Activation Of Polymorphonuclear Neutrophils Research Articles

Vaginal Microbiota and Proinflammatory Status in Patients with Polycystic Ovary Syndrome: An Exploratory Study.

Background/Purpose: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disease most common in patients of childbearing age. This pathology is associated with clinical, metabolic, and reproductive complications. We evaluated the diversity of the vaginal microbiota (VM), the vaginal inflammatory reaction (VIR), the proinflammatory state, and the activation of polymorphonuclear neutrophils (PMN) with the production of neutrophil extracellular traps (NETs). Methods: Thirty-three patients who attended a consultation at the Hospital UTPL-Santa Inés, Loja, Ecuador, from May to August 2023 who were diagnosed with PCOS participated in this study. Blood samples, vaginal discharge, and a survey were obtained. Results: A high number of patients, 23/33 (69.7%), presented altered microbiota in clinical variables associated with PCOS phenotypes A and B, sexual partners (>2), and oligomenorrhoea. A significant statistical association was only observed for sexually transmitted infections at sampling (p = 0.023) and insulin (p = 0.002). All eight cases studied with VIR had PMN/NETotic activity. A high frequency of proinflammatory states was observed in all vaginal microbiota states. Conclusions: These results suggest that the PCOS could trigger a proinflammatory state in the vaginal epithelium independently of the state of the vaginal microbiota. Furthermore, the presence of NETs observed in the cases studied could decrease fertility in these PCOS patients.

Investigation of the rhythmic recruitment of tear neutrophils to the ocular surface and their phenotypes

Hundreds of thousands of polymorphonuclear neutrophils (PMNs) are collected from the ocular surface upon waking, while few are harvested during daytime. This study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs, including changes in IL-8 and C5a in tears, and their phenotypes across different time points in a 24-h cycle. Tear PMNs were collected using a gentle eyewash after 2-h and 7-h of sleep (eye closure, EC) at night, after 2-h EC during the day, and towards the end of the afternoon. Significantly fewer cells were collected after 2-h EC during the day compared to 2-h EC at night. A positive correlation between IL-8 and PMN numbers existed, but not with C5a. Tear PMNs collected after 2-h EC at night were less degranulated and possessed a larger activation potential compared to 7-h EC. Tear PMNs from 7-h EC at night exhibited hyper-segmented nuclei and more NETosis compared to 2 h EC night, indicating an aged and activated phenotype. The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by increased IL-8 in nighttime tears. Higher degranulation and NETs point to the significant activation of tear PMNs on the ocular surface during prolonged eye closure at night.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

Mesenchymal Stromal/Stem Cells Know Best: The Remarkable Complexities of Its Interactions With Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs), the predominant immune cell type in humans, have long been known as first-line effector cells against bacterial infections mainly through phagocytosis and production of reactive oxygen species (ROS). However, recent research has unveiled novel and pivotal roles of these abundant but short-lived granulocytes in health and disease. Human mesenchymal stromal/stem cells (MSCs), renowned for their regenerative properties and modulation of T lymphocytes from effector to regulatory phenotypes, exhibit complex and context-dependent interactions with PMNs. Regardless of species or source, MSCs strongly abrogate PMN apoptosis, a critical determinant of PMN function, except if PMNs are highly stimulated. MSCs also have the capacity to fine-tune PMN activation, particularly in terms of CD11b expression and phagocytosis. Moreover, MSCs can modulate numerous other PMN functions, spanning migration, ROS production, and neutrophil extracellular trap (NET) formation/NETosis, but directionality is remarkably dependent on the underlying context: in normal nondiseased conditions, MSCs enhance PMN migration and ROS production, whereas in inflammatory conditions, MSCs reduce both these functions and NETosis. Furthermore, the state of the MSCs themselves, whether isolated from diseased or healthy donors, and the specific secreted products and molecules, can impact interactions with PMNs; while healthy MSCs prevent PMN infiltration and NETosis, MSCs isolated from patients with cancer promote these functions. This comprehensive analysis highlights the intricate interplay between PMNs and MSCs and its profound relevance in healthy and pathological conditions, shedding light on how to best strategize the use of MSCs in the expanding list of diseases with PMN involvement.

