Granulocytic Subtypes Research Articles

On the label-free analysis of white blood cells by holographic quantitative phase imaging flow cytometry

This study presents an innovative methodology to analyze a blood sample from a healthy donor, providing a quantitative characterization of white blood cells (WBCs). It aims to evaluate the effectiveness of holographic quantitative phase imaging (QPI) flow cytometry (FC) in examining phase-contrast maps at the cellular level, thereby enabling the identification and classification of granulocyte types. Additionally, we demonstrate that an unsupervised method can differentiate granulocyte sub-types, i.e., neutrophils and eosinophils. The results instill strong confidence in the potential future use of QPI FC for liquid biopsies and/or for assessing the heterogeneity of WBCs and, more broadly, to facilitate label-free blood tests.

Neutrophil-like Monocytes Increase in Patients with Colon Cancer and Induce Dysfunctional TIGIT+ NK Cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of immune cells including granulocytic (CD14neg/CD15+/HLA-DRneg) and monocytic subtypes (CD14+/CD15neg/HLA-DRneg). In the present study, we found a population of monocytes expressing the granulocyte marker CD15 that significantly increased in both peripheral blood (PB) and tumoral tissues of patients with colorectal cancer (CRC). Further phenotypical analysis confirmed the granulocytic-like features of this monocyte subpopulation that is associated with an increase in granulocyte-monocyte precursors (GMPs) in the PB of these patients (pts). Mechanistically, this granulocyte-like monocyte population suppressed NK cell activity by inducing TIGIT and engaging NKp30. Accordingly, an increased frequency of TIGIT+ NK cells with impaired functions was found in both the PB and tumoral tissue of CRC pts. Collectively, we provided new mechanistic explanations for tumor immune escape occurring in CRC by showing the increase in this new kind of MDSC, in both PB and CRC tissue, which is able to significantly impair the effector functions of NK cells, thereby representing a potential therapeutic target for cancer immunotherapy.

Fc receptors are key discriminatory markers of granulocytes subsets in people living with HIV-1.

Granulocytes are innate immune cells that play a key role in pathogen elimination. Recent studies revealed the diversity of granulocytes in terms of phenotype and function. In particular, a subset of granulocytes identified as low-density granulocytes (LDG) has been described in physiological conditions and with increased frequencies in several pathological contexts. However, the properties of LDG are still controversial as they vary according to the pathophysiological environment. Here we investigated the heterogeneity of granulocyte populations and the potential differences in phenotype and immunomodulatory capacity between LDG and normal density granulocytes (NDG) in people living with HIV-1 (PLWH). To this end, we developed an optimized method to purify LDG and NDG from a single blood sample, and performed in-depth, comparative phenotypic characterization of both granulocyte subtypes. We also assessed the impact of purification steps on the expression of cell surface markers on LDG by immunophenotyping them at different stages of isolation. We identified 9 cell surface markers (CD16, CD32, CD89, CD62L, CD177, CD31, CD10, CXCR4 and CD172α) differentially expressed between LDG and NDG. Noteworthy, markers that distinguish the two subsets include receptors for the Fc part of IgG (CD16, CD32) and IgA (CD89). Importantly, we also highlighted that the purification procedure affects the expression of several cell surface markers (i.e.CD63, CD66b, …) which must be taken into account when characterizing LDG. Our work sheds new light on the properties of LDG in PLWH and provides an extensive characterization of this granulocyte subset in which Fc receptors are key discriminatory markers.

Phenotypic changes in immune cells induced by granulocyte and monocyte adsorptive apheresis in patients with severe COVID-19: An exvivo study.

