BACKGROUND: Human neutrophil lipocalin (HNL) has been used extensively to differentiate acute bacterial infection from febrile diseases as a biomarker to reflect the activation of the neutrophil. The serum HNL levels in the adult-onset Still's disease (AOSD) patients with and without infection, as well as the healthy controls (HCs), were analyzed statistically in this study to evaluate the value of HNL for the diagnosis of AOSD. METHODS: A total of 129 AOSD patients were enrolled, from whom blood samples were drawn and the AOSD diagnosis was confirmed through the review of the medical records, where the systemic score, demographic characteristics, clinical manifestations, and laboratory parameters were also collected for the patients; in addition, a total of 40 HCs were recruited among the blood donors from the healthcare center with the relevant information collected. The HNL test was done for the blood samples with the enzyme-linked immunosorbent assay and the analyses were done for the correlations of HNL with clinical manifestations and diagnostic effectiveness. RESULTS: The serum HNL increased significantly in the patients with only AOSD as compared with that in the HCs (139.76 ± 8.99 ng/mL vs . 55.92 ± 6.12 ng/mL; P  < 0.001). The serum HNL level was correlated with the white blood cell (WBC) count ( r  = 0.335, P  < 0.001), neutrophil count ( r  = 0.334, P  < 0.001), erythrocyte sedimentation rate ( r  = 0.241, P  = 0.022), C-reactive protein ( r  = 0.442, P  < 0.0001), and systemic score ( r  = 0.343, P  < 0.0001) in the AOSD patients significantly. Patients with fever, leukocytosis ≥15,000/mm 3 , and myalgia in the HNL-positive group were observed relatively more than those in the HNL-negative group ( P  = 0.009, P  = 0.023, and P  = 0.007, respectively). HNL was a more sensitive indicator than ferritin and C-reactive protein (CRP) to differentiate the AOSD patients with bacterial infection from AOSD-only patients, and the Youden index was 0.6 for HNL and 0.29 for CRP. CONCLUSION Serum HNL can be used as a biomarker for the diagnosis of the AOSD, and HNL is also observed to be associated with the disease activity.
Adult ; Humans ; Still's Disease, Adult-Onset/diagnosis* ; C-Reactive Protein/metabolism* ; Neutrophils/metabolism* ; Clinical Relevance ; Biomarkers ; Bacterial Infections

Reduced chemotactic migration of polymorphonuclear neutrophil (PMN) in sepsis patients leads to decreased bacterial clearance and accelerates the progression of sepsis disease. Quantification of PMN chemotaxis in sepsis patients can help characterize the immune health of sepsis patients. Microfluidic microarrays have been widely used for cell chemotaxis analysis because of the advantages of low reagent consumption, near-physiological environment, and visualization of the migration process. Currently, the study of PMN chemotaxis using microfluidic chips is mainly limited by the cumbersome cell separation operation and low throughput of microfluidic chips. In this paper, we first designed an inertial cell sorting chip to achieve label-free separation of the two major cell types by using the basic principle that leukocytes (mainly granulocytes, lymphocytes and monocytes) and erythrocytes move to different positions of the spiral microchannel when they move in the spiral microchannel under different strength of inertial force and Dean's resistance. Subsequently, in this paper, we designed a multi-channel cell migration chip and constructed a microfluidic PMN inertial label-free sorting and chemotaxis analysis platform. The inertial cell sorting chip separates leukocyte populations and then injects them into the multi-channel cell migration chip, which can complete the chemotaxis test of PMN to chemotactic peptide (fMLP) within 15 min. The remaining cells, such as monocytes with slow motility and lymphocytes that require pre-activation with proliferative culture, do not undergo significant chemotactic migration. The test results of sepsis patients ( n=6) and healthy volunteers ( n=3) recruited in this study showed that the chemotaxis index (CI) and migration velocity ( v) of PMN from sepsis patients were significantly weaker than those from healthy volunteers. In conclusion, the microfluidic PMN inertial label-free sorting and chemotaxis analysis platform constructed in this paper can be used as a new tool for cell label-free sorting and migration studies.
Humans ; Chemotaxis ; Neutrophils/metabolism* ; Microfluidics ; Cell Movement ; Sepsis/metabolism*

