first, a proinflammatory milieu was elicited by local administration of classically activated M1 MΦs and in the second by treatment with the C5a receptor (CD88) agonist EP67, which invokes MΦ proinflammatory activity. Early administration of M1-activated MΦs or EP67 significantly attenuated biofilm formation in a mouse model of MRSA catheter-associated infection. Several proinflammatory mediators were significantly elevated in biofilm-infected tissues from MΦ- and EP67-treated animals, revealing effective reprogramming of the biofilm environment to a proinflammatory milieu. A requirement for MΦ proinflammatory activity was demonstrated by the fact that transfer of MyD88-deficient MΦs had minimal impact on biofilm growth. Likewise, neutrophil administration had no effect on biofilm formation. Treatment of established biofilm infections with M1-activated MΦs also significantly reduced catheter-associated biofilm burdens compared with antibiotic treatment. Collectively, these results demonstrate that targeting MΦ proinflammatory activity can overcome the local immune inhibitory environment created during biofilm infections and represents a novel therapeutic strategy.

For a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1-ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

Infections continue to be a serious problem for severely neutropenic oncology and haematopoietic progenitor cell (HPC) transplant patients. Although it is now possible to collect much larger numbers of neutrophils (PMNs) from donors stimulated with granulocyte colony-stimulating factor + corticosteroids, the efficacy of these 'modern' granulocyte/PMN transfusions, with higher doses of PMNs, has not been established by convincing randomized control trials. Accordingly, they cannot be recommended for standard therapy at this time., (© 2012 Blackwell Publishing Ltd.)

Tumor necrosis factor α (TNF-α) has been reported to be a key component of the functional priming, of both myeloid and nonmyeloid cells, that is thought to contribute to the lung's increased susceptibility to injury following shock. Not surprisingly, we found that mice deficient in TNF-α exhibited reduced acute lung injury (ALI) resultant from the combined insults of hemorrhagic shock and sepsis. However, we found that when we adoptively transferred neutrophils from mice expressing TNF-α to neutrophil-depleted mice that lacked TNF-α, they were not able to serve as priming stimulus for the development of ALI. Based on these findings, we proposed that resident lung tissue cells mediate TNF-α priming. To begin to unravel the complex signaling pathway of various resident lung tissue cells in TNF-α-induced priming, we compared the effect of local (intratracheal [i.t.]) versus systemic [intravenous (i.v.)] delivery of TNF-α small interference (siRNA). We hypothesized that alternately suppressing expression of TNF-α in lung endothelial (i.v.) or epithelial (i.t.) cells would produce a differential effect in shock-induced ALI. We found that when in vivo siRNA i.t. or i.v. against TNF-α was administered to C57/BL6 mice at 2 h after hemorrhage, 24 h before septic challenge, that systemic/i.v., but not i.t., delivery of TNF-α siRNA following hemorrhage priming significantly reduces expression of indices of ALI compared with controls. These findings suggest that an absence of local lung tissue TNF-α significantly reduces lung tissue injury following hemorrhage priming for ALI and that pulmonary endothelial and/or other possible vascular resident cells, not epithelial cells, play a greater role in mediating the TNF-α priming response in a mouse model of hemorrhage/sepsis-induced ALI.