Mast Cell Disorders: from Infancy to Maturity

The University of Manchester Research

DOI: 10.1111/all.13657

Document Version Accepted author manuscript

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Citation for published version (APA): Wilcock, A., Bahri, R., Bulfone-paus, S., & Arkwright, P. D. (2018). Mast Cell Disorders: from Infancy to Maturity. Allergy. https://doi.org/10.1111/all.13657

Published in: Allergy

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Download date:05. Oct. 2021 Allergy

Mast Cell Disorders: from Infancy to Maturity

Journal: Allergy

Manuscript ID ALL-2018-00808.R1

Wiley - Manuscript type: Review

Date Submitted by Forthe Peer Review n/a Author:

Complete List of Authors: Wilcock, Amy; University of Manchester, Lydia Becker Institute of Immunology and Inflammation Bahri, Rajia; University of Manchester, Lydia Becker Institute for Immunology and Inflammation Bulfone-Paus, Silvia; University of Manchester, Lydia Becker Institute for Immunology and Inflammation Arkwright, Peter; University of Manchester, Lydia Becker Institute for Immunology and Inflammation

Keywords: mast cells, clinical immunology, genetics

Page 1 of 84 Allergy

1 2 3 ALL-2018-00808 Mast Cell Disorders: from Infancy to Maturity 4 Point-by-point response to reviewer’s comments 5 6 7 The authors would like to thank the reviewer and Editor for their encouraging comments and 8 suggestions regarding our review article. Our detailed point-by-point responses are detailed below. 9 10 Reviewer #1: 11 No comments were received from this reviewer. 12 13 Reviewer #2: 14 I enjoyed reading this manuscript and believe it is a timely and comprehensive review of the subject. I 15 16 do have some queries which I believe require amendment prior to publication. Specifically: 17 18 Comment 1: Page 5, lines 49 to 52. Insect venom sensitivity is known to be associated with an increased 19 prevalence of mastocytosis.For The paper Peer referred to (Zanotti,Review ref 38) has 73% of patients in a selected 20 group with severe anaphylaxis diagnosed as mastocytosis in the absence of a raised baseline mast cell 21 tryptase. Other reports suggest the prevalence is substantially less (eg Bonadonnas 2018 paper). Given 22 this has significant clinical consequences I suggest moderating this statement with the work of 23 Kristensen (Allergy 2017) among others. By convention bone marrow examination is not usually 24 25 recommended where mast cell tryptase is less than 20 (or 11.5 in those with severe anaphylaxis or other 26 features). 27 Response: Thank you for your comments. The text has been amended, taking into account your 28 suggestions. Additional references have been added (Kristensen et al, 2017; Bonadonna & Scaffidi, 29 2018). 30 31 Comment 2: Page 6, Line 39ff. This classification makes no mention of mastocytosis in the skin which is 32 part of the continuum of mastocytosis and is evident in adults. Urticaria Pigmentosa is a pigmented 33 lesion which can urticate. By contrast the description given in this paper is more consistent with Darier’s 34 35 sign. (Hartman et al, JACI 2016) 36 Response: A statement highlighting the fact that urticaria pigmentosa can also occur in adults had been 37 added to the text as requested. 38 39 Comment 3: Page 7, line 12. Mast Cell Activation Syndrome is a disease where its characteristics are yet 40 to be fully defined. This should be mentioned. 41 Response: The authors very much agree with the reviewer and this point has now been added to the 42 text. 43 44 45 Comment 4: Page 9, line 34: Asthma. Evidence that mast cells are involved in asthma is protean. Since 46 imatinib has many effects, it may convey these in asthma by many other pathways – such as eosinophils 47 - than mast cells. But evidence that mast cells are important goes back to the early studies with 48 omalizumab. More recently you may wish to consider Andersson et al Clin Exp Allergy. 2018 and the 49 paper by Hinks and colleagues in Lancet 2015. 50 Response: Thank you for your comment. Reference to the Andersson study has now been added as 51 reference number 80. As Hinks Lancet reference was just a poster abstract, but the authors published 52 53 their full findings in JACI a few months later and this reference (number 82) has now also been included, 54 with pertinent comments added to the text. 55 56 57 58 59 60 Allergy Page 2 of 84

