HELENA Lecture Series: Lung Biology and Disease

Aseptic lung inflammation, mouse models and methods of investigation Tobias Stöger - “Dynamics of pulmonary inflammation”

November 12, 2015

Inflammation, a nonspecific immune response: A basic way in which the body reacts to infection, irritation or other injury, the key feature being warmth, redness, swelling, pain and loss of function.

Inflammatory Stimulation triggered by pathogens (exogenous)

Inflammatory Stimulation triggered by sterile stressors (exogenous or endogenous)

environmental toxins radiation

injury necrosis

drugs (BLM) acid

free radicals

endogen crystal exogen asbestos

smoke

alleregns

Biology of Inflammation

Causes, and physiological and pathological outcomes

modified from Medzhitov R. 2008 NATURE Vol 454 24 July

Inadequate, Imperfect Response

Generic Inflammatory Pathway:

PAMPs: Pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) and other pattern/ pathogen recognition receptors.

Biology of Inflammation

Causes, and physiological and pathological outcomes

modified from Medzhitov R. 2008 NATURE Vol 454 24 July

Inadequate, Imperfect Response

Vasta 2009, Nature Reviews Microbiology 7, 424-438

Pathogen Recognition as Trigger of Inflammation

Pattern-Recognition Receptors (PRR)

PRR recognize exogenous (PAMP) and endogenous (DAMP) Ligands

Mullen et al. Arthritis Research & Therapy (2015) 17:122

PAMP: Pathogen-associated molecular pattern DAMP: Damage associated molecular pattern molecules

Classical triggers of inflammatory signaling

Common pathway for microbial, non-microbial / external and endogenous inducers

Pro-Inflammatory Cytokines, Oxidants, Pathogens…

NF-κB

AP-1

Tran

scrip

tion

Fact

ors

Receptors

Recruitment of Inflammatory Cells

Expression of Pro-Inflammatory Genes:

TNFα, IL1β, IL6, IL8, VCAM1…

self-

perp

etua

ting

infla

mm

ator

y cy

cle

self-perpetuating inflamm

atory cycle

Acute Lung Injury Models

n % Mechanical ventilation 436 30% LPS / Endotoxin 279 19% Live bacteria 224 16% Hyperoxia 175 12% Bleomycin 149 10% Oleic acid 79 5% Cecal ligation & puncture

61 4%

Acid aspiration 38 3%

Total 1,441 100%

Number of papers indexed in PubMed using animal models of acute lung injury from 2003–2007

Matute-Bello, Am J Physiol Lung Cell Mol Physiol. 2008

easily carried out, very reproducible

Headline Conclusions and Lookout Mechanism of Inflammation: Emigration of cells LPS Model of Acute Lung Injury

Rapid immigration of inflammatory cells

Airspace accumulation of inflammatory cells

6 - 12hr

Kinetics of the LPS response over 72 h LPS instilled at a dose of 10 ug/mouse

Method:

Broncho-alveolar lavage (assessment of inflammatory cell count)

&

Mechanism: Leukocyte trafficking

Method: Broncho-alveolar lavage (BAL)

Mouse

BAL after centrifugation

BALF

BAL cells

BAL Fluid (Protein Markers) - total protein (blood–air barrier damage) - LHD (cell damage) - cytokines … BAL Cells - Cytospin preparation (cell differentiation) - FACS - expression analysis bio-protocol.org

healthy asthmatic

Cytospin stained May Grunwald/Giemsa

lavage

Matthay and Zimmerman, Am J Respir Cell Mol Biol, 2005

Cellular Responses and Mediators Contributing to Alveolar–Capillary Membrane Injury

Stramer et al. 2007, Journal of Investigative Dermatology 127, 1009–1017

Cellular dynamics of inflammation/resolution

Time course of inflammation and cell recruitment at side of damage

/ Fibroblasts

Professional Phagocytes (Alveolar Macrophages) Clearance of apoptotic cells by phagocytes

Fluorescent microscopy showing membrane extensions and ingested apoptotic cells (blue) in spacious phagosomes. Erwig and Henson (Cell Death and Diff.) 2008

Clearence of Neutrophils

Sterile particles as trigger of lung inflammation

Inhaled particles can induce lung inflammation

Humans have always been exposed to particulate matter - but particle-quality changed quite recently

2005 1900 0 -5Mio

nano

-1.5Mio

Quartz particles and chronic lung inflammation

Quartz Particle Characteristics and Pulmonary Toxicity Carcinogenic to humans

Distribution frequency of particle size of native (squares) and surface modified (triangles, DQ12-PVNO; circles, DQ12-AL) quartzes were determined by electron microscopy.

Quartz particles generate hydroxyl radicals (·OH) measured by electron spin resonance technique (ESR).

-- DQ12

Prolonged exposure to silica dust (sized between 1-5 microns), causes progressive fibrosis called Silicosis (occupational lung disease).

BAL Analysis (3 – 90d)

Histology of C57BL/6 lungs 2 mo after a single 2.5mg silica (DQ12) administration.

DQ12: sharp-edged crystals fragments 10 µm

Misson et al. AJPLCMP. 2007 Albrecht et al. Am J Respir Cell Mol Biol. 2004

Kulkarni (N Engl J Med, 2006) - correlation between carbon content in alveolar macrophages and reduced lung function

in children - indicates that ambient carbon particles impair the growth of lung function in children.

Kulkarni 2006: N Engl J Med 355

Uptake of Ambient Particles by Alveolar Macrophages

Headline Conclusions and Lookout Intratracheally instilled sterile carbon particles induce acute lung inflammation

Particle Dose / Mouse (BET Surface Area cm2)

S.Takenaka 50 nm

TEM of CP Diagram of a fractal soot particle

BA

L ce

lls:

Stoeger, EHP, 2006

Headline Conclusions and Lookout Inhaled particles induce lung inflammation

Pathways are depending on NP-characteristics

Donaldson 2005 (Particle and Fibre Toxicology)

Asbestos

Fibers

Glass Fibers

Infla

mm

asom

e

IL1β

Nanotubes

Different Time Course of Pulmonary Inflammation Caused by Carbon Nanoparticles and Carbon Nanotubes

0

50

100

150

200

250

300

Co 1 3 7 14 90

BAL

Infla

mm

ator

y Ce

ll Co

unts

[10

E3]

Days After Particle or MHV-68 Challenge

CNT

CNP

*

Ganguly, Part Fibre Toxicol. 2009 Tian, Eur J Pharm Biopharm. 2013 Hirn, et al. in preparation

IT

TEM S.Takenaka

Printex 90 (14nm)

CNP

CNT (4nm x 1-10µm)

CNT

50 nm

Excessive production of inflammatory mediators by ‘frustrated Phagocytosis’ from fiber exposed phagocytes

Frustrated phagocytosis (arrows) of Asbestos Fibers or Carbon Nanotubes

Donaldson et al. Particle and Fibre Toxicology 2010 7:5

Macrophages ‘freak out’ - self-perpetuating inflammatory cycle - ineffective resolution fibrosis carcinogenesis

Chen and Nuñez Nature Reviews Immunology 2010

Sterile fiber / needle shaped particles cause lysosomal destabilization and trigger the inflammasome dependent release of IL-1β

Summary: Mechanisms Triggering Sterile Inflammation

LPS/PAMP DAMP

Receptor Interactions e.g. TLR4 (LPS, HMGB1 …)

Pathway- 0

receptor stimulation

thank you for your attention