Bone Marrow-derived Neutrophils Research Articles

Neutrophil NADPH oxidase regulation by oxidative phosphorylation and accessory glucose metabolic pathways

Neutrophils produce reactive oxygen species (ROS) to extinguish pathogens and regulate intracellular signaling. The major producer of ROS in neutrophils is the NADPH oxidase, which is fueled by NADPH generated via the oxidative pentose phosphate pathway. It remains unclear how other accessory pathways of glucose metabolism and mitochondrial activity influence the oxidative burst in neutrophils. The respiratory burst in bone marrow-derived neutrophils was stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of an inhibitor of glycogen breakdown (glycogen phosphorylase inhibitor; GPi), inhibitors of mitochondrial respiration (antimycin A and rotenone), or an inhibitor of serine biosynthesis (NCT503). Respiratory burst was also measured under conditions of limited glucose and glutamine. ROS production was measured via extracellular flux analysis or via dihydrorhodamine fluorescence. In both male and female mouse neutrophils, inhibition of glycogen breakdown by GPi delayed initiation of the oxidative burst in response to PMA (n = 6, p<0.05, Student’s T-test and Repeated Measure’s ANOVA). Inhibition of mitochondrial oxidative phosphorylation or inhibition of the serine biosynthetic pathway sustained ROS production in PMA-stimulated neutrophils (n = 6, p<0.05, Student’s T-test). Removal of glucose and glutamine showed a truncated oxidative burst (n = 6, p<0.05, Student’s T-test). Ultimately, these findings suggest that glycogen breakdown fuels the initial activation of the oxidative burst, while mitochondrial activity and the serine biosynthesis pathway control the magnitude and duration of neutrophil ROS production. Furthermore, utilization of exogenous glucose and glutamine are necessary for the entire oxidative burst. Future studies will continue to illustrate the mechanisms of neutrophil metabolic plasticity as well as connect their significance in the context of wound healing, exercise, and chronic inflammatory diseases. NIH: R01HL141191, NIH: P01 HL078825. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

B-1a Cells Scavenge NETs to Attenuate Sepsis.

B-1a cells, a regulatory subset of B lymphocytes, produce natural IgM and IL-10. Neutrophil extracellular traps (NETs) play a crucial role in pathogen defense, but their excessive formation during sepsis can cause further inflammation and tissue damage. In sepsis, extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released to induce NET formation. We hypothesize that B-1a cells clear NETs to prevent sepsis-induced injury. Sepsis in mice was induced by injecting 1 × 107 and 5 × 107 CFU E. coli intraperitoneally (i.p.). After 4 and 20 hours, we assessed the number of B-1a cells in the peritoneal cavity using flow cytometry. Our results showed that the number of peritoneal B-1a cells was significantly decreased in E. coli-sepsis mice. Importantly, replenishing B-1a cells via i.p. injection in sepsis mice significantly decreased NETs in peritoneal neutrophils. We also observed a decrease in serum inflammation and injury markers and a significant increase in overall survival rate in B-1a cell-treated septic mice. To understand the mechanism, we co-cultured bone marrow-derived neutrophils (BMDNs) with peritoneal B-1a cells in a contact or non-contact condition using an insert and stimulated them with eCIRP. After 4 hours, we found that eCIRP significantly increased NET formation in BMDNs. Interestingly, we observed that B-1a cells inhibited NETs by 67% in a contact-dependent manner. Surprisingly, when B-1a cells were cultured in inserts, there was no significant decrease in NET formation, suggesting that direct cell-to-cell contact is crucial for this inhibitory effect. We further determined that B-1a cells promoted NET phagocytosis and this was mediated through natural IgM, as blocking IgM receptor attenuated the engulfment of NETs by B-1a cells. Finally, we identified that following their engulfment, NETs were localized into the lysosomal compartment for lysis. Thus, our study suggests that B-1a cells decrease NET content in eCIRP-treated neutrophils and E. coli-sepsis mice.