SARS-CoV-2 causes systemic immune dysfunction, leading to severe respiratory dysfunction and multiorgan dysfunction. Granulocyte and monocyte adsorptive apheresis (GMA) therapy is designed to regulate an excessive inflammatory response and has been proposed as a potential therapeutic strategy for coronavirus disease 2019 (COVID-19). We aimed to investigate a targeted subset of granulocytes and monocytes to be removed after GMA therapy in patients with severe COVID-19 infection. We established an exvivo experimental system to study the effects of GMA. Blood samples were collected into EDTA-treated tubes and a mixture of blood samples and cellulose acetate beads was used in GMA. After GMA, blood samples were removed, and the granulocyte and monocyte subtypes before and after GMA were determined by CyTOF mass cytometry. To analyze mass cytometry data with a self-organizing map, hierarchical clustering was used to determine the appropriate number of metaclusters from t-distributed stochastic neighbor embedding. We included seven patients with severe COVID-19 and four age- and sex-matched volunteers. Granulocyte subsets removed by GMA strongly expressed CD11b, CD16, and CD66b, and weakly expressed CD11c, consistent with mature and activated neutrophils. Monocyte subsets strongly expressed CD14, weakly expressed CD33 and CD45RO, and did not express CD16. These subsets were indicated to promote the release of inflammatory cytokines and activate T cells. The identification of the granulocyte and monocyte subsets removed after GMA in patients with severe COVID-19 may help explain the potential mechanism underlying the effectiveness of GMA in COVID-19 and other inflammatory diseases.

Sputum microbiota and inflammatory subtypes in asthma, COPD, and its overlap

BackgroundAirway microbiota in asthma-COPD overlap (ACO) remains unknown. ObjectiveThis study with ACO-enriched population aimed to clarify airway microbiota in ACO and in mixed granulocytic inflammation, often detected in ACO and chronic airway diseases. MethodsThis is an observational cross-sectional study. Patients with asthma with airflow limitation, ACO and COPD were enrolled. Blood tests, pulmonary function, exhaled nitric oxide and sputum tests were conducted. Sputum microbiota was evaluated using the 16S rRNA gene sequencing technique. ResultsA total of 112 patients (13 asthma, 67 ACO, 32 COPD) were examined. There were no significant differences in α-diversity among the three diseases. The relative abundances of phylum Bacteroidetes, class Bacteroidia, and genus Porphyromonas were associated with decreased eosinophilic inflammation, and were significantly lower in ACO than in COPD. In a comparison of sputum inflammatory subtypes, the proportion of Haemophilus was numerically highest in the mixed granulocytic subtype, followed by the neutrophilic subtype. Likewise, the proportion of Haemophilus was the highest in the intermediate-high (2%–8%) sputum eosinophil group and lowest in the severe (8%≤) eosinophil group. Clinically, Haemophilus proportion was associated with sputum symptoms. Finally, the proportion of Streptococcus was associated with higher blood eosinophil counts and severest airflow limitation. ConclusionBacteroidia and Porphyromonas abundances in sputum are associated with the eosinophil-low phenotype, and ACO may be characterised by a decrease in these taxa. A mild elevation of sputum eosinophil does not preclude the presence of Haemophilus, which should be noted in the management of obstructive airway diseases.

Genome-Scale Methylation Analysis Identifies Immune Profiles and Age Acceleration Associations with Bladder Cancer Outcomes.

Immune profiles have been associated with bladder cancer outcomes and may have clinical applications for prognosis. However, associations of detailed immune cell subtypes with patient outcomes remain underexplored and may contribute crucial prognostic information for better managing bladder cancer recurrence and survival. Bladder cancer case peripheral blood DNA methylation was measured using the Illumina HumanMethylationEPIC array. Extended cell-type deconvolution quantified 12 immune cell-type proportions, including memory, naïve T and B cells, and granulocyte subtypes. DNA methylation clocks determined biological age. Cox proportional hazards models tested associations of immune cell profiles and age acceleration with bladder cancer outcomes. The partDSA algorithm discriminated 10-year overall survival groups from clinical variables and immune cell profiles, and a semi-supervised recursively partitioned mixture model (SS-RPMM) with DNA methylation data was applied to identify a classifier for 10-year overall survival. Higher CD8T memory cell proportions were associated with better overall survival [HR = 0.95, 95% confidence interval (CI) = 0.93-0.98], while higher neutrophil-to-lymphocyte ratio (HR = 1.36, 95% CI = 1.23-1.50), CD8T naïve (HR = 1.21, 95% CI = 1.04-1.41), neutrophil (HR = 1.04, 95% CI = 1.03-1.06) proportions, and age acceleration (HR = 1.06, 95% CI = 1.03-1.08) were associated with worse overall survival in patient with bladder cancer. partDSA and SS-RPMM classified five groups of subjects with significant differences in overall survival. We identified associations between immune cell subtypes and age acceleration with bladder cancer outcomes. The findings of this study suggest that bladder cancer outcomes are associated with specific methylation-derived immune cell-type proportions and age acceleration, and these factors could be potential prognostic biomarkers.

