Neutrophil mRNA Research Articles

TOB1 modulates neutrophil phenotypes to influence gastric cancer progression and immunotherapy efficacy.

The ErbB-2.1(TOB1) signaling transducer protein is a tumor-suppressive protein that actively suppresses the malignant phenotype of gastric cancer cells. Yet, TOB1 negatively regulates the activation and growth of different immune cells. Understanding the expression and role of TOB1 in the gastric cancer immune environment is crucial to maximize its potential in targeted immunotherapy. This study employed multiplex immunofluorescence analysis to precisely delineate and quantify the expression of TOB1 in immune cells within gastric cancer tissue microarrays. Univariate and multivariate Cox analyses were performed to assess the influence of clinical-pathological parameters, immune cells, TOB1, and double-positive cells on the prognosis of gastric cancer patients. Subsequent experiments included co-culture assays of si-TOB1-transfected neutrophils with AGS or HGC-27 cells, along with EdU, invasion, migration assays, and bioinformatics analyses, aimed at elucidating the mechanisms through which TOB1 in neutrophils impacts the prognosis of gastric cancer patients. We remarkably revealed that TOB1 exhibits varying expression levels in both the nucleus (nTOB1) and cytoplasm (cTOB1) of diverse immune cell populations, including CD8+ T cells, CD66b+ neutrophils, FOXP3+ Tregs, CD20+ B cells, CD4+ T cells, and CD68+ macrophages within gastric cancer and paracancerous tissues. Significantly, TOB1 was notably concentrated in CD66b+ neutrophils. Survival analysis showed that a higher density of cTOB1/nTOB1+CD66b+ neutrophils was linked to a better prognosis. Subsequent experiments revealed that, following stimulation with the supernatant of tumor tissue culture, the levels of TOB1 protein and mRNA in neutrophils decreased, accompanied by enhanced apoptosis. HL-60 cells were successfully induced to neutrophil-like cells by DMSO. Neutrophils-like cells with attenuated TOB1 gene expression by si-TOB1 demonstrated heightened apoptosis, consequently fostering a malignant phenotype in AGS and HCG-27 cells upon co-cultivation. The subsequent analysis of the datasets from TCGA and TIMER2 revealed that patients with high levels of TOB1 combined neutrophils showed better immunotherapy response. This study significantly advances our comprehension of TOB1's role within the immune microenvironment of gastric cancer, offering promising therapeutic targets for immunotherapy in this context.

The relationship between unique gut microbiome-derived lipid metabolites and subsequent revascularization in patients who underwent percutaneous coronary intervention

Background and aimsStudies have recently revealed the linoleic acid metabolic pathway of Lactobacillus plantarum, the representative gut bacterium in human gastrointestinal tract, and the anti-inflammatory effects of metabolites in this pathway. However, no clinical trials have evaluated the association between these metabolites and revascularization in patients who underwent percutaneous coronary intervention (PCI). MethodsWe retrospectively reviewed patients who underwent PCI with subsequent revascularization or coronary angiography (CAG) without revascularization. Patients with frozen blood samples at the index PCI and revascularization or follow-up CAG were enrolled. ResultsAmong 701 consecutive patients who underwent PCI, we enrolled 53 patients who underwent subsequent revascularization and 161 patients who underwent follow-up CAG without revascularization. Patients who underwent revascularization showed significantly lower plasma 10-oxo-octadecanoic acid (KetoB) levels (720.5 [551.6–876.5] vs. 818.4 [641.1–1103.6 pg/mL]; p = 0.01) at index PCI. Multivariate logistic regression analysis revealed that decreased plasma KetoB levels at the index PCI were independently associated with subsequent revascularization after PCI (odds ratio; 0.90 per 100 pg/mL increase, 95% confidence interval; 0.82–0.98). Additionally, in vitro experiments showed that the addition of purified KetoB suppressed the mRNA levels of IL-6 and IL-1β in macrophages and IL-1β mRNA in neutrophils. ConclusionsPlasma KetoB level at index PCI was independently associated with subsequent revascularization after PCI, and KetoB could act as an anti-inflammatory lipid mediator in macrophages and neutrophils. The assessment of gut microbiome-derived metabolites may help predict revascularization after PCI.