Monosodium Urate Crystal‐Induced Pyroptotic Cell Death in Neutrophil and Macrophage Facilitates the Pathological Progress of Gout

AbstractMonosodium urate (MSU) crystal deposition in joints can lead to the infiltration of neutrophils and macrophages, and their activation plays a critical role in the pathological progress of gout. However, the role of MSU crystal physicochemical properties in inducing cell death in neutrophil and macrophage is still unclear. In this study, MSU crystals of different sizes are synthesized to explore the role of pyroptosis in gout. It is demonstrated that MSU crystals induce size‐dependent pyroptotic cell death in bone marrow‐derived neutrophils (BMNs) and bone marrow‐derived macrophages (BMDMs) by triggering NLRP3 inflammasome‐dependent caspase‐1 activation and subsequent formation of N‐GSDMD. Furthermore, it is demonstrated that the size of MSU crystal also determines the formation of neutrophil extracellular traps (NETs) and aggregated neutrophil extracellular traps (aggNETs), which are promoted by the addition of interleukin‐1β (IL‐1β). Based on these mechanistic understandings, it is shown that N‐GSDMD oligomerization inhibitor, dimethyl fumarate (DMF), inhibits MSU crystal‐induced pyroptosis in BMNs and J774A.1 cells, and it further alleviates the acute inflammatory response in MSU crystals‐induced gout mice model. This study elucidates that MSU crystal‐induced pyroptosis in neutrophil and macrophage is critical for the pathological progress of gout, and provides a new therapeutic approach for the treatment of gout.

Matrix mechanics regulate the polarization state of bone marrow-derived neutrophils through the JAK1/STAT3 signaling pathway.

Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. STATEMENT OF SIGNIFICANCE.

Photopolymerizable Hydrogel for Enhanced Intramyocardial Vascular Progenitor Cell Delivery and Post-Myocardial Infarction Healing.

Cell transplantation success for myocardial infarction (MI) treatment is often hindered by low engraftment due to washout effects during myocardial contraction. A clinically viable biomaterial that enhances cell retention can optimize intramyocardial cell delivery. In this study, a therapeutic cell delivery method is developed for MI treatment utilizing a photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. Human vascular progenitor cells, capable of forming functional vasculatures upon transplantation, are combined with an in situ photopolymerization approach and injected into the infarcted zones of mouse hearts. This strategy substantially improves acute cell retention and promoteslong-term post-MI cardiac healing, including stabilized cardiac functions, preserved viable myocardium, and reduced cardiac fibrosis. Additionally, engrafted vascular cells polarize recruited bone marrow-derived neutrophils toward a non-inflammatory phenotype via transforming growth factor beta (TGFβ) signaling, fostering a pro-regenerative microenvironment. Neutrophil depletion negates the therapeutic benefits generated by cell delivery in ischemic hearts, highlighting the essential role of non-inflammatory, pro-regenerative neutrophils in cardiac remodeling. In conclusion, this GelMA hydrogel-based intramyocardial vascular cell delivery approach holds promise for enhancing the treatment of acute myocardial infarction.

EXTRACELLULAR CIRP INHIBITS NEUTROPHIL APOPTOSIS TO PROMOTE ITS AGING BY UPREGULATING SERPINB2 IN SEPSIS.