OBJECTIVE: To investigate the predictive value of pancreatitis activity scoring system (PASS) combined with Neutrophil to lymphocyte ratio (NLR) and C-reactive protein (CRP) for infected pancreatic necrosis (IPN) in patients with severe acute pancreatitis (SAP). METHODS: Clinical data of SAP patients admitted to the First Affiliated Hospital of Zhengzhou University from January 2020 to January 2023 were retrospectively collected, including basic information, vital signs at admission, first laboratory indexes within 48 hours of admission. The PASS scores at admission and 24, 48 and 72 hours after admission were calculated. According to the diagnostic criteria of IPN, the patients were divided into the non-IPN group and the IPN group, and the independent risk factors of SAP complicating IPN were determined by using univariate analysis and multifactorial Logistic regression. The receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of NLR, CRP, and PASS score, alone and in combination for IPN in patients with SAP. RESULTS: A total of 149 SAP patients were enrolled, including 102 in the non-IPN group and 47 in the IPN group. The differences in PASS score at each time point, NLR, CRP, procalcitonin (PCT), blood urea nitrogen, blood chloride, and days of hospitalization between the two groups were statistically significant. Multifactorial Logistic regression analysis showed that 72 hours admission PASS score [odds ratio (OR) = 1.034, 95% confidence interval (95%CI) was 1.005-1.065, P = 0.022], NLR (OR = 1.284, 95%CI was 1.139-1.447, P = 0.000), and CRP (OR = 1.015, 95%CI was 1.006-1.023, P = 0.001) were independent risk factors for IPN in patients with SAP. ROC curve analysis showed that the area under the ROC curve (AUC) of the PASS score at 72 hours of admission, NLR, and CRP alone in predicting IPN in SAP patients were 0.828, 0.771, and 0.701, respectively. The AUC of NLR combined with CRP, PASS combined with NLR, and PASS combined with CRP were 0.818, 0.895, and 0.874, respectively. The combination of PASS score at 72 hours after admission, NLR, and CRP had a better predictive ability for IPN in patients with SAP (AUC = 0.922, 95%CI was 0.877-0.967), and the sensitivity was 72.3% when the cut-off value was 0.539. CONCLUSIONS The predictive value of the PASS score at 72 hours after admission, NLR and CRP in combination for IPN in SAP patients is better than that of the combination of each two and individual detection and has better test efficacy.
Humans ; Pancreatitis, Acute Necrotizing/diagnosis* ; C-Reactive Protein/metabolism* ; Acute Disease ; Neutrophils/metabolism* ; Retrospective Studies ; ROC Curve ; Lymphocytes ; Prognosis

Objective To explore the role of autophagy, apoptosis of neutrophils and neutrophils extracellular traps (NET) formation in systemic lupus erythematosus (SLE). Methods Thirty-six patients with SLE were recruited as research subjects, and 32 healthy controls matched accordingly were enrolled as control subjects. The expression levels of microtubule associated protein 1 light chain 3B (LC3B), autophagy-related gene5(ATG5), P62, B-cell lymphoma 2(Bcl2), Bcl2-related X protein (BAX) in neutrophils were detected by Western blot analysis. Flow cytometry was employed to analyze the expression of LC3B on neutrophils. The expression level of myeloperoxidase(MPO) in plasma was estimated by ELISA. Furthermore, neutrophils were cultured in vitro and stimulated by 100 nmol/L rapamycin and 10 μg/mL lipopolysaccharide (LPS) for 6 hours, respectively. And then, the expression levels of LC3B, ATG5, P62, Bcl2 and BAX in neutrophils were detected by Western blot analysis. The level of MPO in culture supernatant was detected by ELISA. The change of fluorescence intensity of NET in culture supernatant was assayed by Sytox^TM Green staining combined with fluorescence spectrophotometry. Results Compared with healthy controls, the levels of autophagy and apoptosis of neutrophils and NET formation in SLE patients were increased. The level of apoptosis and NET formation was positively associated with neutrophil autophagy. The level of autophagy showed an increase but had no effect on apoptosis and NET formation for neutrophil stimulated by rapamycin. The levels of autophagy and NET formation also increased with no significant effect on apoptosis for neutrophil induced by LPS. Conclusion The autophagy, apoptosis and NET formation of neutrophils increase in SLE patients. The activation of autophagy and NET in neutrophils possibly result from the inflammatory internal environment in SLE patients.
Humans ; Neutrophils ; Extracellular Traps/metabolism* ; Lipopolysaccharides/pharmacology* ; bcl-2-Associated X Protein/metabolism* ; Sirolimus/pharmacology* ; Lupus Erythematosus, Systemic ; Autophagy