The levels of peripheral blood TNF-α, Decorin and neutrophils MAPK1 mRNA levels of patients with preeclampsia and their clinical significance

Objective This study analyzed the levels of peripheral blood tumor necrosis factor-α (TNF-α), Decorin (DCN) and Mitogen-activated protein kinase 1 (MAPK1) mRNA in neutrophils of patients with preeclampsia and their correlations, in order to provide more thoughts on the diagnosis and treatment of clinical patients. Methods 81 patients with preeclampsia who had regular prenatal checkups and delivered in our hospital from June 2020 to April 2022 were analyzed, including 26 patients with early-onset and 55 patients with late-onset, and 50 pregnant women with normal pregnancy who had prenatal checkups and delivered in our hospital during the same period were selected as the control group. Record the clinical data of patients, record the expression of peripheral blood TNF-α, DCN and neutrophils MAPK1 mRNA of patients with early-onset, late-onset and the control group, and record the correlation between DCN level, MAPK1 mRNA expression and TNF-α level of patients with preeclampsia. Results The diastolic and systolic blood pressures were significantly higher in early-onset and late-onset patients, and the gestational week of delivery was significantly lower in early-onset and late-onset patients, respectively (p < .05); there was no statistically significant difference in the average age, BMI, average pregnancy time, and average births between the three groups (p > .05). The expressions of peripheral blood TNF-α, DCN, and neutrophils MAPK1 mRNA of the early-onset and late-onset groups were all higher than those in the control group (p < .05); and the expressions of TNF-α, DCN, and MAPK1 mRNA in the peripheral blood of the early-onset group were all higher than those in the late-onset group (p < .05); Pearson correlation analysis showed that DCN level and TNF-α level in patients with preeclampsia were positively correlated (r = 0.98639, p < .05); Neutrophils MAPK1 mRNA expression and TNF-α level were positively correlated (r = 0.9611, p < .05). Conclusion TNF-α, DCN and neutrophils MAPK1 mRNA were all highly expressed in the peripheral blood of patients with pre-eclampsia and were more significantly elevated in patients with early-onset preeclampsia, and the expression levels of DCN and MAPK1 mRNA were positively correlated with TNF-α levels. It is possible that all three factors are involved in the pathogenesis of preeclampsia, and are expected to be used as indicators for early prediction and diagnosis.

The expression and clinical significance of neutrophil myeloperoxidase in patients with myeloperoxidase-antineutrophil cytoplasmic antibody associated vasculitis

Objective: To investigate the expression and clinical significance of neutrophil myeloperoxidase (MPO) in patients with MPO-antibody associated vasculitis (AAV). Methods: Thirty-six newly diagnosed MPO-AAV patients who were hospitalized in the First Affiliated Hospital, Anhui Medical University from July 2018 to June 2021 were enrolled,and 36 age and sex matched healthy subjects were selected as controls. Neutrophil MPO level was detected by flow cytometry (FCM) and MPO mRNA was tested by real time quantitative polymerase chain reaction (RT-qPCR) in all subjects. Serum complement fragment C5 (C5a) and MPO in both groups and serum MPO-anti-antineutrophilic cytoplasmic antibody(ANCA) in MPO-AAV group were measured by enzyme linked immunosorbent assay (ELISA), while the disease activity was evaluated by Birmingham vasculitis activity score-V3 (BVAS-V3). Results: Compared with the heathy control group, the expression of MPO mRNA in neutrophils, serum MPO and complement C5a in MPO-AAV group were significantly higher[MPO mRNA:30.2±11.5 vs. 1.9±0.6, P<0.001;MPO:(112.0±68.7) IU/L vs. (87.4±22.9) IU/L, P=0.01; C5a:(187.3±90.3) ng/ml vs. (107.3±31.1) ng/ml, P<0.001; respectively], while the mean fluorescence intensity (MFI) of MPO in neutrophils were significantly lower [ 1 343.3±723.4 vs. 2 868.0±1 136.5, P<0.001]. In MPO-AAV group, the expression of neutrophil MPO mRNA was positively correlated with serum MPO-ANCA and MPO levels (r=0.537, P=0.001 and r=0.358, P=0.032; respectively). Multiple regression analysis suggested that neutrophil MPO mRNA expression was positively correlated with serum MPO-ANCA level (β=0.695, P=0.006); neutrophil MPO level was negatively correlated with serum MPO-ANCA, MPO and complement C5a levels (r=-0.335, P=0.046; r=-0.372, P=0.026; r=-0.577, P<0.001; respectively). Further, neutrophil MPO level was negatively correlated with serum complement C5a level (β=-0.374, P=0.043). BVAS-V3 was positively correlated with MPO mRNA expression in neutrophils, serum MPO-ANCA, MPO and complement C5a (r=0.598, P<0.001; r=0.599, P<0.001; r=0.537, P=0.001; r=0.415, P=0.012; respectively) and negatively correlated with MPO level in neutrophils (r=-0.342, P=0.041). In multiple regression analysis it suggested that BVAS-V3 was positively correlated with MPO mRNA expression in neutrophils (β=0.511, P=0.002). Conclusion: In MPO-AAV patients, MPO synthesis and release in neutrophils are both significantly increased, which might be influenced by serum MPO-ANCA and C5a, respectively. Furthermore, MPO synthesis activity in neutrophils is an independent factor related to disease activity.

