ECCMID/ICC 2011 May 07 , 2011 - Milan Library in the lung Lecture Pieter S. Hiemstra author Onlineby

Laboratory for Respiratory © Cell and Immunology, Department of Pulmonology, Leiden University Medical Center, Leiden, TheESCMID Netherlands Antimicrobial peptides and proteins in human airway secretions Library Antimicrobial pppeptides Antimicrobial peptides: • α- and β- -Antimicrobial • (CAP-18/LL-37) -Pro- and anti-inflammatory -Immunomodulatory Antimicrobial proteins Lecture-Wound repair enhancing • •secrethhliA2tory phospholipase A2author • bactericidal permeability-increasingOnlineby protein (BPI) • serprocidins (,© G, azurocidin) • cationic serine proteinase inhibitors (SLPI, elafin) • • PLUNC proteins? • surfactantESCMID proteins Antibacterial activity of antimicrobial pppeptides Library

Lecture

EM images of Pseudomonas aeruginosa PAO1 exposed for 1.5 or 3 h ours to the ov ine ca thauthoreli c idin SMAP29 Onlineby Featur es of an timi cr obi al peptides ©: Anttcobaecassimicrobial mechanisms: • amino acid composition • disruption of membrane integrity • amphipathicity • intracellular activity of •cation ic ch arge translocated peptides •size ESCMID Brogden, Nat Rev Microbiol. 2005 The local environment regulates the antimicrobial activity of AMPs in the lung Library • salt concentration •pH • degradation by microbialLecture and host proteases • inhibition by products released from dead cells (F-actin, DNA) author •mucins Onlineby • bacterial polysaccharides© • glycosaminoglycans

ESCMID and epithelial cells are the main (but not the only) cellular source of antim icro bia l pep tides Library • phagocytes •epithelial cells • but also mast cells, T Lecturecells etc. author Onlineby ©

ESCMID Airway epithelial cell functions in host defense against infection Library Antimicrobial peptides, proteins and reactive oxygen and nitrogen intermediates Mucociliary clearance Lecture

Surface author BiBarrier epithelium Onlineby © Crosstalk with other Cytokines, cell types, including: chemokines • dendritic cells and growth factors • fibroblasts • airway smoothESCMID Submucosal muscle cells gland Regulation of epithelial production of antimicrobial peptides Library Microbial exposure Tissue injury LectureVitamin D and repair author Onlineby © Transcription factors Production of ESCMID antimicrobial peptides granules – rich sources of neutrophil serine proteinases and antimicrobial peptides Library azurophilic : elastase cathepsin G Lectureprotiteinase 3 lysozyme neutrophilauthor defensins (HNP1-4) Onlineby :© lysozyme lactoferrin CAP-18/LL-37 ESCMID Role of antimicrobial peptides in neutroppyhil antimicrobial activity Library

1. Intracellular killing in phagolysosome 2. Ex traceLecturell u lar killing a fter neutrophil 3.Format authorio n o f neut rop hil ex trace llu la r traps (NETs) following novel ROS dependent Onlinecellby death program ©

ESCMID Neutrophil extracellular traps (NET): a mechanism to capture and kill micro-organisms Library

Lecture author Onlineby ©

ESCMID Brinkmann and Zychlinsky, Nat Rev Microbiol 2007 Modulation of antimicrobial peptide expression in chronic inflammatory lung disease

Library Inflammation, Genetic polymorphisms injury and repair Cigarette smoke Lecture Production of antimicrobialauthor pppeptides Onlineby © Air pollution Microbial exposure Sunlight exposure (it(vitami n D) ESCMID Levels of AMPs in bronchoalveolar lavage fluid in stable lung transplant recipients: idllifitiltfitthincreased levels in presence of a positive culture for a respiratory pathogen Library

Antimicrobial No pathogens Pathogens (n=8) P value peptide (n=25) LL-37 ( ng/ ml) 0(00 (0-16) Lecture9(09 (0-34) 0.002 HNP1-3 (ng/ml) 2 (0-1092)author 2498 (2-3212) <0.001 hBD-2 (pg/ml)Online 114 (0-868)by 481 (0-2450) 0.004 © SLPI (ng/ml) 133 (56-545) 506 (1-1296) 0.001

Elafin (pg/ml ) 411 (0-15541) 1264 (0-12117) 0.602 ESCMID Anderson et al, ERJ 2008 Development of bronchiolitis obliterans syndrome (BOS) after lung transplantation and antimicrobial polypeptides Library • BAL characteristics: Similar pattern in sputum during COPD – ↑exacerbations with NTHi or M. catarrhalis: – SLPI ↓ LectureLactoferrin = –elafin ═ Lysozyme ↓ – hBD-2 ═ authorSLPI ↓ – neutrophil defensinsOnline (HNP1-3)by LL-37↑ ↑ – hCAP18/LL-37 ↑ © Parameswaran et al, 2011

ESCMID Anderson et al, ERJ 2008 Possible consequences of aberrant exppyression/activity of AMPs Library INSUFFICIENT EXCESSIVE

Lecture author • increased susceptibilityOnlineby • inflammation to infection • increased (Th1) • iiddimpaired wound repai i?©r? (t)i(auto)immun it?ity?

