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RATIONALE: Research studies typically quantify acute respiratory exacerbation episodes (AECOPD) among people with chronic obstructive pulmonary disease (COPD) based on self-report elicited by survey questionnaire. However, AECOPD quantification by self-report could be inaccurate, potentially rendering it an imprecise tool for identification of those with exacerbation tendency. OBJECTIVE: Determine the agreement between self-reported and health records-documented quantification of AECOPD and their association with airway inflammation. METHODS: We administered a questionnaire to elicit the incidence and severity of respiratory exacerbations in the three years preceding the survey among current or former heavy smokers with or without diagnosis of COPD. We then examined electronic health records (EHR) of those with COPD and those without (tobacco-exposed persons with preserved spirometry or TEPS) to determine whether the documentation of the three-year incidence of moderate to very severe respiratory exacerbations was consistent with self-report using Kappa Interrater statistic. A subgroup of participants also underwent bronchoalveolar lavage (BAL) to quantify their airway inflammatory cells. We further used multivariable regressions analysis to estimate the association between respiratory exacerbations and BAL inflammatory cell composition with adjustment for covariates including age, sex, height, weight, smoking status (current versus former) and burden (pack-years). RESULTS: Overall, a total of 511 participants completed the questionnaire, from whom 487 had EHR available for review. Among the 222 participants with COPD (70 ± 7 years-old; 96% male; 70 ± 38 pack-years smoking; 42% current smoking), 57 (26%) reported having any moderate to very severe AECOPD (m/s-AECOPD) while 66 (30%) had EHR documentation of m/s-AECOPD. However, 42% of those with EHR-identified m/s-AECOPD had none by self-report, and 33% of those who reported m/s-AECOPD had none by EHR, suggesting only moderate agreement (Cohens Kappa = 0.47 ± 0.07; P

BACKGROUND: Endometriosis is considered as a systemic disease with the presence of proinflammatory cytokines in the circulation, which drives hypercoagulable state of endometriosis. Currently, endometriosis is classified into four stages: I (minimal), II (mild), III (moderate) and IV (severe). The aim of this study is to investigate the correlations between inflammatory markers and coagulation factors in patients diagnosed of endometriosis with stage IV. METHODS: This retrospective case-control study included 171 endometriosis patients with stage IV and 184 controls. Continuous data were expressed by mean ± standard deviation. Mann-Whitney U and χ2 tests were used to compare the medians and frequencies among the groups. Spearman analysis was conducted to determine the correlation among the measured parameters. The diagnostic values of the parameters differentiating endometriomas were tested by receiver operating characteristic (ROC) curve. RESULTS: The time of activated partial thromboplastin time (APTT) was decreased and the concentration of fibrinogen (FIB) and neutrophil-to-lymphocyte ratio (NLR) were increased in women of endometriosis with stage IV. The APTT were negatively correlated with NLR while the concentrations of FIB were positively correlated with NLR. The ROC analysis showed that the Area under the curve (AUC) of FIB was 0.766 (95% confidence interval:0.717-0.814) with sensitivity and specificity reaching 86.5 and 60.9%, respectively. The AUC of CA125 and CA199 was 0.638 (95% confidence interval: 0.578-0.697), 0.71 (95% confidence interval: 0.656-0.763) with sensitivity and specificity reaching 40.9 and 91.8%, 80.7 and 56.5% respectively. The combination of these factors showed the highest AUC of 0.895 (0.862-0.927) with sensitivity of 88.9% and specificity of 77.7%. CONCLUSION: In the present study, we found that inflammatory factors showed significant correlation with APTT or FIB in endometriosis with stage IV. Moreover, the coagulation factors combined with CA125 and CA199 were more reliable for identifying the endometriosis with stage IV.

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ4 exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ4-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ4. In Gβ4 knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

UNLABELLED: Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

In sub-Saharan Africa, multidrug-resistant non-typhoidal Salmonella serovars are a common cause of fatal bloodstream infection. Malnutrition is a predisposing factor, but the underlying mechanisms are unknown. Here we show that vitamin A deficiency, one of the most prevalent micronutrient deficits afflicting African children, increases susceptibility to disseminated non-typhoidal Salmonella disease in mice and impairs terminal neutrophil maturation. Immature neutrophils had reduced expression of Slc11a1, a gene that encodes a metal ion transporter generally thought to restrict pathogen growth in macrophages. Adoptive transfer of SLC11A1-proficient neutrophils, but not SLC11A1-deficient neutrophils, reduced systemic Salmonella burden in Slc11a1-/- mice or mice with vitamin A deficiency. Loss of terminal granulopoiesis regulator CCAAT/enhancer-binding protein ϵ (C/EBPϵ) also decreased neutrophil-mediated control of Salmonella, but not that mediated by peritoneal macrophages. Susceptibility to infection increased in Cebpe-/- Slc11a1+/+ mice compared with wild-type controls, in an Slc11a1-expression-dependent manner. These data suggest that SLC11A1 deficiency impairs Salmonella control in part by blunting neutrophil-mediated defence.

PURPOSE: Evaluate the safety and efficacy of efbemalenograstim alfa for neutrophil support in breast cancer patients undergoing myelosuppressive chemotherapy in a phase 2, dose-finding, open-label study (NCT01648322, ClinicalTrials.gov, 2012-07-19). METHODS: 232 patients received up to 4 cycles of chemotherapy, 141 patients with docetaxel + cyclophosphamide (TC) and 91 patients with docetaxel + doxorubicin + cyclophosphamide (TAC). Patients were randomized to efbemalenograstim alfa (80, 240, or 320 µg/kg [TC]; 240 or 320 µg/kg [TAC]) or pegfilgrastim (6 mg) on Day 2 of each cycle. RESULTS: Efbemalenograstim alfa was non-inferior to pegfilgrastim in duration of moderate and severe neutropenia (absolute neutrophil count [ANC]  2.0 × 109/L after the expected ANC nadir) was 2.0-2.4 and 1.9 days for TAC patients treated with efbemalenograstim alfa and pegfilgrastim, respectively. No significant difference was found between any dose of efbemalenograstim alfa and pegfilgrastim in TAC Cycle 1 for incidence of moderate to severe neutropenia (76%-77% of patients) or incidence of severe neutropenia (ANC

BACKGROUND: Pulmonary hypertension (PH) in dogs with myxomatous mitral valve disease (MMVD) is caused by increased pulmonary venous pressure. Thrombosis, vascular remodeling, and vasoconstriction mediated by platelets could exacerbate PH. HYPOTHESIS: Dogs with PH will exhibit a hypercoagulable state, characterized by increased platelet activation, platelet-leukocyte, and platelet-neutrophil aggregate formation. ANIMALS: Eleven dogs (≥3.5 kg) diagnosed with MMVD and PH and 10 dogs with MMVD lacking PH. METHODS: Prospective cohort ex vivo study. All dogs underwent echocardiographic examination, CBC, 3-view thoracic radiographs, and heartworm antigen testing. Severity of PH and MMVD were assessed by echocardiography. Viscoelastic monitoring of coagulation was assessed using thromboelastography (TEG). Platelet activation and platelet-leukocyte/platelet-neutrophil interactions were assessed using flow cytometry. Plasma serotonin concentrations were measured by ELISA. RESULTS: Unstimulated platelets from dogs with MMVD and PH expressed more surface P-selectin than MMVD controls (P = .03). Platelets from dogs with MMVD and PH had persistent activation in response to agonists. The number of platelet-leukocyte aggregates was higher in dogs with MMVD and PH compared with MMVD controls (P = .01). Ex vivo stimulation of whole blood resulted in higher numbers of platelet-neutrophil aggregates in dogs with MMVD and PH (P = .01). Assessment of hypercoagulability based on TEG or plasma serotonin concentrations did not differ between groups. CONCLUSION AND CLINICAL IMPORTANCE: Platelet hyperresponsiveness and increased platelet-neutrophil interaction occur in dogs with MMVD and PH, suggesting that platelets play a role of in the pathogenesis of PH. Clinical benefits of antiplatelet drugs in dogs with MMVD and PH require further investigation.