Human primary neutrophil mRNA does not contaminate human resolving macrophage mRNA after efferocytosis

The ingestion of apoptotic corpses by macrophages, a process called efferocytosis, is a crucial step in inflammation resolution, since it alters macrophage phenotype toward a pro-resolving profile to foil inflammation and to favor tissue repair. Up to now, the resolving macrophages remain poorly characterized, especially in humans. Global investigations, like RNA sequencing, would be very helpful to unravel some features of these elusive cells. Nonetheless, these inquiries may be challenging in a single-species model, since the fate of ingested mRNA remains unknown and may hinder any subsequent mRNA investigations in the phagocyte. A full human model consisting of primary human neutrophil and primary human monocyte-derived macrophage co-culture was set up several decades ago to mimic in vitro the efferocytosis process. However, to our knowledge, this model has not been characterized as a suitable model to perform global mRNA investigations. Indeed, the extent of ingested neutrophil mRNA contamination has not been assessed in resolving macrophages. This work answers to this crucial question. Indeed, based on the protocols presented in this article, we demonstrate that neutrophil mRNA is severely degraded and is not able to cross-contaminate resolving macrophage mRNA, contrary to apoptotic human peripheral blood derived mononuclear cell (PBMC) or apoptotic leukemic Jurkat cell mRNA. Moreover, this allogenic co-culture system does not favor neither neutrophil activation nor macrophage pro-inflammatory cytokine release. Collectively, we highlight that this model of primary human neutrophil and primary human monocyte-derived macrophage co-culture is the best model for mRNA investigations in human resolving macrophages to help improving our knowledge on these crucial cells.

The Effect of Hemodialysis on the Expression of CXCL8 and its mRNA in Neutrophils of the Patients with Chronic Renal Failure

The Effect of Hemodialysis on the Expression of CXCL8 and its mRNA in Neutrophils of the Patients with Chronic Renal Failure

Strain-Specific Impact of Fusobacterium nucleatum on Neutrophil Function.

Neutrophil function is critical for initiation and progression of infecto-inflammatory diseases. Key quorum-sensing plaque bacteria, such as Fusobacterium nucleatum, act as bridging species between early and late colonizer pathogens, such as Porphyromonas gingivalis, as the biofilm ages and periodontal inflammation increases. This study is designed to determine impact of different F. nucleatum strains on neutrophil function. Cells of human promyelocytic leukemia cell line-60 were differentiated into neutrophil-like cells and cultured with F. nucleatum strains of subspecies (ssp.) nucleatum ATCC 25586, ssp. polymorphum ATCC 10953, and ssp. vincentii ATCC 49256. Neutrophil phagocytosis of F. nucleatum strains and neutrophil apoptosis were analyzed by flow cytometry. Superoxide generation was measured by cytochrome C reduction in the presence and absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 μM) stimulation. Proinflammatory cytokine release was determined after 2, 6, and 24 hours of culture in the presence/absence of different F. nucleatum strains. Expression of Toll-like receptor (TLR)2, TLR4, and nuclear factor (NF)-kappa B mRNA levels were analyzed using real-time quantitative polymerase chain reaction. Each experiment was repeated at least three times in triplicate. Data were analyzed using analysis of variance followed by post hoc Bonferroni correction. All strains of F. nucleatum significantly increased phagocytic capacity of neutrophils. Neutrophil phagocytosis of F. nucleatum ssp. polymorphum was significantly greater than that of F. nucleatum ssp. vincentii and ssp. nucleatum (P <0.001). F. nucleatum ssp. nucleatum and ssp. polymorphum significantly blocked fMLP-induced superoxide generation (P <0.001). Although F. nucleatum vincentii also reduced superoxide generation (25%), the impact was not as strong as that of ssp. nucleatum (83%) and ssp. polymorphum (100%). All F. nucleatum strains stimulated significant increase in neutrophil apoptosis compared with control (P <0.001) and significantly increased expression of NF-κB mRNA in neutrophils (P <0.05). Levels of interleukin-8 and tumor necrosis factor-α produced by neutrophils were significantly increased in all F. nucleatum groups compared with control (P <0.001). These findings suggest that different strains of F. nucleatum impact neutrophil function in different ways. Two of three subspecies blocked neutrophil superoxide generation in response to a secondary stimulus, preventing oxidative killing by neutrophils. The direct role of bridging species in pathogenesis of periodontitis may be greater than previously suspected in which they create a favorable environment for pathogenic transition of the dental ecosystem.

