Title Page

Mitochondria-induced formation of neutrophil

extracellular traps is enhanced in the elderly
via Toll-like receptor 9
 Introduction
• Neutrophils are a type of white blood cell that plays a crucial role in
defending the body against pathogens.

• One of their key weapons is neutrophil extracellular traps (NETs).

• NETs are web-like structures made of DNA and proteins that trap and
kill harmful microbes like bacteria.
• NETs can also be a double-edged sword. While they are essential for
fighting infection, their formation during sterile inflammation
(inflammation without an infection) can be detrimental.

• This presentation explores the complex role of NETs and introduces a

new aspect: the influence of aging on NET formation.

• NET formation is a powerful tool in the neutrophil's arsenal within the

immune system.
• While NETs effectively eliminate pathogens, their uncontrolled release
can disrupt the delicate balance of the immune response, leading to
autoimmune reactions where the body attacks healthy tissues.

• The concept of sterile inflammation highlights the complexity of the

immune system, which can sometimes react to non-infectious stimuli
in ways that cause harm.

• Understanding the factors that influence NET formation, particularly

in the context of aging, is crucial for developing new strategies to
manage chronic inflammatory diseases.
 Background
Neutrophils in Immune System:

• Neutrophils are key in battling infections.

• They use neutrophil extracellular traps (NETs) to trap pathogens.

• NETs are web-like structures made of DNA and proteins.

• NETs form during sterile inflammation, a form of inflammation without an

active infection.
NETs: A Double-Edged Sword

• NETs can damage healthy tissues in their web, contributing to chronic

inflammatory conditions.

• Understanding NET formation mechanisms is crucial for developing

new therapeutic strategies.

• Recent research suggests mitochondria might initiate NET formation,

but details remain under investigation.
Toll-like Receptor 9 (TLR9) Function

• Acts as immune cell sensor.

• Detects foreign DNA pattern.

• Triggers immune response, potentially NET formation.

 Methodology
Clinical Subjects
• Recruit patients and healthy volunteers (n = 81) from hospitals or
universities.
• Divide participants into cohorts based on age or immune status:
• Healthy adults (control group, n = 11)
• Children (n = 16)
• Elderly (60-79 years old, n = 16)
• Very elderly (≥80 years old, n = 11)
• Pregnant women (n = 15)
• Patients with rheumatoid arthritis (n = 12)
Experimental Animals

• Purchase young (2-3 months old, n = 11) and old (18-26 months old, n
= 15) male C57/BL6J mice.

• House mice under controlled conditions (light/dark cycle, temperature,

humidity) with free access to water and food.

• Obtain ethical approval for the animal experiment.

Blood Collection and Neutrophil Isolation

• Collect peripheral blood from participants via venipuncture.

• Separate plasma and neutrophils using centrifugation steps.

• Count neutrophils and resuspend them in culture medium for further

use.
TLR9-mediated NF-κB Signaling Reporter Cells Cultivation and
Assay
• Use human TLR9-expressing HEK-Dual reporter cells and control cells.

• Stimulate reporter cells with diluted patient plasma to detect NF-κB

activation.

• Analyze cell culture supernatants for NF-κB reporter activity.

• Include negative and positive controls in the analysis.

Bacteria Cultivation
• Grow Escherichia coli bacteria and measure their optical density for
quantification.

Isolation of Mitochondria
• Isolate mitochondria from human placental tissue or mouse liver.
• Follow the same procedure for both human and mouse mitochondria
isolation.
• Assess mitochondrial purity and quantity using qPCR.
Detection of NETs by Live-Cell Microscopy
• Plate isolated neutrophils on a culture plate with culture medium or
patient plasma.
• Stain neutrophils with Hoechst 33342 and SYTOX Green for live-cell
imaging.
• Use a Cytation 7 Cell Imaging Multi-Mode Reader to capture images
kinetically.
• Analyze images to quantify NET formation based on SYTOX Green
positivity and size criteria.
• Verify NETs with anti-citrullinated histone H3 antibody staining
Detection of NETs by Fluorescence Microscopy
• Seed neutrophils on poly-L-lysine-coated coverslips and stimulate
them for NET formation.
• Fix and permeabilize cells, followed by blocking with FBS solution.
• Stain cells with anti-citrullinated histone H3 antibody and SYTOX
Green.
• Use an Axiolab 5 fluorescence microscope to capture images.
• Identify NETs based on DNA morphology and histone H3
citrullination.
Biochemical Analyses
• Measure cytokine concentrations in plasma using LEGENDplex
Human Inflammation Panel.
• Quantify myeloperoxidase (MPO), neutrophil elastase (NE), and
citrullinated histone H3 in plasma using ELISA kits.
• Analyze relative nucleosome concentration and 8-hydroxy-2′-
deoxyguanosine (8-OHdG) using ELISA kits.
• Measure absorbance using a Synergy H4 Hybrid Reader and calculate
analyte concentrations.
Quantification of MPO-DNA Complexes

• Coat a high-binding plate with anti-human MPO antibody and block

with BSA solution.
• Incubate the plate with patient plasma, anti-DNA antibody, and TMB
substrate.
• Measure absorbance at 450 nm and normalize data to in vitro NET
standards.
ecDNA Quantification
• Isolate extracellular DNA (ecDNA) from plasma using a QIAamp 96
DNA Blood kit.
• Quantify ecDNA concentration using a fluorometric Accublue High
Sensitivity dsDNA Quantitation kit.
• Measure fluorescence and calculate ecDNA concentrations based on
the provided standard.
DNase Activity of Plasma
• Measure DNase activity using an in-house fluorometric method.
• Incubate plasma samples with isolated DNA and SYTOX Green stain.
• Measure fluorescence before and after incubation to calculate DNase
activity.
• Express activity in Kunitz units/mL based on an RNase-free DNase