Background: Sepsis reduces neutrophil apoptosis. As the result, neutrophils may become aged, exacerbating inflammation and tissue injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern to promote inflammation and tissue injury in sepsis. SerpinB2, a serine protease inhibitor, has been shown to inhibit apoptosis. We hypothesize that eCIRP upregulates SerpinB2 to promote aged neutrophil subset by inhibiting apoptosis in sepsis. Methods: We stimulated bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice with 1 μg/mL of recombinant mouse CIRP (i.e., eCIRP) and assessed cleaved caspase-3 and SerpinB2 by western blotting. Apoptotic neutrophils were assessed by Annexin V/PI. Bone marrow-derived neutrophils were stimulated with 1 μg/mL eCIRP and treated with or without PAC-1 (caspase-3 activator) and aged neutrophils (CXCR4 hi CD62L lo ) were assessed by flow cytometry. To induce sepsis, we performed cecal ligation and puncture in WT or CIRP -/- mice. We determined the percentage of aged neutrophils and SerpinB2 + neutrophils in blood and spleen by flow cytometry. Results: We found that cleaved caspase-3 levels were increased at 4 h of PBS treatment compared with 0 h but decreased by eCIRP treatment. Extracellular cold-inducible RNA-binding protein reduced apoptotic cells after 20 h of treatment. Extracellular cold-inducible RNA-binding protein also increased the frequencies of aged neutrophils compared with PBS after 20 h, while PAC-1 treatment reduced aging in eCIRP-treated BMDNs. Extracellular cold-inducible RNA-binding protein significantly increased the expression of SerpinB2 at protein levels in BMDNs at 20 h. In WT mice, the frequencies of aged and SerpinB2 + neutrophils in blood and spleen were increased after 20 h of cecal ligation and puncture, while in CIRP -/- mice, aged and SerpinB2 + neutrophils were significantly decreased compared with WT mice. We also found that aged neutrophils expressed significantly higher levels of SerpinB2 compared with non-aged neutrophils. Conclusions: eCIRP inhibits neutrophil apoptosis to increase aged phenotype by increasing SerpinB2 expression in sepsis. Thus, targeting eCIRP could be a new therapeutic strategy to ameliorate inflammation caused by neutrophil aging in sepsis.

Leptin/obR signaling exacerbates obesity-related neutrophilic airway inflammation through inflammatory M1 macrophages

BackgroundObesity-related asthma is a kind of nonallergic asthma with excessive neutrophil infiltration in the airways. However, the underlying mechanisms have been poorly elucidated. Among the adipokines related to obesity, leptin is related to the inflammatory response. However, little is understood about how leptin acts on the leptin receptor (obR) in neutrophilic airway inflammation in obesity-associated asthma. We explored the inflammatory effects of leptin/obR signaling in an obesity-related neutrophilic airway inflammation mouse model.MethodsWe established a neutrophilic airway inflammation mouse model using lipopolysaccharide (LPS)/ovalbumin (OVA) sensitization and OVA challenge (LPS + OVA/OVA) in lean, obese, or db/db (obR deficiency) female mice. Histopathological, bronchoalveolar lavage fluid (BALF) inflammatory cell, and lung inflammatory cytokine analyses were used to analyze airway inflammation severity. Western blotting, flow cytometry, reverse transcription‐polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the underlying mechanisms. In vitro bone marrow‐derived macrophage (BMDM) and bone marrow-derived neutrophil experiments were performed.ResultsWe found that the serum leptin level was higher in obese than in lean female mice. Compared to LPS/OVA + OVA-treated lean female mice, LPS/OVA + OVA-treated obese female mice had higher peribronchial inflammation levels, neutrophil counts, Th1/Th17-related inflammatory cytokine levels, M1 macrophage polarization levels, and long isoform obR activation, which could be decreased by the obR blockade (Allo-Aca) or obR deficiency, suggesting a critical role of leptin/obR signaling in the pathogenesis of obesity-related neutrophilic airway inflammation in female mice. In in vitro experiments, leptin synergized with LPS/IFN-γ to promote the phosphorylation of the long isoform obR and JNK/STAT3/AKT signaling pathway members to increase M1 macrophage polarization, which was reversed by Allo-Aca. Moreover, leptin/obR-mediated M1 macrophage activity significantly elevated CXCL2 production and neutrophil recruitment by regulating the JNK/STAT3/AKT pathways. In clinical studies, obese patients with asthma had higher serum leptin levels and M1 macrophage polarization levels in induced sputum than non-obese patients with asthma. Serum leptin levels were positively correlated with M1 macrophage polarization levels in patients with asthma.ConclusionsOur results demonstrate leptin/obR signaling plays an important role in the pathogenesis of obesity-related neutrophilic airway inflammation in females by promoting M1 macrophage polarization.Graphical abstract

Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow-derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture-induced sepsis but not in CIRP-/- mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non-APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ-mediated hyper-NETosis.