Inherent bioelectrical parameters of hundreds of thousands of single leukocytes based on impedance flow cytometry.

As label-free biomarkers, bioelectrical properties of single cells have been widely used in hematology analyzers for 3-part differential of leukocytes, in which, however, instrument dependent bioelectrical parameters (e.g., DC/AC impedance values) rather than inherent bioelectrical parameters (e.g., diameter Dc , specific membrane capacitance Csm and cytoplasmic conductivity σcy ) were used, leading to poor comparisons among different instruments. In order to address this issue, this study collected inherent bioelectrical parameters from hundreds of thousands of white blood cells based on a home-developed impedance flow cytometry with corresponding 3-part differential of leukocytes realized. More specifically, leukocytes were separated into three major subtypes of granulocytes, monocytes and lymphocytes based on density gradient centrifugation. Then these separated cells were aspirated through a constriction-microchannel based impedance flow cytometry where inherent bioelectrical parameters of Dc , Csm and σcy were quantified as 9.8 ± 0.7μm, 2.06 ± 0.26 μF/cm2 , and 0.34 ± 0.05 S/m for granulocytes (ncell =134,829); 10.4 ± 1.0μm, 2.45 ± 0.48 μF/cm2 , and 0.42 ± 0.08 S/m for monocytes (ncell =40,226); 8.0 ± 0.5μm, 2.23 ± 0.34 μF/cm2 , and 0.35 ± 0.08 S/m for lymphocytes (ncell =129,193). Based on these inherent bioelectrical parameters, neural pattern recognition was conducted, producing a high "classification accuracy" of 93.5% in classifying these three subtypes of leukocytes. These results indicate that as inherent bioelectrical parameters, Dc , Csm , and σcy can be used to electrically phenotype white blood cells in a label-free manner.

P077 Different granulocyte subsets are involved in the pathogenesis in Crohn’s disease fistula

Abstract Background Early in inflammation granulocytes migrate from the circulation into inflamed areas and rapidly accumulate. They form an important role in first defense as well as the orchestration of downstream immune responses. In the circulation, several different subsets of granulocytes have been described, for example immature, intermediate and mature granulocytes. Surprisingly, despite their abundance and prominent function, this cell type is rarely studied from a tissue perspective, most likely due to their short lifespan ex vivo. One example of gastrointestinal disease with an extremely high number of granulocytes is the occurrence of perianal fistula which contain a considerable amount of granulation tissue. As curettage of this tissue is part of the treatment, the fistula tract is good source to study tissue derived granulocyte subsets. The aim of this study was to characterize granulocyte subsets in the fistula tract. Methods Curettage material of perianal fistula tracts was obtained during surgical intervention from patients with CD (n=15) and cryptoglandular fistulas (n=5). From 2 CD patients venous blood was taken and a ficoll step was performed. Single-cell suspensions were stained with a 35-antibody panel, including CD66, CD14, CD33, CD16 and CD11b. Samples were analyzed using mass cytometry (CyTOF). Results Granulocytes, characterized as CD45+CD66a+ cells, were the main cellular component of the fistula tract (64%±24) compared to other immune cells. This was more pronounced in cryptoglandular fistula compared to CD fistula (81% vs. 67%; p=0.019). Within these cells we could differentiate between 4 subsets, mature granulocytes (CD33+CD11b+CD16+), intermediate (CD33intCD11b+CD16int), immature granulocytes (CD33intCD11b-CD16int) and eosinophils (CD33-CD11b-CD16-). Immature granulocytes were the most abundant in de fistula tract (31%±15), closely followed by the mature (30%±23) and intermediate (23±9). No significant difference was seen in the percentages of granulocyte subsets between CD and cryptoglandular fistula. Conclusion Here, we show the presence of different subtypes of granulocytes in fistula derived granulation tissue. We show a heterogeneous group of cells ranging from immature neutrophils to mature neutrophils. Due to their high number in active ulcerating tissue, further evaluation of granulocyte subsets and function could be an interesting target for medical treatment options.

Single-cell RNA-seq profiling of individual Biomphalaria glabrata immune cells with a focus on immunologically relevant transcripts.