BackgroundInfection with the protozoan parasite Toxoplasma gondii leads to the formation of lifelong cysts in neurons that can have devastating consequences in the immunocompromised. In the immunocompetent individual, anti-parasitic effector mechanisms and a balanced immune response characterized by pro- and anti-inflammatory cytokine production establishes an asymptomatic infection that rarely leads to neurological symptoms. Several mechanisms are known to play a role in this successful immune response in the brain including T cell production of IFNγ and IL-10 and the involvement of CNS resident cells. This limitation of clinical neuropathology during chronic infection suggests a balance between immune response and neuroprotective mechanisms that collectively prevent clinical manifestations of disease. However, how these two vital mechanisms of protection interact during chronic Toxoplasma infection remains poorly understood.Main textThis study demonstrates a previously undescribed connection between innate neutrophils found chronically in the brain, termed "chronic brain neutrophils" (CBNeuts), and neuroprotective mechanisms during Toxoplasma infection. Lack of CBNeuts during chronic infection, accomplished via systemic neutrophil depletion, led to enhanced infection and deleterious effects on neuronal regeneration and repair mechanisms in the brain. Phenotypic and transcriptomic analysis of CBNeuts identified them as distinct from peripheral neutrophils and revealed two main subsets of CBNeuts that display heterogeneity towards both classical effector and neuroprotective functions in an age-dependent manner. Further phenotypic profiling defined expression of the neuroprotective molecules NRG-1 andErbB4 by these cells, and the importance of this signaling pathway during chronic infection was demonstrated via NRG-1 treatment studies.ConclusionsIn conclusion, this work identifies CBNeuts as a heterogenous population geared towards both classical immune responses and neuroprotection during chronic Toxoplasma infection and provides the foundation for future mechanistic studies of these cells.

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1β (IL-1β). However, the role of IL-1β in intestinal defense against Salmonella remains unclear. Here, we show that IL-1β production is detrimental during Salmonella infection. Mice lacking IL-1β (IL-1β -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1β -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1β induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1β loss prevented mortality in Salmonella-infected IL-1β -/- mice. Finally, we found that IL-1β expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1β signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1β signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis.

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity.

Photodynamic therapy (PDT) effectively kills cancer cells and initiates immune responses that promote anticancer effects locally and systemically. Primarily developed for local and regional cancers, the potential of PDT for systemic antitumor effects [in situ photo‐vaccination (ISPV)] remains underexplored. This study investigates: (1) the comparative effectiveness of paclitaxel (PTX) prodrug [Pc‐(L‐PTX)2] for PDT and site‐specific PTX effects versus its pseudo‐prodrug [Pc‐(NCL‐PTX)2] for PDT combined with checkpoint inhibitors; (2) mechanisms driving systemic antitumor effects; and (3) the prophylactic impact on preventing cancer recurrence. A bilateral tumor model was established in BALB/c mice through subcutaneous injection of CT26 cells. Mice received the PTX prodrug (0.5 μmole kg−1, i.v.), and tumors were treated with a 690‐nm laser (75 mW cm−2 for 30 min, drug‐light interval 0.5 h, light does 135 J cm−1), followed by anti‐CTLA‐4 (100 μg dose−1, i.p.) on days 1, 4, and 7. Notable enhancement in both local and systemic antitumor effectiveness was observed with [Pc‐(L‐PTX)2] compared to [Pc‐(NCL‐PTX)2] with checkpoint inhibitor. Immune cell depletion and immunohistochemistry confirmed neutrophils and CD8+ T cells are effectors for systemic antitumor effects. Treatment‐induced immune memory resisted newly rechallenged CT26, showcasing prophylactic benefits. ISPV with a PTX prodrug and anti‐CTLA‐4 is a promising approach for treating metastatic cancers and preventing recurrence. [ABSTRACT FROM AUTHOR]

As frontline cells, the precise recruitment of neutrophils is crucial for resolving inflammation and maintaining the homeostasis of the organism. Increasing evidence suggests the pivotal role of neutrophil chemotaxis in cancer progression and metastasis. Here, we collected clinical data and peripheral blood samples from patients with tumours to examine the alterations in the neutrophil quantity and chemotactic function using the Cell Chemotaxis Analysis Platform (CCAP). Transcriptome sequencing data of pan-cancer were obtained from The Cancer Genome Atlas (TCGA). Using the least absolute shrinkage and selection operator (LASSO) Cox regression model, we selected a total of 29 genes from 155 neutrophil- and chemotaxis-related genes to construct the ChemoScore model. Meanwhile, nomogram-based comprehensive model was established for clinical application. Furthermore, immunofluorescence (IF) staining was employed to assess the relationship between the neutrophils infiltrating and the survival outcomes of tumours. In this observational study, the chemotactic function of neutrophils was notably diminished in patients. The establishment and validation of ChemoScore suggested neutrophil chemotaxis to be a risk factor in most tumours, whereby higher scores were associated with poorer survival outcomes and were correlated with various immune cells and malignant biological processes. Moreover, IF staining of tumour tissue substantiated the adverse correlation between neutrophil infiltration and the survival of patients with lung adenocarcinoma (P = 0.0002) and colon adenocarcinoma (P = 0.0472). Taken together, patients with tumours demonstrated a decrease in chemotactic function. ChemoScore potentially prognosticates the survival of patients with tumours. Neutrophil chemotaxis provides novel directions and theoretical foundations for anti-tumour treatment. [ABSTRACT FROM AUTHOR]

Summary We investigated the effect of Ropeginterferon alfa‐2b (Ropeg) versus phlebotomy‐only (Phl‐O) on the neutrophil‐to‐lymphocyte ratio (NLR) in 126 patients randomized in the low‐polycythaemia vera (PV) phase II trial. Patients with a baseline NLR ≥3.5 vs. <3.5 had a longer history of PV, were more likely to have splenomegaly, higher JAK2V617F variant allele frequency (VAF) (56% vs. 20% p = 0.001) and more proliferative disease. Ropeg was superior to Phl‐O in reducing NLR (p = 0.008), and the reduction was strongly influenced by the reduction in neutrophils and less by a change in lymphocytes (−59% and −14% respectively). This effect was associated with the achievement of the low‐PV primary end‐point (p = 0.021), symptom reduction and reduction in JAK2 VAF. Interestingly, the reduction in JAK2 VAF from baseline was linearly associated with the reduction in NLR. Patients who failed Phl‐O at 12 months had characteristics that distinguished them from responders, including very high NLR and resistance to cross‐over to 100 μg Ropeg every 2 weeks suggesting higher escalated doses of Ropeg. In conclusion, the study provides evidence that NLR can serve as a valuable biomarker to assess and guide treatment with Ropeg in the early stage of low‐risk PV patients. [ABSTRACT FROM AUTHOR]

Neutrophils collectively migrate to sites of injury and infection. How these swarms are coordinated to ensure the proper level of recruitment is unknown. Using an ex vivo model of infection, we show that human neutrophil swarming is organized by multiple pulsatile chemoattractant waves. These waves propagate through active relay in which stimulated neutrophils trigger their neighbors to release additional swarming cues. Unlike canonical active relays, we find these waves to be self-terminating, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-terminating behavior. We observe near-constant levels of neutrophil recruitment over a wide range of starting conditions, revealing surprising robustness in the swarming process. This homeostatic control is achieved by larger and more numerous swarming waves at lower cell densities. We link defective wave termination to a broken recruitment homeostat in the context of human chronic granulomatous disease. [Display omitted] • Neutrophil swarming is organized by pulsatile, self-extinguishing waves of LTB4 • NADPH-oxidase activation is critical for relay wave self-extinction • Neutrophils adjust the size and number of waves to homeostatically control recruitment • Chronic granulomatosis disease neutrophils generate undamped relay waves that do not extinguish Strickland et al. find that human neutrophils organize pulsatile, self-extinguishing waves of leukotriene B4 to ensure robust homeostatic recruitment. Self-extinguishing waves are dependent on the activity of NADPH oxidase, and deficiency in this pathway leads to a reduced ability to extinguish relay waves. [ABSTRACT FROM AUTHOR]