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

Copolymer functional groups modulate extracellular trap accumulation and inflammatory markers in HL60 and murine neutrophils

Undesirable host responses to implants commonly lead to impaired device function. As the first immune cell to respond to inflammation, activated neutrophils release antimicrobials and neutrophil extracellular traps (NETs) that prime microenvironments for macrophages and other infiltrating cells. This research aims to understand how functional groups in copolymers of isodecyl acrylate (IDA) that are known to modulate healing in vivo, modulate neutrophil cells. Phorbol myristate acetate-activated HL60 cells and bone marrow-derived murine neutrophils (BMDN) were incubated with coatings of IDA copolymerized with, methacrylic acid (MAA films), methyl methacrylate (MM films), or MM functionalized with hexamethylenediamine (HMD films). Cells incubated on HMD films resulted in increased accumulation of NETs at the film’s surface in comparison to other copolymers because of increased adhesion of HL60 onto HMD films or increased rates of NETosis from BMDN. Overall, lower inflammation was observed with cells on MAA films. HL60 cells had no increase in classical inflammatory markers such as tumor necrosis factor alpha and intracellular adhesion molecule-1, whereas HL60 on HMD films had increases in these same markers. Taken together, these studies give important insights into how neutrophils interact differently with functionalized copolymers and the proteins that adsorb to them, with MAA (carboxyl groups) leading to behavior associated with lower inflammation and HMD (amine groups) with higher inflammation and accumulation of NETs.

Abstract 3647: Anti-tumor actions of neutrophils are mediated via STAT5 inhibition

Abstract The overall patient survival rate drops dramatically when localized prostate cancer (PCa) progresses to bone metastatic prostate cancer (BM-PCa). The treatment for BM-PCa is limited to palliative therapy, and it is currently incurable. Neutrophils are the most prevalent immune cell in the bone, and we have found that PCa enhances their recruitment and infiltration into the prostate tumor-bone microenvironment of patients with BM-PCa. Additionally, we observed that bone marrow neutrophils directly induce apoptosis of PCa cells both in vitro and in vivo and that this is mediated by the inhibition of STAT5, a transcription factor that promotes PCa progression. Moreover, STAT5 has also been found to contribute to the development of resistance to standard care androgen deprivation therapy. Neutrophils specifically target and kill only cells that express STAT5, as stable knockdown of STAT5 in BM-PCa cells makes them resistant to neutrophil-mediated cytotoxicity, while the overexpression of STAT5 in STAT5-negative PCa cells, which are generally resistant to killing by neutrophils, sensitizes them to neutrophil-mediated killing. Furthermore, transcriptomic analyses derived from the STAT5 knockdown and STAT5 overexpressing cells suggest that STAT5 expression in PCa induces pro-inflammatory signaling and that neutrophil-mediated cytotoxicity may be exerted via STAT5-induced IL-1 and/or IL-6. Preliminary data in our laboratory show that STAT5-induced production of IL-1 by PCa cells leads to their death by neutrophils, as knocking out the IL-1 receptor on neutrophils reduces their cytotoxic potential against STAT5-positive PCa cells. Interestingly, in addition to killing BM-PCa cells, we have demonstrated that bone marrow-derived neutrophils are also capable of killing pancreatic cancer cells. Further delineation of the role of neutrophils in STAT5 signaling in BM-PCa growth holds promise for enhancing neutrophil cytotoxicity in the bone which could lead to novel therapeutic options for bone metastatic prostate cancer. Citation Format: Sanjana Rajgopal, Massar Alsamraae, Diane Costanzo-Garvey, Leah Cook. Anti-tumor actions of neutrophils are mediated via STAT5 inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3647.