Endothelin a Receptor (ETRA) Blockade Inhibits Leukocyte Adhesion to the Vascular Endothelium in Sickle Cell Disease

▪Objective:Recurrent acute episodes of vaso-occlusion (VOC) are one of the hallmarks of sickle cell disease (SCD). Leukocytes are in an activated state in SCD and their recruitment to the vascular endothelium marks a critical event in the pathophysiology of the vaso-occlusive process. Endothelin-1 (ET-1), a potent vasoconstrictive and pro-inflammatory endogenous peptide, is elevated in steady state SCD, increases during acute VOC crisis, and remains elevated post crisis. This study asked whether the ET-1-ETRA signaling pathway is involved in promoting leukocyte adhesion in a hypoxia-reoxygenation challenged mouse model of SCD. Secondly, we wondered whether ET-1 is able to directly activate and increase the proadhesive phenotype of the peripheral leukocyte population.Methods: Animals:We used the UAB knockout-transgenic mouse model of SCD for our studies (Ryan, 1997). Experimental groups consisted of homozygous (SCD-/-), heterozygous (SCD+/-), and C57BL/6 control mice. Intravital Microscopy:Bosentan (BOS; dual ETRA/B antagonist) or Ambrisentan (AMB; specific ETRA antagonist) was administered by medicated chow for 8 weeks. Prior to imaging, animals were exposed to 5 hrs of 8% O2 followed by 2 hrs of reoxygenation in room air. Leukocytes were stained in vivo with a PE-conjugated anti-CD45 antibody. Mice were anesthetized and the calvarial bone marrow microvascular network was observed using an intravital microscope with water-immersion objectives. Images were analyzed off-line for leukocyte velocity, adhesion, and flow dynamics (Image Pro-Plus 5.0). Superoxide/Cytokine/Integrin measurements: Leukocytes were isolated from freshly harvested peripheral blood (SCD-/- and controls) and exposed to ET-1 or vehicle. ROS production was measured by luminescence (Lumistar Galaxy Luminometer). Intracellular cytokine and surface integrin expression was analyzed by flow cytometry. Neutrophil Adhesion Assay: Peripheral neutrophils were purified by a Ficoll gradient from SCD-/- mice and controls. Following ex vivo incubation with ET-1 or vehicle, the purified neutrophils were added to pre-activated (thrombin) rat PMVECs. After washing with PBS, the cells were fixed and stained with MPO to identify adherent neutrophils.Results: Intravital microscopy revealed that ETR blockade with BOS, but not AMB, enhanced WBC rolling and adhesion at steady state without altering flow velocity, suggesting an important role for ETRB signaling at baseline in SCD-/- animals. Following a hypoxia/reoxygenation protocol, both antagonists were equally effective in decreasing WBC rolling and adhesion in SCD-/- animals. Both ETR antagonists were able to correct H/R-induced decreases in blood flow velocity to near baseline values. Next we asked if ET-1 is capable of activating WBCs by first measuring levels of receptor mRNA in sorted peripheral neutrophils and monocytes. Next we measured intracellular cytokine, surface integrins and ROS production following ex vivo exposure to ET-1. Results indicated a very low amount of ETRA mRNA in neutrophils of SCD-/- mice and controls. ETRB mRNA was predominantly expressed in monocytes of SCD-/- mice. ET-1 ex vivo stimulation failed to increase cytokine, integrin and ROS levels beyond baseline in sickle neutrophils and monocytes. Functionally, more neutrophils from SCD-/- mice adhered to pre-activated endothelial cells than controls; however, ex vivo ET-1 stimulation failed to increase adhesion.Summary and Conclusions: Our results demonstrate that selective ETRA blockade is sufficient to abrogate H/R-induced leukocyte-endothelial interactions in a preclinical model of SCD. While the dual ETRA/B antagonist produced similar results after H/R, its proadhesive effect at steady state suggests an important role for the endothelin B receptor under baseline conditions. Although the long-term significance of these findings remains to be elucidated, at this early stage our results suggest a potential therapeutic advantage for a selective ETRA treatment strategy in SCD. The lack of response of the leukocyte population to direct ET-1 stimulation suggests that ET-1 targets primarily the vascular endothelium to promote leukocyte adhesion. In conclusion, this study established a potentially important role for clinically available ETRA antagonists in the treatment portfolio for patients with SCD. DisclosuresGutsaeva:Grifols: Research Funding.

Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress

Organic trace mineral (ORG) supplementation to dairy cows in substitution of sulfate (INO) sources has been associated with improvement in immune function during stressful states such as the peripartal period. However, the effect of supplemental ORG during pregnancy on the neonatal calf is unknown. Therefore, our aim was to investigate the effects of ORG supplementation during late pregnancy on the immune system and growth of the neonatal calf. Of specific interest was the evaluation of inflammation-related microRNA (miRNA) and target gene expression in blood neutrophils as indicators of possible nutritional programming. Forty multiparous cows were supplemented for 30d prepartum with 40mg/kg of Zn, 20mg/kg of Mn, 5mg/kg of Cu, and 1mg/kg of Co from either organic (ORG) or sulfate (INO) sources (total diet contained supplemental 75mg/kg of Zn, 65mg/kg of Mn, 11mg/kg of Cu, and 1mg/kg of Co, and additional Zn, Mn, and Co provided by sulfates), and a subset of calves (n=8/treatment) was used for blood immunometabolic marker and polymorphonuclear leukocyte (PMNL) gene and miRNA expression analyses. Samples were collected at birth (before colostrum feeding), 1d (24h after colostrum intake), and 7 and 21d of age. Data were analyzed as a factorial design with the PROC MIXED procedure of SAS. No differences were detected in BW, but maternal ORG tended to increase calf withers height. Calves from INO-fed cows had greater concentrations of blood glucose, GOT, paraoxonase, myeloperoxidase, and reactive oxygen metabolites. Antioxidant capacity also was greater in INO calves. The PMNL expression of toll-like receptor pathway genes indicated a pro-inflammatory state in INO calves, with greater expression of the inflammatory mediators MYD88, IRAK1, TRAF6, NFKB, and NFKBIA. The lower expression of miR-155 and miR-125b in ORG calves indicated the potential for maternal organic trace minerals in regulating the PMNL inflammatory response at least via alterations in mRNA and miRNA expression. Overall, these results indicate that maternal nutrition with organic trace minerals could alter the neonatal innate immune response at least in part via changes in gene and miRNA expression. Further studies involving inflammatory challenges during the neonatal period should be performed to determine the functional benefit of maternal organic trace minerals on the neonatal immune response.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production.Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts.We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures:No relevant conflicts of interest to declare.

Effect of conventional and intensified milk replacer feeding programs on performance, vaccination response, and neutrophil mRNA levels of Holstein calves

This study compared conventional and intensified milk replacer feeding regimens on growth, intake, respiratory and fecal scores, vaccination response, and neutrophil mRNA levels. Holstein calves were randomly assigned to a 10-wk study on d 2 of life. Treatments were conventional (CON; n=8) and intensified (INT; n=7) milk replacer feeding programs. Conventional calves were fed a 20.8% crude protein and 21.0% fat milk replacer at 1.25% of birth body weight (BW) from wk 1 to 6 of life and 0.625% of birth BW during wk 7. A 29.3% crude protein and 16.2% fat milk replacer was fed to INT calves at 1.5% of birth BW during wk 1, 2% of current BW from wk 2 to 6, and 1% of current BW during wk 7. All calves were given milk replacer twice daily during wk 1 to 6, once daily during wk 7, and were weaned completely during wk 8. Calf starter intake was measured daily through wk 8. Body weight and withers height were measured weekly. Fecal and respiratory scores were recorded twice daily at feeding. Calves were vaccinated against ovalbumin at the end of wk 1, 3, and 5. Blood samples were collected at the end of wk 1, 3, 5, and 8 for analysis of serum anti-ovalbumin IgG concentration and for isolation of neutrophils. Quantitative PCR was used to measure neutrophil mRNA levels of 7 functionality genes. Treatment did not affect total DMI or anti-ovalbumin IgG response. Intensified milk replacer feeding increased average daily gain, protein intake, fat intake, and feed efficiency compared with the CON feeding program. Compared with CON calves, INT calves had greater fecal scores, indicating looser feces and greater respiratory scores, indicating more respiratory problems. Calves assigned to the INT treatment had increased neutrophil mRNA levels of L-selectin, and at wk 8, neutrophil cytosolic factor 1 was increased and toll-like receptor 4 tended to be increased compared with CON calves. This suggests greater activation of neutrophils in INT calves postweaning, but differences were relatively small and levels of the other 4 genes were unaffected. An INT milk replacer feeding program increased growth, fecal scores, and respiratory scores preweaning, increased mRNA levels of 2 neutrophil genes postweaning, and did not affect vaccination response.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression

BackgroundDelayed neutrophil apoptosis may be an important factor in the persistent inflammation associated with chronic obstructive pulmonary disease (COPD). Bcl-2 family proteins are important regulators of neutrophil apoptosis. We determined the mRNA levels of pro-apoptotic Bak and anti-aptototic Bcl-xl and Mcl-1 members of the Bcl-2 family in unstimulated peripheral blood neutrophils from patients with mild to moderate COPD and compared these to neutrophils from healthy controls.MethodsNeutrophils were isolated from peripheral blood samples of 47 COPD patients (smokers: N = 24) and 47 healthy controls (smokers: N = 24). Percentages of apoptotic cells were determined at 4, 24, and 36 h for unstimulated neutrophils cultured in vitro. Neutrophil mRNA expression of Bak, Bcl-xl, and Mcl-1 was determined by real-time polymerase chain reaction (PCR). FEV1 (% predicted) and FVC were determined by spirometry and correlations between mRNA levels and lung function parameters were determined.ResultsThe percentages of apoptotic cells among unstimulated neutrophils from COPD patients were significantly lower compared to cells from controls after 4, 24, and 36 h in culture; smoking history had only a minimal effect on these differences. Unstimulated neutrophils from COPD patients had significantly lower Bak mRNA expression and higher expressions of Bcl-xl and Mcl-1 mRNA than cells from healthy controls. Again, smoking history had only a minimal effect on these trends. Bak mRNA expression was significantly positively correlated with both % predicted FEV1 and the FEV1/FVC ratio, while Bcl-xl and Mcl-1 mRNA expressions were significantly negatively correlated with %predicted FEV1 and the FEV1/FVC ratio.ConclusionsThe genes for pro-apoptotic Bak, and anti-apoptotic Bcl-xl and Mcl-1 may be important in regulating the delayed neutrophil apoptosis observed in COPD, which may contribute to COPD pathogenesis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1605269445677066

Strain-dependent induction of neutrophil histamine production and cell death byPseudomonas aeruginosa

Airway diseases often feature persistent neutrophilic inflammation and infection. In cystic fibrosis bronchitis, for example, Pseudomonas aeruginosa is isolated frequently. Previously, this laboratory revealed that neutrophils become major sources of histamine in mice with tracheobronchitis caused by the wall-less bacterium Mycoplasma pulmonis. To test the hypothesis that more-broadly pathogenic P. aeruginosa (which expresses cell wall-associated LPS and novel toxins) has similar effects, we incubated naïve mouse neutrophils with two strains of P. aeruginosa. Strain PAO1 greatly increased neutrophil histamine content and secretion, whereas strain PA103 depressed histamine production by killing neutrophils. The histamine-stimulating capacity of PAO1, but not PA103-mediated toxicity, persisted in heat-killed organisms. In PAO1-infected mice, lung and neutrophil histamine content increased. However, PAO1 did not alter production by mast cells (classical histamine reservoirs), which also resisted PA103 toxicity. To explore mechanisms of neutrophil-selective induction, we measured changes in mRNA encoding histidine decarboxylase (rate-limiting for histamine synthesis), probed involvement of endotoxin-TLR pathways in Myd88-deficient neutrophils, and examined contributions of pyocyanin and exotoxins. Results revealed that PAO1 increased histamine production by up-regulating histidine decarboxylase mRNA via pathways largely independent of TLR, pyocyanin, and type III secretion system exotoxins. PAO1 also increased histidine decarboxylase mRNA in neutrophils purified from infected lung. Stimulation required direct contact with neutrophils and was blocked by phagocytosis inhibitor cytochalasin D. In summary, Pseudomonas-augmented histamine production by neutrophils is strain-dependent in vitro and likely mediated by up-regulation of histidine decarboxylase. These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine.

