1. Allergy, atopy and asthma Rich Locksley Micro 204 December 2014

2. Allergy and asthma: cells and cytokines timeline Eos, basos, mast cells Paul Ehrlich Anaphylaxis Charles Richet 1879 1913 1921 1967 1983 1986 1991 1992 1998 2002 2009- Prausnitz-Kustner IgE Ishizaka/Be nnich/Johan sson Th2 cytokines in asthma lung biopsies IL-4 ko mice IL-13 ko mice IL-4/5/13 ko mice IL-5 ko mice Th2 cells Mosmann, Coffman IL-4, IL-5, IL-13  IL-5,  IL-13

3. Global burden of allergic pathology Parasitic helminth infections Intestinal nematodes (>2 billion) Tissue filaria (>120 million) Tapeworms (>2 million) Schistosomes (>200 million) Major causes of liver failure, bladder cancer, elephantiasis, blindness, epilepsy (post-reproductive morbidity) Asthma and Allergy >300 million worldwide Asthma prevalence increased 75% from 1980-1994 >70% asthma patients have allergy 1:8-12 US children >$20 billion/yr health care costs US Developed Countries Developing Countries

4. A word about helminths Account for ~80% of all individual animals on earth Estimates up to 10 6 species Three major, two minor clades - all includes parasitic species 2.9 billion humans estimated infected Universal in rural subtropical environments typical of human evolution Essentially universal in feral vertebrates (mammals, birds, fish, reptiles, amphibians) Genome size ~ 20,000 Parkinson J, et al. 2004. A transcriptome analysis of the phylum Nematoda. Nature Genet 36:1259- 67. If type 2 immunity protects us from helminths, it doesn’t do a very good job… Maybe helminths exploit the pathway for other things…

5. Ana - phylaxis: “Backwards Protection” XX Richet: 1913 Nobel Prize

6. Allergy An adverse immune response to environmental antigens (allergens). Operational definition: IgE Manifestations: Anaphylaxis, Eczema, Hayfever, Asthma Many Allergens Acquired Via the Mucosa Insect venoms Pollens Mite feces Animal danders Food Haptenylated drugs (penicillin)

7. IgE = “Reagin” or “Reaginic Antibodies” PCA: Passive Cutaneous Anaphylaxis Atopic to Antigen X Serum (IgE) (Ishizaka, 1966) Non-Atopic  IgE Antigen X Immediate Hypersensitivity

8. Stages of Allergy - I. “Wheal & Flare” PGD 2 LTC 4 LTD 4 LTE 4 Allergen Fc  RI IgE Post-capillary Venule Edema Histamine Early Phase Reaction - minutes

9. CCR3 44 Eosinophils Stages of Allergy - II. Late Phase Reaction - hours 44CCR8 Th2 TNF PGD 2 IL-4 IL-13 IL-5 IL-3 IL-9 44 Basophils Eotaxins 1, 2, 3 MCP 2, 3, 4 RANTES

10. CCR3 44 Eosinophils Stages of Allergy - II. Late Phase Reaction - hours 44CCR8 Th2 PGD 2 IL-13 IL-5 IL-9 44 Basophils Eotaxins 1, 2, 3 MCP 2, 3, 4 RANTES Mast cell ILC2 cell GM-CSF TNF

11. Stages of Allergy - III. Chronic Phase Reaction - wks - yrs TGF-  ; Toxic Granules 1. Subepithelial Fibrosis 2. Smooth Muscle Hypertrophy 3. Mucosal and Mast Cell Hyperplasia

12. Fc  RI: Key Player Lyn Syk P PP IgE K d = 10 -10 M    AA PLA-2 PGD2 LTC 4 LAT PI(3)K GADS/SLP-76 VAV Ras Cytoskeleton MAPK TNF IL-4 Granule Exocytosis PLC  1 Ca ++ Rab Btk/ItkPIP3 +++

