Immunology of Upper Airway & Treatment of Allergic Rhinitis

1. Cummings Chapter 40: Immunology of the Upper Airway and Pathophysiology and Treatment of Allergic Rhinitis Chapter 41: Physiology of Olfaction December 3, 2014 Joshua Au

2. Key Points • Immune system divided between the innate and adaptive systems. • Review of innate and adaptive immunity • Allergic Rhinitis (AR) is a Type I, IgE dependent, mast cell-mediated immune reaction • AR impairs quality of life • Treatment of AR

3. Innate Immunity • Immediateforeign recognition via pattern recognition receptors • Barrier mechanisms - epithelial cell layers, mucus layers, epithelial cilia • Soluble proteins and small bioactive molecules that activate cells - cytokines, chemise, enzymes • Activated phagocytes - PMN, macrophages.

4. Toll Like Receptors • Identified first in drosophila. • Transmembrane proteins with extracellular repeated leucine rich motifs. Intracellular component similar to IL-1 receptor induces cell signaling. • Located on monocytes, B cells, T cells, and dendritic cells. • General specificity: Lipopolysaccharides (LPS) of Gram Negative bacteria activate TLR4. Microbial lipoproteins activate TLR2. TLR9 activated by bacterial DNA. TLR5 bacterial flagella. TLR3 ds- RNA - induces IFN-a IFNb for antiviral response. sRNA activate TLR7 TLR8.

5. Antimicrobial Peptides AMPS • Directly kill microbes • Cathelicidins - found in neutrophil granules - undergo extracellular proteolytic cleavage - release C-terminal peptide- LL-37. Antimicrobial to pseudomonas, E Choli, Staph. Chemoattractant for mast cells, PMN, monocytes, T cells, induce histamine release. • Defensins • alpha - expressed by PMN, nasal epithelial cells. Oxygen independent killing of phagocytes microorganisms, increase expression of TNF-a, and IL 1. • Beta - chemotactic, histamine release

6. Adaptive Immunity • Antigen specific receptors on T and B lymphocytes by gene rearrangement of T cell receptor and B cell receptors. Small number of cells with specificity, increases with proliferation • Self tolerance - recognize self and attach non self. Human leukocyte antigen complex and Major histocompatibility complex Chromosome 6. MHB class I (HLA-A, B, C) Class II (HLA-DR, DQ, DP).

7. MHC • MHC-I on most somatic cells - present endogenous antigens to cytotoxic T cells, allowing recognition of virus infected cells. When recognized by TCR - T cell proliferates and matures (CD8+). Killing only targeted cells. • MHCII expressed only on immunocompetent antigen presenting cells (APCs) - macrophages, monocytes, dendritic cells, B lymphocytes. Unregulated by IFN-gamma, present exogenous antigens to CD4+ T helper cells at sites of inflammation.

8. Cell Differentiation • Pluripotent stem cells to myeloid or lymphoid cells • Lymphoid cells - T cells (80%) TCR recognize APCs, thymus, B cells (10%) membrane bound immunoglobulin, bone marrow, NK cells large granular lymphocytes - no TCR or Ig, recognize virus infected or tumor cells with complex cell surface receptors (10%), bone marrow. • Myeloid - erythrocytes, neutrophils, monocytes, eosinophils, basophils, mast cells - differentiation in bone marrow.

9. T Cell Development • Leave bone marrow to reach thymus. Recombination of TCR gene segments of alpha, beta, gamma, delta. • Ligand dependent selections when bind peptide loaded MHC complex. If not bound they are eliminated by apoptosis, and those that react too strongly to self antigens also eliminated by apoptosis. Positive and negative selection.

10. T Cell Differentiation • CD4 T helper cells (65%) activate humoral (B cell) immunity and delayed type hypersensitivity. Two types: • TH1 - specialize in macrophage activation by IFN gamma production and contact dependent stimulation in delayed type hypersensitivity. directed by IFN generated by innate response • TH2 - Recognize MHC II, CD4 cell stimulate B cells by IL4, CD40 and CD40 ligand interaction. Important in IgE production and eosinophilic inflammation - allergy and helminth clearance. • CD8 cytotoxic T Cells (35%)- granzymes - apoptosis - and perforin - membrane disruption, targeted cell killing. Fas binding - target cell apoptosis (negative selection).