[The mechanism of Xuebijing injection in preventing and treating lung injury induced by cardiopulmonary bypass by regulating the apoptosis of alveolar polymorphonuclear neutrophil].

To investigate the protective effect of Xuebijing injection on acute lung injury (ALI) associated with cardiopulmonary bypass (CPB) by regulating the apoptosis of polymorphonuclear neutrophils (PMN). Thirty male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), CPB model group (CPB group) and Xuebijing pretreatment group (XBJ group) according to the random number table method, with 10 rats in each group. Rats in the CPB group and XBJ group undergoing CPB procedures for 60 minutes. Rats in the Sham group did not undergo CPB. Rats in the XBJ group received intraperitoneal injection of 4 mL/kg Xuebijing injection 2 hours before CPB. Rats in the Sham group and CPB group were injected with an equal amount of normal saline. 4 hours after CPB, arterial blood was collected for blood gas analysis to calculate respiratory index (RI), and lung tissue of rats was collected for determination of lung index (LI) and pulmonary water containing rate. PMN in bronchoalveolar lavage fluid (BALF) were collected and the activity of caspase-3 was detected. The apoptosis rate was detected by flow cytometry. The expressions of microRNA-142-3p (miR-142-3p) and FoxO1 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The protein expression of FoxO1 was detected by Western blotting. In addition, HL-60 cells were divided into control oligonucleotide transfection group, miR-142-3p mimics transfection group, and miR-142-3p inhibitor transfection group. After 48 hours of transfection, the activity of miR-142-3p binding to FoxO1 was detected using dual luciferase reporter genes. Compared with Sham group, RI, LI and pulmonary water containing rate were significantly increased in CPB group. The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly decreased, the expression of miR-142-3p was decreased, and the expression of FoxO1 protein was increased. However, compared with CPB group, RI, LI and pulmonary water containing rate were significantly decreased in XBJ group [RI: 0.281±0.066 vs. 0.379±0.071, LI: 4.50±0.26 vs. 5.71±0.42, pulmonary water containing rate: (80.31±32.50)% vs. (84.59±3.41)%, all P < 0.01]. The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly increased [caspase-3 activity: 0.350±0.021 vs. 0.210±0.014, apoptosis rate: (15.490±1.382)% vs. (8.700±0.701)%, both P < 0.01], the expression of miR-142-3p was significantly up-regulated (2-ΔΔCt: 2.61±0.17 vs. 0.62±0.05, P < 0.01), and the protein expression of FoxO1 was decreased [FoxO1/GAPDH (relative expression level): 0.81±0.04 vs. 1.22±0.06, P < 0.01]. However, there was no statistically significant difference in FoxO1 mRNA expression among the three groups. The bioinformatics analysis results showed that miR-142-3p can bind to the FoxO1 3'untranslated region (3'UTR). In HL-60 cells, compared with control oligonucleotide transfection group, the transfection of miR-142-3p mimics could reduce the expression of FoxO1 protein [FoxO1/GAPDH (relative expression level): 0.48±0.06 vs. 1.00±0.05, P < 0.01], however, the transfection of miR-142-3p inhibitor increased the expression of FoxO1 protein [FoxO1/GAPDH (relative expression level): 1.37±0.21 vs. 1.00±0.05, P < 0.05]. But, transfection with miR-142-3p mimics or inhibitor had no effect on FoxO1 mRNA expression. The luciferase reporter gene showed that miR-142-3p could bind to the FoxO1 3'UTR to inhibit FoxO1 expression. Xuebijing injection may promote the apoptosis of pulmonary alveolar PMN through the miR-142-3p/FoxO1 axis, and play a role in the prevention and treatment of CPB-induced ALI.