13. Mouse = Human Mouse IgG1/Fc  RIII on mast cells can trigger pathway (not present on human mast cells) Human APC, Eosinophils:    2 Fc  RI IgE-linked antigen presentation in human

14. How do perivascular mast cells acquire serum IgE? …..they reach in and take it Mast cell (green) around a blood vessel (white outline) extending a process into the lumen to bind an IgE-coated bead. Cheng et al, Immunity 38:166-75, 2013

15. Mast Cell Granules Histamine: Vasodilator Edema* Heparin: Bonds constituents (negative charge) Neutral Serine Proteases: Tryptase All Chymase Carboxypeptidase Cathepsin G C.T. > mucosal Knock-out: no mast cell granules *Dudeck A et al, Immunity 34:973-84, 2011

16. Myeloid Cell Eicosanoids Ca ++ IgE PLA 2 AA FLAP 5-LO* LTA 4 LTB 4 LTC 4 LTD 4 LTE 4 LTC 4 synthase; in asthma Mast Cells Eosinophils Basophils AAM  LTA 4 Hydrolase PMN M  BLT1 BLT2 chemoattractant Transpeptidase Dipeptidase CysLT1,CysLT2 (SRS-A) Smooth Muscle Contraction Endothelial NO Mucus Secretion * Mouse ko: anaphylaxis Cell Recruitment

17. Leukotriene Receptors BLT1 LTB4 ( K d ~0.5 nM ) Gene duplicationMyeloid, lymphoid cells Tissue 14q (h)recruitment BLT2 LTB4 ( K d ~23 nM ), More widely other eicosanoids CysLT1 LTD4>C4=E4 X (h) PBL, lung smooth muscle, Vascular lung macrophages, relaxation; smooth small intestine muscle contraction; cell activation CysLT2 LTC4=LTD4>E4 13q14 (h) PBL, lung, heart, brain Cell activation

18. Mast Cell PGD2 Ca ++ IgE PLA-2 AA PGD2 COX DP1** K d ~ 1.5 nM (airway epi, basophils,smooth muscle lung, GI) **Mouse ko = lung inflammation, bronchial hyperreactivity *Mouse ko = skin allergic hypersensitivity Bronchoconstriction Chemokines (Eotaxins, MCPs, MDC) in asthma CRTh2 (DP2)* K d ~ 2.5 nM (Th2, eo, baso,ILC2, lung smooth muscle, GI)

19. Eosinophil Granules Specific Primary Major Basic Protein Eosinophil Cationic Protein (RNase3) Eosinophil Peroxidase Eosinophil-derived Neurotoxin (Rnase 2) Lysophospholipase (Charcot-Leyden crystals) CYTOTOXICITY (multi-system toxicity in patients with idiopathic HES)

20. Eosinophils and airway remodeling Less subepithelial and interstitial collagen deposition in sensitized mice lacking eosinophils (Humbles et al., Science 305:1776, 2004)

21. Asthma Prevalence: >300 million worldwide 1/6 U.S. children Cost in U.S. > $6 billion/yr ing Developed > Nondeveloped Countries Hygiene Hypothesis Reflects developmental evolution of mucosal/regulatory immune system, possibly through effects on establishing the commensal flora ?Worms, ectoparasites, childhood infections

22. Asthma: an epidemic in the absence of infection… Inverse relationship between infections and immune disorders Bach JF. 2002. N Engl J Med. 347:911.

23. Asthma Triad 1. Reversible episodes airway obstruction ( mucus, eosinophils, T cells with IL-4, IL-5, IL-13, GM-CSF, etc.) 2. Chronic airway inflammation ( T cells, eosinophils with type 2 cytokines; chronic subepithelial, epithelial changes with mucus cell hyperplasia) 3. Bronchial hyperreactivity with provocative agents

24. The global epidemic of asthma Masoli et al., The global burden of asthma: executive summary of the GINA dissemination committee report. Allergy 59:469-78, 2004