690: Toll-like receptor 1 and Toll-like receptor 5 expression is down-regulated on maternal neutrophils in preeclampsia

690: Toll-like receptor 1 and Toll-like receptor 5 expression is down-regulated on maternal neutrophils in preeclampsia

Influence of dietary antioxidants and fatty acids on neutrophil mediated bacterial killing and gene expression in healthy Beagles

Dietary (n-3) fatty acids from fish oil have been used to modulate immune function in many mammalian species. Together, dietary antioxidants and behavioral enrichment have been shown to enhance neutrophil phagocytosis in geriatric Beagle dogs. The purpose of this study was to further investigate the effects of vitamins E and C, in combination with dietary fish oil, on neutrophil mediated bacterial killing, and on transcript levels of selected neutrophil mRNA. Fifty adult Beagle dogs were randomized into five dietary treatment groups for 60 days. All foods were complete and balanced and met the nutrient profiles of AAFCO for adult dogs. For 60 days before study initiation, dogs consumed a pretrial food that contained 74 IU/kg vitamin E and 0 mg/kg vitamin C. The five experimental foods were confirmed by analytical methods to contain ≥640 IU/kg vitamin E and 130 mg/kg vitamin C (as fed). Experimental foods ranged from low levels of EPA and DHA (pretrial food and lowest experimental food had 0.01% EPA and no detectable DHA) to the highest experimental food with 0.25% EPA and 0.17% DHA. Ex vivo bactericidal activity of activated, peripheral-blood neutrophils against Lactococcus lactis was determined after 1 h incubation. Bactericidal activity was calculated as a percentage of control values (bacteria incubated in media without neutrophils). Transcript levels of genes involved in neutrophil-mediated immune functions were determined by real-time qPCR. Dogs in all treatment groups had increased serum vitamin E concentration ( P < 0.01). After consuming experimental food for 60 days, neutrophils from dogs in all 5 treatment groups also had increased bactericidal activity ( P < 0.01). Dietary fish oil however, had no effect on bactericidal activity. Stepwise multiple regression analysis demonstrated that the change in neutrophil mediated bacterial killing was significantly correlated to changes in gene expression of interleukin-8 receptor (IL-8R), interleukin converting enzyme (ICE), and myeloperoxidase (MPO; r 2 = 0.33; P = 0.003). When stepwise multiple regression analysis was performed considering each mRNA as a dependent variable and change in selected individual and summed fatty acid concentrations as independent variables, change in the ratio of saturated fatty acids (SFA) to polyunsaturated fatty acids (PUFA) was significant ( P ≤ 0.05) in the mRNA regression analyses for IL-8R, ICE, MPO, and cyclooxygenase-2. In summary, circulating neutrophils from dogs fed diets enriched in vitamins E and C had significantly increased bactericidal activity as well as altered gene expression. Change in SFA to PUFA ratio also altered neutrophil gene expression.

The effect of dexamethasone on clock gene mRNA levels in bovine neutrophils and lymphocytes

Circadian rhythms are driven by oscillating expression of a family of transcription factors called clock genes. In rodents, clock genes drive circadian rhythms in white blood cell function, and glucocorticoids are believed to regulate these rhythms. Little is known about circadian rhythms of cattle white blood cells. The objectives of this study were: (1) to quantify mRNA levels of clock genes in neutrophils and lymphocytes over 24 h in healthy steers; and (2) to quantify effects of dexamethasone on clock gene mRNA levels in bovine neutrophils and lymphocytes. We hypothesized that bovine neutrophils and lymphocytes would display 24 h variations in clock gene mRNA levels and that those patterns would be disrupted by glucocorticoid treatment. Six Holstein steers were injected with 0 or 0.10 mg/kg body weight dexamethasone according to a crossover design. Neutrophils and lymphocytes were collected from jugular blood at 0, 4, 8, 12, 16, 20, and 24 h relative to treatment administration. Neutrophil and lymphocyte mRNA levels of the clock genes Clock, Bmal1, Per1, Per2, Cry1, Cry2, Rev-erbα, and CK1ɛ were quantified. For neutrophils, an interaction between treatment and time was found for Clock, Cry1, and CK1ɛ. Time affected Clock, Per1, Cry1, Rev-erbα, and CK1ɛ. For all of those genes except Per1, neutrophils from control steers displayed 24 h changes of mRNA levels characteristic of circadian regulated cells. The dexamethasone treatment increased neutrophil mRNA levels of Per1, decreased Clock, Cry1, Cry2, and Rev-erbα, and tended to decrease Bmal1. These results suggest that circadian rhythms have the potential to impact bovine neutrophil function, and that glucocorticoid-induced disruption of neutrophil circadian rhythms may contribute to periparturient immunosuppression. For lymphocytes, an interaction between treatment and time was observed for Per1 and tended to occur for Per2 and Cry2. Although time affected Per1 and Rev-erbα, distinct 24 h patterns of lymphocyte clock gene mRNA levels were not evident as they were in neutrophils. Treatment increased Per1 and decreased Cry2, but the magnitude of the treatment effect was small. In summary, 24 h patterns in clock gene mRNA levels were observed in bovine neutrophils and to some degree in lymphocytes, and these patterns were disrupted by dexamethasone administration. Although further research is needed, individual variation in white blood cell circadian rhythms and glucocorticoid responsiveness may help to explain individual differences in periparturient disease susceptibility.

