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Granulocyte-lineage cells are important innate immune effectors across all vertebrates. Named for conspicuous secretory granules, granulocytes have historically been studied for their antimicrobial roles. Although versions of these cells are found in all vertebrate species examined to date, disparate environmental and physiological pressures acting on distinct vertebrate classes have shaped many of the facets dictating granulocyte biology. Immune pressures further determine granulopoietic constraints, ultimately governing granulocyte functions. For amphibians that inhabit pathogen-rich aquatic environments for some or all their lives, their unique granulocyte biologies satisfy many of their antimicrobial needs. Amphibians also occupy an intermediate position in the evolution of vertebrate immune systems, using combinations of primitive (e.g., subcapsular liver) and more recently evolved (e.g., bone marrow) tissue sites for hematopoiesis and specifically, granulopoiesis. The last decade of research has revealed vertebrate granulocytes in general, and amphibian granulocytes in particular, are more complex than originally assumed. With dynamic leukocyte phenotypes, granulocyte-lineage cells are being acknowledged for their multifaceted roles beyond immunity in other physiological processes. Here we provide an overview of granulopoiesis in amphibians, highlight key differences in these processes compared to higher vertebrates, and identify open questions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Objectives: Stroke-induced immunosuppression (SIIS) increases the risk of poststroke infections. We aimed to determine whether failed versus successful thrombolytic therapy (TT) resulted in SIIS-associated changes in peripheral granulocyte markers at 1 week following the insult., Methods: We collected peripheral blood samples from 19 patients with acute ischemic stroke undergoing TT within 6 h after the onset of their first symptoms and 7 days after the insult. Age-matched controls were sampled on one occasion. We compared the expression of CD15 and CD64 on monocytes, granulocytes, and lymphocytes using flow cytometry., Results: The proportion of granulocytes and CD15+ granulocytes was comparable between controls and stroke patients at both time points. While the proportion of CD15bright granulocytes was also comparable, the mean fluorescence intensity (MFI) of CD15 on this subset was reduced in stroke patients by day 7 but was overall higher at both time points compared to controls. The MFI of CD15 on granulocytes was lower in stroke patients with failed TT than in those with successful TT 1 week after the insult., Conclusions: Our current results indicate that TT may not only acutely reduce the systemic inflammatory response following stroke but may also play a role in reversing SIIS at a later stage following the insult, as reflected by the higher expression of the CD15 marker on granulocytes following successful TT., (© 2022 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Despite extensive research, there is still no treatment that would lead to remission in all patients with rheumatoid arthritis as our understanding of the affected site, the synovium, is still incomplete. Recently, single-cell technologies helped to decipher the cellular heterogeneity of the synovium; however, certain synovial cell populations, such as endothelial cells or peripheral neurons, remain to be profiled on a single-cell level. Furthermore, associations between certain cellular states and inflammation were found; whether these cells cause the inflammation remains to be answered. Similarly, cellular zonation and interactions between individual effectors in the synovium are yet to be fully determined. A deeper understanding of cell signalling and interactions in the synovium is crucial for a better design of therapeutics with the goal of complete remission in all patients., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice., Competing Interests: Disclosures:   D.M. Lowe reported personal fees from Merck, non-financial support from CSL Behring, and non-financial support from Fujifilm Chemical Co. outside the submitted work. No other disclosures were reported., (© 2021 Bohrer et al.)

Aging is one of the most prominent risk factors for heart failure. Myeloid-derived suppressor cells (MDSCs) accumulate in aged tissue and have been confirmed to be associated with various aging-related diseases. However, the role of MDSCs in the aging heart remains unknown. Through RNA-seq and biochemical approaches, we found that granulocytic MDSCs (G-MDSCs) accumulated significantly in the aging heart compared with monocytic MDSCs (M-MDSCs). Therefore, we explored the effects of G-MDSCs on the aging heart. We found that the adoptive transfer of G-MDSCs of aging mice to young hearts resulted in cardiac diastolic dysfunction by inducing cardiac fibrosis, similar to that in aging hearts. S100A8/A9 derived from G-MDSCs induced inflammatory phenotypes and increased the osteopontin (OPN) level in fibroblasts. The upregulation of fibroblast growth factor 2 (FGF2) expression in fibroblasts mediated by G-MDSCs promoted antisenescence and antiapoptotic phenotypes of fibroblasts. SOX9 is the downstream gene of FGF2 and is required for FGF2-mediated and G-MDSC-mediated profibrotic effects. Interestingly, both FGF2 levels and SOX9 levels were upregulated in fibroblasts but not in G-MDSCs and were independent of S100A8/9. Therefore, a novel FGF2-SOX9 signaling axis that regulates fibroblast self-renewal and antiapoptotic phenotypes was identified. Our study revealed the mechanism by which G-MDSCs promote cardiac fibrosis via the secretion of S100A8/A9 and the regulation of FGF2-SOX9 signaling in fibroblasts during aging.