The immune cells of the snail Biomphalaria glabrata are classified into hyalinocyte and granulocyte subtypes. Both subtypes are essential for the proper functioning of the snail immune response, which we understand best within the context of how it responds to challenge with the human parasite Schistosoma mansoni. Granulocytes are adherent phagocytic cells that possess conspicuous granules within the cell cytoplasm. Hyalinocytes, on the other hand, are predominantly non-adherent and are known to produce a handful of anti-S. mansoni immune effectors. While our understanding of these cells has progressed, an in-depth comparison of the functional capabilities of each type of immune cell has yet to be undertaken. Here, we present the results of a single-cell RNA-seq study in which single granulocytes and hyalinocytes from S. mansoni-susceptible M-line B. glabrata and S. mansoni-resistant BS-90 B. glabrata are compared without immune stimulation. This transcriptomic analysis supports a role for the hyalinocytes as producers of immune effectors such as biomphalysin and thioester-containing proteins. It suggests that granulocytes are primarily responsible for producing fibrinogen-related proteins and are armed with various pattern-recognition receptors such as toll-like receptors with a confirmed role in the anti-S. mansoni immune response. This analysis also confirms that the granulocytes and hyalinocytes of BS-90 snails are generally more immunologically prepared than their M-line counterparts. As the first single-cell analysis of the transcriptional profiles of B. glabrata immune cells, this study provides crucial context for understanding the B. glabrata immune response. It sets the stage for future investigations into how each immune cell subtype differs in its response to various immunological threats.

Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation.

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

Mosquito cellular immunity at single-cell resolution.

Hemocytes limit the capacity of mosquitoes to transmit human pathogens. Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti mosquito vectors. Our data reveal the functional diversity of hemocytes, with different subtypes of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A previously unidentified cell type in An. gambiae, which we term "megacyte," is defined by a specific transmembrane protein marker (TM7318) and high expression of lipopolysaccharide-induced tumor necrosis factor-α transcription factor 3 (LL3). Knockdown experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify and validate two main hemocyte lineages and find evidence of proliferating granulocyte populations. This atlas of medically relevant invertebrate immune cells at single-cell resolution identifies cellular events that underpin mosquito immunity to malaria infection.

Immune Alterations Following Neurological Disorders: A Comparison of Stroke and Seizures.

Background: Granulocytes and monocytes are the first cells to invade the brain post stroke and are also being discussed as important cells in early neuroinflammation after seizures. We aimed at understanding disease specific and common pathways of brain-immune-endocrine-interactions and compared immune alterations induced by stroke and seizures. Therefore, we compared granulocytic and monocytic subtypes between diseases and investigated inflammatory mediators. We additionally investigated if seizure type determines immunologic alterations.Material and Methods: We included 31 patients with acute seizures, 17 with acute stroke and two control cohorts. Immune cells were characterized by flow cytometry from blood samples obtained on admission to the hospital and the following morning. (i) Monocytes subpopulations were defined as classical (CD14++CD16−), (ii) intermediate (CD14++CD16+), and (iii) non-classical monocytes (CD14dimCD16+), while granulocyte subsets were characterized as (i) “classical granulocytes” (CD16++CD62L+), (ii) pro-inflammatory (CD16dimCD62L+), and (iii) anti-inflammatory granulocytes (CD16++CD62L−). Stroke patient's blood was additionally drawn on days 3 and 5. Cerebrospinal fluid mitochondrial DNA was quantified by real-time PCR. Plasma High-Mobility-Group-Protein-B1, metanephrine, and normetanephrine were measured by ELISA.Results: HLA-DR expression on monocytes and their subpopulations (classical, intermediate, and non-classical monocytes) was reduced after stroke or seizures. Expression of CD32 was increased on monocytes and subtypes in epilepsy patients, partly similar to stroke. CD32 and CD11b regulation on granulocytes and subpopulations (classical, anti-inflammatory, pro-inflammatory granulocytes) was more pronounced after stroke compared to seizures. On admission, normetanephrine was upregulated in seizures, arguing for the sympathetic nervous system as inducer of immune alterations similar to stroke. Compared to partial seizures, immunologic changes were more pronounced in generalized tonic-clonic seizures.Conclusion: Seizures lead to immune alterations within the immediate postictal period similar but not identical to stroke. The type of seizures determines the extent of immune alterations.