Abstract 5871: Pan-cancer myeloid cell analysis at the single cell level reveals the influence of distinct organ sites in myeloid cell phenotypes and support targeting S100A4 to reverse immune suppression

Abstract With abundant pro-tumorigenic myeloid cells and few tumoricidal tumor-infiltrating lymphocytes (<5%), GBM is representative of “immune cold” tumors. As such, many different types of immunotherapies have failed to show significant benefits for most glioma patients. Hence, a better understanding of drivers of the immune suppressive microenvironment in GBM and other immune cold tumors is urgently needed to guide future immunotherapy development and application. We recently analyzed 201,986 human glioma and immune cells from 44 tissue fragments from 18 human glioma patients, and present a comprehensive and high-resolution cellular, molecular, and spatial heterogeneity atlas of human glioma. We report an extensive spatial and molecular heterogeneity of glioma and immune cells within the same patient. In addition, we discovered that cell:cell communication between glioma:myeloid cells is considerably more robust than glioma:T-cells, indicating that myeloid cells form a communication hub in vivo. To gain a deeper understanding of these important immune cells, we analyzed 83,479 glioma-infiltrating myeloid cells and identified 9 molecularly distinct myeloid subtypes: 3 microglia subtypes, 3 bone marrow-derived macrophage (BMDM) subtypes, MDSCs, neutrophils, and dendritic cells. Notably, we found that five of these myeloid cell subtype gene signatures are significant predictors of glioma patient survival, independent of glioma cell mutational profiles or gene expression patterns. Leveraging our dataset, we also identified a novel immunotherapy target that is highly expressed in immune-suppressive macrophages and T cells but not in anti-tumor leukocytes: S100A4. We provide both in vitro and in vivo evidence that S100a4 deletion in stromal cells is sufficient to reprogram the immune microenvironment and significantly extend the survival of two independent glioma models. To broaden the potential impact of targeting S100A4 as a selective modulator of immune suppressive leukocytes, we compared the molecular signatures of glioma-associated myeloid cells to those from 12 other cancer types and peripheral blood myeloid cells. We found that S100A4 expression pattern is highly consistent among all tumor types, where its expression is highest in the monocytes and MDSCs and low in most DCs and tissue-resident macrophages. Our preliminary analysis also shows that myeloid cells in gliomas are molecularly distinct from corresponding cell types in other cancers, strongly indicating the role brain microenvironment in influencing the infiltrating BMDM maturation and polarization. Citation Format: Nourhan Abdelfattah, Parveen Kumar, Caiyi Wang, Jia-Shiun Leu, David Baskin, William Flynn, Ruli Gao, Kumar Pichumani, Omkar Ijare, Stephanie Wood, Suzanne Powell, David Haviland, Brittany Parker Kerrigan, Frederick Lang, Sujit Prabhu, Kristin Huntoon, Wen Jiang, Betty Kim, Joshy George, Kyuson Yun. Pan-cancer myeloid cell analysis at the single cell level reveals the influence of distinct organ sites in myeloid cell phenotypes and support targeting S100A4 to reverse immune suppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5871.

Extracellular Acidification Augments NLRP3-Mediated Inflammasome Signaling in Macrophages.

Inflammation is a series of host defense processes in response to microbial infection and tissue injury. Inflammatory processes frequently cause extracellular acidification in the inflamed region through increased glycolysis and lactate secretion. Therefore, the immune cells infiltrating the inflamed region encounter an acidic microenvironment. Extracellular acidosis can modulate the innate immune response of macrophages; however, its role for inflammasome signaling still remains elusive. In the present study, we demonstrated that macrophages exposed to an acidic microenvironment exhibited enhanced caspase-1 processing and IL-1β secretion compared with those under physiological pH. Moreover, exposure to an acidic pH increased the ability of macrophages to assemble the NLR family pyrin domain containing 3 (NLRP3) inflammasome in response to an NLRP3 agonist. This acidosis-mediated augmentation of NLRP3 inflammasome activation occurred in bone marrow-derived macrophages but not in bone marrow-derived neutrophils. Notably, exposure to an acidic environment caused a reduction in the intracellular pH of macrophages but not neutrophils. Concordantly, macrophages, but not neutrophils, exhibited NLRP3 agonist-mediated translocation of chloride intracellular channel protein 1 (CLIC1) into their plasma membranes under an acidic microenvironment. Collectively, our results demonstrate that extracellular acidosis during inflammation can increase the sensitivity of NLRP3 inflammasome formation and activation in a CLIC1-dependent manner. Thus, CLIC1 may be a potential therapeutic target for NLRP3 inflammasome-mediated pathological conditions.

