Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial by inhibiting neutrophil extracellular traps

IntroductionNeutrophil extracellular traps (NETs) clear pathogens but may contribute Q8 pathogenically to host inflammatory tissue damage during sepsis. Innovative therapeutic agents targeting NET formation and their potentially harmful collateral effects remain understudied.MethodsWe investigated a novel therapeutic agent, neonatal NET-Inhibitory Factor (nNIF), in a mouse model of experimental sepsis – cecal ligation and puncture (CLP). We administered 2 doses of nNIF (1 mg/ kg) or its scrambled peptide control intravenously 4 and 10 hours after CLP treatment and assessed survival, peritoneal fluid and plasma NET formation using the MPO-DNA ELISA, aerobic bacterial colony forming units (CFU) using serial dilution and culture, peritoneal fluid and stool microbiomes using 16S rRNA gene sequencing, and inflammatory cytokine levels using a multiplexed cytokine array. Meropenem (25 mg/kg) treatment served as a clinically relevant treatment for infection.ResultsWe observed increased 6-day survival rates in nNIF (73%) and meropenem (80%) treated mice compared to controls (0%). nNIF decreased NET formation compared to controls, while meropenem did not impact NET formation. nNIF treatment led to increased peritoneal fluid and plasma bacterial CFUs consistent with loss of NET-mediated extracellular microbial killing, while nNIF treatment alone did not alter the peritoneal fluid and stool microbiomes compared to vehicle-treated CLP mice. nNIF treatment also decreased peritoneal TNF-a inflammatory cytokine levels compared to scrambled peptide control. Furthermore, adjunctive nNIF increased survival in a model of sub-optimal meropenem treatment (90% v 40%) in CLP-treated mice.DiscussionThus, our data demonstrate that nNIF inhibits NET formation in a translationally relevant mouse model of sepsis, improves survival when given as monotherapy or as an adjuvant with antibiotics, and may play an important protective role in sepsis

Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH

Alprenolol-biotin binding in subsets of peripheral blood cells is decreased in PAH as compared to controls.

<p>p-value represents Student’s t test comparison of study groups.</p

Alprenolol-biotin binding in circulating endothelial cells (CEC) correlates with TAPSE.

<p>ρ represents Spearman's rank correlation coefficient with corresponding p value.</p

Increased endothelial microparticles in pulmonary arterial hypertension (A) and correlation with TAPSE (B).

<p><b>A)</b> Percentage of endothelial microparticles (Ec-MPs) as defined by CD144⁺ microparticles (MPs) among annexin-V⁺ MPs was increased in PAH. p-value represents Student’s t test comparison of study groups. <b>B</b>) Percentage of Ec-MPs correlates with TAPSE. ρ represents Spearman's rank correlation coefficient with corresponding p value.</p

Study population characteristics.

<p>Study population characteristics.</p

Gating strategy for flow cytometry assay.

<p><b>A)</b> Gating strategy for hematopoietic peripheral blood cell subsets. Artifact exclusion included time gating (1), aggregate exclusion (2), and DAPI gating for cells in G₀/G₁ (3). FSC vs SSC gating was used to discriminate polymorphonuclear cells (PMN) and mononuclear cells (MNC) (4). CD3 was used as a T-cell maker (5) and CD19 as a B-cell marker (6). CD45 gating of the CD3^-CD19^- MNC (<b>7</b>) based on fluorescence-minus one (FMO) control (<b>8</b>), followed by CD34 and CD133 gating (<b>9</b>) based on FMO control (<b>10</b>) were used to select hematopoietic progenitor cells (HPC). Alprenolol binding was then assessed based on FMO control (<b>11</b>). <b>B)</b> Gating strategy for circulating endothelial cells (CEC). Artifact exclusion included time gating (<b>1</b>), aggregate exclusion (<b>2</b>), and DAPI gating for cells in G₀ and G₁ (<b>3</b>). CD45^- gating of the CD3^-CD19^- population was based on FMO control (<b>4</b>). CD34⁺ gating was used to define CEC (<b>5</b>).</p

Gating strategy for urinary endothelial cell microparticles.

<p>Sequential gating was used to quantify the percentage of endothelial-derived microvesicles among annexin-V positive microparticles in the urine. Small Angle Light Scatter (SALS) and Large Angle Lights Scatter (LALS) were used to gate the microvesicle population (<b>A</b>). Annexin-V⁺ events were selected on a LALS/Green fluorescence channel plot (<b>B</b>). Annexin-V⁺ region was defined based on unstained sample (<b>D</b>). CD144 (VE-cadherin)⁺ events in the annexin-V⁺ gate were defined on an LALS/orange fluorescence channel (<b>C</b>) based on an FMO control missing CD144 (<b>E</b>) and isotype matched IgG staining (<b>F</b>). Samples were acquired at a flow rate of 6.01 μL/min for 2 minutes. At least 5000 annexin-V⁺ CD144-PE⁺ microparticles were acquired.</p

Alprenolol-biotin binding of human PBCs specific for the βAR.

<p><b>A)</b> Titration of the alprenolol-biotin probe demonstrated an optimal concentration of 25 μg/mL. Values represent MFI for WBC population. <b>B)</b> The titration curve shows that binding is a saturable and specific process. <b>C)</b> Decay curve for alprenolol binding. Values represent mean ± SE of 2 experiments each with N = 3. <b>D)</b> Isoproterenol, another ligand for βAR, displaced alprenolol from the receptor in a dose-dependent fashion. <b>E)</b> Losartan, an angiotensin II receptor antagonist (scramble competitor) did not affect binding of the alprenolol probe. Mean ± SE of 2 experiments are shown.</p

Antibodies and probes used for flow cytometric assay.

<p>Antibodies and probes used for flow cytometric assay.</p