Background & Aims: Magnesium (Mg 2+ ) is able to modulate the differentiation and proliferation of cells. Mg 2+ restriction can trigger neutrophilia, but the processes that result in this change have yet to be investigated and are not fully understood. Hematopoiesis is a complex process that is regulated by many factors, including cytokines and growth factors, and is strongly influenced by nutrient availability. In this context, our objective was to investigate the impact of the short-term restriction of dietary Mg 2+ on bone marrow hematopoietic and peripheral blood cells, especially in processes related to granulocyte differentiation and proliferation., Methods: Male C57BL/6 mice were fed a Mg 2+ restricted diet (50 mg Mg 2+ /kg diet) for 4 weeks. Cell blood count and bone marrow cell count were evaluated. Bone marrow cells were also characterized by flow cytometry. Gene expression and cytokine production were evaluated, and a colony-forming cell assay related to granulocyte differentiation and proliferation was performed., Results: Short-term dietary restriction of Mg 2+ resulted in peripheral neutrophilia associated with an increased number of granulocytic precursors in the bone marrow. Additionally, Mg 2+ restriction resulted in an increased number of granulocytic colonies formed in vitro. Moreover, the Mg 2+ restricted group showed increased expression of CSF3 and CEBPα genes as well as increased production of G-CSF in association with increased expression of STAT3 protein., Conclusion: Short-term dietary restriction of Mg 2+ induces granulopoiesis by increasing G-CSF production and activating the CEBPα and STAT-3 pathways, resulting in neutrophilia in peripheral blood., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Introduction: Endometriosis is a chronic systemic disease, which influence negatively the quality of life of affected women and responsible for infertility and chronic pelvic pain. Pathophysiology of the disease is still enigmatic, but insufficient immune surveillance may play a role in it. Peripheral natural immune cell function is rarely examined. The aim of the study was to examine phagocyte function of peripheral neutrophil granulocytes and monocytes, whether this phagocytic activity is affected by the presence or removal of endometriotic lesions in women with endometriosis., Material and Methods: Twenty-six preoperative, 13 postoperative samples from women with endometriosis, 23 samples from healthy women, 14 pre- and postoperative samples from the surgical control group were enrolled. Cells were isolated from peripheral blood samples, marked and evaluated for the phagocytosis index with immunofluorescent microscope after phagocyting the zymosane molecules., Results: Phagocyte function of monocytes and neutrophil granulocytes decreased significantly women with endometriosis before surgery compared to healthy controls. However, 7 days after surgery the postoperative values showed significant improvement compared to the preoperative results of women with endometriosis. This increment reached the values of the healthy women. In the surgical control group no difference was detected between the pre- and postoperative outcomes., Discussion: Decreased phagocyte function of the examined cells, which can be the result of the circulating immunosuppressive factors, may play a role in the deficient clearance of ectopic endometrial tissue. Based on the postoperative results, these immunosuppressive factors may be reduced or eliminated 7 days after surgery in women with endometriosis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE., Methods: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively., Results: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM., Conclusions: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The differentiation of distinct leukocyte subsets is governed by lineage-specific growth factors that elicit disparate expression of transcription factors and markers by the developing cell populations. For example, macrophages (Mφs) and granulocytes (Grns) arise from common granulocyte-macrophage progenitors in response to distinct myeloid growth factors. In turn, myelopoiesis of the Xenopus laevis anuran amphibian appears to be unique to other studied vertebrates in several respects while the functional differentiation of amphibian Mφs and Grns from their progenitor cells remains poorly understood. Notably, the expression of colony stimulating factor-1 receptor (CSF-1R) or CSF-3R on granulocyte-macrophage progenitors marks their commitment to Mφ- or Grn-lineages, respectively. CSF-1R is activated by the colony stimulating factor-1 (CSF-1) and interleukin (IL-34) cytokines, resulting in morphologically and functionally distinct Mφ cell types. Conversely, CSF-3R is ligated by CSF-3 in a process indispensable for granulopoiesis. Presently, we explore the relationships between X. laevis CSF-1-Mφs, IL-34-Mφs and CSF-3-Grns by examining their expression of key lineage-specific transcription factor and myeloid marker genes as well as their enzymology. Our findings suggest that while the CSF-1- and IL-34-Mφs share some commonalities, the IL-34-Mφs possess transcriptional patterns more akin to the CSF-3-Grns. IL-34-Mφs also possess robust expression of dendritic cell-associated transcription factors and surface marker genes, further underlining the difference between this cell type and the CSF-1-derived frog Mφ subset. Moreover, the three myeloid populations differ in their respective tartrate-resistant acid phosphatase, specific- and non-specific esterase activity. Together, this work grants new insights into the developmental relatedness of these three frog myeloid subsets., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy., (© 2020 Mehmeti-Ajradini et al.)

Over the years of evolution, thousands of different animal species have evolved. All these species require an immune system to defend themselves against invading pathogens. Nevertheless, the immune systems of different species are obviously counteracting against the same pathogen with different efficiency. Therefore, the question arises if the process that was leading to the clades of vertebrates in the animal kingdom-namely mammals, birds, amphibians, reptiles, and fish-was also leading to different functions of immune cells. One cell type of the innate immune system that is transmigrating as first line of defense in infected tissue and counteracts against pathogens is the neutrophil granulocyte. During the host-pathogen interaction they can undergo phagocytosis, apoptosis, degranulation, and form neutrophil extracellular traps (NETs). In this review, we summarize a wide spectrum of information about neutrophils in humans and animals, with a focus on vertebrates. Special attention is kept on the development, morphology, composition, and functions of these cells, but also on dysfunctions and options for cell culture or storage.

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all- trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 ( TRIM25 ) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation., Competing Interests: The authors declare no competing interest.

Background: A healthy immune system plays a particularly important role in newborns, including in calves that are far more susceptible to infections (viral, bacterial and other) than adult individuals. Therefore, the present study aimed to evaluate the influence of HMB on the chemotactic activity (MIGRATEST® kit), phagocytic activity (PHAGOTEST® kit) and oxidative burst (BURSTTEST® kit) of monocytes and granulocytes in the peripheral blood of calves by flow cytometry., Results: An analysis of granulocyte and monocyte chemotactic activity and phagocytic activity revealed significantly higher levels of phagocytic activity in calves administered HMB than in the control group, expressed in terms of the percentage of phagocytising cells and mean fluorescence intensity (MFI). HMB also had a positive effect on the oxidative metabolism of monocytes and granulocytes stimulated with PMA (4-phorbol-12-β-myristate-13-acetate) and Escherichia coli bacteria, expressed as MFI values and the percentage of oxidative metabolism., Conclusion: HMB stimulates non-specific cell-mediated immunity, which is a very important consideration in newborn calves that are exposed to adverse environmental factors in the first weeks of their life. The supplementation of animal diets with HMB for both preventive and therapeutic purposes can also reduce the use of antibiotics in animal production.