PIR-B negatively regulates Trk receptors to mediate neurite growth inhibition by myelin

Severe congenital neutropenia (SCN) or Kostmann’s syndrome is characterized by a stop in differentiation of myeloid progenitor cells at the myelocytic or promyelocytic stage. The pathophysiology of SCN is still unclear. We previously showed that the tyrosine kinase JAK2 is phosphorylated and activated in neutrophils from patients with severe congential neutropenia. We investigated the role of tyrosine phosphatases in this disease. Expression of the SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2 was analyzed in myeloid cells from patients with SCN in comparison to healthy donors. We investigated tyrosine phosphatase expression in myeloid cells at the protein level by Western blot analysis using polyclonal antisera against SHP-1 and SHP-2. Whereas SHP-1 and SHP-2 were hardly detectable in neutrophils from healthy donors, neutrophils from patients with SCN revealed high amounts of these two proteins in Western blot analyses. Reverse transcriptase-polymerase chain reaction and Northern blot analyses demonstrated no dramatic differences of SHP-1 mRNA in neutrophils from congenital neutropenia patients as compared to healthy donors. SHP-2 mRNA was hardly detectable in the neutrophils from patients and in normal neutrophils. Increased expression of SHP protein correlated with elevated activity of both SHP-1 and SHP-2 in neutrophils of patients with SCN. Taken together, these data indicate differential regulation for SHP-1 and SHP-2 at the protein level in neutrophils from SCN patients in comparison to healthy donors. We suggest that overexpression of SHP-1 and SHP-2 protein in neutrophils and not in mononuclear cells from patients with SCN might be related to the disease, e.g., by defective dephosphorylation of proteins involved in intracellular signaling pathways.

Identification and characterization of the first large deletion of the MYH9 gene associated with MYH9 disorders

MYH9 disorders are autosomal dominant macrothrombocytopenias with leukocyte inclusion bodies. Single point mutations in the protein-coding sequence of the MYH9 gene are the most common cause. So far no large gene deletion/insertion and splicing defects have been reported. Conventional DNA sequencing of each MYH9-coding exon showed no abnormalities in a patient. Reverse transcription- polymerase chain reaction (PCR) amplification and sequencing of neutrophil mRNA identified an inframe deletion of exon 25. Further long-range PCR amplification of genomic DNA revealed a deletion of 1220 nucleotides including entire exon 25. Immunoblot analysis showed a small, abnormal protein in neutrophils but not in platelets. This is the first report of a large deletion of the MYH9 gene leading to the development of MYH9 disorders.

Lithium down-regulates the expression of CXCR4 in human neutrophils

The CXC chemokine receptor CXCR4 and its unique ligand SDF-1 (stromal-derived factor-1) play critical roles for the retention of hematopoietic cells within the bone marrow (BM) and for their mobilization into the circulation. Lithium often produces neutrophilia in psychiatric patients, but the mechanism of mobilization related to neutrophilia has not been fully clarified. We showed here that lithium dose-dependently reduces the levels of surface CXCR4 protein and mRNA in neutrophils, but not in lymphocytes. The chemotactic migration of neutrophils in response to SDF-1 was reduced after a pre-incubation with lithium. We provide evidence that lithium down-regulates the CXCR4 expression of neutrophils and it attenuates their responsiveness to SDF-1. Our studies support the concept that down-regulation of CXCR4 is one of the mechanisms by which causes neutrophilia.