1 2 3 Comment 5: Page 13 line 45. The role of cardiac mast cells should be mentioned here as significant 4 myocardial damage can cause elevated mast cell tryptase 5 6 Response: The effect of mast cells on the myocardium are quite complex. Both damaging and protective 7 effects have been documented depending on the disease model. This has been highlighted and a 8 number of additional references added. 9 10 Comment 6: Table 1: Cutaneous mastocytosis is not just found in children. Mast cell activation 11 syndrome: whilst TPSAB1 has been found in some cases, this is not found in all: this needs some 12 qualification. 13 Response: The text of this table has been refined as recommended. 14 15 16 Additional response: Reference numbering and citations have been updated. 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 3 of 84 Allergy

1 2 3 4 1 Mast Cell Disorders: from Infancy to Maturity 5 6 7 2 A. Wilcock, R. Bahri, S. Bulfone-Paus, P. D. Arkwright 8 9 10 3 Lydia Becker Institute of Immunology and Inflammation, University of Manchester, United 11 12 13 4 Kingdom 14 15 16 5 17 18 19 6 Correspondence Dr PeterFor Arkwright, Peer Department Review of Paediatric Allergy and Immunology, Royal 20 21 7 Manchester Children’s Hospital, Oxford Road, Manchester, M13 9WL, United Kingdom, email 22 23 24 8 address: [email protected], telephone: + 44 161 701 0678 25 26 27 9 Keywords: mast cell, mastocytosis, tryptase, gene duplication, pediatrics, asthma, inflammatory 28 29 10 bowel disease 30 31 32 11 Abbreviations: BPD bronchopulmonary dysplasia, CD Crohn’s disease, CM cutaneous 33 34 12 mastocytosis, CMP common myeloid precursor, CSU chronic spontaneous urticaria, HIE 35 36 37 13 hypoxic ischemic encephalopathy, HSC hematopoietic stem cell, IBD inflammatory bowel 38 39 14 disease, MCAS mast cell activation syndrome, MC – mast cell, MCp mast cell precursor, 40 41 15 RDBPCT randomized double-blind, placebo-controlled trial, ROP retinopathy of prematurity, 42 43 44 16 SIDS sudden infant death syndrome, TRPA1 transient receptor potential ankyrin 1, UC 45 46 17 ulcerative colitis 47 48 49 18 Author contributions Drs Wilcock and Arkwright initiated and produced the first draft of the 50 51 19 manuscript, which was then critically refined and reviewed by all authors prior to submission. 52 53 54 Competing Interests None declared. 55 20 56 57 58 59 60 Allergy Page 4 of 84

1 2 3 21 Funding No funding was received for this review article. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 5 of 84 Allergy

1 2 3 22 ABSTRACT 4 5 6 23 Mast cells are typically linked to immediate hypersensitivity and anaphylaxis. This review looks 7 8 9 24 beyond this narrow role, focusing on how these cells have evolved and diversified via natural 10 11 25 selection promoting serine protease gene duplication, augmenting their innate host defence 12 13 26 function against helminths and snake envenomation. Plasticity of mast cell genes has come at a 14 15 16 27 price. Somatic activating mutations in the mast cell growth factor KIT gene causes cutaneous 17 18 28 mastocytosis in young children, and systemic mastocytosis with a more guarded prognosis in 19 For Peer Review 20 29 adults who may also harbor other gene mutations with oncogenic potential as they age. Allelic 21 22 23 30 TPSAB1 gene duplication associated with higher basal mast cell tryptase is possibly one of the 24 25 31 commonest autosomal dominantly inherited multi-system diseases affecting the skin, 26 27 32 gastrointestinal tract, circulation, musculoskeletal system and affect. 28 29 30 33 Mast cells are also establishing a new-found importance in severe asthma, and in 31 32 33 34 remodelling of blood vessels in cancer and atherosclerotic vascular disease. Furthermore, recent 34 35 35 evidence suggests that mast cells sense changes in oxygen tension, particularly in neonates, and 36 37 36 that subsequent degranulation may contribute to common lung, eye and brain diseases of 38 39 37 prematurity classically associated with hypoxic insults. One hundred and forty years since Paul 40 41 42 38 Ehrlich’s initial description of “mastzellen”, this review collates and highlights the complex and 43 44 39 diverse roles that mast cells play in health and disease. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 6 of 84