Background: Ischemic preconditioning (IPC) protects the myocardium against ischemia/reperfusion injury. Evidence suggests that hyperglycemia inhibits IPC-induced cardioprotection. The effects of hyperglycemia initiated during different phases of IPC on myocardial injury were characterized with emphasis on apoptosis and aggregation of polymorphonuclear granulocytes (PMN)., Methods: Male Wistar rats were subjected to 35 min of myocardial ischemia and 2 h of reperfusion. Control animals were not further treated. IPC was induced by three cycles of 3 min ischemia and 5 min of reperfusion before major ischemia. Hyperglycemia (blood glucose more than 22.2 mmol/L) was induced by glucose administration with or without IPC during different phases (trigger- (before ischemia), mediator- (during ischemia), early reperfusion-phase). One additional group received an anti-PMN-antibody before ischemia. Infarct size was quantified by triphenyltetrazolium chloride staining. Cytochrome C release and B-cell lymphoma two (Bcl-2) expression were assessed by western blot analysis. Poly-ADP-Ribose staining and PMN accumulation were quantified with immunohistochemistry and histochemistry., Results: IPC reduced infarct size compared with control. Hyperglycemia completely abolished IPC-induced cardioprotection independent of the time point of initiation. Hyperglycemia before and during major ischemia but without IPC also slightly reduced infarct size. IPC reduced the accumulation of PMNs. This effect was reversed by hyperglycemia during trigger- and mediator-phase but not by hyperglycemia during reperfusion. Hyperglycemia alone had no effect on PMN accumulation. In all treatment groups, signs of myocardial apoptosis were reduced compared with control. IPC alone, combined with hyperglycemia and anti-PMN treatment, reduced apoptosis by a Bcl-2-associated mechanism. Hyperglycemia alone reduced apoptosis by a Bcl-2-independent pathway., Conclusion: Hyperglycemia inhibits IPC-induced cardioprotection independent of its onset. Furthermore, hyperglycemia prevents apoptosis and IPC-induced reduction of PMN aggregation.

Purpose: Immunotherapy is a new standard first-line treatment for non-small cell lung cancers (NSCLC) with high programmed cell death-ligand 1 (PD-L1) expression (≥ 50%) and second-line treatment regardless of PD-L1 status, though not all patients benefit from this approach. Much effort is ongoing to identify robust prognostic and predictive biomarkers of response to immune checkpoint inhibitors, overcoming PD-L1 that appears limited in its ability to discriminate patient candidates to this new class of anticancer agents. The purpose of this research study is to identify potential new biomarkers for immunotherapy in lung cancer., Methods: Fifty-three consecutive patients with advanced NSCLC treated with nivolumab were enrolled in the study. All the patients received a blood analysis looking for the relationship between different populations of baseline white blood cells and granulocytic myeloid-derived suppressor cells (Gr-MDSC) detected by flow cytometry, to identify and characterize patients with poor likelihood of benefit from nivolumab in NSCLC second-line setting, regardless of clinical feature and PDL1 expression., Results: Univariate analysis showed that high baseline levels of Gr-MDSC and low baseline CD8/Gr-MDSC ratio are associated with significantly better (P = 0.02) response to immunotherapy treatment. Log-rank tests suggested a significant improvement in OS and PFS with high baseline levels of Gr-MDSC levels (≥ 6 cell/μl), low absolute neutrophil count (< 5840/μl), high eosinophil count (> 90 /μl), and NLR < 3. The multivariate analysis showed a statistically significant improvement for PFS (P = 0.003) and OS (P = 0.05) in favour of the identified good prognostic Gr-MDSC-linked asset group, compared with the poor prognosis group., Conclusion: The role of Gr-MDSC appears interesting as a potential biomarker in NSCLC patients receiving immune-checkpoint inhibitors. Further analyses are needed to confirmed and study in deep the role of these particular cells and their role in cancer response and progression during ICI therapy.

Common X-linked genetic polymorphisms are expected to alter cellular responses affecting males and females differently through sex-linked inheritance pattern as well as X chromosome (ChrX) mosaicism and associated ChrX skewing, which is unique to females. We tested this hypothesis in ex vivo lipopolysaccharide and phorbol ester-stimulated polymorphonuclear granulocytes (PMNs) and monocytes from healthy volunteers (n = 51). Observations were analyzed after stratification by sex alone or the presence of variant IRAK1 haplotype a common X-linked polymorphism with previously demonstrated major clinical impacts. Upon cell activation, CD11b, CD45, CD66b, CD63, and CD14 expression was markedly and similarly elevated in healthy males and females. By contrast, PMN and monocyte activation measured by CD11b, CD66b, and CD63 was increased in variant-IRAK1 subjects as compared with WT. Stratification by IRAK1 genotype and sex showed similar cell activation effect on variant-IRAK1 subjects and an intermediate degree of cell activation in heterozygous mosaic females. The increased membrane expression of these proteins in variant-IRAK1 subjects was associated with similar or increased intersubject but uniformly decreased intrasubject cell response variabilities as compared with WT. We also tested white blood cell ChrX skewing in the healthy cohort as well as in a sample of female trauma patients (n = 201). ChrX inactivation ratios were similar in IRAK1 WT, variant, and heterozygous healthy subjects. Trauma patients showed a trend of blunted ChrX skewing at admission in homozygous variant-IRAK1 and heterozygous mosaic-IRAK1 female subjects as compared with WT. Trauma-induced de novo ChrX skewing was also depressed in variant-IRAK1 and mosaic-IRAK1 female trauma patients as compared with WT. Our study indicates that augmented PMN and monocyte activation in variant-IRAK1 subjects is accompanied by decreased intrasubject cellular variability and blunted de novo ChrX skewing in response to trauma. A more pronounced cell activation of PMNs and monocytes accompanied by decreased response variabilities in variant-IRAK1 subjects may be a contributing mechanism affecting the course of sepsis and trauma and may also impact sex-based outcome differences due to its X-linked inheritance pattern and high prevalence.