As an important part of tumor microenvironment, neutrophils are poorly understood due to their spatiotemporal heterogeneity in tumorigenesis. Here we defined, at single-cell resolution, CD44-CXCR2- neutrophils as tumor-specific neutrophils (tsNeus) in both mouse and human gastric cancer (GC). We uncovered a Hippo regulon in neutrophils with unique YAP signature genes (e.g., ICAM1, CD14, EGR1) distinct from those identified in epithelial and/or cancer cells. Importantly, knockout of YAP/TAZ in neutrophils impaired their differentiation into CD54+ tsNeus and reduced their antitumor activity, leading to accelerated GC progression. Moreover, the relative amounts of CD54+ tsNeus were found to be negatively associated with GC progression and positively associated with patient survival. Interestingly, GC patients receiving neoadjuvant chemotherapy had increased numbers of CD54+ tsNeus. Furthermore, pharmacologically enhancing YAP activity selectively activated neutrophils to suppress refractory GC, with no significant inflammation-related side effects. Thus, our work characterized tumor-specific neutrophils in GC and revealed an essential role of YAP/TAZ-CD54 axis in tsNeus, opening a new possibility to develop neutrophil-based antitumor therapeutics.
Humans ; Animals ; Mice ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Stomach Neoplasms/pathology* ; Neutrophils/pathology* ; Signal Transduction/genetics* ; YAP-Signaling Proteins ; Tumor Microenvironment ; Hyaluronan Receptors/genetics*

OBJECTIVE: To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). METHODS: Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. RESULTS: Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. CONCLUSIONS CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.
Animals ; Humans ; Extracellular Traps ; Neutrophils ; Interleukin-8/metabolism* ; Dermatophagoides farinae ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Epithelial Cells ; Interferon-gamma/metabolism* ; Tetradecanoylphorbol Acetate/pharmacology*

OBJECTIVE: To evaluate the value of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) for assessing disease activity in patients with rheumatoid arthritis (RA) treated with tofacitinib. METHODS: This retrospective study was conducted among 98 RA patients in active stage treated with tofacitinib in Third Xiangya Hospital and 100 healthy control subjects from the Health Management Center of the hospital from 2019 to 2021. We collected blood samples from all the participants for measurement of erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and other blood parameters 1 month before and 6 months after tofacitinib treatment. We further evaluated PLR and NLR before and after tofacitinib treatment in the RA patients, and analyzed their correlations with RA disease activity. RESULTS: PLR and NLR increased significantly in RA patients as compared with the healthy controls. In the RA patients, PLR and NLR were positively correlated with the levels of hs- CRP, ESR, IL- 6, Disease Activity Score of 28 joints-ESR (DAS28-ESR), anti-cyclic citrullinated peptide (CCP), and rheumatoid factor (RF) before and after tofacitinib treatment. Tofacitinib treatment for 6 months significantly decreased hs-CRP, ESR, IL-6, CCP, RF and DAS28-ESR levels in the RA patients. CONCLUSION NLR and PLR can be useful biomarkers for assessing disease activity in RA patients treated with tofacitinib.
Humans ; Neutrophils ; Retrospective Studies ; C-Reactive Protein/analysis* ; Interleukin-6/metabolism* ; Arthritis, Rheumatoid ; Lymphocytes