Isolation of Murine Bone Marrow-Derived Neutrophils for Infection Modeling.

West Nile virus (WNV) is a single-stranded-RNA flavivirus that can cause neurological illnesses. The ability of WNV to cause neurological illnesses is dependent on the virus' ability to gain access into the brain. However, the mechanisms by which WNV enters the brain are elusive, with evidence that neutrophils act as vehicles for viral delivery. To determine the role of neutrophils in WNV central nervous system delivery, modified protocols for the isolation and migration of neutrophils from mouse bone marrow for in vitro WNV infection modeling is described.

S100A9 induces reactive oxygen species-dependent formation of neutrophil extracellular traps in abdominal sepsis

Recent evidence suggests that targeting S100A9 reduces pathological inflammation in abdominal sepsis. Herein, we investigated the role of S100A9 in neutrophil extracellular trap (NET) formation in septic lung damage. NETs were detected by electron microscopy in the lung and by confocal microscopy in vitro. Stimulation of isolated mouse bone marrow-derived neutrophils with S100A9 triggered formation of NETs. Blocking TLR4 and RAGE reduced S100A9-induced generation of NETs and DNA-histone complexes. Moreover, S100A9 challenge increased generation of reactive oxygen species (ROS) in bone marrow neutrophils. Co-incubation with the NADPH oxidase inhibitor not only decreased ROS formation but also attenuated induction of DNA-histone complexes in S100A9-stimulated neutrophils. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in male C57BL/6 mice. Administration of the S100A9 inhibitor ABR-238901 decreased CLP-induced formation of NETs in lungs and DNA-histone complexes in plasma. In addition, transmission electron microscopy revealed that S100A9 was abundantly expressed on NETs in the lungs in CLP mice. By use of intravital microscopy, we found that local injection of NETs increased leukocyte adhesion and migration in the mouse cremaster muscle microvasculature. Notably, treatment with ABR-238901 attenuated NET-induced leukocyte adhesion and extravasation in the cremaster muscle, suggesting that NET-associated S100A9 promotes leukocyte recruitment in vivo. Taken together, these novel findings suggest that S100A9 triggers ROS-dependent formation of NETs via TLR4 and RAGE signaling in neutrophils. Moreover, S100A9 regulates both formation of NETs and NET-induced leukocyte recruitment in vivo. Thus, targeting S100A9 might be useful to ameliorate lung damage in abdominal sepsis.

Neutrophils as major drivers of increased atherosclerosis in a murine model of chronic colitis