Bacteremia is a hallmark of invasive Streptococcus suis infections of pigs, often leading to septicemia, meningitis, or arthritis. An important defense mechanism of neutrophils is the generation of reactive oxygen species (ROS). In this study, we report high levels of ROS production by blood granulocytes after intravenous infection of a pig with high levels of S. suis -specific antibodies and comparatively low levels of bacteremia. This prompted us to investigate the working hypothesis that the immunoglobulin-mediated oxidative burst contributes to the killing of S. suis in porcine blood. Several S. suis strains representing serotypes 2, 7, and 9 proved to be highly susceptible to the oxidative burst intermediate hydrogen peroxide, already at concentrations of 0.001%. The induction of ROS in granulocytes in ex vivo -infected reconstituted blood showed an association with pathogen-specific antibody levels. Importantly, inhibition of ROS production by the NADPH oxidase inhibitor apocynin led to significantly increased bacterial survival in the presence of high specific antibody levels. The oxidative burst rate of granulocytes partially depended on complement activation, as shown by specific inhibition. Furthermore, treatment of IgG-depleted serum with a specific IgM protease or heat to inactivate complement resulted in >3-fold decreased oxidative burst activity and increased bacterial survival in reconstituted porcine blood in accordance with an IgM-complement-oxidative burst axis. In conclusion, this study highlights an important control mechanism of S. suis bacteremia in the natural host: the induction of ROS in blood granulocytes via specific immunoglobulins such as IgM., (Copyright © 2020 American Society for Microbiology.)

Psychological stress may be linked to cancer incidence; however, more direct evidence is required to support this viewpoint. In this study, we investigated the effects of stress on immunosurveillance against cancer cells using a previously established examination stress model. We showed that the cancer killing activity (CKA) of granulocytes (also known as polymorphic nuclear cells, PMNs) is sharply reduced during examination stress stimulation in some donors who are psychologically sensitive to examination stress, with the concentration of plasma stress hormones (cortisone, epinephrine, and norepinephrine) increasing accordingly. The effects of stress hormones on immune cell CKA were also investigated under two in vitro co-incubation conditions, with all three hormones found to exert inhibitory effects on the CKA of PMNs and mononuclear cells. We showed that stress triggered the release of stress hormones which had profound inhibitory effects on the innate anticancer functions of PMNs. These results provide a possible explanation for the relationship between psychological stress and cancer incidence.

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease., Competing Interests: The authors have declared that no competing interests exist.

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation., (Copyright © 2019 Ui Mhaonaigh, Coughlan, Dwivedi, Hartnett, Cabral, Moran, Brennan, Doyle, Hughes, Lucey, Floudas, Fearon, McGrath, Cormican, De Bhailis, Molloy, Brady and Little.)

The present study was undertaken to estimate the approximate size of nuclear regions occupied by nucleolar bodies during the cell differentiation and maturation. The differentiation and maturation of human leukemic granulocytic cells in patients suffering from the chronic phase of the chronic granulocytic leukemia (CML) represented a convenient model for such study because of the large number of cells for the diameter measurements at the single cell level. Early and advanced differentiation or maturation stages of these cells are well defined and nucleolar bodies and nuclear outlines are easily seen by simple cytochemical methods for the visualization of RNA and silver stained proteins in smear preparations. During the cell differentiation and maturation, the estimated size of the nuclear region occupied by nucleolar bodies decreased in both untreated and treated patients with the anti-leukemic therapy. However, the size reduction of nucleolar bodies in differentiated and mature cells was larger than that of the nucleus. In addition, the results also indicated that the nuclear region occupied by nucleolar bodies was characteristic for each differentiation and maturation stage of the granulocytic cell lineage and was not substantially influenced by the anti-leukemic therapy of CML patients.

The immune response relies on the integration of cell-intrinsic processes with cell-extrinsic cues. During infection, B cells vacate the marrow during emergency granulopoiesis but return upon restoration of homeostasis. Here we report a novel glycosylation-mediated crosstalk between marrow B cells and hematopoietic progenitors. Human B cells secrete active ST6GAL1 sialyltransferase that remodels progenitor cell surface glycans to suppress granulopoiesis. In mouse models, ST6GAL1 from B cells alters the sialylation profile of bone marrow populations, and mature IgD+ B cells were enriched in sialylated bone marrow niches. In clinical multiple myeloma, ST6GAL1 abundance in the multiple myeloma cells negatively correlated with neutrophil abundance. These observations highlight not only the ability of medullary B cells to influence blood cell production, but also the disruption to normal granulopoiesis by excessive ST6GAL1 in malignancy., Competing Interests: EI, ML, YZ, SN, KH, JL No competing interests declared, (© 2019, Irons et al.)

Background: Neutrophils and eosinophils are multifunctional granulocytes derived from common myelocytic-committed progenitor cells. Severe congenital neutropenia 1 (SCN1) caused by ELANE mutations is a rare disease characterized by very low numbers of circulating neutrophils. Little is known about the functional characteristics of the SCN1 granulocytes, except that eosinophilia has been noticed in both bone marrow and peripheral blood. In this study, we profiled the number and function of granulocytes in patients suffering from SCN1., Methods: Nine patients diagnosed with SCN1 were enrolled in this study and absolute counts of eosinophils and neutrophils from bone marrow aspirates and peripheral blood samples were analysed. In addition, Ficoll-Paque enriched granulocytes from patients and healthy controls were analysed for specific eosinophil and neutrophil markers using flow cytometry and for NADPH-oxidase activity-profile by chemiluminescence., Results: Our data demonstrate a skewed granulocyte population in SCN1 patients dominated by eosinophils in both bone marrow and peripheral blood. The latter was detected only by blood smear examination, but not by automated blood analysers. Furthermore, we show that the SCN1 eosinophils exerted normal production of reactive oxygen species generated by the NADPH-oxidase, however the response was profoundly different from that of healthy control neutrophils., Conclusions: SCN1 patients with ELANE mutations suffer from neutropenia yet display eosinophilia in the bone marrow and blood, as revealed by smear examination but not by automatic blood analysers. The SCN1 eosinophils are functionally normal regarding production of reactive oxygen species (ROS). However, the ROS profile produced by eosinophils differs drastically from that of neutrophils isolated from the same blood donor, implying that the eosinophilia in SCN1 cannot compensate for the loss of neutrophils regarding ROS-mediated functions.