SUN-134 Estrogen Induces Granulocytic Myeloid Derived Suppressor Cell Production to Potentially Modulate Lymphangioleiomyomatosis (LAM) Tumor Progression

Affecting almost exclusively women, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by estrogen-sensitive metastatic smooth muscle cell-like adenomas that grow slowly, resulting in cystic lung change and loss of pulmonary function. LAM tumors are caused by mutations in tuberous sclerosis complex 1 or 2 genes (TSC1 or TSC2). Mutations in TSC1 or TSC2 genes results in deficient inhibitory regulation of the mammalian target of rapamycin complex 1 (mTORC1), which in turn leads to increased mTORC1 activity and cell proliferation. There is no consensus amongst LAM researchers regarding the origin of these estrogen receptor-positive smooth muscle like LAM cells; however, we previously reported inactivation of TSC2 in the mouse uterus results in notable LAM features in the setting of primary myometrial tumors. Approximately 50% of the TSC2-null mice had metastatic myometrial tumors present in the lung, suggesting that LAM tumor cells might in fact originate from the uterus, thus explaining the female sexual dimorphism, the estrogen sensitivity, and the metastatic nature of the LAM tumors.Interestingly, flow cytometry revealed large numbers of granulocytic myeloid derived suppressors cells (G-MDSCs) in the blood and myometrial tumors of uterine-specific Tsc2 null mice. MDSCs are known to accumulate in the setting of chronic inflammation caused by trauma, infection, and various cancers. This granulocytic subtype not only has the capacity to suppress anti-tumor immune cells of the tumor microenvironment, but also directly promotes tumor cell malignant neoplasicity. We found that Tsc2-null myometrial tumors required MDSCs for normal progression, as MDSC depletion or inhibition of MDSC recruitment reduced tumor growth. We have showed that these tumors expressed estrogen receptors and were exquisitely sensitive to estrogen, while other studies demonstrate that G-MDSCs are also positively influenced by estradiol. Therefore, we hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen also stimulates tumor growth by promoting MDSC production in the bone marrow. We have developed a technique to stimulate MDSC production from mouse bone marrow. Using this strategy, we found that estradiol is indeed a potent promotor of G-MDSC production. These effects occurred in both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ERα; knockout mice, we showed that ERα; is necessary for promoting a G-MDSC fate for immature myeloid cells and precursors. Thus, estradiol may have duel effects in LAM, both directly promoting tumor growth and indirectly upregulating MDSC production, which in turn promotes tumor growth. We propose that these estrogen effects on MDSC production are not limited to LAM and may be important regulators of tumor growth in many tissues.

Guided migration analyses at the single-clone level uncover cellular targets of interest in tumor-associated myeloid-derived suppressor cell populations

Myeloid-derived suppressor cells (MDSCs) are immune cells that exert immunosuppression within the tumor, protecting cancer cells from the host’s immune system and/or exogenous immunotherapies. While current research has been mostly focused in countering MDSC-driven immunosuppression, little is known about the mechanisms by which MDSCs disseminate/infiltrate cancerous tissue. This study looks into the use of microtextured surfaces, coupled with in vitro and in vivo cellular and molecular analysis tools, to videoscopically evaluate the dissemination patterns of MDSCs under structurally guided migration, at the single-cell level. MDSCs exhibited topographically driven migration, showing significant intra- and inter-population differences in motility, with velocities reaching ~40 μm h−1. Downstream analyses coupled with single-cell migration uncovered the presence of specific MDSC subpopulations with different degrees of tumor-infiltrating and anti-inflammatory capabilities. Granulocytic MDSCs showed a ~≥3-fold increase in maximum dissemination velocities and traveled distances, and a ~10-fold difference in the expression of pro- and anti-inflammatory markers. Prolonged culture also revealed that purified subpopulations of MDSCs exhibit remarkable plasticity, with homogeneous/sorted subpopulations giving rise to heterogenous cultures that represented the entire hierarchy of MDSC phenotypes within 7 days. These studies point towards the granulocytic subtype as a potential cellular target of interest given their superior dissemination ability and enhanced anti-inflammatory activity.