Abstract Background Recent findings showed a higher risk of premature atherosclerosis and cardiovascular events in patients with inflammatory bowel disease (IBD) especially during acute flare of the chronic disease. The underlying mechanisms remain to be defined since traditional risk factors such as hypercholesterinemia are not present in these patients. Purpose The present study aimed to unravel the underlying pathomechanisms of enhanced atherogenesis and -progression in a murine model of colitis. Methods Chronic colitis was induced in male Apolipoprotein E-deficient mice (Apoe−/−) using dextran sodium sulphate (DSS) in drinking water for 2,3 or 5 cycles, while the control group received regular drinking water. Each cycle consisted of 6 days DSS application and two weeks of recovery. Aortic atherosclerotic plaque burden was determined by en face Oil Red O staining and immune cell subsets were analysed by flow cytometry in the circulation, the bone marrow, and the aorta. Neutrophil depletion was performed via i.p injection of a Ly6G-depleting antibody or respective isotype control. Bone marrow-derived neutrophils were further analysed by transcriptome analysis. Results Similar to IBD patients, mice with chronic colitis exhibited an increased aortic plaque burden after 15 weeks of treatment despite the absence of classical risk factors. Over time, both circulating and aortic neutrophils showed an oscillatory detection pattern with the first significant increase after the 2nd DSS administration whereby the second increase after the 3rd DSS cycle was even stronger. Also, pro-inflammatory cytokines were elevated in the plasma and specifically G-CSF showed the same oscillatory pattern with increased plasma level already after the 2nd DSS administration and an even stronger increase after the 3rd thereby pointing towards alterations in bone marrow hematopoiesis. In line, flow cytometric analyses confirmed a greater rise of hematopoietic stem and even myeloid progenitor cells compared with the 2nd DSS application in IBD mice after 3rd treatment. scRNA-Seq analysis of progenitor cells revealed changes in cell differentiation towards neutrophils and upregulation of proinflammatory genes in isolated neutrophils of DSS-treated mice. These neutrophils showed also a more adhesive phenotype revealed by increased mRNA expression of Glg1 and Selplg. Accordingly, also Sele mRNA was increased in the aorta. The reduction of circulating neutrophils by an anti-Ly6G antibody during the acute phases of colitis reduced the aortic plaque burden compared to isotype treaded mice. Conclusion The current findings suggest detrimental effects of chronic colitis on atherogenesis and -progression in Apoe−/− mice via increased differentiation of myeloid cells into neutrophils and the promotion of a more adhesive and proinflammatory phenotype. These modified neutrophils may act as initiators of atherogenesis by promoting the invasion of immune cells into the aortic wall. Funding Acknowledgement Type of funding sources: None.

Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis.

Severe coronavirus disease (COVID-19) is accompanied by acute respiratory distress syndrome and pulmonary pathology, and is presented mostly with an inflammatory cytokine release, a dysregulated immune response, a skewed neutrophil/lymphocyte ratio, and a hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19-related mortality, the limitation of the use of vaccine against immunocompromised individuals, those with comorbidity, and emerging variants remains a concern. In the current study, we investigate for the first time the efficacy of the Glycyrrhiza glabra (GG) extract, a potent immunomodulator, against SARS-CoV-2 infection in hamsters. Prophylactic treatment with GG showed protection against loss in body weight and a 35%–40% decrease in lung viral load along with reduced lung pathology in the hamster model. Remarkably, GG reduced the mRNA expression of pro-inflammatory cytokines and plasminogen activator inhibitor-1 (PAI-1). In vitro, GG acted as a potent immunomodulator by reducing Th2 and Th17 differentiation and IL-4 and IL-17A cytokine production. In addition, GG also showed robust potential to suppress ROS, mtROS, and NET generation in a concentration-dependent manner in both human polymorphonuclear neutrophils (PMNs) and murine bone marrow-derived neutrophils (BMDNs). Taken together, we provide evidence for the protective efficacy of GG against COVID-19 and its putative mechanistic insight through its immunomodulatory properties. Our study provides the proof of concept for GG efficacy against SARS-CoV-2 using a hamster model and opens the path for further studies aimed at identifying the active ingredients of GG and its efficacy in COVID-19 clinical cases.

Pannexin1 channel-dependent secretome from apoptotic tumor cells shapes immune-escape microenvironment

Apoptotic cell death is a critical step in organism development and tissue homeostasis. Apoptotic cells affect immune cell activities in normal tissues. It is not clear whether similar cell death machinery causes tumor environments to evade anti-tumor immune responses. Here, using a mouse transplant model, we found a large number of tumor cells undergoing intrinsic apoptosis in tumors derived from the 4T1 breast cancer cell line, where neutrophils significantly accumulated. Interestingly, these apoptotic 4T1 tumor cells directly induced neutrophil extracellular traps (NETs) in a pannexin 1 (Panx1) channel-dependent manner, and knockdown of Panx1 in 4T1 cells led to a reduction in tumor size. Spermidine released through Panx1 from apoptotic 4T1 cells induced NETs in bone marrow-derived neutrophils in vitro. In addition, inhibition of spermidine synthesis suppressed tumor growth in the mouse transplant model. Collectively, our data suggested a new immune-escape mechanism for tumors by Panx1-mediated secretome from intrinsic apoptotic cells, which may provide a new therapeutic target for cancer.