Background: Patients with neutropenia or granulocyte dysfunction may require granulocyte transfusions for adequate immune restoration. High-molecular-weight hydroxyethyl starch (HES) is the most commonly used sedimentation agent to enhance granulocyte collection efficiency. However, authorities recently restricted the use of HES due to its unfavorable risk-benefit profile. As modified fluid gelatin (MFG) is already used as an alternative sedimentation agent, we tested the hypothesis that MFG is not inferior to HES in terms of the functionality and viability of granulocytes., Study Design and Methods: Granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15%, or 30% MFG (Gelafundin) or HES (Hespan), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ROS) production, neutrophil extracellular trap formation (NETosis), antigen expression, and viability were subsequently investigated in vitro., Results: Relative to the controls, all three concentrations of HES compared to only 15% and 30% MFG lowered migration distances, and the 15% and 30% concentrations of both sedimentation agents reduced track straightness. HES resulted in lower CD11b expression and higher CD62L expression compared to MFG and the controls, whereas the differences for CD66b did not reach statistical significance. No significant differences in the timing of ROS production or NETosis, or in neutrophil viability or respiratory burst were observed., Conclusion: These results indicate that MFG is not inferior to HES in terms of granulocyte function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with HES., (© 2019 AABB.)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of low-density granulocytes (LDGs) with a heightened capacity for spontaneous NETosis, but the contribution of LDGs to SLE pathogenesis remains unclear. To characterize LDGs in human SLE, gene expression profiles derived from isolated LDGs were characterized by weighted gene coexpression network analysis, and a 92-gene module was identified. The LDG gene signature was enriched in genes related to neutrophil degranulation and cell cycle regulation. This signature was assessed in gene expression datasets from two large-scale SLE clinical trials to study associations between LDG enrichment, SLE manifestations, and treatment regimens. LDG enrichment in the blood was associated with corticosteroid treatment as well as anti-dsDNA, low serum complement, renal manifestations, and vasculitis, but the latter two of these associations were dependent on concomitant corticosteroid treatment. In addition, LDG enrichment was associated with enrichment of gene signatures induced by type I IFN and TNF irrespective of corticosteroid treatment. Notably, LDG enrichment was not found in numerous tissues affected by SLE. Comparison with relevant reference datasets indicated that LDG enrichment is likely reflective of increased granulopoiesis in the bone marrow and not peripheral neutrophil activation. The results have uncovered important determinants of the appearance of LDGs in SLE and have emphasized the likely role of LDGs in specific aspects of lupus pathogenesis., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Introduction: Cell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34 + quantification; (ii) percentage of CD34 + viability and (iii) evaluation of HSC functional ability to form colony forming unit-granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34 + , using a Fluorescent Labeled Inhibitor of CAspases (FLICA)., Methods: Caspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34 + , CD3 + , CD14 + and CD56 + cells. Viable 7AAD - /FLICA + cells were then correlated with various parameters., Results: We showed a significant caspase pathway activation, with 23% CD34 + /7AAD - /FLICA + cells after thawing, compared with the 2% described in fresh CD34 + cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3 + , CD56 + and CD14 + cells. We also report a significant correlation between the rate of CD34 + /7AAD - /FLICA + cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004)., Discussion: Our results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3 + and CD56 + subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI)., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

In the parasitoid/polydnavirus/host system, polydnaviruses protect larva development in the host hemocoel by suppressing the host immune response. However, the negative effects on the parasitoid and the strategy of the parasitoid to deal with this disadvantage are still unknown. Microplitis bicoloratus bracovirus induces granulocyte apoptosis to suppress immune responses, resulting in an apoptotic haemolymph environment in which immature M. bicoloratus larva develop. Here, we determined the transcriptional profiles of immature M. bicoloratus across five time-points throughout the immature developmental process from egg to third instar. Dynamic gene expression pattern analysis revealed clear rapid changes in gene expression characteristic of each developmental stage, indicating faster sequential unambiguous functional division during development. Combined with the proteome of the host haemolymph, immature parasitoids likely secreted a Cu/Zn superoxide dismutase to reduce reactive oxygen species generation by symbiotic bracovirus. These data established a basis for further studies of parasitoid/host interactions and identified a novel positive self-protection mechanism for the parasitoid., (Copyright © 2018. Published by Elsevier Ltd.)

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response, resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation, systemic immune activation, and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic, functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L, CD16, and CD80, consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-γ via the production of reactive oxygen species. Furthermore, CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Bacterial and fungal infections still remain an important cause of mortality in patients with hematological malignancies and in recipients of hematopoietic stem cell transplants (HSCT) especially in developing countries like India. Granulocyte transfusions (GTX) from healthy donors may lead to early clearance of index infection and thus prevent mortality. The aim of the present study was to evaluate the efficacy of GTX in combating life-threatening infections and preventing mortality in patients of hematological disorders/recipients of HSCT with severe neutropenia. This study was a prospective, observational analysis of patients with different hematological disorders/recipients of HSCT, who received GTX from January 2014 to December 2017. All patients had an Absolute neutrophil Count (ANC) < 0.5 x 109/L and a life threatening sepsis defined by presence of hemodynamic instability/ impending septic shock/ continuous high fever despite the use of the highest line of antimicrobials. A total of 143 granulocyte collections were done for 66 infectious episodes (IEs) in 60 patients. Multidrug resistant organisms (MDROs) were observed in 47/66 IEs (71.2%) and fungal infections were seen in 9/66 IEs (13.6%). Resolution of index infection after GTX was seen in 45/66 IEs (68.2%), and the 30 day overall survival (OS) was 67.7%. OS was significantly higher in patients who received GTX within 7 days of neutropenic sepsis (p = 0.01). Patients with MDROs who received early GTX therapy had a better OS as compared to those who received late GTX (p = 0.02). GTX were well tolerated and only 6 patients' developed mild features of transfusion related acute lung injury (TRALI) which was managed conservatively, and 1 patient demonstrated hypocalcemic tetany. GTX may be of particular relevance in countries like India, where the incidence of infections is very high in neutropenic patients and there is an increasing emergence of MDROs., Competing Interests: The authors have declared that no competing interests exist.

