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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