Impaired polymorphonuclear neutrophil functions in diabetics

Background and objectives: Polymorphonuclear neutrophils (PMN) are the first line of host resistance against infections. Diabetics are prone to both bacterial and fungal infections. The present study evaluated the phagocytic and killing activity of PMN in diabetics. Material and methods: Females aged 30 to 50 years with and without diabetes mellitus were enrolled. Functions of PMN were assessed by determining the phagocytic rate, phagocytic index and killing of C. albicans by PMN. Results: A total of 36 diabetic patients and 15 age matched non-diabetic healthy individuals were enrolled. Phagocytosis and killing of C. albicans by PMN were significantly (p&lt;0.05) lower in patients with diabetes mellitus compared to non-diabetic healthy individuals (86.5±14.6 vs. 94.5±4.2; 56.7±23.8 vs. 81.5±24.2). Conclusion: Phagocytic and killing functions of PMN were significantly reduced in patients with diabetes mellitus. IMC J Med Sci. 2024; 18(1):008. DOI: https://doi.org/10.55010/imcjms.18.008 *Correspondence:Tanzinah Nasrin, Microbiologist, Quality Control Laboratory, Department of Fisheries, Ministry of Fisheries and Livestock, Dhaka, Bangladesh. Email: tanzinahn8@gmail.com

Characterization of TREM-1 Signaling in Human Neutrophils By Kinome Array and RNA-Seq

The Triggering Receptor Expressed on Myeloid Cells (TREM)-1 is a member of the Immunoglobulin superfamily and an activating receptor mainly expressed on myeloid cells. TREM-1 ligation leads to immediate activation of polymorphonuclear neutrophils (PMN) resulting in degranulation and release of reactive oxygen species as well as various effector molecules. Beyond its role in acute and chronic inflammatory processes, TREM-1 is also involved in cancer emergence and progression probably by alteration of the tumor associated neutrophils (TAN) and macrophages (TAM). Advanced information about the TREM-1 signaling cascade may reveal novel therapeutic approaches, as various kinases can specifically be targeted with kinase inhibitors. Therefore, we investigated the protein tyrosine kinome (PTK) and serine threonine kinome (STK) of human PMN after TREM-1 activation. To gain further insights beyond the signaling cascade, we performed RNAseq to validate the TREM-1 mediated activation and to reveal the TREM-1 mediated transcriptome. Purified PMN from healthy donors were stimulated with a monoclonal TREM-1 antibody compared to an isotype matched control for 20 minutes (kinome activity) or 60 minutes (RNAseq). For kinomics, PamChip arrays (PamGene) were used to quantify the kinome activity and upstream kinase analysis (UKA) was used to predict the kinase activity. The final data analysis and the UKA was performed by PamGene. A median final score (depending on the significance and the specificity of the kinase activity compared to the control group) &amp;gt; 1.2 was defined as specific kinase activity according to the manufacturer's recommendations. For transcriptomics, Novogene performed library preparation and sequencing, and data analysis was performed with CLC. Differentially expressed genes were defined as FDR adjusted p value ≤ 0.05 and fold change ≥2. Gene set enrichment analysis was performed with Enrichr using the libraries Reactome 2022, WikiPathway 2021 Human, and GO Biological Process 2023 and significance was defined as adjusted p value ≤ 0.05. We identified the activation of 31 PTKs and 38 STKs upon TREM-1 ligation. The kinase statistic revealed changes in the activation state of the kinases present in the TREM-1 treated cells compared to the untreated control group ranging from 2.4 to 5.5 (PTK) and from 1.2 to 1.6 (STK) suggesting more TREM-1 mediated activity alteration of the PTKs. Interestingly, there were no downregulated kinase activities after receptor ligation. Out of the PTK family, we identified ten Src family kinases as well as the Syk family member ZAP70 and other kinases such as BTK, already previously shown to abrogate TREM-1 mediated PMN activation. The RNAseq after TREM-1 ligation revealed 542 differentially expressed genes from which 102 genes were upregulated and 440 genes were downregulated. Gene set enrichment analysis revealed upregulation of genes related to nuclear events (kinase and transcription factor activaton, R-HSA-198725) and cell chemotaxis (GO:0060326) validating the activation of several kinases after TREM-1 ligation. We found increased expressions of genes involved in adipogenesis (WP236) and lung fibrosis (WP3624) suitably to the observed increased activity of Src family kinases that are described to be involved in lung fibrosis. Moreover, some upregulated genes related to NGF-stimulated transcription (R-HSA-9031628) or regulation of transcription in response to stress (GO:0043618) in which the MAPK are known to be involved. However, we did not observe clearly increased MAPK activity. Besides upregulation, TREM-1 ligation led to decreased expression of several genes involved in (positive) regulation of cytokine production involved in inflammatory response (GO:1900017 and GO:1900015) and signaling by interleukins (R-HSA-449147). Taken together, the results contribute to the knowledge about TREM-1 in inflammatory disorders and reveal approaches of its role in cancer. This study revealed various kinases possibly involved in early TREM-1 signaling of neutrophils that may be suitable targets to inhibit TREM-1 mediated cell activation. Moreover, we linked the TREM-1 mediated kinase activity to the resulting gene expression. Further studies are needed to validate the results in vitro and in vivo and and to identify suitable pharmacological inhibitors to modulate TREM-1 mediated activities in inflammatory conditions and cancer.