In the present work, the effects of the neonicotinoid imidacloprid formulation on the total hemocyte counts (THC) and differential hemocyte counts (DHC) were investigated in foraging workers of the stingless bee Melipona quadrifasciata under (or not) the challenge with the bacteria Escherichia coli. The THC was not altered with the insecticide exposure and/or bacterial infection. However, the DHC of the bees changed with the imidacloprid exposure and/or bacterial infection. The number of prohemocytes (stem cells) increased in bees exposed to imidacloprid, but it did not change after the bacterial infection. The number of plasmatocytes (phagocytic cells) increased in imidacloprid-exposed and uninfected bees and decreased in infected bees regardless of the exposure to imidacloprid. On the other hand, there was a reduction of granulocytes, the most active immune cells, after imidacloprid exposure and an increase of granulocytes after the infection. Previous studies have shown that the neonicotinoids exposure can impair the immune system of bees. Our findings showed that the relative number of granulocytes declined with imidacloprid exposure, but the overall capacity of hemocyte responses in terms of total numbers after bacterial infection persisted even after the insecticide exposure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Defining new prognostic biomarkers has become one of the most promising perspectives for the long-term care of patients with juvenile idiopathic arthritis (JIA). The new efficient indicators of disease activity and potential response to treatment are crucial in establishing new therapeutic plans in accordance with the "treat to target" strategy. One of the most studied proteins is called S100A12, which is an alarmin specific for granulocytes, considered as a marker of their activity., Materials and Methods: Study involved 80 patients diagnosed with JIA. Children with systemic subtype were not included in the study. In 53 cases, blood samples were obtained in two time points. Results from the study group were compared to 29 age- and sex-matched healthy individuals., Results: Serum S100A12 levels were higher in JIA than in healthy controls at the study baseline (11.67 ± 6.59 vs. 6.01 ± 2.33 ng/ml). There were no significant differences in S100A12 values between assessed subtypes of JIA. The highest concentrations were observed in patients within a disease flare. S100A12 levels were not dependent from using glucocorticosteroids. The studied protein appeared to be an efficient biomarker for JIA patients: 100% specificity as a diagnostic marker (cut-off level: 10.73 ng/ml) and 100% sensitivity as an indicator of exacerbations within a 3-month observation (cut-off level: 5.48 ng/ml)., Conclusions: S100A12 may become an important factor influencing decisions on aggressiveness of JIA therapy. Further elaboration on the clinical algorithm is necessary for that protein to be included in everyday practice.

Acute exercise evokes an almost instantaneous lymphocytosis, followed by sustained lymphopenia that occurs within just 30-60 min after exercise cessation. The aim of this study was to characterize the immediate (order of minutes) post-exercise kinetics of lymphocyte and monocyte egress, and to determine whether this egress is associated with heart rate recovery following a single bout of steady state dynamic exercise. Eleven healthy subjects cycled for 30-min at ~70% of their estimated peak power. Blood samples were collected from an intravenous catheter before exercise, during exercise (E) at +15 and +30 min, and during passive recovery (R) at exactly +1, +2, +3, +4, +5 and +10 min after exercise cessation. Complete blood counts and flow cytometry were used to enumerate total monocytes, lymphocytes: CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, NK-cells and γδ T-cells in whole blood. Both lymphocytes and monocytes displayed rapid egress kinetics, by R+3 the total numbers of all cell types examined were significantly lower than E+30. NK-cells egressed more rapidly than other lymphocyte subtypes, followed by CD8+, γδ, and then CD4+ T-cells. Further, the egress of NK-cells, CD4+, and CD8+ T-cells positively correlated with heart rate recovery after exercise cessation. In conclusion, lymphocyte and monocyte egress is rapid and occurs within minutes of exercise recovery, underscoring both the importance of collection time for post exercise blood samples, and the use of intravenous catheters to capture peak cell mobilization. The rate of egress may be dependent on how quickly hemodynamic equilibrium is restored on cessation of exercise and is, therefore, likely to be influenced by individual fitness levels., (Copyright © 2018. Published by Elsevier Inc.)

Despite advances in our understanding of the mechanisms underlying the progression of chronic kidney disease and the development of fibrosis, only limited efficacious therapies exist. The calcium binding protein S100A8/A9 is a damage-associated molecular pattern which can activate Toll-like receptor (TLR)-4 or receptor for advanced glycation end-products (RAGE). Activation of these receptors is involved in the progression of renal fibrosis; however, the role of S100A8/A9 herein remains unknown. Therefore, we analysed S100A8/A9 expression in patients and mice with obstructive nephropathy and subjected wild-type and S100A9 knock-out mice lacking the heterodimer S100A8/A9 to unilateral ureteral obstruction (UUO). We found profound S100A8/A9 expression in granulocytes that infiltrated human and murine kidney, together with enhanced renal expression over time, following UUO. S100A9 KO mice were protected from UUO-induced renal fibrosis, independently of leucocyte infiltration and inflammation. Loss of S100A8/A9 protected tubular epithelial cells from UUO-induced apoptosis and critical epithelial-mesenchymal transition steps. In-vitro studies revealed S100A8/A9 as a novel mediator of epithelial cell injury through loss of cell polarity, cell cycle arrest and subsequent cell death. In conclusion, we demonstrate that S100A8/A9 mediates renal damage and fibrosis, presumably through loss of tubular epithelial cell contacts and irreversible damage. Suppression of S100A8/A9 could be a therapeutic strategy to halt renal fibrosis in patients with chronic kidney disease., (© 2018 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Bladder cancer (BC) is a disease arising from the malignant cells of the urinary bladder. Myeloid-derived suppressor cells (MDSCs) expand broadly and have strong immunosuppressive activities in the cancer microenvironment. Determining how to inhibit the negative effects of MDSCs requires immediate attention. In this study, we found that granulocytic-MDSCs (G-MDSCs), which constitute one of the two types of MDSCs, were significantly increased in BC tissues compared with those in the adjacent bladder tissues. There was a robust negative correlation between the G-MDSCs and the CD8 + T cells in the BC tissues. In this study, we attempted to identify pharmacological approaches to eliminate MDSCs and restore T cell anti-tumor activities. It is necessary to explore a method to eliminate the detrimental effects of MDSCs. Cisplatin, a chemotherapy medication used to treat BC, not only rapidly kills proliferating cancer cells but also affects the tumor immune microenvironment. However, the mechanism underlying this phenomenon is largely unknown. In this study, we found that Cisplatin directly inhibited the proliferation and induced the apoptosis of T24 cells (a BC cell line), as well as decreased the percentage of the G-MDSCs in the population of peripheral blood mononuclear cells (PBMCs), which restored the expansion of the CD8 + T cells. In the C3H/He mouse BC model, Cisplatin treatment inhibited the progression of BC and effectively decreased the proportion of G-MDSCs. These results suggest that Cisplatin treatment enhances the anti-tumor function of CD8 + T cells by decreasing G-MDSCs. This finding provides a new perspective for Cisplatin treatment to prevent the progression of BC, particularly in patients with abnormally high levels of G-MDSCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cellular age distribution of hierarchically organized tissues can reveal important insights into the dynamics of cell differentiation and self-renewal and associated cancer risks. Here, we examine the effect of progenitor compartments with varying differentiation and self-renewal capacities on the resulting observable distributions of replicative cellular ages. We find that strongly amplifying progenitor compartments, i.e. compartments with high self-renewal capacities, substantially broaden the age distributions which become skewed towards younger cells with a long tail of few old cells. For several of these strongly amplifying compartments, the age distribution becomes virtually independent of the influx from the stem cell compartment. By contrast, if tissues are organized into many downstream compartments with low self-renewal capacity, the shape of the replicative cell distribution in more differentiated compartments is dominated by stem cell dynamics with little added variation. In the limiting case of a strict binary differentiation tree without self-renewal, the shape of the output distribution becomes indistinguishable from that of the input distribution. Our results suggest that a comparison of cellular age distributions between healthy and cancerous tissues may inform about dynamical changes within the hierarchical tissue structure, i.e. an acquired increased self-renewal capacity in certain tumours. Furthermore, we compare our theoretical results to telomere length distributions in granulocyte populations of 10 healthy individuals across different ages, highlighting that our theoretical expectations agree with experimental observations., (© 2018 The Authors.)