25. A disease of persistent Th2-associated airway inflammation

26. Innate cytokines in Type 2 immunity 21 Kb Sullivan et al, Nature Immunology 2011: N. brasiliensis S. mansoni

27. Type 2 immunity as a confluence of innate and adaptive responses to epithelial insults… …and why no Tregs? (prominent in helminth infection) Locksley, Cell 140:777-783, 2010: ILC2 cells

28. Group 1: T-bet + IFNγ Group 2: GATA3 hi Group 3: RORγt + IL-5 IL-13 IL-17A IL-22 Ex-RORγt IEL ILC1 LTi NK-22 ILC1cNK IFNγ Natural helper cells Nuocytes Ih2 ILC1 ILC2 ILC3 Innate lymphoid cells, 2014… Th1 V Th2 V Th17 Th22 V V Ex- Th17 V

29. Type 2 immune responses Anti-helminth immunity Allergic pathology ?Treg homeostasis Tissue homeostasis Basal eosinophilopoiesis Mucosal integrity ?Organ repair Metabolic homeostasis White adipose tissue ?Beige adipose ?Adaptive thermogenesis Unexpected roles for ILC2s (and probably Th2s)…

30. ILC2s sustain resident eosinophils and AAMs needed for metabolic homeostasis Wu et al, Science 2011; Molofsky et al, J Exp Med 2013 Role for eosinophils and AAMs in brown fat (adaptive thermogenesis) and ‘beige’ fat consistent with role for ILC2s Nguyen et al, Nature 2011; Qui et al, Cell 2014; Rao et al, Cell 2014; Lee et al, Cell (in press)

31. ILC2s integrate diverse signals to dynamically regulate tissue microenvironments IL-25, IL-33, TSLP IL-13 VIP Neuropeptide transmitters IL-5 Circulating eosinophils AAMs Local eosinophils CysLTs IL-2, IL-7, IL-9, TL1A IL-9, GM-CSF Amphiregulin Cytokines Lipid mediators Treg homeostasis IFN-  AAM

32. Birds of a feather…

33. Eggs Pharynyx Do worms exploit type 2 immunity for their own metabolic and tissues needs? Chitin in egg shells and pharyngeal feeding tube

34. Moffat MM et al, NEJM 363:1211-21, 2012 Asthma GWAS Part of ILC2 / Th2 signature Asthma GWAS

35. Mouse 'Asthma' OVA X 3-5 BAL: Eos, Th2 Histology: Mucus Cell Hyperplasia, Inflammation (BALT’s) Physiology: Airway Hyperreactivity

36. Therapy of human asthma Approved in clinic Steroids and beta-agonists (long- and short-acting) - mainstay 5-LO inhibitors (LTA4 synthesis blockers) CysLTI antagonists (LTD4 blockers) Monoclonal humanized anti-IgE mAb (E25) Anti-IL-5 (decrease disease in high-eo subsets, usually steroid- resistant) Anti-IL-13 (in Th2-high signature asthmatics) In development/testing: anti-TSLP (clinical trial promising) Suplatast tosilate (suppresses IL-4, IL-5 from Th2 cells) CDP840, phosphodiesterase type 4 inhibitor IL-4 receptor, IL-4/13 inhibitors, anti-IL-5R OX40L blockade anti-TNF (steroid resistant asthma), IL-17 inhibitors anti-IL-33

37. Topics for discussion Asthma reaches prevalence levels of 8%-12% in some urban Westernized populations. Allergic asthma is predominantly a disease of childhood. Consider the impact of genetics, environment and development in constructing a model to explain the immunologic underpinnings explaining the increasing prevalence of asthma. How would you proceed to validate and/or intervene therapeutically based on your conclusions? Asthma is an integrated tissue response involving innate and adaptive cell cross- talk. Delineate the relevant cell types, the pathways believed to drive the tissue pathology, and identify shared ‘nodes’ of intervention designed to disrupt the cytokine/cellular network in order to restore homeostasis. Consider possible ‘off- target’ side-effects, even in other tissues.