1. Microbes and Allergic Disease Cathryn Nagler-Anderson Massachusetts General Hospital Harvard Medical School

2. 1. Examine what we know about how the body discriminates between harmful pathogens and innocuous foreign substances (antigens). 2. Introduce the mucosal immune system, the portal through which most foreign substances and microbes enter the body. 3. Present some work from our laboratory on the influence of intestinal microbes on allergic disease.

3. There are two arms to the immune response to any foreign substance (antigen) entering the body: the innate immune response the adaptive immune response

4. Innate Immune Response rapidly deployed - first line defense encoded in genome - conserved from flies to humans broad recognition of microbial“patterns” no memory of past encounter with antigen

5. Adaptive Immune Response exquisitely specific recognition of millions of different potential antigens requires gene rearrangement to generate antigen specific receptors long-term memory of past antigen encounter

6. Pattern recognition receptors (PRR) of the innate immune system see pathogen associated molecular patterns (PAMPS) PAMPS are typically highly conserved, essential components of microbial structure or metabolism

7. Based on Nat. Rev. Immunol. 2001; 1: 135 The Toll-like receptor (TLR) family of Pattern Recognition Receptors TLR-6 TLR-9 MD-2 CD14 TLR-4 TLR-2 TLR-5 TLR-3 TLR-1 Peptidoglycan (G+) Lipoprotein Lipoarabinomannans Zymosan (Yeast) GPI protein LPS(G-) RSV F dsRNA Flagellin Bacterial CpG DNA

8. Antigen specific receptors on T cells and B cells characterize the adaptive immune response. T cells - cell mediated immunity B cells - antibody mediated (humoral) immunity

9. T cells see peptide fragments of antigen presented by MHC proteins on antigen presenting cells Antigen presenting cell (APC) MHC T cell T cell receptor

10. Two signals are required for T cell activation Costimulatory molecules on APC provide the second signal MHC TCR

11. Adjuvant - a substance that activates the innate immune system to produce signals required for an adaptive immune response Adjuvants potentiate T cell immunity by: 1. Enhancing expansion of antigen specific T cells 2. Altering antigen presentation (upregulation of costimulation). 3. Influencing differentiation of T cell subsets.

12. Adjuvants

13. Pathogens IL-12, IL-18 TH1 APC T TLRs Antigen presentation Costimulatory molecules IFN-  Antigen presentation without activation of the innate immune system leads to non-responsiveness (anergy) APC T TLRs Antigen presentation Costimulatory molecules Soluble Proteins Anergy IL-12, IL-18 Cathryn Nagler- Anderson:

14. Th2Th1 IL-2 IFN-  TNF cell mediated responses IL-4 IL-5 IL-13 help antibody responses CD4 + T helper cells

15. Th 1 Th 2 Gram - negative bacteria Helminthic parasites Degree of polarization Th1 Th2 Bacteria (tuberculosis) Viruses (measles) Helminthic Parasites (worms) Most immune responses have both a Th1 and Th2 component. The T cell response to some infectious agents becomes polarized to one Th subset.

16. Work in our laboratory has examined the influence of two types of microbes on the development of food allergy Intestinal worms (helminths) Intestinal (commensal) bacteria

17. Antigens enter through mucosal surfaces Right subclavian vein Superior vena cava Liver Stomach Mesenteric nodes Peyer’s patches Appendix Trachea Left subclavian vein Mediastinal lymph nodes Lungs Heart Spleen Thoracic duct Large intestine Small intestine Oesophagus

18. Gut-associated lymphoid tissue Peyer’s patch Villus epithelium

19. The induction of non- responsiveness to orally administered antigens (including food) is called oral tolerance.

20. Microbes living in the gut influence the immune response to a food antigen through their interactions with the innate and adaptive immune system.