Transendothelial migration (TEM) of lymphocytes and neutrophils is associated with the ability of their deformable nuclei to displace endothelial cytoskeletal barriers. Lamin A is a key intermediate filament component of the nuclear lamina that is downregulated during granulopoiesis. When elevated, lamin A restricts nuclear squeezing through rigid confinements. To determine if the low lamin A expression by leukocyte nuclei is critical for their exceptional squeezing ability through endothelial barriers, we overexpressed this protein in granulocyte-like differentiated HL-60 cells. A 10-fold higher lamin A expression did not interfere with chemokinetic motility of these granulocytes on immobilized CXCL1. Furthermore, these lamin A high leukocytes exhibited normal chemotaxis toward CXCL1 determined in large pore transwell barriers, but poorly squeezed through 3 μm pores toward identical CXCL1 gradients. Strikingly, however, these leukocytes successfully completed paracellular TEM across inflamed endothelial monolayers under shear flow, albeit with a small delay in nuclear squeezing into their sub-endothelial pseudopodia. In contrast, CXCR2 mediated granulocyte motility through collagen I barriers was dramatically delayed by lamin A overexpression due to a failure of lamin A high nuclei to translocate into the pseudopodia of the granulocytes. Collectively, our data predict that leukocytes maintain a low lamin A content in their nuclear lamina in order to optimize squeezing through extracellular collagen barriers but can tolerate high lamin A content when crossing the highly adaptable barriers presented by the endothelial cytoskeleton., (©2018 Society for Leukocyte Biology.)

The aim of this study was to assess modulatory effects of dietary supplements mannan oligosaccharide (MOS) and clinoptilolite (CPL) as potential alternatives to antibiotic growth promoters (AGP) given to 4-week old pigs at weaning (Day 0) on their innate/adaptive immunity by determining: alterations in C-reactive protein (CRP) and haptoglobin (HpG) serum levels, efficiency of blood monocytes (MO) and neutrophilic granulocytes (GR) for in vitro phagocytosis (PHC)/microbicidity (MBC) and proportion of extrathymic double positive CD4 CD8 (CD4+CD8+) T cells throughout 35 days of the study. Neither MOS nor CPL changed the serum concentrations of CRP, whereas that of HpG was significantly increased in the CPL supplemented pigs (p<0.05) at Day 35. Activity of PHA of GR was significantly increased by both dietary supplements (p<0.05) from Day 7 to Day 35. Also, the GR from pigs fed with both supplements had significantly increased MBC at Day 7 (p<0.05), but at Day 35 such an increase was observed only for CPL. The in vitro PHC/MBC of MO did not change in either group of supplemented pigs. The pigs supplemented with MOS had a significantly higher proportion of CD4+CD8+ T lymphocytes at Day 28 (p<0.05). Although both supplements showed a promising ability to stimulate rather innate than adaptive cellular immunity, it does not appear that any solely applied natural substance such as MOS or CPL in the current study could be a competitive alternative to conventional AGP for improving health and promoting growth in weaned pigs., (Copyright© by the Polish Academy of Sciences.)

Preterm delivery is the leading cause of perinatal morbidity and mortality. Among the most important complications in preterm infants are peri- or postnatal infections. Myeloid-derived suppressor cells (MDSC) are myeloid cells with suppressive activity on other immune cells. Emerging evidence suggests that granulocytic MDSC (GR-MDSC) play a pivotal role in mediating maternal-fetal tolerance. The role of MDSC for postnatal immune-regulation in neonates is incompletely understood. Until the present time, nothing was known about expression of MDSC in preterm infants. In the present pilot study, we quantified GR-MDSC counts in cord blood and peripheral blood of preterm infants born between 23 + 0 and 36 + 6 weeks of gestation (WOG) during the first 3 months of life and analysed the effect of perinatal infections. We show that GR-MDSC are increased in cord blood independent of gestational age and remain elevated in peripheral blood of preterm infants during the neonatal period. After day 28 they drop to nearly adult levels. In case of perinatal or postnatal infection, GR-MDSC accumulate further and correlate with inflammatory markers C-reactive protein (CRP) and white blood cell counts (WBC). Our results point towards a role of GR-MDSC for immune-regulation in preterm infants and render them as a potential target for cell-based therapy of infections in these patients., (© 2017 British Society for Immunology.)