Tas2R signaling enhances mouse neutrophil migration via a ROCK-dependent pathway.

Type-2 bitter taste receptors (Tas2Rs) are a large family of G protein-coupled receptors that are expressed in the oral cavity and serve to detect substances with bitter tastes in foods and medicines. Recent evidence suggests that Tas2Rs are also expressed extraorally, including in immune cells. However, the role of Tas2Rs in immune cells remains controversial. Here, we demonstrate that Tas2R126, Tas2R135, and Tas2R143 are expressed in mouse neutrophils, but not in other immune cells such as macrophages or T and B lymphocytes. Treatment of bone marrow-derived neutrophils from wild-type mice with the Tas2R126/143 agonists arbutin and d-salicin led to enhanced C-X-C motif chemokine ligand 2 (CXCL2)-stimulated migration in vitro, but this response was not observed in neutrophils from Tas2r126/135/143-deficient mice. Enhancement of CXCL2-stimulated migration by Tas2R agonists was accompanied by increased phosphorylation of myosin light chain 2 (MLC2) and was blocked by pretreatment of neutrophils with inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK), but not by inhibitors of the small GTPase RhoA. Taken together, these results demonstrate that mouse neutrophils express functional Tas2R126/143 and suggest a role for Tas2R126/143-ROCK-MLC2-dependent signaling in the regulation of neutrophil migration.

Bone marrow is the preferred source for isolation of rat neutrophils and the subsequent acquisition of neutrophil extracellular traps.

BackgroundNeutrophil extracellular traps (NETs) are a network structure with DNA as skeleton scaffolding a wide range of antimicrobial proteins, which have been shown to contribute to the pathogenesis, persistence, and progression of many chronic inflammatory diseases. The method for isolation of human or mouse NETs has been well-established. However, in some diseases such as atrial fibrillation, the model can only be established in rats. While most related studies isolate rat neutrophils from the peripheral blood, which is insufficient for further acquisition of NETs. Despite the consumptiveness of rat peripheral blood neutrophil isolation, bone-marrow deprived neutrophil is rarely employed and has not been compared to peripheral neutrophil with its NETs secreting capability.MethodsIn the current study, a different bone-marrow-oriented strategy was described and conducted. Based on centrifugal program settings, a one-step method and a two-step method was developed and compared. The purity of the isolated neutrophils was determined by Wright’s staining and the viability was detected by flow cytometry. NETosis is the specialized cell death of neutrophils accompanied with NETs formation and its degree of rat neutrophils was analyzed by phase contrast microscopy, fluorescence microscopy, and Celigo whole view analysis. The Picogreen dsDNA Assay Kit was used to determine the concentration of NETs obtained from the NET-rich supernatants. The levels of secreted NETs in rat peripheral blood and bone marrow were compared.ResultsApproximately 0.5×108–1×108 neutrophils could be obtained from the bone marrow of a single rat, with viability above 90%, which was comparable to that of neutrophils isolated from humans and mice. The final concentration of NETs obtained from the supernatant ranged from 8–12 µg/mL. The secretion of NETs from bone marrow-derived neutrophils showed a similar trend to polymorphonuclear (PMN) leukocytes. In addition, the extrusion of the intracellular matrix was incomplete during NETosis, and rat NETs showed weak cross-linking capability for forming large-scale net-like structures.ConclusionsThe bone marrow-oriented strategy for isolating rat neutrophils is accessible and repeatable. NETs formation capability is similar between rat bone-marrow and peripheral blood neutrophils.