Although the role of low-density granulocytes (LDGs), neutrophils in the peripheral blood mononuclear cell (PBMC) fraction, and neutrophil extracellular traps (NETs) in assessing lupus disease severity is acknowledged, data specific to childhood-onset lupus remains scarce. This study analyzed 46 patients with childhood-onset systemic lupus erythematosus (82.6% females, mean age 14.5 ± 0.3 years), including 26 cases with normal complement levels and 20 with low complement levels, along with 20 healthy adult volunteers. Key parameters that distinguished healthy volunteers from lupus patients and differentiated between lupus patients with low and normal complement were serum interferon (IFN)-α, serum citrullinated histone 3 (CitH3), and extracellular traps (ETs) in LDGs. However, NETs (assessed by nuclear staining morphology), LDG abundance, and other parameters (such as endotoxemia, cytokines, and double-stranded (ds) DNA) did not show such differentiation. When lipopolysaccharide (LPS) was administered to LDGs in the PBMC fraction, it induced ETs in both low and normal complement groups, indicating the inducible nature of ETs. In adult healthy volunteers, activation by recombinant IFN-α or dsDNA in isolated neutrophils induced LDGs and NETs (identified using immunofluorescent staining for CitH3, myeloperoxidase, and neutrophil elastase) at 45 min and 3 h post-stimulation, respectively. Additionally, approximately half of the LDGs underwent late apoptosis at 3 h post-stimulation, as determined by flow cytometry analysis. Activation by IFN-α or dsDNA in LDGs also led to a more pronounced expression of CD66b, an adhesion molecule, compared to regular-density neutrophils, suggesting higher activity in LDGs. In conclusion, IFN-α and/or dsDNA in serum may transform regular-density neutrophils into LDGs before progressing to NETosis and apoptosis, potentially exacerbating lupus severity through cell death-induced self-antigens. Therefore, LDGs and ETs in LDGs could provide deeper insights into the pathophysiology of childhood-onset lupus. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) infection induces a marked influx of neutrophils into the lungs, which intensifies the severity of tuberculosis (TB). The metabolic state of neutrophils significantly influences their functional response during inflammation and interaction with bacterial pathogens. However, the effect of Mtb infection on neutrophil metabolism and its consequent role in TB pathogenesis remain unclear. In this study, we examined the contribution of glycolysis and fatty acid metabolism on neutrophil responses to Mtb HN878 infection using ex-vivo assays and murine infection models. We discover that blocking glycolysis aggravates TB pathology, whereas inhibiting fatty acid oxidation (FAO) yields protective outcomes, including reduced weight loss, immunopathology, and bacterial burden in lung. Intriguingly, FAO inhibition preferentially disrupts the recruitment of a pathogen-permissive immature neutrophil population (Ly6Glo/dim), known to accumulate during TB. Targeting carnitine palmitoyl transferase 1a (Cpt1a)-a crucial enzyme in mitochondrial β-oxidation-either through chemical or genetic methods impairs neutrophils' ability to migrate to infection sites while also enhancing their antimicrobial function. Our findings illuminate the critical influence of neutrophil immunometabolism in TB pathogenesis, suggesting that manipulating fatty acid metabolism presents a novel avenue for host-directed TB therapies by modulating neutrophil functions. Author summary: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is a significant global health issue. Neutrophils, immune cells that rapidly enter the lungs during Mtb infection, contribute both to fighting the infection and exacerbating lung damage. Their metabolic state, especially the way they produce energy, is crucial for their function during TB. However, the specific impact of Mtb on neutrophil metabolism and its role in TB progression has been unclear. In this study, we investigated the roles of glycolysis and fatty acid metabolism in neutrophil responses to Mtb infection. We found that inhibiting glycolysis worsens TB outcomes, while blocking fatty acid oxidation (FAO) provides protection, reducing lung damage, bacterial load, and overall disease severity. Notably, FAO inhibition specifically disrupts the accumulation of an immature neutrophil population that is more susceptible to Mtb, thereby improving infection control. Targeting carnitine palmitoyl transferase 1a (Cpt1a), a key enzyme in fatty acid metabolism, impaired neutrophil migration to infection sites and enhanced their antimicrobial activity. These findings suggest that modulating neutrophil metabolism, particularly through FAO inhibition, could be a promising strategy for developing host-directed TB therapies. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) continues to be a major health concern in today's world. Despite conflictive findings, evidence supports systemic inflammation's impact on CRC patients' survival rates. Therefore, this study aims to assess the prognostic role of the innate immune system in patients with CRC. Method: A total of 449 patients were included, with a 5-year follow-up period, and absolute neutrophil counts and their related ratios were measured. Results: The non-survival group had increased levels of white blood cells, neutrophils (both p<0.001), and monocytes (p=0.038), compared to the survival group, along with other neutrophil-related ratios. We observed increased mortality risk in patients in the highest tertile of white blood cells [HR=1.85 (1.09-3.13), p<0.05], neutrophils [HR=1.78 (95% CI: 1.07-2.96), p<0.05], and monocytes [HR=2.11 (95% CI: 1.22-3.63)], compared to the lowest tertile, after adjusting for all clinicopathological variables. Random forest analysis identified neutrophils as the most crucial variable in predicting survival rates, having an AUC of 0.712, considering all clinicopathological variables. A positive relationship between neutrophil counts and metastasis was observed when neutrophil counts are considered continuous (b=0.92 (0.41), p<0.05) and tumor size (width) when neutrophils were considered as logistic variable (T1 vs T3) [OR=1.42, (95% CI: 1.05-1.98), p<0.05]. Conclusion: This study offers comprehensive insights into the immune factors that impact the prognosis of CRC, emphasizing the need for personalized prognostic tools. [ABSTRACT FROM AUTHOR]

Among several quantitative trait loci involved in tuberculosis (TB) control in mice, one was mapped within the chromosome 17 segment occupied by the H2 complex and another within the chromosome 3 segment comprising the S100A8/9 genes, which encode neutrophil inflammatory factor S100A8/9. Previously, we developed a panel of H2‐congenic mouse strains differing by small segments of the major histocompatibility complex Class II (MHC‐II) region from TB‐susceptible H2j mice transferred onto the genetic background of the TB‐resistant C57BL/6 (H2b) strain. Susceptible B6.I‐9.3 mice differ from B6 progenitors by the alleles of their only classical MHC‐II H2‐Aβ gene. The goals of the present study were to: (i) comprehensively characterise the differences in TB‐related phenotypes between mice of the two strains and (ii) decipher interactions between the H2‐Aβ and S100A8/9 genes. Here, we describe the dynamics of TB‐related phenotypes differentiating B6.I‐9.3 and B6 mice (colony forming units counts, histopathology, lung immune cell infiltration and cytokine profiles). We show that disproportionally diminished CD4+ T‐cell population, an enlarged S100A8/9‐positive neutrophil population and higher S100A8/9 serum levels in B6.I‐9.3 mice collectively form the 'susceptible' phenotype before infection. An increase in IL‐17 and a decrease in intrferon‐gamma production by CD4+ T‐cells in these mice provide a mechanistic explanation of this phenotype. Using F2 segregation analysis, we show that the number of S100A8/9‐producing neutrophils in lungs and spleens and the proportion of Th17 CD4+ T‐cells in lungs are significantly lower in the presence of the MHC‐II dominant 'resistant' b allele compared to the recessive 'susceptible' j/j genotype. This provides direct genetic evidence that MHC‐II‐regulated CD4+ T‐cell landscapes determine neutrophil abundance before infection, an important pathogenic factor in TB immunity. [ABSTRACT FROM AUTHOR]

Background and Objective Methods Results Conclusion Severe asthma is a heterogeneous disease with subtype classification according to dominant airway infiltrates, including eosinophilic (Type 2 high), or non‐eosinophilic asthma. Non‐eosinophilic asthma is further divided into paucigranulocytic or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in the airways. Severe non‐eosinophilic asthma has few effective treatments and many patients do not qualify for biologic therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease and has proven a valuable therapeutic target. We hypothesized that the CFTR may also play a role in non‐eosinophilic asthma.Patient‐derived human bronchial epithelial cells (hBECs) were isolated and differentiated at the air‐liquid interface. Single cell RNA‐sequencing (scRNAseq) was used to identify epithelial cell subtypes and transcriptional activity. Ion transport was investigated with Ussing chambers and immunofluorescent quantification of ionocyte abundance in human airway epithelial cells and murine models of asthma.We identified that hBECs from patients with non‐eosinophilic asthma had reduced CFTR function, and did not differentiate into CFTR‐expressing ionocytes compared to those from eosinophilic asthma or healthy donors. Similarly, ionocytes were also diminished in the airways of a murine model of neutrophilic‐dominant but not eosinophilic asthma. Treatment of hBECs from healthy donors with a neutrophilic asthma‐like inflammatory cytokine mixture led to a reduction in ionocytes.Inflammation‐induced loss of CFTR‐expressing ionocytes in airway cells from non‐eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target. [ABSTRACT FROM AUTHOR]