Background: High-mobility group AT-hook 2 (HMGA2) may serve as an architectural transcription factor, and it can regulate a range of normal biological processes including proliferation and differentiation. Upregulation of HMGA2 expression is correlated to the undifferentiated phenotype of immature leukaemic cells. However, the underlying mechanism of HMGA2-dependent myeloid differentiation blockage in leukaemia is unknown., Methods: To reveal the role and mechanism of HMGA2 in differentiation arrest of myeloid leukaemia cells, the quantitative expression of HMGA2 and homeobox A9 (HOXA9) was analysed by real-time PCR (qRT-PCR). The regulatory function of HMGA2 in blockage of differentiation in human myeloid leukaemia was investigated through in vitro assays (XTT assay, May-Grünwald-Giemsa, flow cytometry analysis and western blot)., Results: We found that the expression of HMGA2 and HOXA9 was reduced during the process of granulo-monocytic maturation of acute myeloid leukaemia (AML) cells, knockdown of HMGA2 promotes terminal (granulocytic and monocytic) differentiation of myeloid leukaemia primary blasts and cell lines, and HOXA9 was significantly downregulated in leukaemic cells with knockdown of HMGA2. Downregulation of HOXA9 in myeloid leukaemia cells led to increased differentiation capacity in vitro., Conclusions: Our data suggest that increased expression of HMGA2 represents a possible new mechanism of myeloid differentiation blockage of leukaemia. Aberrant expression of HMGA2 may enhance HOXA9-dependent leukaemogenesis and myeloid leukaemia phenotype. Disturbance of the HMGA2-HOXA9 pathway is probably a therapeutic strategy in myeloid leukaemia.

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

For biomedical applications, it is important to know, which kinds of blood cells can capture quantum dots (QDs). The maximum accumulation of QDs was found for the monocyte fraction of leukocytes, the minimum binding of QDs was observed for lymphocytes. It was found that CdSe/ZnS-MPA QDs are actively absorbed by the cells and have more expressed toxicity. The classical mechanism of the phagocytosis of QDs was revealed for neutrophils, when the QDs are located in phagolysosomes. The capture of QDs by neutrophil granulocytes has resulted in a destruction of certain types of QDs. The interaction of the neutrophils with the QDs has resulted in the death of the cells by one of the following cell death mechanisms: necrosis, apoptosis, autophagy, NETos, or mummification. The aggregation of the QDs manifested as an increase of the hydrodynamic diameter of the QDs was found to occur under the influence of serum and under the influence of blood cells (lymphocytes and neutrophils) in a serum-free medium., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Circular RNAs (circRNAs) are pervasively expressed circles of non‑coding RNAs. Even though many circRNAs have been reported in humans, their expression patterns and functions remain poorly understood. In this study, we employed a pipeline named RAISE to detect circRNAs in RNA‑seq data. RAISE can fully characterize circRNA structure and abundance. We evaluated inter-individual variations in circRNA expression in humans by applying this pipeline to numerous non‑poly(A)-selected RNA‑seq data. We identified 59,128 circRNA candidates in 61 human liver samples, with almost no overlap in the circRNA of the recruited samples. Approximately 89% of the circRNAs were detected in one or two samples. In comparison, 10% of the linear mRNAs and non‑coding RNAs were detected in each sample. We estimated the variation in other tissues, especially the circRNA high-abundance tissues, in advance. Only 0.5% of the 50,631 brain circRNA candidates were shared among the 30 recruited brain samples, which is similar to the proportion in liver. Moreover, we found inter- and intra-individual diversity in circRNAs expression in the granulocyte RNA‑seq data from seven individuals sampled 3 times at one-month intervals. Our findings suggest that careful consideration of inter-individual diversity is required when extensively identifying human circRNAs or proposing their use as potential biomarkers and therapeutic targets in disease.

High-quality relationships have been shown to be beneficial for physical and mental health. This study examined overall relationship satisfaction and perceived stress as well as daily reports of partner support, partner conflict, and physical intimacy obtained over the course of one week in a sample of 129 high and low stress mothers. Telomere length was examined in whole blood, as well as the two cell subpopulations: peripheral blood mononuclear cells (PBMCs) and granulocytes. Telomerase activity was measured in PBMCs. Analyses revealed no statistically significant associations of telomere length with current relationship satisfaction, daily support or conflict, or perceived stress. In contrast, women who reported any sexual intimacy during the course of the week had significantly longer telomeres measured in whole blood and PBMCs, but not in granulocytes. These relationships held covarying for age, body mass index, perceived stress, the relationship indices, and caregiver status. Sexual intimacy was not significantly related to PBMC telomerase activity. These data provide preliminary data that sexual intimacy is associated with longer telomere length. Future studies investigating these associations are warranted., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Mutations of the splicing factor-encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor-associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe 2+ . We showed that HO-1 knock-out mice (HO-1 -/- ) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1. The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1 -/- mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1 -/- mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPβ (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPβ expression and activity in HO-1 -/- myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1 -/- granulocytes were functionally competent as determined by oxidative burst capacity. In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPβ protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia., (Copyright © 2017. Published by Elsevier GmbH.)

Granulopoiesis is accelerated during Gram-negative bacterial infection through activation of toll-like receptor 4 (TLR4). In this study, we tested whether activation of TLR2 promotes granulopoiesis by using the well-known TLR2 agonist, peptidoglycan (PGN). Neutrophils in bone marrow and spleen, and plasma granulocyte colony-stimulating factor (G-CSF) were increased in mice that had received intraperitoneal PGN administration. Incorporation of BrdU into bone marrow neutrophils increased, demonstrating that PGN accelerated granulopoiesis. Treatment of bone marrow cells (BMCs) with PGN increased neutrophils in vitro and promoted the secretion of G-CSF from Ly-6G - Ly-6C + monocytes. The accelerated granulopoiesis caused by PGN was not seen in TLR2-deficient and MyD88-deficient BMCs. Additionally, PGN induced G-CSF production in human umbilical vein endothelial cells. These findings demonstrate that PGN promotes the secretion of G-CSF from monocytes and endothelial cells, leading to the acceleration of granulopoiesis. Our results illustrate that bacterial recognition by TLR2 facilitates granulopoiesis during Gram-positive bacterial infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)