LncRNA HCG18 loaded by polymorphonuclear neutrophil-secreted exosomes aggravates sepsis acute lung injury by regulating macrophage polarization.

Polymorphonuclear neutrophils (PMNs) exert significant roles in septic acute lung injury (ALI). Accumulating evidence suggests that PMN-derived exosomes (PMN-exo) are a novel subcellular entity that is the fundamental link between PMN-driven inflammation and tissue damage. However, the role of PMN-exo in septic ALI and the underlying mechanisms remain unclear. Tumor necrosis factor-α (TNF-α), a key regulator of innate immunity in septic ALI, was used to induce PMN activation in vitro. Using an in vitro co-culture system, the rat alveolar macrophage cell line NR8383 was co-cultured with TNF-α-stimulated PMN-released exosomes (TNF-α-exo) to further confirm the results of the in vitro studies and explore the underlying mechanisms involved. A septic lung injury model was established by cecal ligation and puncture surgery, and PMN-exo were injected into septic mice through the tail vein, and then lung injury, inflammatory release, macrophage polarization, and apoptosis were examined. The results reported that TNF-α-exo promoted the activation of M1 macrophages after i.p. injection in vivo or co-culture in vitro. Furthermore, TNF-α-exo affected alveolar macrophage polarization by delivering HCG18. Mechanistic studies indicated that HCG18 mediated the function of TNF-α-exo by targeting IL-32 in macrophages. In addition, tail vein injection of si-HCG18 in septic mice significantly reduced TNF-α-exo-induced M1 macrophage activation and lung macrophage death, as well as histological lesions. In conclusion, TNF-α-exo-loaded HCG18 contributes to septic ALI by regulating macrophage polarization. These findings may provide new insights into novel mechanisms of PMN-macrophage polarization interactions in septic ALI and may provide new therapeutic strategies for patients with sepsis.

Melanoma-derived soluble mediators modulate neutrophil biological properties and the release of neutrophil extracellular traps

Polymorphonuclear neutrophils (PMNs) are the main effector cells in the inflammatory response. The significance of PMN infiltration in the tumor microenvironment remains unclear. Metastatic melanoma is the most lethal skin cancer with an increasing incidence over the last few decades. This study aimed to investigate the role of PMNs and their related mediators in human melanoma. Highly purified human PMNs from healthy donors were stimulated in vitro with conditioned media (CM) derived from the melanoma cell lines SKMEL28 and A375 (melanoma CM), and primary melanocytes as controls. PMN biological properties (chemotaxis, survival, activation, cell tracking, morphology and NET release) were evaluated. We found that the A375 cell line produced soluble factors that promoted PMN chemotaxis, survival, activation and modification of morphological changes and kinetic properties. Furthermore, in both melanoma cell lines CM induced chemotaxis, activation and release of neutrophil extracellular traps (NETs) from PMNs. In contrast, the primary melanocyte CM did not modify the biological behavior of PMNs. In addition, serum levels of myeloperoxidase, matrix metalloprotease-9, CXCL8/IL-8, granulocyte and monocyte colony-stimulating factor and NETs were significantly increased in patients with advanced melanoma compared to healthy controls. Melanoma cell lines produce soluble factors able to “educate” PMNs toward an activated functional state. Patients with metastatic melanoma display increased circulating levels of neutrophil-related mediators and NETs. Further investigations are needed to better understand the role of these “tumor-educated neutrophils” in modifying melanoma cell behavior.

The Role of Diabetic Choroidopathy in the Pathogenesis and Progression of Diabetic Retinopathy.