Potential role of myeloid-derived suppressor cells in pulmonary fibrosis

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recruitment of bone marrow-derived myeloid cells is implicated in lung fibrosis by promoting fibrosis via paracrine mechanisms. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSC) in IPF patients. More recently, the importance of MDSC is suggested by the observation of MDSC accumulation in IPF lungs. The objective of this study was to assess the accumulation/expansion of MDSC in bleomycin-induced mouse model of pulmonary fibrosis and human IPF, and investigate their potential role in lung myofibroblast differentiation. Flow cytometry analysis showed that MDSCs in lung were increased in a mouse model of lung fibrosis, as well as in peripheral blood samples from IPF patients. The increase of the monocytic subtype of MDSC (M-MDSC) was greater than that for the granulocytic subtype (G-MDSC) in the peripheral blood samples from patients with IPF. In vitro co-culture of normal mouse lung fibroblasts with sorted bone marrow (BM)-derived MDSCs using trans-well inserts revealed paracrine activation of lung fibroblasts by activated M-MDSC, but not GMDSC, as manifested by significantly elevated mRNA levels of α-smooth muscle actin and TGFβ mRNAs. Both quiescent M-MDSC and G-MDSC failed to stimulate fibroblast activation or myofibroblast differentiation. Interestingly, the level of TGFβ mRNA in M-MDSC was higher than G-MDSC. These findings indicated that in pulmonary fibrosis, BM-derived MDSCs were mobilized and recruited to the lung, and the recruited M-MDSC subpopulation subsequently played a paracrine role by activating resident lung fibroblasts and myofibroblast differentiation.

A retrospective study of clinical features of cough variant asthma in Chinese adults

BackgroundClinical features of cough variant asthma (CVA) in Chinese adults are largely uncertain.MethodsA total of 303 patients newly diagnosed as uncontrolled asthma (symptom control and future risk of adverse outcomes), including 175 CVA and 128 classic asthma (CA), were enrolled in this retrospective survey. Clinical features including basic characteristics, pulmonary function, airway hyperresponsiveness (AHR) and cell counts of induced sputum, were compared retrospectively. All patients were classified into four inflammatory subtypes based on the counts of induced sputum eosinophils and neutrophils as eosinophilic (E), neutrophilic (N), mixed granulocytic (M), and paucigranulocytic (P) subtypes. Inflammatory subtype distribution was also compared.ResultsCompared with CA patients, CVA patients were younger (P = 0.009), had a higher prevalence of female patients (P = 0.001), higher parameter values of baseline pulmonary function (P ≤ 0.01 for all), shorter duration of disease (P = 0.002), lower AHR (P = 0.001) and lower sputum eosinophil% (P = 0.009). There was a difference in the AHR distribution as the percentage of moderate and severe AHR in CVA was significantly lower than in CA (41.72% VS 64.70%, P = 0.001). The inflammatory subtype distribution was different as the proportion of E and M subtypes in CVA was lower than in CA (56.0% vs 67.19%, P = 0.049). The proportion of subtype P was the lowest and subtype M was the highest in both CVA and CA patients. There was a similar negative correlation of sputum eosinophil% with AHR in CVA and CA (r = − 0.337, P < 0.0001 and r = − 0.27, P = 0.026, respectively), and a positive correlation between sputum eosinophil% and improvement rate of FEV1 after inhalation of bronchodilator (ΔFEV1%) (r = 0.33, P = 0.01).ConclusionsCVA patients showed a better pulmonary function and lower airway inflammation in contrast to CA patients, which may participate in the pathogenesis of chronic cough in CVA.

2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)-induced myeloid derived suppressor cells (MDSC) have heightened metabolic activity which correlates with microRNA and gene expression involved in immunosuppression