11. T Cell Differentiation • TH17 cells - inversely related to Th2 eosinophilic direction. Mobilize neutrophil recruitment. • Treg - suppress T cell responses, prevent autoimmunity. Develop in thymus high affinity for IL2 receptor (CD25).

12. B Cell Development • Develop in bone marrow. • Pro Bcell to Pre B cell noted by recombinationn of Mu heavy chain of Ig. Kappa or Lambda light chain development links with heavy chain to IgM at surface for immature B cell. • B cell response - provides humoral immunity from extracellular pathogens through production of antibodies, neutralize pathogens and toxins, facilitate opsonization, activate complement.

13. B Cell Function • IgM produced in absence of infection, lower affinity - first line defense against bacterial infection. Primary infection - production of high affinity specific Abs. • Dendritic cells present Ag in lymphoid organs via MHCII to Bcell • B cell migrates to T-B cell interface where they encounter primed TH cells binding CD40L (Tcell) to CD40 (Bcell)- activate B cell, promoting Ig class switch • Cytokine specific - IL10 -> IgG1, IgG3. IL 3, 13 -> IgE. TGF beta -> IgA, IFN gamma -> IgG2. • Activated B cells to follicle initiate germinal center and differentiate to Ig secreting plasma cells for 2-3 weeks. Some cells exit germinal centers and provide memory.

14. Other Cell Types • Natural Killer cells - large granular lymphocytes –not restricted to recognition of target cells by MHC. Inhibited by MHC I molecules presenting self antigens. • Kill cells that have down regulated MHC I expression - old or infected by viruses (avoid Tcell killing). • Monocytes and macrophages – APCs • Names depend on location - Langerhans cells epidermis, Kupffer cells liver, microglial cells CNS, dendritic cells in lymphatic tissue. • Express MHC I and MHCII recognition of antigen to TCR, Phagocytose microbes - facilitated by opsonization, coats foreign material with antibodies, phagocytose, vacillate infused with lysosomal enzymes which destroys with nitric oxide.

15. Other Cell Types • Neutrophils - 60% of leukocytes • Produce reactive oxygen species, phagocytosis, cytokines for inflammation. • Eosinophils - 2-5% leukocytes • Contains granules - major basic protein, eosinophil derived neurotoxin - against helminths. Stimulate histamine release. Induce bronchoconstriction, airway hyperreactivity. • Basophils and Mast cells • Cell surface IgEreceptors- Type Ihypersensitivity responses and response to helminths. Release histamine - cause tissue inflammation, edema, smooth muscle contraction. • Platelets -Produced from megakaryocytes.

16. Lymphoid Organs • Primary lymphoid organs • thymus and bone marrow - where lymphocytes differentiate and mature • Secondary lymphoid organs • mature lymphocytes reside and immune responses are generated • Systemic immune system- spleen and LNs • Mucosal immune system - Peyer’s patches, tonsil, intraepithelial lymphocytes.

17. Lymph Nodes • Receive antigens from blood and lymph • Cortex - B cells predominate in germinal centers expressing IgM or IgD.Ig class switching, development of memory B cells. • Parametrical region - contains T cells, macrophages, dendritic cells, B cells. • Medulla - center - Bcells, T cells, macrophages, plasma cells - secrete Ig to efferent lymphatics.

18. Mucosal Immune System • Tonsils - Adenoids, palatine tonsils, lingual tonsils • Develop in childhood and involute around puberty • Crypts maximize surface area exposure to pharynx. • Lobubles B cells; surrounding lymphoid tissue contian T cells, macrophages, dendritic cells, B cells. • Peyers Patches - jejunum and ileum mucosa facilitate Ag presentation from intestinal lumen to T and B cells. • Lamina propria beneath epithelium secrete IgA – neutralize toxins

19. Humoral Immune Response • T cell dependent • Follicular B cell presents MHC antigen to T cell that promotes class switching, Ig production, memory. • T cell independent • B cell response - Large antigens with repeating determinants like carbohydrates in capsules and cell walls of bacteria cause activation and secretion of IgM.