Diabetic choroidopathy was first described on histopathological specimens of diabetic eyes. This alteration was characterized by the accumulation of PAS-positive material within the intracapillary stroma. Inflammation and polymorphonuclear neutrophils (PMNs) activation are crucial elements in choriocapillaris impairment. The evidence of diabetic choroidopathy in vivo was confirmed with multimodal imaging, which provides key quantitative and qualitative features to characterize the choroidal involvement. The choroid can be virtually affected in each vascular layer, from Haller's layer to the choriocapillaris. However, the damage on the outer retina and photoreceptor cells is essentially driven by a choriocapillaris deficiency, which can be assessed through optical coherence tomography angiography (OCTA). The identification of characteristic features of diabetic choroidopathy can be significant for understanding the potential pathogenic and prognostic implications in diabetic retinopathy.

FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs' coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

Pathophysiology and clinical manifestations of immune complex vasculitides.

Immune complex (IC) vasculitides present inflammations of vessel walls associated with perivascular deposition of immunoglobulins (Igs), mostly ICs. They encompass systemic and skin-limited variants of IgA vasculitis (IgAV), cryoglobulinemic vasculitis (CV), rheumatoid, lupus, and hypocomplementemic vasculitides, serum sickness cutaneous IgM/IgG (non-IgA) vasculitis, and recurrent macular (hypergammaglobulinemic or exertion-induced) vasculitis. Serum sickness and CV fulfill the criteria of a type III hypersensitivity immune reaction as large lattices of the IC precipitate at vessel walls and activate polymorphonuclear neutrophils (PMNs). Immunoglobulin-A vasculitis differs with regard to the causes of perivascular deposition of ICs since here many IgA1 molecules are hypoglycosylated (Gd-IgA1), which appears to facilitate their perivascular deposition in skin and mesangium (via e.g. CD71). The reasons for increased generation of immunoglobulins or formation of IC and their perivascular deposition in either skin or systemic organs are different and not fully explored. A common denominator of OC vasculitides is the activation of PMNs near the vessel wall via Fcy or Fcα receptors. Acute episodes of IgAV additionally require PMNs to become preactivated by IgA1 or by IC already in circulation. This intravascular priming results in increased adherence and subsequently vessel-destructive NETosis when they encounter IgA deposited at the vessel walls. Binding of IgA1 to PMNs in blood stream is associated with increased serum levels of hypogalactosidated IgA1. The characteristic clinical picture of IgAV (and also of so-called IgG/IgM vasculitis) comprises palpable or retiform purpura with a clear predilection for lower legs, probably due to stasis-related reduction in blood velocity, while in other IC vasculitides, additional factors influence the sites of vasculitides. Our knowledge of distinct forms and different pathophysiological pathways of IC vasculitides may lead to in efficacious or targeted therapies. Antibodies to complement components or intestinal budesonide for IgAV are promising agents (the latter suppresses the pathophysiologically related IgA nephropathy by reducing the generation of mucosal IgA.

Itraconazole and neutrophil interactions in the immune-inflammatory response of paracoccidioidomycosis using a murine air pouch infection model

Paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis (Pb), is a severe mycosis, prevalent in tropical countries. The presence of polymorphonuclear neutrophils (PMN) in lesions is conspicuous, indicating their central role in innate immunity through the direct killing of Pb and the production of cytokines that activate acquired immunity in the presence of itraconazole (Itra). The toxicity and direct antifungal activity of Itra on Pb in splenocyte co-cultures were evaluated in vitro. Itra showed no toxic effect but marked antifungal activity against Pb. Purified PMN were obtained by the subcutaneous (SC) injection of Pb into mice. Results showed the effect of Itra on the size of the air pouch produced, the cellular population that migrated to the infection site, protein, and mitochondrial metabolism patterns, production of ROS an NO, and the number of cytokines synthesized. Lower doses (3 and 10 mg/kg) of Itra did not affect the cellular profile but led to a lower influx of viable more active PMN, and increased production of ROS and proteins. At a dose of 50 mg/kg the PMN profile remained unchanged along with all other parameters analyzed remained unaltered. Decreases in most cytokine levels were inversely proportional to the Itra concentration. Lower Itra concentrations may elicit activation of the immune response because the combined effects of therapy and immune response are needed, while the higher dose does not require it. Itra also promotes the activation of the cytokines which elicit PMN activation and consequently the resolution of Pb18 infection in the air pouch.

Systemic Activation of Neutrophils by Immune Complexes Is Critical to IgA Vasculitis.