Familial Mediterranean fever (FMF) is characterized by inflammatory attacks due to overactivation of pyrin inflammasome. This study aimed to investigate the reliability of S100A8/A9, neopterin, and matrix metalloproteinase 3 (MMP3) at monitoring subclinical inflammation and disease activity, and at differentiating FMF attacks from appendicitis, the most common misdiagnosis among FMF patients. Blood samples (n = 75), comprising from FMF patients during an attack (n = 20), the same FMF patients during the attack-free period (n = 14), patients with appendicitis (n = 24), and healthy volunteers (n = 17) were obtained. Duplicate determinations of S100A8/A9, neopterin, and MMP-3 levels were conducted using the enzyme-linked immunosorbent assay (ELISA). FMF patients with and without attack and patients with appendicitis had significantly elevated S100A8/A9 levels compared to healthy volunteers (P-values: < 0.001, 0.036, 0.002, respectively). Patients with appendicitis and FMF patients with and without attack had significantly increased serum neopterin levels compared to healthy volunteers (P-value: < 0.001). MMP3 levels were significantly higher among patients with appendicitis and FMF patients during attack compared to healthy controls (P-values: < 0.001, 0.001). Serum levels of S100A8/A9, neopterin, and MMP3 were increased significantly during attacks compared to attack-free periods among FMF patients (P-values: 0.03, 0.047, 0.007). S100A8/A9 emerges as a valuable marker for monitoring disease activity. Neopterin and S100A8/A9 might help physicians to monitor subclinical inflammation during the attack-free periods of FMF patients. MMP3 might aid in diagnosing FMF attacks when distinguishing between attack and attack-free periods is challenging. S100A8/A9 emerges as a valuable marker for monitoring disease activity. Neopterin and S100A8/A9 might help physicians to monitor subclinical inflammation during the attack-free periods of FMF patients. MMP3 might aid in diagnosing FMF attacks when distinguishing between attack and attack-free periods is challenging. Graphical Abstract [ABSTRACT FROM AUTHOR]

The article discusses the role of the chemokine CCL28 in modulating neutrophil responses during infection with mucosal pathogens. The study found that CCL28 promotes neutrophil accumulation in the gut during Salmonella infection and in the lung during Acinetobacter infection. Neutrophils isolated from the infected mucosa express CCL28 receptors on their surface, and CCL28 stimulation enhances neutrophil antimicrobial activity. The absence of CCL28 in mice confers protection against Acinetobacter infection but makes them susceptible to Salmonella infection. These findings highlight the importance of CCL28 in the immune response to bacterial infections. The document also provides information on the materials and methods used in the study, as well as references to related scientific articles. [Extracted from the article]

Objectives Systemic lupus erythematosus (SLE) is a complex autoimmune disease with varying symptoms and multi-organ damage. Relapse-remission cycles often persist for many patients for years with the current treatment. Improved understanding of molecular changes caused by SLE flare and intensive treatment may result in more targeted therapies. Methods RNA sequencing was performed on peripheral blood mononuclear cells (PBMCs) from 65 SLE patients in flare, collected both before (SLE1) and after (SLE2) in-hospital treatment, along with 15 healthy controls (HC). Differentially expressed genes (DEGs) were identified among the three groups. Enriched functions and key molecular signatures of the DEGs were analysed and scored to elucidate the transcriptomic changes during treatment. Results Few upregulated genes in SLE1 vs HC were affected by treatment (SLE2 vs SLE1), mostly functional in interferon signalling (IFN), plasmablasts and neutrophils. IFN and plasmablast signatures were repressed, but the neutrophil signature remained unchanged or enhanced by treatment. The IFN and neutrophil scores together stratified the SLE samples. IFN scores correlated well with leukopenia, while neutrophil scores reflected relative cell compositions but not cell counts. Conclusions In-hospital treatment significantly relieved SLE symptoms with expression changes of a small subset of genes. Notably, IFN signature changes matched SLE flare and improvement, while enhanced neutrophil signature upon treatment suggested the involvement of low-density granulocytes (LDG) in disease development. [ABSTRACT FROM AUTHOR]

Objectives: Carcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is an adhesion molecule that acts as a coinhibitory receptor in the immune system. We previously demonstrated that CEACAM1 is predominantly expressed on peripheral blood neutrophils in patients with RA. The aim of the present study was to investigate the effects of Janus kinase inhibitors (JAKi) on cytokine-activated human neutrophils and CEACAM1 expression. Methods: Peripheral blood neutrophils were obtained from healthy subjects. Isolated neutrophils were stimulated with tumor necrosis factor-alpha (TNF-α) or granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of JAKi. The expression of CEACAM1 in peripheral blood neutrophils was analyzed by flow cytometry. Protein phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 was assessed by western blot using phospho-specific antibodies. Results: We found that TNF-α-induced CEACAM1 expression was marginally suppressed after pretreatment with pan-JAK inhibitor, tofacitinib. Moreover, TNF-α induced STAT1 and STAT3 phosphorylation at the late stimulation phase (4 to 16 h). The expressions of CEACAM1 on neutrophils were markedly up-regulated by GM-CSF not by interleukin (IL)-6 stimulation. All JAKi inhibited GM-CSF-induced CEACAM1 expressions on neutrophils, however, the inhibitory effects of baricitinib were larger compared to those of tofacitinib or filgotinib. Moreover, CEACAM1 was marginally upregulated in interferon (IFN)-γ stimulated neutrophils. Similarly, JAKi inhibited IFN-γ-induced CEACAM1 expressions on neutrophils. Conclusions: We demonstrated that JAKi prevent GM-CSF-induced CEACAM1 expression in neutrophils, and JAKi-induced inhibition depends on their selectivity against JAK isoforms. These findings suggest that JAKi can modulate the expression of CEACAM1 in cytokine-activated neutrophils, thereby limiting their activation. [ABSTRACT FROM AUTHOR]

Background: Neutrophils are key players in the innate immune system, actively migrating to sites of inflammation in the highly energetic process of chemotaxis. In this study, we focus on the role of acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the synthesis of triglycerides, the major form of stored energy, in neutrophil chemotaxis. Methods and results: Using a mouse model of psoriasis, we show that DGAT1-deficiency reduces energy-demanding neutrophil infiltration to the site of inflammation, but this inhibition is not caused by decreased glycolysis and reduced ATP production by neutrophils lacking DGAT1. Flow cytometry and immunohistochemistry analysis demonstrate that DGAT1 also does not influence lipid accumulation in lipid droplets during inflammation. Interestingly, as has been shown previously, a lack of DGAT1 leads to an increase in the concentration of retinoic acid, and here, using real-time PCR and publicly-available next-generation RNA sequencing datasets, we show the upregulation of retinoic acid-responsive genes in Dgat1KO neutrophils. Furthermore, supplementation of WT neutrophils with exogenous retinoic acid mimics DGAT1-deficiency in the inhibition of neutrophil chemotaxis in in vitro transwell assay. Conclusions: These results suggest that impaired skin infiltration by neutrophils in Dgat1KO mice is a result of the inhibitory action of an increased concentration of retinoic acid, rather than impaired lipid metabolism in DGAT1-deficient mice. [ABSTRACT FROM AUTHOR]

The article focuses on a 5-month-old infant with a rapidly expanding rash and fever, initially misdiagnosed as cellulitis, but later identified as a potential spider bite. Topics include her clinical presentation and progression of symptoms, the initial treatment with antibiotics, and the findings of the blackish skin lesion with expanding erythema and subsequent evaluation in the emergency department.