20. Immunoglobulins • Variable potion - heavy and light chain - binds to Ag. • Constant portion -heavy chain - responsible for effector function binding HC receptors and activate complement. • IgG- 75% monomer - fix complement, neutralization, opsonization, bacteriolysis, agglutination, hemolysis • IgM- 10% pentamer - earliest Ag receptor on B cells. early humoral phase. efficient complement fixing. replaced by IgG • IgA - 15% monomeric or dimeric. mucosal immunity - excreted. neutralize substances and prevent systemic access. • IgE0.004% monomer - effects mast cells, basophils causing immediate hypersensitivity reactions. • IgD0.2% monomer - membrane bound on B cell surfaces.

21. Complement • Cleavage of C3 • - C3a fragment - anaphylatoxin, mast cell degranulation, edema, phagocytic recruiment. • C3b binds antigen • assembles membrane attack complex (MAC - C6,7,8). • Deficiency - pyogenic infections • deficiency of serum inhibitor of C1 - mast cell independent angioedema.

22. Hypersensitivity Response • Type I - Mast cell mediated - IgE-dependent (anaphylaxis) or IgE-independent (iodine contrast) • Type II - cytotoxic antibody - IgG and IgM bind to Ag of target cell, destruction of cells - opsonization, complement, cell lysis. Autoimmune hemolytic anemia. • Type III - Immune complex - Ig and Ag form immune complex deposited in normal tissues, activate complement, inflammation, neutrophil influx, tissue injury – vasculitis, lupus, RA • Type IV - delayed hypersensitivity - T cell mediated, antibody independent. PPD.

23. Allergic Rhinitis • Clinical hypersensitivity of the nasal mucosa to foreign substances through IgE Ab. • Prevalence 10-20% affects 20-40 million • Genetics - Atopy predisposition to respond to environmental allergens with production of IgE occurs only in 13% in children without atopic parents, 29% in one atopic parents or sibling and 47% with both parents. • 4-6x more likely in pts with asthma • Sleep loss, learning, working deficiencies - productivity loss $593 per year. Health care costs $2-5 billion/yr.

24. Hygiene Hypothesis • Observation - Increasing allergy, autoimmune disease, insulin dependent diabetes in developed countries with concomitant decrease in infections due to vaccinations, antibiotics, hygiene. • Infection response: TH1 cytokines IL2 and IFN gamma, Allergy: TH2 cytokines IL4 and 5. Down reg of TH1 and upreg of TH2. • Decrease in antigenic stimulation from decreased infections decrease IL10 and decrease Treg activity which help to down regulate TH1 and TH2. So increased TH1 and Th2.

25. Pathophysiology of AR • Ag deposited on nasal mucosa engulfed by APCs and presented to helper T cells. Th2 CD4+ cells secrete IL4,5, 13 -> B cells produce IgE -> recruit eosinophils. • IgE bind mast cells and basophils and sensitize the nasal mucosa. Subsequent exposure - Membrane IgE receptors for Ag causing mast cells degranulation • Early response - pruritus, sneezing, rhinorrhea, nasal congestion. Increased mediators histamine, kinins, tryptase, PGD2, leukotriene C4 • Late response 4-10 hrs – histamine – nasal congestion

26. Evaluation and Diagnosis • Symptoms • Temporality – Seasonal, Perennial, Episodic; Intermittent vs Persistent • Severity • Physical Exam – MEE, erythema/edema of conjuntiva/NC; septal devation, CRS • Tests – Skin testing – Wheal <15min +IgE • Limitations - antihistamine , children not able to tolerate, eczema dermatographism preclude interpretation, systemic reactions may occur • Total IgE elevated 40% in AR. Less sensitive than skin testing

27. Therapy • Avoidance • Antihistamine – Sneezing, rhinorrhea, not congestion • Phenylephrine, Afrin congestion, rhinitis medicamentosa • Ipratroprium – anticholinergic – rhinorrhea • Mast cell stabilizers – Cromolyn, montelukast • Intranasal steroids – Most potent – Decreased TH2 • Efficacy after 8 hrs • Systemic steroids – refractory pts, side effects