21. Life cycle of an intestinal worm, Heligmosomoides polygyrus L1 L2 L3 Small Intestine L4 9 - 11 Days wall Lumen Adult Eggs 24 - 36 h 2 - 6 Days

22. H. polygyrus establishes a chronic, non- invasive niche in the intestinal lumen From Telford et al, Parasite Immunol. 1998: 20, 601

23. 0 1000 2000 3000 4000 5000 6000 IL-5 PBS fed OVA fed PBS fed OVA fed Non-infected Infected Oral Ag primes for a Th2 response in helminth infected mice 0 500 1000 1500 2000 pg/ml (+SE) IFN  PBS fed OVA fed PBS fed OVA fed Non-infected Infected Shi et al, J. Immunol. 1998, 160: 2449 ( Th1 ) ( Th2 )

24. Response to different forms of antigen presented to the GALT leads to systemic nonresponsiveness or immunity Antigen specific reduction in proliferative capacity Antigen specific clonal expansion 1st Ag challenge T cell proliferation and activation Subsequent challenges Profound reduction in proliferative capacity; functional non-responsiveness due to failure to clonally expand antigen specific clones and/or cell death Clonal expansion enhanced by each antigen challenge leads to productive immune response Soluble Ag Ag + adjuvant, microbes

25. Allergic disease (Th2) Helminth Infection (Th2) Microbial Infection (Th1) The relationship between infection and allergic disease In developed countries: High incidence of allergic disease Low incidence of infectious diseases (Th1) Low infection with worms (helminths) In developing countries: Low incidence of allergic disease High incidence of infectious diseases (Th1) High infection with worms (helminths)

26. Pathogens IL-12, IL-18 TH1 APC T TLRs Antigen presentation Costimulatory molecules IFN-  Helminths, Allergens IL-4, IL-10 ? TH2 APC T TLRs Antigen presentation Costimulatory molecules IL-4, IL,5 IL-13 ? ? ? IFN-  IL-4 TLR signaling regulates the development of Th1 and Th2 cells

27. The Hygiene Hypothesis Allergy and asthma have increased during the last 20-50 years due to reduced exposure to childhood bacterial and viral infections brought about by improvements in vaccination and sanitation. In the absence of these Th1 polarizing stimuli mucosal immune responses fail to overcome their inherent Th2 bias and become slanted in the direction of allergy.

28. (N. Engl. J. Med. 2001, 344: 30) Does helminth infection induce an allergic response? Interleukin-13

29. A model for food allergy An allergic response to a food antigen (peanut) can be induced in C3H/HeJ mice by repeated oral administration of peanut plus a mucosal adjuvant (cholera toxin) An allergic response is measured by: 1. Symptoms of anaphylaxis (itching,wheezing, labored respiration, convulsions) 2. Elevated levels of plasma histamine 3. Induction of peanut specific IgE

30. Peanut IgE ng/ml 0 100 200 300 400 500 0 2500 5000 7500 10000 ∆ - Histamine ng/ml PN/HP PBSPBS/CT PNPN/CT HP/PBS Helminth infection (HP) does not induce an allergic response to a food antigen Bashir et al J. Immunol. 2002, 169:3284

31. 0 250 500 750 1000 PN/CT PN HP/PN/CT HP/PBS PBS/CT Peanut IgE ng/ml 0 1000 2000 3000 4000 ∆ - Histamine ng/ml Helminth infection protects against peanut allergy Bashir et al J. Immunol. 2002, 169:3284

32. Counter-regulation hypothesis TH1 TH2 Organ specific auto- immune disease Allergic disease INFLAMMATION IL-10 (TGF-  ) Inhibitory Adapted from Wills-Karp, et al Nat.Rev.Immuno.1, 2001:69 Helminths Viruses Bacteria Protozoa Commensal gut Microflora Commensal gut Microflora Protozoa Viruses Bacteria CD4 + regulatory T cells CD8 + T cells B cells Dendritic cells Monocytes/ macrophages Epithelial cells

33. PN-IgE ng/ml  - Histamine C3H/HeJ C3HeB/FeJ C3H/HeOuJ CBA/J 0 2500 5000 7500 10000 0 1000 2000 3000 Mice lacking TLR-4, the receptor for bacterial LPS, are highly susceptible to an allergic response to peanut plus CT

34. PN-IgE ng/ml C3H/HeJ C3HeB/FeJ FeJ + Antibiotics  - Histamine 0 1000 2000 3000 0 1000 2000 3000 Removal of gut bacteria by antibiotic treatment induces an allergic response to peanut in TLR-4 positive mice

35. T T B B T T B B B B T Immature (or mature?) DC in GALT (PP, MLN?) Tr1 Th3 IL-10 TGF-  IL-10 TLR-4 IFN-  IgE Allergy is induced in the absence of TLR-4 signaling by luminal bacteria