Pelvic organ prolapse (POP), a downward descent of the vagina and/or uterus through the vaginal canal, is a prevalent condition affecting up to 40% of women. Several risk factors of POP have been identified, including childbirth, connective tissue defects, and chronic intra-abdominal pressure; however, the underlying etiologies of POP development are not fully understood, leading to a high burden on patients and the healthcare systems. The uterosacral ligaments are key support structures of the uterus and upper vagina. Our previous work describes observed histopathological changes in uterosacral ligament (USL) tissue and demonstrates the presence of neutrophils in a subgroup of POP individuals. This presence of neutrophils prompted an examination for the presence of a broader spectrum of inflammatory cell types in the USL. Immunohistochemical staining was performed to identify neutrophils, lymphocytes, macrophages, and mast cells outside of the vasculature. All 4 inflammatory cell types were increased in the POP-HQ system-defined POP-Inflammatory (POP-I) phenotype USL tissue relative to the USL tissues of control or other POP-HQ phenotypes. Focal T-lymphocyte and macrophage co-accumulations were observed in the arterial walls from some patients of the POP-vascular (POP-V) phenotype suggesting previous arterial injury. In addition, 1 control and 2 POP-V subjects' USLs contained arterial wall foamy macrophages, evidence of atherosclerosis. These findings further support a complex etiology for POP and indicate that personalized approaches to preventing and treating the condition may be warranted. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to establish a population pharmacokinetic (PK) model to evaluate the dynamic relationship between the concentrations of total and unbound paclitaxel, and the exposure-response analysis of albumin-bound paclitaxel (nab-paclitaxel) after pegylated recombinant human granulocyte colony-stimulating factor (PEG-G-CSF) administration in patients with metastatic breast cancer. Methods: A total of 653 concentrations corresponding to total paclitaxel and 334 concentrations corresponding to unbound paclitaxel were analyzed in 24 subjects who randomized received a single 260 mg/m2 dose of two nab-paclitaxel formulations with a 21–35-day washout period. PEG-G-CSF was administered to all the patients in each cycle to prevent neutropenia. The exposure-response relationships were evaluated using the exposure to total, albumin-coated, and unbound paclitaxel, as well as the reduction in neutrophil count. The exposure data were analyzed using nonlinear mixed-effect modeling. A linear regression model was used to test the statistical significance of the correlation between percentage of reduction in neutrophil count and exposure. Results: The PK characteristics of total paclitaxel were described using a three-compartment model with first-order elimination, and a mechanism-based model incorporating linear release of nab-paclitaxel and the saturated binding of unbound paclitaxel to plasma components was established. The release ratio of paclitaxel from nab-paclitaxel was estimated to be 4.60% and the maximum unbound fraction (2.76%) was reached at the end of the infusion. The study found that a longer duration of total paclitaxel concentration > 0.19 µmol/L was significantly correlated with a reduction in neutrophil count (r2 = 0.23, P = 0.00062). Specifically, a duration of > 8.6 h was a predictor of a decreased neutrophil count. Conclusion: The decrease in neutrophils induced by nab-paclitaxel was significantly correlated with the duration above a total paclitaxel concentration of 0.19 µmol/L despite the use of PEG-G-CSF. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate the predictive role of inflammatory biomarkers in the development of contrast-induced acute kidney injury (CI-AKI) in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). The inflammatory biomarkers assessed were: platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), derived neutrophil-to-lymphocyte ratio (dNLR), neutrophil-to-lymphocyte*platelet ratio (NLPR), systemic inflammatory index (SII), and systemic inflammation response index (SIRI). Overall, 950 patients undergoing PCI were enrolled. The frequency of CI-AKI was 15.2% (n = 144). The levels of NLR, MLR, NLPR, SII, and SIRI were higher in the CI-AKI group than in the Non–CI-AKI group (P <.05). The addition of NLR ≥2.96, dNLR ≥2.08, NLPR ≥.012, SII ≥558.04, and SIRI ≥1.13 to the Mehran score model significantly increased the area under the curve (P <.05). Multivariable logistic regression analyses indicated that inflammatory biomarkers were significantly associated with CI-AKI, including NLR ≥2.96 (OR = 1.588, P =.017), dNLR ≥2.08 (OR = 1.686, P =.007), SII ≥558.04 (OR = 1.521, P =.030), and SIRI ≥1.13 (OR = 1.601, P =.017). Therefore, inflammation is associated with the development of CI-AKI, and preoperative hematological inflammatory markers could predict the risk of CI-AKI in ACS patients undergoing PCI. [ABSTRACT FROM AUTHOR]

Inflammation contributes to the development of thrombosis, but the mechanistic basis for this association remains poorly understood. Innate immune responses and coagulation pathways are activated in parallel following infection or injury, and represent an important host defense mechanism to limit pathogen spread in the bloodstream. However, dysregulated proinflammatory activity is implicated in the progression of venous thromboembolism and arterial thrombosis. In this review, we focus on the role of myeloid cells in propagating thromboinflammation in acute inflammatory conditions, such as sepsis and coronavirus disease 2019 (COVID-19), and chronic inflammatory conditions, such as obesity, atherosclerosis, and inflammatory bowel disease. Myeloid cells are considered key drivers of thromboinflammation via upregulated tissue factor activity, formation of neutrophil extracellular traps (NETs), contact pathway activation, and aberrant coagulation factor–mediated protease-activated receptor (PAR) signaling. We discuss how strategies to target the intersection between myeloid cell–mediated inflammation and activation of blood coagulation represent an exciting new approach to combat immunothrombosis. Specifically, repurposed anti-inflammatory drugs, immunometabolic regulators, and NETosis inhibitors present opportunities that have the potential to dampen immunothrombotic activity without interfering with hemostasis. Such therapies could have far-reaching benefits for patient care across many thromboinflammatory conditions. [ABSTRACT FROM AUTHOR]

A substantial increase in colorectal cancer (CRC)–associated fatalities can be attributed to tumor recurrence and multidrug resistance. Traditional treatment options, including radio- and chemotherapy, also exhibit adverse side effects. Ancient treatment strategies that include phytochemicals like resveratrol are now widely encouraged as an alternative therapeutic option. Resveratrol is the natural polyphenolic stilbene in vegetables and fruits like grapes and apples. It inhibits CRC progression via targeting dysregulated cancer-promoting pathways, including PI3K/Akt/Kras, targeting transcription factors like NF-κB and STAT3, and an immunosuppressive tumor microenvironment. In addition, combination therapies for cancer include resveratrol as an adjuvant to decrease multidrug resistance that develops in CRC cells. The current review discusses the biology of resveratrol and explores different mechanisms of action of resveratrol in inhibiting CRC progression. Further, the detrimental role of resveratrol on the immunosuppressive tumor microenvironment of CRC has been discussed. This review illustrates clinical trials on resveratrol in different cancers, including resveratrol analogs, and their efficiency in promoting CRC inhibition. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) and placental neutrophil reverse transmigration (r-TM) are implicated in the pathogenesis of pre-eclampsia (PE). However, the role of the comorbidity of PE and human immunodeficiency virus (HIV) infection in placental neutrophil r-TM and serum NETs remains unknown. Human placental tissue (n = 160) and serum (n = 80) samples were obtained post-ethical approval and divided by pregnancy type and HIV status and across the study population. Immunohistochemistry and morphometry were performed to localize and quantify junctional adhesion molecule-C (JAM-C) expression as an inverse marker of neutrophil r-TM within placental villi. An enzyme-linked immunosorbent assay (ELISA) was performed to quantify the concentration of citrullinated histone H3 (cit-H3) as a marker of NETs. GraphPad Prism (version 8.0.2) was used to compare the results, and a p value of p < 0.05 was considered statistically significant. The localization of JAM-C was observed on the syncytiotrophoblasts (STBs) and endothelial cells of placental villi. The immunoexpression of JAM-C was elevated in PE vs. normotensive (N) placentae. In the exchange villi, JAM-C immunoexpression was higher in the N+ve vs. N-ve group. However, in PE comorbid HIV infection, JAM-C expression was lower in the PE+ve vs. PE-ve group. Citrullinated histone-H3 concentration was lower in the N+ve vs. N-ve group but elevated in early-onset PE (EOPE)+ve vs. late-onset PE (LOPE)+ve group. These results indicate that PE and HIV-infected placentae individually express elevated JAM-C, manifesting in less neutrophil r-TM. However, in exchange villi of PE comorbid with HIV infection reduced JAM-C enhances neutrophil r-TM, thus supporting the synergistic effect of PE comorbid with HIV. [ABSTRACT FROM AUTHOR]