28. Immunotherapy • Subcutaneous immunotherapy SCIT -effective in pollen allergy - rise in IgG, inc in IgG and IgA in nasal secretion, reduced basophil reactivity of basophils, reduced lymphocyte responsiveness to allergens, recuded inflammatory cells in nasal mucosa, shift from TH2 to TH1, spress seasonal rise in IgE. Onset of action is slow 12 weeks increasing over 1 year, requires compliance - multiple visits. Duration is 3-5 yrs. • Sublingual immunotherapy (SLIT) - extracts administered sublingually. Ease of administration - local side effects - itching and swelling of lips. Not approved by FDA.

29. COCLIA • R2 • 4.1.3 • What are the different types of hypersensitivity reactions? Give examples of each? • 4.1.4 • What are the different immunoglobulin types and what is their typical structure?

30. Hypersensitivity Response • Type I - Mast cell mediated - IgE-dependent (anaphylaxis) or IgE-independent (iodine contrast) • Type II - cytotoxic antibody - IgG and IgM bind to Ag of target cell, destruction of cells - opsonization, complement, cell lysis. Autoimmune hemolytic anemia. • Type III - Immune complex - Ig and Ag form immune complex deposited in normal tissues, activate complement, inflammation, neutrophil influx, tissue injury – vasculitis, lupus, RA • Type IV - delayed hypersensitivity - T cell mediated, antibody independent. PPD.

31. Immunoglobulins • Variable potion - heavy and light chain - binds to Ag. • Constant portion -heavy chain - responsible for effector function binding HC receptors and activate complement. • IgG- 75% monomer - fix complement, neutralization, opsonization, bacteriolysis, agglutination, hemolysis • IgM- 10% pentamer - earliest Ag receptor on B cells. early humoral phase. efficient complement fixing. replaced by IgG • IgA - 15% monomeric or dimeric. mucosal immunity - excreted. neutralize substances and prevent systemic access. • IgE0.004% monomer - effects mast cells, basophils causing immediate hypersensitivity reactions. • IgD0.2% monomer - membrane bound on B cell surfaces.

32. COCLIA • R3 • 4.1.6 • What are medical therapy options for allergic rhinitis (different classes of medications and their effects)? • 4.1.7 • What are the common allergens and the times of year (seasonal vs. perennial) that patients are most affected?

33. Therapy • Avoidance • Antihistamine – Sneezing, rhinorrhea, not congestion • Phenylephrine, Afrin congestion, rhinitis medicamentosa • Ipratroprium – anticholinergic – rhinorrhea • Mast cell stabilizers – Cromolyn, montelukast • Intranasal steroids – Most potent – Decreased TH2 • Efficacy after 8 hrs • Systemic steroids – refractory pts, side effects

34. Allergens

35. COCLIA • R4 • 4.1.12 • Review humoral vs. cell-mediated immunity. What are natural killer cells? • B cell/Ab vs T cell • Natural Killer cells - large granular lymphocytes –not restricted to recognition of target cells by MHC. Inhibited by MHC I molecules presenting self antigens. • Kill cells that have down regulated MHC I expression - old or infected by viruses (avoid Tcell killing).

36. COCLIA • 4.1.16 • Describe a typical history and physical findings in a patient with allergic rhinitis.

37. Evaluation and Diagnosis • Symptoms • Temporality – Seasonal, Perennial, Episodic; Intermittent vs Persistent • Severity • Physical Exam – MEE, erythema/edema of conjuntiva/NC; septal devation, CRS • Tests – Skin testing – Wheal <15min +IgE • Limitations - antihistamine , children not able to tolerate, eczema dermatographism preclude interpretation, systemic reactions may occur • Total IgE elevated 40% in AR. Less sensitive than skin testing

38. COCLIA • R5 • 4.1.18 • Educate us on immunotherapy…SET vs. RAST based treatment. • 4.1.17 • Describe MQT and how it is used.