ESCMID Processing of the cathelicidin hCAP-18/LL-37 Library

signal SCTE LL-37 peptide cathelin Lecture

AauthorLL-38 Onlinegastricsinby Nasal lavage fluid hCAP-18© hCAP-18

ESCMID LL-37 Selected activities of LL-37 in the lung

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hCAP-18 Proteinase 3 Antimicrobial activity LPS and LTA neutralisation Survival Lecture LL-37 Dendritic cell maturation author by Angiogenesis Chemotaxis Online © Cell death and survival Epithelial Activation of wound repair epithelial cells and smooth muscl e cell s EGFR activation ESCMID Updated from Tjabringa et al, MAPK activation Pulm Pharm Ther 2005 IL-8 release Macrophage differentiation and heterogeneity

• Secrete pro- mφ1 Library mφ1 inflammatory GM-CSF cy tokines (e. g. IL-12, IL-18, IL-23, IL-6 ) • Good antigen- presenting Pro-inflammatory Lecture capacity macrophage • Promotes Th1- author immunity Onlineby CD14+ M-CSF © mφ2 • IL-10 secretion mφ2 • CD163 expression • Poor antigen- presenting capacity ESCMID Anti-inflammatory • Promote Th- macrophage regulatory cells LL-37 affects M-CSF-driven macrophage differentiation: morphology, FACS analysis and cytokine production

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Lecture author Onlineby LL-37 skews macrophage © differenti a tion t oward s the pro-inflammatory Mf1 phenotype

ESCMID Van der Does et al J Immunol 2010 Library

Vitamin D as a majorLecture regulator of hCAP18/LL-37 synthesis by macrophages and epithelialauthor cells Onlineby ©

ESCMID Vitamin D: a multifunctional vitamin

• Major regulator of bone health andLibrary calcium htihomeostasis • Also implicated in: – Cancer Lecture – Autoimmune disease – Diabetes author – Metabolic syndromeOnlineby – Cardiovascular disease© – Infections – …….. ESCMID Synthesis of vitamin D3

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Lecture author Onlineby ©

ESCMID Deeb et al, Nat. Rev. Cancer 2007 Role of vitamin D induced antimicrobial peptide production in anti-mycobacterial activity of and macrophages Library

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ESCMID Adams et al Nat Clin Pract Endocrinol Metabol 2008 Locally activated vitamin D3 induces expression of cathelicidin hCAP18/LL-37 in airway epithelial cells Library

Lecture author Cell lysates Onlineby ©Induct i on ofhCAP18/LL-37requir es active conversion of 25D3 into 1,25D3. as shown by the effect of the 1α- hydroxylase inhibitor, itraconazole ESCMID Hansdottir et al, J Immunol 2008 Library OliOnline L ect ure Lib rary

Slide withheldLecture author at requestOnline ofby author ©

ESCMID Library OliOnline L ect ure Lib rary

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ESCMID Increased killing of Pseudomonas aeruginosa PAO1 by apical washing from ALI-PBEC cultured for 2 weeks in presence of IL-4orIL4 or IL-13 Library

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OO 50000author * 2 wk ALI PA ± IL-4 or IL-13 Onlineby Apical wash 0 - 10201020 + PAO1 © IL-4 (ng/ml) IL-13 (ng/ml)

ESCMIDCFU Zuyderduyn et al, Respiratory Research 2011 AMPs and treatment of infections

Library Advantages Limitations • Broad-spectrum antimicrobial • Complex biological fluids may activity inhibit activity • Active against multidrug- • Degradation by host and resistant bacteria Lecturemicrobial proteases • Low acquisition of resistance • Rapid renal clearance • SithddSynergy with drugs and •authorHos t ce ll tox ic ity endogenous mechanisms by • Pro-inflammatory activity • Binding and neutralisationOnline of endtdotox in ©

Strategies • Topical administration of synthetic or recombinant AMPs •ESCMID Increasing endogenous production (vitamin D, butyrate) Development of an LL-37-derived synthetic antimicrobial peptide with decreased pro- inflammatory activity Library • P60.4Ac is a 24-amino acid antimicrobial peptide derived from the C-terminus of LL-37 with amino acid substitutions introduced to enhance stability and amphipaticity

•Compared to LL-37,,py P60.4Ac displays: Lecture – potent LPS-neutralizing

– antimi crobi al acti vit y author

– decreased neutrophil Onlineby chemot acti c acti vit y ©

• Phase I and phase II trials using local application in chronic otitis media confirm its therapppeutical potential ESCMID Nell et al, Peptides 2005 Conclusions

• Both local deficient as well as excessive expressionLibrary of AMPs may contribute to chronic lung inflammation and infection • Local conditions may affect the (antimicrobial) activity of AMPs • In addition to its role in host defense against infection, LL-37 has a wide variety of effects on inflammatory, immune and structural cells in the lung (includingLecture epithelial cells and airway smooth muscle cells) • AMP-based treatment of inflammatory author and infectious lung disease feasible by enhancing their expression or application of peptides Onlineby – active against multidrug resistant “superbugs” – balance between pro-inflammatory© and antimicrobial activity

ESCMID Acknowledgements Library

Lecture author Onlineby Airway epithelial© cell studies - 2011 Respiratory Cell Biology and Immunology Lab Dept of Parasitology, LUMC Dept. of Pulmonology, LUMC, Hermelijn Smits Leiden, The Netherlands Gimano Amatngalim, Katrien Eger, Jasmijn Schrumpf, Galapagos NV, Leiden, NL Marianne vanESCMID Sterkenburg, Renate Verhoosel, Jamil Aarbiou Emily van ‘t Wout, Suzanne Zuyderduyn Annemarie Lekkerkerker