A prompt diagnosis of urinary tract infection (UTI) is necessary to minimize its symptoms and limit sequelae. The current UTI screening by urine test strip analysis and microscopic examination has suboptimal diagnostic accuracy. A definitive diagnosis of UTI by urine culture takes two to three days for the results. These limitations necessitate a need for better biomarkers for the diagnosis and subsequent management of UTI in children. Here, we review the value of currently available UTI biomarkers and highlight the potential of emerging biomarkers that can facilitate a more rapid and accurate UTI diagnosis. Of the newer UTI biomarkers, the most promising are blood procalcitonin (PCT) and urinary neutrophil gelatinase-associated lipocalin (NGAL). PCT can provide diagnostic benefits and should be considered in patients who have a blood test for other reasons. NGAL, which is on the threshold of clinical care, needs more research to address its scope and utilization, including point-of-care application. Employment of these and other biomarkers may ultimately improve UTI diagnosis, guide UTI therapy, reduce antibiotic use, and mitigate UTI complications. [ABSTRACT FROM AUTHOR]

To unravel the heterogeneity and function of microenvironmental neutrophils during intervertebral disc degeneration (IDD). Single-cell RNA sequencing (scRNA-seq) was utilized to dissect the cellular landscape of neutrophils in intervertebral disc (IVD) tissues and their crosstalk with nucleus pulposus cells (NPCs). The expression levels of macrophage migration inhibitory factor (MIF) and ACKR3 in IVD tissues were detected. The MIF/ACKR3 axis was identified and its effects on IDD were investigated in vitro and in vivo. We sequenced here 71520 single cells from 5 control and 9 degenerated IVD samples using scRNA-seq. We identified a unique cluster of neutrophils abundant in degenerated IVD tissues that highly expressed MIF and was functionally enriched in extracellular matrix organization (ECMO). Cell-to-cell communication analyses showed that this ECMO-neutrophil subpopulation was closely interacted with an effector NPCs subtype, which displayed high expression of ACKR3. Further analyses revealed that MIF was positively correlated with ACKR3 and functioned via directly binding to ACKR3 on effector NPCs. MIF inhibition attenuated degenerative changes of NPCs and extracellular matrix, which could be partially reversed by ACKR3 overexpression. Clinically, a significant correlation of high MIF/ACKR3 expression with advanced IDD grade was observed. Furthermore, we also found a positive association between MIF+ ECMO-neutrophil counts and ACKR3+ effector NPCs density as well as higher expression of the MIF/ACKR3 signaling in areas where these two cell types were neighbors. These data suggest that ECMO-neutrophil promotes IDD progression by their communication with NPCs via the MIF/ACKR3 axis, which may shed light on therapeutic strategies. [ABSTRACT FROM AUTHOR]

Objective: Inflamatory processes play an important role in the etiology of psychiatric disorders. The aim of this study was to evaluate inflammatory complete blood count (CBC) parameters in substance use disorder (SUD). Methods: The study included 90 patients with SUD and 90 healthy controls (HC). The Student t-test was used to compare CBC parameters between the groups. The Kruskal – Wallis test was used to compare the data on the basis of substance types. Results: Leukocyte, lymphocyte, and monocyte levels were higher in SUD group than HC. Leukocyte and neutrophil levels of patients with opioid use disorder (OUD) were higher than HC. Platelet levels and neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and systemic immune inflammation index values were higher in patients with OUD than in patients with non-opioid substance use disorder (nOUD). Moreover, lymphocyte and monocyte levels were higher in patients with nOUD than in HC. Monocyte levels were increased in patients with cannabinoid use disorder compared to HC. Conclusion: The results suggested that inflammatory CBC parameters play an important role in the etiology of SUD according to type of substance. However, there is not enough data for supporting the clinical use of these parameters.. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) is a bacterial pathogen that exclusively colonizes the human gastric mucosa and can cause persistent infection. In this process, H. pylori employs various strategies to avoid recognition by the human immune system. These range from passive defense strategies (e.g., altered LPS or flagellin structures) that prevent recognition by pattern recognition receptors (PRRs) to more active approaches, such as inhibition of IL-2 secretion and proliferation of T cells via VacA. Despite the growing evidence that H. pylori actively manipulates the human immune system for its own benefit, the direct interaction of H. pylori with immune cells in situ is poorly studied. Here, we present a novel intravital imaging model of the murine stomach gastric mucosa and show for the first time the in situ recruitment of neutrophils during infection and a direct H. pylori-macrophage interaction. For this purpose, we applied multiphoton intravital microscopy adapted with live drift correction software (VivoFollow) on LysM-eGFP and CX3CR1-eGFP reporter mice strains in which specific subsets of leukocytes are fluorescently labeled. Multiphoton microscopy is proving to be an excellent tool for characterizing interactions between immune cells and pathogens in vivo. Author summary: This study focuses on the dynamics of H. pylori infection and its interaction with myeloid immune cells, using an innovative in vivo multiphoton imaging approach to observe real-time events in the infected gastric mucosa. In contrast to conventional methods, this novel technique allows us to dynamically investigate the host immune response. Our results show the spatio-temporal distribution of neutrophils and the internalization of H. pylori by macrophages over time. Using intravital multiphoton microscopy enhanced with live drift correction software and utilizing LysM-eGFP and CX3CR1-eGFP reporter mice, we established an intravital imaging model for the gastric mucosa of mice. Notably, we observe a significant increase in neutrophil infiltration into the gastric submucosa during early infection, indicating a rapid response of the innate immune system within the gastric crypts. However, in the chronic infection phase, a contrasting decrease in neutrophil influx is seen, indicating an adaptive innate immune response with fewer neutrophils infiltrating the tissue at an increased rate. Importantly, our data suggest that H. pylori is dependent on macrophages to proliferate and colonize the gastric mucosa during chronic infection. [ABSTRACT FROM AUTHOR]

Millions of people worldwide are affected by neurodegenerative diseases and cognitive impairment, which includes dementia, while there are only symptomatic treatments available for this syndrome at present. However, several important prospective drug targets have been identified in recent years that can potentially arrest or even reverse the progression of neurodegenerative diseases. Their natural or synthetic ligands are currently in the experimental stage of drug development. In vitro and preclinical (e.g. using animal models) studies confirm their therapeutic potential, but clinical trials often fail or produce conflicting results. Here, we first review the complexity and typology of dementia, followed by the discussion of currently available treatments, and, finally, some novel molecular and cellular approaches to this problem. [ABSTRACT FROM AUTHOR]

Background: This study explores the potential role of Thioredoxin-interacting protein (TXNIP) silencing in endothelial colony-forming cells (ECFCs) within the scope of age-related comorbidities and impaired vascular repair. We aim to elucidate the effects of TXNIP silencing on vasculogenic properties, paracrine secretion, and neutrophil recruitment under conditions of metabolic stress. Methods: ECFCs, isolated from human blood cord, were transfected with TXNIP siRNA and exposed to a high glucose and β-hydroxybutyrate (BHB) medium to simulate metabolic stress. We evaluated the effects of TXNIP silencing on ECFCs' functional and secretory responses under these conditions. Assessments included analyses of gene and protein expression profiles, vasculogenic properties, cytokine secretion and neutrophil recruitment both in vitro and in vivo. The in vivo effects were examined using a murine model of hindlimb ischemia to observe the physiological relevance of TXNIP modulation under metabolic disorders. Results: TXNIP silencing did not mitigate the adverse effects on cell recruitment, vasculogenic properties, or senescence induced by metabolic stress in ECFCs. However, it significantly reduced IL-8 secretion and consequent neutrophil recruitment under these conditions. In a mouse model of hindlimb ischemia, endothelial deletion of TXNIP reduced MIP-2 secretion and prevented increased neutrophil recruitment induced by age-related comorbidities. Conclusions: Our findings suggest that targeting TXNIP in ECFCs may alleviate ischemic complications exacerbated by metabolic stress, offering potential clinical benefits for patients suffering from age-related comorbidities. [ABSTRACT FROM AUTHOR]