In IgA vasculitis (IgAV) perivascular deposition of IgA1 immune complexes (IgA-ICs) is traditionally considered the fundamental trigger for polymorphonuclear neutrophil (PMN)-mediated damage. We propose that IgA-IC deposition, although mandatory, is not sufficient alone for IgAV. Serum IgA-IC levels and IgA-IC binding to PMNs were quantified in IgAV patients and controls. Activation of PMNs was evaluated by neutrophil extracellular trap (NET) release, adherence, and cytotoxicity assays and in a flow system to mirror conditions at postcapillary venules. In vitro results were related to findings in biopsies and a mouse vasculitis model. During acute IgAV flares we observed elevated serum levels of IgA-ICs and increased IgA-IC binding to circulating PMNs. This IgA-IC binding primed PMNs with consequent lowering of the threshold for NETosis, demonstrated by significantly higher release of NETs from PMNs activated in vitro and PMNs from IgAV patients with flares compared with surface IgA-negative PMNs after flares. Blocking of FcαRI abolished these effects, and complement was not essential. In the flow system, marked NETosis only occurred after PMNs had adhered to activated endothelial cells. IgA-IC binding enhanced this PMN tethering and consequent NET-mediated endothelial cell injury. Reflecting these in vitro findings, we visualized NETs in close proximity to endothelial cells and IgA-coated PMNs in tissue sections of IgAV patients. Inhibition of NET formation and knockout of myeloperoxidase in a murine model of IC vasculitis significantly reduced vessel damage in vivo. Binding of IgA-ICs during active IgAV primes PMNs and promotes vessel injury through increased adhesion of PMNs to the endothelium and enhanced NETosis.

Interaction of sperm cells with the female reproductive tract in cattle: Focus on neutrophil extracellular trap formation

After insemination of cows, either naturally or artificially, the deposition of semen into the vagina or uterus results in an immune reaction which is based on polymorphonuclear neutrophil activity. Sperm must be resistant to immune system actions of the female for an adequate time to allow fertilization to occur. Neutrophils, however, either directly phagocytize sperm through cell–cell attachment or entrap sperm cells in neutrophil extracellular traps (NETs), structures consisting of neutrophil nuclear DNA and associated proteins. In this review article, the interaction of neutrophils and sperm cells in t cattle will be described, with a special focus on the formation of neutrophil extracellular traps (NETs).

Glycolysis, monocarboxylate transport, and purinergic signaling are key events in Eimeria bovis-induced NETosis.

The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis—an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)—is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.

Polymorphonuclear neutrophil activation by Src phosphorylation contributes to HLA-A2 antibody-induced transfusion-related acute lung injury

Human leukocyte antigen (HLA)-A2 antibody contributes to the pathogenesis of transfusion-related acute lung injury (TRALI) via polymorphonuclear neutrophil (PMN) activation, but the signaling pathways involved this process remain largely undefined. In this study, we sought to study the signaling pathways involved in the pathogenesis of HLA-A2-induced TRALI. Lipopolysaccharide (LPS), and the plasma from the HLA-A2 antibody-positive donors were utilized to establish a rat model of TRALI. Human pulmonary endothelial cells (HPMECs) were in vitro co-cultured with HLA-A2 antibody-treated PMNs and then treated with LPS to induce a cytotoxicity model. The effects of HLA-A2 antibody on HPMEC injury were evaluated in this model. Besides, dasatinib was used to block the Src phosphorylation to explore whether Src involved in the TRALI or HPMEC injury induced by HLA-A2 antibody. The HLA-A2 antibody plus LPS induced TRALI and stimulated PMN activation in rats. HLA-A2 antibody-induced TRALI could be attenuated via depletion of PMN. HLA-A2 antibody activated NF-κB and NLRP3 inflammasome. In addition, HLA-A2 antibody aggravated the HPMEC injuries and the release of PMN surfaces makers, but dasatinib treatment reversed this effect, indicating that HLA-A2 antibody activated PMNs and exacerbated TRALI by stimulating phosphorylation of Src followed by activation of NF-κB and NLRP3 inflammasome, which was validated in vivo. In summary, HLA-A2 induced PMNs by activating NF-κB/NLRP3 inflammasome via phosphorylated-Src elevation, thereby exacerbating TRALI. This study highlights promising target for the treatment of antibody-mediated TRALI.

