Aseptic lung inflammation, mouse models and methods of investigation

Transcription

1 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

2 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.

3 Inflammatory Stimulation triggered by pathogens (exogenous)

4 Inflammatory Stimulation triggered by sterile stressors (exogenous or endogenous) radiation environmental toxins smoke drugs (BLM) acid endogen crystal exogen asbestos alleregns free radicals necrosis injury

5 Biology of Inflammation Causes, and physiological and pathological outcomes Generic Inflammatory Pathway: PAMPs: Pathogen-associated molecular patterns recognized by tolllike receptors (TLRs) and other pattern/ pathogen recognition receptors. Inadequate, Imperfect Response modified from Medzhitov R NATURE Vol July

6 Biology of Inflammation Causes, and physiological and pathological outcomes Inadequate, Imperfect Response modified from Medzhitov R NATURE Vol July

7 Pathogen Recognition as Trigger of Inflammation Pattern-Recognition Receptors (PRR) Vasta 2009, Nature Reviews Microbiology 7,

8 PRR recognize exogenous (PAMP) and endogenous (DAMP) Ligands PAMP: Pathogen-associated molecular pattern DAMP: Damage associated molecular pattern molecules Mullen et al. Arthritis Research & Therapy (2015) 17:122

9 Classical triggers of inflammatory signaling Common pathway for microbial, non-microbial / external and endogenous inducers Pro-Inflammatory Cytokines, Oxidants, Pathogens Receptors self-perpetuating inflammatory cycle Transcription Factors AP-1 NF-κB Expression of Pro-Inflammatory Genes: TNFα, IL1β, IL6, IL8, VCAM1 self-perpetuating inflammatory cycle Recruitment of Inflammatory Cells

10 Acute Lung Injury Models

11 Number of papers indexed in PubMed using animal models of acute lung injury from n % Mechanical ventilation % LPS / Endotoxin % Live bacteria % Hyperoxia % Bleomycin % Oleic acid 79 5% Cecal ligation & puncture 61 4% Acid aspiration 38 3% easily carried out, very reproducible Total 1, % Matute-Bello, Am J Physiol Lung Cell Mol Physiol. 2008

12 LPS Model of Acute Lung Injury Mechanism of Inflammation: Conclusions Headline Emigration and Lookout of cells Rapid immigration of inflammatory cells Airspace accumulation of inflammatory cells Kinetics of the LPS response over 72 h LPS instilled at a dose of 10 ug/mouse 6-12hr

13 Method: Broncho-alveolar lavage (assessment of inflammatory cell count) & Mechanism: Leukocyte trafficking

14 Method: Broncho-alveolar lavage (BAL) lavage Mouse BAL after centrifugation Cytospin stained May Grunwald/Giemsa healthy asthmatic BAL Fluid (Protein Markers) - total protein (blood air barrier damage) - LHD (cell damage) - cytokines BALF BAL cells BAL Cells - Cytospin preparation (cell differentiation) - FACS - expression analysis bio-protocol.org

15 Cellular Responses and Mediators Contributing to Alveolar Capillary Membrane Injury Matthay and Zimmerman, Am J Respir Cell Mol Biol, 2005

16 Cellular dynamics of inflammation/resolution Time course of inflammation and cell recruitment at side of damage / Fibroblasts Stramer et al. 2007, Journal of Investigative Dermatology 127,

17 Professional Phagocytes (Alveolar Macrophages) Clearance of apoptotic cells by phagocytes Clearence of Neutrophils Fluorescent microscopy showing membrane extensions and ingested apoptotic cells (blue) in spacious phagosomes. Erwig and Henson (Cell Death and Diff.) 2008

18 Sterile particles as trigger of lung inflammation

19 Inhaled particles can induce lung inflammation Humans have always been exposed to particulate matter - but particle-quality changed quite recently nano -5Mio -1.5Mio

20 Quartz particles and chronic lung inflammation

21 Quartz Particle Characteristics and Pulmonary Toxicity Carcinogenic to humans DQ12: sharp-edged crystals fragments BAL Analysis (3 90d) -- DQ12 10 µm Prolonged exposure to silica dust (sized between 1-5 microns), causes progressive fibrosis called Silicosis (occupational lung disease). Distribution frequency of particle size of native (squares) and surface modified (triangles, DQ12- PVNO; circles, DQ12-AL) quartzes were determined by electron microscopy. Histology of C57BL/6 lungs 2 mo after a single 2.5mg silica (DQ12) administration. Quartz particles generate hydroxyl radicals ( OH) measured by electron spin resonance technique (ESR). Misson et al. AJPLCMP Albrecht et al. Am J Respir Cell Mol Biol. 2004

22 Uptake of Ambient Particles by Alveolar Macrophages 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

23 Intratracheally instilled Conclusions Headline sterile and Lookout carbon particles induce acute lung inflammation TEM of CP Diagram of a fractal soot particle S.Takenaka 50 nm BAL cells: Particle Dose / Mouse (BET Surface Area cm 2 ) Stoeger, EHP, 2006

24 Inhaled particles induce lung inflammation Conclusions Headline and Lookout Pathways are depending on NP-characteristics Fibers Asbestos IL1β Inflammasome Glass Fibers Nanotubes Donaldson 2005 (Particle and Fibre Toxicology)

25 Different Time Course of Pulmonary Inflammation Caused by Carbon Nanoparticles and Carbon Nanotubes BAL Inflammatory Cell Counts [10E3] Co IT Printex 90 (14nm) CNP TEM S.Takenaka 50 nm Days After Particle or MHV-68 Challenge CNT (4nm x 1-10µm) CNT * CNT CNP Ganguly, Part Fibre Toxicol Tian, Eur J Pharm Biopharm Hirn, et al. in preparation

26 Excessive production of inflammatory mediators by frustrated Phagocytosis from fiber exposed phagocytes Frustrated phagocytosis (arrows) of Asbestos Fibers or Carbon Nanotubes Macrophages freak out - self-perpetuating inflammatory cycle - ineffective resolution fibrosis carcinogenesis Donaldson et al. Particle and Fibre Toxicology :5

27 Sterile fiber / needle shaped particles cause lysosomal destabilization and trigger the inflammasome dependent release of IL-1β Chen and Nuñez Nature Reviews Immunology 2010

28 Summary: Mechanisms Triggering Sterile Inflammation Pathway- 0 receptor stimulation LPS/PAMP DAMP Receptor Interactions e.g. TLR4 (LPS, HMGB1 )

29 thank you for your attention