1 2 3 40 1 FOREWORD 4 5 6 7 41 Scientists and clinicians alike recognize allergen-induced, IgE-dependent mast cell (MC) 8 9 42 degranulation leading to immediate hypersensitivity reactions and anaphylaxis as the sine qua 10 11 43 non of MC dysfunction. This aspect of MC dysfunction has been extensively explored in 12 13 14 44 previous original research articles and reviews and will not be covered here. The aim of this 15 16 45 review is to summarize our current knowledge of primary and secondary MC disorders focusing 17 18 46 on those not associated with acute allergic reactions, thus providing the reader with a broader and 19 For Peer Review 20 21 47 more complete understanding of the role of this immune cell in health and disease. It also aims to 22 23 48 highlight how an appreciation of MC dysfunction from early infancy to old age can improve our 24 25 49 understanding of this broad and complex range of disorders. Diversification of MC function 26 27 50 coincided with extensive duplication of tryptase and other serine protease genes.1 Gene 28 29 30 51 duplication resulting in archetypal membrane-bound proteases being secreted into the milieu led 31 32 52 to their systemic effects, and may have been promoted by a survival advantage, with secreted 33 34 53 MC proteases rapidly degrading and neutralizing potentially deadly Hymenoptera and snake 35 36 2,3 37 54 toxins. 38 39 40 55 2 WHAT ARE MAST CELLS? 41 42 43 56 MCs first evolved 500 million years ago in Ascidians (sea squirts), providing host innate 44 45 4 46 57 immunity against bacteria and parasites. Over the millennia, MCs gained additional functions 47 48 58 regulating inflammation, wound healing, coagulation, adaptive immunity and acute allergic 49 50 59 responses.5,6 MCs are non-proliferating long-lived sedentary immune tissue cells. They 51 52 60 differentiate from common myeloid precursors (CMPs) in the bone marrow. Immature MC 53 54 55 61 precursors (MCp) with proliferative potential leave the bone marrow to home in on epithelial 56 57 58 59 60 Page 7 of 84 Allergy

1 2 3 62 tissues in contact with the external environment such as the skin, respiratory and gastrointestinal 4 5 7-10 6 63 tracts via specific integrin and chemokine receptors (Figures 1 & 2). They also lodge in 7 8 64 perivascular spaces and connective tissues surrounding nerves and then terminally differentiate 9 10 65 into non-proliferating mature MCs expressing secretory granules.11 11 12 13 66 When activated, MCs release inflammatory mediators from their storage granules 14 15 16 67 (histamine, chymotrypsin-related serine proteases such as tryptase, chymase, carboxypeptidase), 17 18 68 as well as via phospholipid membrane metabolism (platelet activating factor, leukotrienes, 19 For Peer Review 20 69 prostaglandins) and after de novo synthesis (cytokines (TNF, IL-4) and chemokines (IL-8, 21 22 12,13 23 70 monocyte chemoattractant protein 1 (MCP-1)). Mature human MCs are classically divided 24 25 71 into two subpopulations. MCTC expressing tryptase, chymase, carboxypeptidase and cathepsin, 26 27 72 predominate in connective tissue and skin. In contrast, MCT expressing tryptase but no other 28 29 14,15 30 73 serine proteases, predominate in healthy lung parenchyma and gut mucosa. Inflammatory 31 32 74 cytokines such as IL-4 can alter the balance of MCT and MCTC in the lungs, leading to a 33 34 16-18 75 predominance of MCTC in asthmatics. These observations suggest a degree of plasticity and 35 36 76 interconversion between these two MC subtypes depending in the microenvironment (Figure 2). 37 38 39 40 77 Recent studies have demonstrated functional and potentially important clinical 41 42 78 differences between these MC subgroups. MCT are activated by cross-linking of surface FcRI 43 44 79 leading to classical IgE mediated hypersensitivity reactions. In contrast, MC express high 45 TC 46 47 80 levels of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which activate 48 49 81 cytoplasmic calcium release via a phospholipase C pathway.19 MRGPRX2 can be activated by 50 51 82 endogenous peptides such as substance P, anaphylatoxins C3a and C5a, and VIP, as well as 52 53 20-23 54 83 drugs such as morphine, vancomycin, sulfamethoxazole and cisatracurium. Activation of 55 56 84 MRGPRX2 on MCTC induces a rapid but less extensive release of granule contents characteristic 57 58 59 60 Allergy Page 8 of 84