39. SET vs RAST • Skin Endpoint Titration (SET) aka Intradermal Dilutional Testing (IDT) • Sequential administration of fivefold diluted antigens • Wheal >7mm in <10min, positive • Low systemic response, in office, reproducible, high cost • Radioallergosorbent Test (RAST) React serum with serios of known allergens, radiolabeled anti-IgE identifies specific antigen-IgE complexes (older) • Takes weeks, more expensive than skin

40. MQT – Modified Quantitative Testing Highly correlated with IDT, lower cost, less time.

41. Cummings Chapter 41: Physiology of Olfaction December 3, 2014 Joshua Au

42. Key Points • Olfactory epithelium – peudostratified columnar neuroepithelium with supporting cells, bipolar olfactory receptor neurons. • Receptor neurons coalesce into bundles CNI through the cribriform plate to olfactory bulb • Most common reasons for olfactory loss are CRS, polyps, URI, head trauma, neurologic disorders. • Treatment for olfactory disorders are limited.

43. Anatomy of Olfaction • Nasal cavity airflow – 50% middle meatus, 35% inferior meatus, 15% olfactory region. • Odorant molecules are trapped by mucus • Bipolar Neuron at posterior superior NC activated by stimuli through dendrites and cilia. Synapse to olfactory bulb at skull base. • Olfactory epithelium 1cm2 bilaterally of pseudostratified columnar epithelium on a vascular lamina propria. • Four cell types: ciliated olfactory receptors, microvillar cells, supporting (sustentacular) cells, basal cells.

44. Neural Anatomy of Olfaction • Olfactory Bulb – frontal cortex. Primary nerves synapses at glomeruli. Possible mapping of the bulb into spatial pattern to the glomerular activation elicited by odorants. • Olfactory connections in the brain – Central connections between the olfactory tubercle, amygdala, hypothalamus – connections between food intake, temperature regulation, sleep, vision, memory, taste. • Common Chemical sense – Free nerve endings of trigeminal, glossopharyngeal, vagus provide additional chemoreceptivity to the respiratory tract.

45. Olfactory Transduction and Coding • Odorant molecule dissolved in mucus, bound by soluble binding proteins, esp hydrophobic molecules; bind to receptor proteins on surface olfactory cilia. cAMP and IP3 pathways transduce signal to depolarize the cell. Electrical signal to olfactory bulb • Adrian’s Proposal • Olfactory Odor Map – Four zones – each contains different olfactory receptor subtypes • Odor memory 1 yr vs visual memory 5 mo.

46. Stimulation and Measurement of Olfaction • Detection threshold – most dilute concentration of particular odorant detectable. • Test restest reliability low. • Identification test – name correctly. Suprathreshold test • Simple detection test – smell alcohol pad • Objective tests • Electro-olfactogram – electrode on olfactory epithelium detect negative voltage shift with odorant stimulation • Brain evoked potentials – summated percutaneous brain electrical activity averaged after exposure to odorant.

47. Factors affecting Olfaction • Obstruction • Head Trauma • Aging • Congenital Disfuction • Toxic Exposures • Neoplasm • Neurologic disorders • Surgery

48. Diagnosis and Management • Dx – H&P, endoscopy, radiology. Biopsy considered research tools. • Management – at level of disruption – obstruction treatable. Other non-anatomic etiologies have no proven therapy. • Reassure patient – regarding food, appreciate in other ways than taste – texture, color. Safety to gases.

49. COCLIA • R2 4.3.1Describe the basic physiology of olfaction. • 4.3.6What is the differential diagnosis of anosmia? • R3 4.3.5What is the alcohol sniff test? • R4 4.3.3What is the mechanism by which individual odorants chemicals are encoded as distinct? • R5 4.3.12How does septoplasty and turbinate reduction influence olfaction?

50. 4.3.1 Describe the basic physiology of olfaction. • Odorant molecule dissolved in mucus, bound by soluble binding proteins, esp hydrophobic molecules; bind to receptor proteins on surface olfactory cilia. cAMP and IP3 pathways transduce signal to depolarize the cell. Electrical signal to olfactory bulb