Background. Systemic sclerosis (SSc) is an autoimmune disease characterized by inflammation, progressive vasculopathy, and fibrosis of skin and internal organs. The aim of the study was to evaluate extended inflammatory parameters (EIP) in patients with SSc in comparison to the control group of healthy subjects. Methods. A total of 28 patients with SSc and 29 healthy controls (HCs) were included in the study. The following EIP parameters were analyzed: neutrophil reactive intensity (NEUT‐RI), neutrophil granularity intensity (NEUT‐GI), antibody‐synthesizing lymphocytes (AS‐LYMP), and reactive lymphocytes (RE‐LYMP). Results. Patients with SSc showed significantly higher values of parameters determining neutrophil reactivity and neutrophil granularity when compared to HCs (respectively, 49.16 FI vs. 44.33 FI, p < 0.001, and 152.01 SI vs. 147.51 SI, p < 0.001). Moreover, patients with SSc had higher absolute numbers of RE‐LYMP than HCs (0.69 × 103/µl vs. 0.04 × 103/µl, p < 0.001). Importantly, significant correlations between the RE‐LYMP and either IL‐6 (R = 0.447, p < 0.001) or ESR (R = 0.532, p < 0.001) were found among patients with SSc. Conclusions. Changes in NEUT‐RI, NEUT‐GI, and RE‐LYMP levels positively correlate with inflammation in SSc and, thus, could potentially be used as an additional reliable inflammatory biomarker to assess inflammation in this disease. [ABSTRACT FROM AUTHOR]

Introduction: Crohn's disease (CD) is characterized by chronic inflammation and intestinal fibrosis leading to lifelong complications. However, the disease pathogenesis remains elusive, and the therapeutic options are limited. Here, we investigated the interaction between neutrophils and intestinal fibroblasts in the development of CD immunofibrosis, a disease mechanism predisposing to inflammatory and fibrotic complications. Methods: Peripheral neutrophils, enriched neutrophil extracellular traps (eNETs), serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies from CD, ulcerative colitis (UC) patients, and healthy individuals (HI), were studied. Transcriptome analysis of neutrophils, multi-cytokine profiling and cell-based functional assays at mRNA/protein level were performed. Results: Compared to UC, PIFs from CD patients, independently to the presence of strictures, displayed a distinct pro-fibrotic phenotype characterized by negative Krüppellike Factor-2 (KLF2) and increased cellular communication network factor-2 (CCN2) expression leading to collagen production. In both UC and CD, PIFs-derived IL-8 acted as a culprit chemoattractant for neutrophils in the intestine, where CD neutrophils were accumulated close to fibrotic lesions. Functionally, only CD neutrophils via eNETs induced a CD-like phenotype in HI PIFs, suggesting their fibrotic plasticity. High IFNa in serum and IFα-responsive signature in peripheral neutrophils were observed in CD, distinguishing it from UC. Moreover, CD serum stimulated the release of fibrogenic eNETs from neutrophils in an IFNa-dependent manner, suggesting the priming role of IFNa in circulating neutrophils. Inhibition of eNETs or JAK signaling in neutrophils or PIFs prevented the neutrophil-mediated fibrotic effect on PIFs. Furthermore, both serum IFNa levels and mRNA levels of key IFN signaling components in neutrophils were wellcorrelated with CD severity. Conclusions: This study reveals the important role of the IFNa/neutrophil/fibroblast axis in CD immunofibrosis, suggesting candidate biomarkers and putative therapeutic targets. [ABSTRACT FROM AUTHOR]

Extracorporeal membrane oxygenation (ECMO), as an extracorporeal life support technique, can save the lives of reversible critically ill patients when conventional treatments fail. However, ECMO-related acute organ injury is a common complication that increases the risk of death in critically ill patients, including acute kidney injury, acute brain injury, acute lung injury, and so on. In ECMO supported patients, an increasing number of studies have shown that activation of the inflammatory response plays an important role in the development of acute organ injury. Cross-cascade activation of the complement system, the contact system, and the coagulation system, as well as the mechanical forces of the circuitry are very important pathophysiological mechanisms, likely leading to neutrophil activation and the production of neutrophil extracellular traps (NETs). NETs may have the potential to cause organ damage, generating interest in their study as potential therapeutic targets for ECMO-related acute organ injury. Therefore, this article comprehensively summarized the mechanism of neutrophils activation and NETs formation following ECMO treatment and their actions on acute organ injury. [ABSTRACT FROM AUTHOR]

Neutrophils utilize a variety of metabolic sources to support their crucial functions as the first responders in innate immunity. Here, through in vivo and ex vivo isotopic tracing, we examined the contributions of different nutrients to neutrophil metabolism under specific conditions. Human peripheral blood neutrophils, in contrast to a neutrophil-like cell line, rely on glycogen storage as a major metabolic source under resting state but rapidly switch to primarily using extracellular glucose upon activation with various stimuli. This shift is driven by a substantial increase in glucose uptake, enabled by rapidly increased GLUT1 on cell membrane, that dominates the simultaneous increase in gross glycogen cycling capacity. Shifts in nutrient utilization impact neutrophil functions in a function-specific manner: oxidative burst depends on glucose utilization, whereas NETosis and phagocytosis can be flexibly supported by either glucose or glycogen, and neutrophil migration and fungal control are enhanced by the shift from glycogen utilization to glucose utilization. This work provides a quantitative and dynamic understanding of fundamental features in neutrophil metabolism and elucidates how metabolic remodeling shapes neutrophil functions, which has broad health relevance. [ABSTRACT FROM AUTHOR]

AbstractIntroductionMethodsResultsConclusionPaediatric brain tumours (PBT) are the most common cause of death among all childhood cancers. The neutrophil to lymphocyte ratio (NLR) has been shown to prognosticate many adult cancers. There is a paucity of literature on the NLR in PBTs. This study aims to study the link between PBTs and the NLR by comparing the preoperative serum NLR in children under 16 with brain tumours with their outcome in terms of grade of brain tumour and overall survival.This is a retrospective case control study. The NLRs were compared between patients with benign or malignant PBTs and patients who were alive or dead. Receiver-operating characteristic (ROC) curve analyses were performed and Youden indexes were calculated to evaluate the predictive potential of the NLR. A cut-off point of NLR > 4 was selected for the calculation of odds ratios.A total of 515 patients were included in this study. 53.8% were male. 66.2% had benign PBTs. 81.0% were alive at the time of the study. Patients with malignant PBTs had a higher NLR compared to patients with benign PBTs (p = 0.0066**). There was no difference in the NLR between patients who were dead compared to those who were alive (p = 0.1682 ns). The NLR had a Youden’s index of 0.1567 to predict malignant PBTs and 0.1285 to predict survival.A high NLR was associated with an increased odds of having a malignant PBT but a reliable cut-off point was not identified and the underlying mechanisms for this remain unknown. The NLR is a poor diagnostic biomarker due to its poor overall sensitivity and specificity. More research is required to further study the role of immunity in PBTs. [ABSTRACT FROM AUTHOR]

Background: Neutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries. Results: Using this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization. Conclusions: In summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Neutrophils (polymorphonuclear leukocytes, PMNs) are the most abundant subtype of white blood cells and are among the main actors in the inflammatory response. Psoriatic arthritis (PsA) is a chronic inflammatory disease affecting both the axial and peripheral joints. Typically associated with psoriasis, PsA can also affect multiple systems and organs, including the nails and entheses. Despite the involvement of PMNs in PsA, their specific role in the disease remains poorly understood. This study aimed to characterize the biological functions of PMNs and neutrophil-related mediators in PsA patients. Materials and methods: 31 PsA patients and 22 healthy controls (HCs) were prospectively recruited. PMNs were isolated from peripheral blood and subjected to in vitro stimulation with lipopolysaccharide (LPS), N-Formylmethionyl-leucylphenylalanine (fMLP), tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate (PMA), or control medium. Highly purified peripheral blood PMNs (>99%) were evaluated for activation status, reactive oxygen species (ROS) production, phagocytic activity, granular enzyme and neutrophil extracellular traps (NETs) release. Serum levels of matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO), TNF, interleukin 23 (IL-23), and interleukin 17 (IL-17) were measured by ELISA. Serum Citrullinated histone H3 (CitH3) was measured as a NET biomarker. Results: Activated PMNs from PsA patients displayed reduced activation, decreased ROS production, and impaired phagocytic activity upon stimulation with TNF, compared to HCs. PMNs from PsA patients also displayed reduced granular enzyme (MPO) and NET release. Serum analyses revealed elevated levels of MMP-9, MPO, TNF, IL-23, IL-17, and CitH3 in PsA patients compared to HCs. Serum CitH3 levels positively correlated with MPO and TNF concentrations, and IL-17 concentrations were positively correlated with IL-23 levels in PsA patients. These findings indicate that PMNs from PsA patients show reduced in vitro activation and function, and an increased presence of neutrophil-derivedmediators (MMP-9, MPO, TNF, IL-23, IL-17, and CitH3) in their serum. Conclusions: Taken together, our findings suggest that PMNs from PsA patients exhibit an "exhausted" phenotype, highlighting their plasticity and multifaceted roles in PsA pathophysiology. [ABSTRACT FROM AUTHOR]