Abstract Myeloid-derived suppressor cells (MDSCs) are bone marrow (BM)-derived cells consisting of monocytic (M-MDSC) or granulocytic (G-MDSC) subtypes with immunosuppressive activity. In the current study, we examined the metabolism and functions of MDSCs induced by TCDD a highly toxic environmental pollutant. Inasmuch, we injected mice with either vehicle or 10μg/kg TCDD interpersonally and found that TCDD increased both M-MDSCs and G-MDSCs in the peritoneal exudate when compared to controls. We determined the cell bioenergetics in TCDD-induced MDSCs when compared to naïve BM-MDSCs. For this purpose, we examined the mitochondrial oxygen consumption rate (OCR) and found that TCDD-induced MDSCs had heightened basal respiration, ATP-linked respiration, proton (H+) leak, maximal respiration, spare respiratory capacity as well as total proton efflux rate (PER) which means more suppressive function when compared to BM-MDSCs. We next examined whether the heightened metabolic potential in TCDD-induced MDSCs correlated with the microRNA and gene expression. MiRNA array analysis demonstrated that in TCDD-induced MDSCs, certain miRNAs (e.g. mir-543-3p, mir-150-5p) were downregulated which target immunosuppressive genes involved in MDSC functions, such as ARG2, IL-10, STAT-3, and PIM1. Next, we investigated the role of miRNA-150-5p in IL-10, PIM1, and STAT3 expression as well as miRNA-543-3p effects on ARG2 using transfection experiments. We found that MDSCs transfected with mature miRNA-150-5p and miRNA-543-3p were downregulated whereas inhibitors upregulated the expression of respective targets. In summary, our data revealed that TCDD modulates miRNA expression which can impact gene expression and metabolism in MDSC.

TET2 expression is a potential prognostic and predictive biomarker in cytogenetically normal acute myeloid leukemia.

TET2 (Ten-Eleven Translocation 2) gene is a member of TET family that can modify DNA through catalyzing the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). Although TET2 mutation has been disclosed in a variety of hematopoietic malignancies, the prognostic implication of TET2 expression in acute myeloid leukemia (AML) remains largely elusive. In this study, real-time quantitative PCR was carried out to detect the level of TET2 transcript in 134 de novo AML patients and 35 healthy donors. TET2 mRNA level was significantly down-regulated in AML patients compared with controls (p = 0.010). Among the French-American-British (FAB) subtypes, the incidence of TET2 under-expression in M0/M1 subtypes was significantly higher than in the other subtypes M2/M3/M4/M5/M6 (p = 0.017), and also markedly higher than in the other granulocyte subtypes M2/M3 (p = 0.005). TET2 low-expressed patients showed a significantly higher frequency of NPM1 mutations than TET2 high-expressed patients. Although there was no significant difference in complete remission rate between two groups (low and high TET2 expression), patients with low TET2 expression had markedly shorter overall survival (OS) in both non-M3 and cytogenetically normal AML (CN-AML) (p = 0.016 and 0.044, respectively). Furthermore, multivariate analysis confirmed the prognostic value of TET2 expression on OS among CN-AML patients (p = 0.049). Importantly, TET2 expression in complete remission (CR) time was significantly higher than newly diagnosis time (p = 0.001), and was returned to lower level when in relapse time (p < 0.001). These findings indicated that down-regulation of TET2 expression was a common event and acted as a prognostic and predictive biomarker in CN-AML patients.

Granulocytes as modulators of dendritic cell function.

Effector T cell development is directly driven by APCs, in particular, by antigen-primed dendritic cells (DCs). Depending on the pathogenic stimulus and the microenvironment, DCs induce proliferation and polarization of naive CD4+ T cells into different effector subsets, such as Th1, Th2, Th17, or regulatory T cells (Tregs). During inflammation, DCs are found in close proximity to other innate immune cells, including all granulocyte subtypes, which potentially influence the immunomodulatory capacities of DCs. Neutrophils, eosinophils, and basophils are rapidly recruited into infected tissues where their main function is to eliminate invading pathogens. Mast cells are tissue-resident granulocytes that also contribute to host defense against pathogens but have, thus far, primarily been associated with their detrimental roles in allergic diseases. Although granulocytes have always been considered essential in innate immunity, their ability to influence the development of adaptive immunity has long been overlooked. This view is now changing, as multiple studies showed significant modulating effects of granulocytes on key players of adaptive immunity, including DCs and lymphocytes. Neutrophils, eosinophils, basophils, and mast cells regulate recruitment and activation of DCs through the release of mediators or via direct cell-cell contact, thereby influencing antigen-specific T cell responses. In this review, we will summarize the current knowledge on the impact of granulocytes on DC functioning and the subsequent putative consequences of this cross-talk on T cell proliferation and polarization. Together, this overview underscores the importance of granulocyte-DC communication to establish optimal immune responses.

Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1–5, 6–10, 11–15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of β-glucan receptors for phagocytosis.