Neutrophils are predominant leukocytes in the circulation, which are essential for killing invading pathogens via the activation of effector responses and the production of reactive oxygen species (ROS), also named as "oxidative burst." When infected, activated neutrophils fight bacteria, fungi, and viruses through oxidative burst, phagocytosis, degranulation, and the production of neutrophil extracellular traps (NETs) in a neutrophil death process named as "NETosis" (Mutua and Gershwin, 2021). NETs, consisting of DNA fibers decorated with modified histones and numerous antimicrobial proteins from cytoplasmic granules and the nucleus, can either be beneficial or detrimental (Mutua and Gershwin, 2021). Several pathways can lead to this death process. In response to various stimuli, NETosis traps and clears pathogens, facilitating phagocytosis by other neutrophils and phagocytes. However, excessive NETosis often results in disease due to increasing the pro-inflammatory response and perpetuating the inflammatory condition (Hellebrekers et al., 2018; Hidalgo et al., 2019; Klopf et al., 2021). Accordingly, inhibiting aberrant NETosis may alleviate the severity of various autoimmune and inflammatory diseases.
DNA ; Extracellular Traps/metabolism* ; Neutrophils/metabolism* ; Reactive Oxygen Species/metabolism* ; Respiratory Burst

Humans ; Computational Biology ; Neutrophils/metabolism* ; Sepsis/metabolism* ; Signal Transduction/genetics* ; Immunosuppression Therapy ; Gene Expression Profiling

OBJECTIVE: To investigate the clinical value of neutrophil CD64 index in hematological malignancies with pulmonary infection. METHODS: The cohort study method was used to retrospectively analyze the clinical data of 125 patients with hematological malignancies and pulmonary infections who were treated in The Third Affiliated Hospital of Soochow University. All the patients were divided into four stages according to the diagnosis and treatment process: non-infected stage (T1), the symptoms of infection had appeared before using antibiotics (T2), one week after anti-infective treatment (T3), and after stopping antibiotics (T4). CD64 index, C-reactive protein (CRP), blood cell count, and immune cell level were compared before and after infection (T1 vs T2), the correlation between CD64 index and other indicators were explored, the change trends of the significantly different indicators in the course of the disease were observed, and the diagnostic efficacy of CD64 index and CRP were compared. The surviving patients were followed up for whether reinfection occurred within 30 days after discharge, and the re-examination results of indices before discharge (in stage of T4) between reinfected and non-reinfected patients were compared to find the risk factors of reinfection. RESULTS: Before and after infection, the CD64 index, CRP, CD14⁺HLA-DR⁺, CD4⁺, and lymphocyte counts were significantly different (all P<0.05). There was a negative correlation of CD64 index with CD14⁺HLA-DR⁺ (r=-0.395, P<0.001), a negative correlation with CD3⁺ (r=-0.1.87, P=0.047), and a negative correlation with lymphocyte count (r=-0.230, P=0.006), while a positive correlation with CRP(r=0.313, P<0.001). The area under the curve of CD64 index, CRP, and CD64 index combined with CRP was 0.790 (95%CI: 0.711-0.868), 0.754(95%CI: 0.667-0.841), and 0.835(95%CI: 0.762-0.907), respectively; the sensitivity was 59.6%, 72.7%, and 74.7%, the specificity was 89.2%, 73.0%, and 78.4%, and the cut-off value was 0.488, 0.457, and 0.531, respectively. There were only two re-examination indexes showed significantly different before discharge between reinfected patients and non-reinfected patients: CD14⁺HLA-DR⁺ (F=8.524, P=0.004) and CD64 index (F=9.993, P=0.002). The increase of CD64 index was an independent risk factor for reinfection within 30 days after discharge from the hospital (HR=1.790, 95%CI: 1.343-2.386, P<0.001). CONCLUSION CD64 index has diagnostic value in patients with hematological malignancies and pulmonary infection, and its specificity is higher than that of CRP. The combination of the two indicators can improve the diagnostic sensitivity. CD64 index has a predictive value for reinfection within 30 days after infection treatment.
Anti-Bacterial Agents/therapeutic use* ; Biomarkers ; C-Reactive Protein/metabolism* ; Cohort Studies ; Hematologic Neoplasms/metabolism* ; Humans ; Neutrophils/metabolism* ; Receptors, IgG/metabolism* ; Reinfection ; Retrospective Studies