Inhibition of Recruitment and Activation of Neutrophils by Pyridazinone-Scaffold-Based Compounds.

In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing exacerbated cell responses and tissue damage, novel therapeutic agents are sought. 4aa and 4ba, two pyridazinone-scaffold-based phosphodiesterase-IV inhibitors are compared in vitro to zardaverine for their ability to: (1) modulate production of pro-inflammatory mediators, reactive oxygen species (ROS), and phagocytosis; (2) modulate degranulation by PMNs after transepithelial lung migration. Compound 4ba and zardaverine were tested in vivo for their ability to limit tissue recruitment of PMNs in a murine air pouch model. In vitro treatment of lipopolysaccharide-stimulated PMNs with compounds 4aa and 4ba inhibited the release of interleukin-8, tumor necrosis factor-α, and matrix metalloproteinase-9. PMNs phagocytic ability, but not ROS production, was reduced following treatment. Using a lung inflammation model, we proved that PMNs transmigration led to reduced expression of the CD16 phagocytic receptor, which was significantly blunted after treatment with compound 4ba or zardaverine. Using the murine air pouch model, LPS-induced PMNs recruitment was significantly decreased upon addition of compound 4ba or zardaverine. Our data suggest that new pyridazinone derivatives have therapeutic potential in inflammatory diseases by limiting tissue recruitment and activation of PMNs.

Gold Nanorods Induce Endoplasmic Reticulum Stress and Autocrine Inflammatory Activation in Human Neutrophils.

Gold nanorods (AuNRs) are promising agents for diverse biomedical applications such as drug and gene delivery, bioimaging, and cancer treatment. Upon in vivo application, AuNRs quickly interact with cells of the immune system. On the basis of their strong intrinsic phagocytic activity, polymorphonuclear neutrophils (PMNs) are specifically equipped for the uptake of particulate materials such as AuNRs. Therefore, understanding the interaction of AuNRs with PMNs is key for the development of safe and efficient therapeutic applications. In this study, we investigated the uptake, intracellular processing, and cell biological response induced by AuNRs in PMNs. We show that uptake of AuNRs mainly occurs via phagocytosis and macropinocytosis with rapid deposition of AuNRs in endosomes within 5 min. Within 60 min, AuNR uptake induced an unfolded protein response (UPR) along with induction of inositol-requiring enzyme 1 α (IREα) and features of endoplasmic reticulum (ER) stress. This early response was followed by a pro-inflammatory autocrine activation loop that involves LOX1 upregulation on the cell surface and increased secretion of IL8 and MMP9. Our study provides comprehensive mechanistic insight into the interaction of AuNRs with immune cells and suggests potential targets to limit the unwanted immunopathological activation of PMNs during application of AuNRs.

Molecular Basis for Paradoxical Activities of Polymorphonuclear Neutrophils in Inflammation/Anti-Inflammation, Bactericide/Autoimmunity, Pro-Cancer/Anticancer, and Antiviral Infection/SARS-CoV-II-Induced Immunothrombotic Dysregulation.

Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cells in the circulation. These cells act as the fast and powerful defenders against environmental pathogenic microbes to protect the body. In addition, these innate inflammatory cells can produce a number of cytokines/chemokines/growth factors for actively participating in the immune network and immune homeostasis. Many novel biological functions including mitogen-induced cell-mediated cytotoxicity (MICC) and antibody-dependent cell-mediated cytotoxicity (ADCC), exocytosis of microvesicles (ectosomes and exosomes), trogocytosis (plasma membrane exchange) and release of neutrophil extracellular traps (NETs) have been successively discovered. Furthermore, recent investigations unveiled that PMNs act as a double-edged sword to exhibit paradoxical activities on pro-inflammation/anti-inflammation, antibacteria/autoimmunity, pro-cancer/anticancer, antiviral infection/COVID-19-induced immunothrombotic dysregulation. The NETs released from PMNs are believed to play a pivotal role in these paradoxical activities, especially in the cytokine storm and immunothrombotic dysregulation in the recent SARS-CoV-2 pandemic. In this review, we would like to discuss in detail the molecular basis for these strange activities of PMNs.