Objectives: To assess the impact of topical papaverine administration in complete and incomplete colonic anastomosis, by examining bursting pressure, hydroxyproline concentration, collagen content, inflammation levels, inflammatory cell infiltration, neoangiogenesis, and necrosis grades. Methods: We performed an experimental study on rats, in which they were divided into the following 4 groups of 16 subjects each. Group 1 [complete anastomosis (CA) without papaverine (CA -P) group], Group 2 [CA with papaverine (CA +P) group], Group 3 [incomplete anastomosis (ICA) without papaverine (ICA -P) group], and Group 4 [ICA with papaverine (ICA +P) group]. Results: The lymphocyte infiltration score of the ICA +P3 (day 3) group was significantly higher compared to the ICA -P3 group (p = 0.018). The median Ehrlich–Hunt score (p = 0.012), inflammation score (p = 0.026), and neutrophil infiltration score (p = 0.041) of the CA +P7 (day 7) group were significantly lower than the corresponding data of the CA -P7 group. Additionally, the necrosis score of the ICA +P7 group was significantly lower than that of the ICA -P7 group (p = 0.014). Conclusions: Data from the current study reveal that, although topical papaverine seems to suppress inflammation in anastomosis tissue and reduce necrosis at 7 days, definite conclusions regarding its impact on anastomotic leak cannot be drawn without further studies investigating anastomotic wound healing and anastomotic leak, preferably with both shorter- and longer-term evaluations. [ABSTRACT FROM AUTHOR]

Simple Summary: Equine laminitis is a serious condition causing severe pain and lameness in horses, often leading to euthanasia. This study developed a lab model to better understand laminitis using keratinocytes which were exposed to conditions simulating the disease. This research showed that adding muscle-derived stem cells helped protect and restore the keratinocytes' metabolism. These results highlight the potential of stem cell therapy in treating laminitis, offering new hope for managing this debilitating disease in horses. Laminitis in horses is a crippling condition marked by the deterioration of the dermal–epidermal interface, leading to intense lameness and discomfort, often necessitating euthanasia. This study aimed to establish an in vitro model of laminitis using a continuous keratinocyte cell line exposed to anoxia–reoxygenation and an activated neutrophil supernatant. A significant decrease in the keratinocytes' metabolism was noted during the reoxygenation period, indicative of cellular stress. Adding muscle-derived mesenchymal stem/stromal cells during the reoxygenation demonstrated a protective effect, restoring the keratinocytes' metabolic activity. Moreover, the incubation of the keratinocytes with either an activated neutrophil supernatant or myeloperoxidase alone induced increased keratinocyte myeloperoxidase activity, which was modulated by stem cells. These findings underscore the potential of muscle-derived mesenchymal stem/stromal cells in mitigating inflammation and restoring keratinocyte metabolism, offering insights for future cell therapy research in laminitis treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: People living with cancer are at a higher risk of developing a venous thromboembolism (VTE). A VTE may interrupt anticancer therapy, which increases the risk of mortality. Cancer treatment guidelines recommend the use of preventive anticoagulants only in those at high risk of a VTE, identified by a valid risk assessment tool, such as the Khorana score. However, the Khorana score at its original cut-off value of 3 points has a low sensitivity and discriminatory capability in lung cancer patients, and the updated Khorana score with a 2-point cut-off value lacks validation in this population. Other risk scores, such as the PROTECHT, CONKO, and COMPASS-CAT scores, have not been validated in large lung cancer cohorts. The aim of this study was to evaluate these four existing risk scores in this patient population. We validated the Khorana score with a cut-off value of 2 points and the CONKO score with a cut-off value of 2 points. Background: The purpose of this study was to evaluate the discriminatory capability of the Khorana, PROTECHT, CONKO, and COMPASS-CAT scores in ambulatory patients with lung cancer. Methods: This retrospective cohort study included 591 patients with newly diagnosed lung cancer. A symptomatic or incidental VTE occurred in 108 patients. Results: The Khorana score at a 2-point threshold had a discriminatory capability with an odds ratio (OR) of 1.80 and an AUC of 0.57 for 6 months, and an OR of 1.51 and an AUC of 0.55 for 12 months. The CONKO score at a 2-point threshold had a stronger discriminatory capability for both 6 months and 12 months with ORs of 3.00 and 2.13, and AUCs of 0.63 and 0.59, respectively. Additionally, higher white blood cell counts, higher neutrophil counts, hypoalbuminaemia, and not undergoing lung surgery were related to VTE occurrence (p < 0.05). Conclusions: The Khorana score with the 2-point threshold was validated in ambulatory patients with lung cancer, with the results indicating a decline in its discriminatory capability over time (at 12 months vs. 6 months from diagnosis). The CONKO score at the original 2-point threshold showed a stronger discriminatory capability but further validation with a larger sample size is recommended. The identified predictors should be further investigated in future research. [ABSTRACT FROM AUTHOR]

Simple Summary: Neutrophil granulocytes in the vicinity of malignant tumors are referred to as tumor-associated neutrophils (TANs). Knowledge about the role of TANs in the disease progression of early hormone-receptor-positive breast cancer is limited but essential to the development of new immunotherapies and biomarkers. In this work, we counted immunohistochemically stained TANs in sections of 144 early-stage breast cancer tumors and correlated these results with disease-free survival. Our results indicate that not only intratumoral TANs but also those in adjacent normal tissue and in sentinel lymph nodes were associated with shorter disease-free survival. Combined analysis with other immune cells from previous studies revealed that intratumoral TANs were only associated with prognosis in tumors that did not express an unfavorable macrophage polarization profile, providing a clinical example of the known interactions between different types of immune cells within the tumor microenvironment. This indicates that future research in the field should evaluate the two types of immune cells together. Background: Tumor-associated neutrophils (TANs) are important modulators of the tumor microenvironment with opposing functions that can promote and inhibit tumor progression. The prognostic role of TANs in early luminal breast cancer is unclear. Methods: A total of 144 patients were treated for early-stage hormone-receptor-positive breast cancer as part of an Accelerated Partial Breast Irradiation (APBI) phase II trial. Resection samples from multiple locations were processed into tissue microarrays and sections thereof immunohistochemically stained for CD66b+ neutrophils. CD66b+ neutrophil density was measured separately in the stromal and intraepithelial compartment. Results: High stromal and intraepithelial CD66b+ TAN density was a negative prognostic factor in central tumor samples. In addition, neutrophil density in adjacent normal breast tissue and lymph node samples also correlated with reduced disease-free survival. TAN density correlated with CD163+ M2-like tumor-associated macrophage (TAM) density, which we analyzed in a previous study. TANs were a negative prognostic factor in tumors with an elevated M1/M2 TAM ratio, while this impact on patient outcome was lost in tumors with a low M1/M2 ratio. A combined multivariate analysis of TAM and TAN density revealed that only TAM polarization status was an independent prognostic factor. Conclusions: CD66b+ neutrophils were a negative prognostic factor in early-stage luminal breast cancer in single-marker analysis. Combined analysis with TAMs could be necessary to correctly evaluate their prognostic impact in future studies. TAN recruitment might act as a compensatory mechanism of immunoevasion and disease progression in tumors that are unable to sufficiently attract and polarize TAMs. [ABSTRACT FROM AUTHOR]