1 2 3 85 of anaphylactoid reactions.19 MRGPRX2 activation may also be important in chronic 4 5 24 6 86 spontaneous urticaria. In addition, chymase released from MCTC directly converts angiotensin I 7 8 87 to angiotensin II, possibly contributing to vascular remodelling in asthma, atherosclerosis and 9 10 88 aortic aneurysms.25-27 11 12 13 14 89 3 PRIMARY MAST CELL DISORDERS 15 16 17 90 Primary MC disorders can broadly be divided into two groups (Table I, Figure 3).28 Mastocytosis 18 19 91 is the clonal proliferation Forof MCs, usuallyPeer due to Review a sporadic somatic activating mutation in the 20 21 29,30 22 92 MC growth factor receptor c-kit/CD117. Activating KIT mutations may not only promote 23 24 93 MC proliferation and survival, but also make MCs more sensitive to degranulation.31,32 Primary 25 26 94 MC activation syndrome (MCAS) is characterized by exaggerated release of MC granule 27 28 33 29 95 contents without evidence of clonal proliferation. Recently findings suggest that in many cases, 30 31 96 MCAS may be due to excessive allelic gene duplication, particularly of the -tryptase gene 32 33 97 TPSAB1.34,35 34 35 36 37 98 3.1 Mastocytosis - clonal MC proliferation 38 39 40 99 Mastocytosis is a rare, usually sporadic clonal disease of MCs. A family history is present in 41 42 100 only 4% of patients. MCs may accumulate in the bone marrow, spleen, skin, and/or 43 44 101 gastrointestinal tract. Adults typically present with raised MC tryptase, flushing, abdominal pain, 45 46 47 102 diarrhoea, muscle aches and pains, osteoporosis, hypotensive episodes, neurological and 48 49 103 psychiatric disturbances (systemic mastocytosis). Urticaria pigmentosa characterized by 50 51 104 pigmented brown patches which urticate, particularly after rubbing the skin or changes in 52 53 105 temperature, although more common in young children is also part of the clinical disease 54 55 56 106 spectrum in adults. In 90% of cases, this is associated with somatic D816V activating mutations 57 58 59 60 Page 9 of 84 Allergy

1 2 3 107 of the MC growth factor receptor KIT gene in bone marrow MC progenitors.30 The clinical 4 5 36 6 108 course ranges from indolent to malignant (acute or chronic MC leukemia, MC sarcoma). 7 8 109 Adults with Hymenoptera venom anaphylaxis should also be investigated for systemic 9 10 110 mastocytosis, even if baseline MC tryptase is normal and there are no skin signs.37 The reported 11 12 prevalence of underlying clonal mast cell disease in venom allergy varies greatly from 7% to 13 111 14 15 112 94%, but is higher in patients suffering from hypotension and syncope,38,39 Some series suggest 16 17 113 the prevalence may be as high as 77%.38 Adults with mastocytosis who are venom allergic may 18 19 114 require life-long venom immunotherapyFor Peer as and alsoReview in adults suffering from anaphylactic 20 21 39 22 115 reactions have been reported after discontinuation of immunotherapy. The latter observation . 23 24 116 suggests that these patients may require life-long venom immunotherapy.40 Although bone 25 26 117 marrow examination is conventionally not recommended for patients where their MC tryptase 27 28 29 118 mast cell tryptase is less than 20ng/ml (or 11.5ng/ml in those with severe anaphylaxis or other 30 31 119 features suggestive of mastocytosis), normal MC tryptase does not exclude the diagnosis. KIT 32 33 120 D816V mutations has been detected in the blood of 92% of adults with Hymenoptera venom- 34 35 induced anaphylaxis, reducing the need for more invasive tests in some patients.41 36 121 37 38 39 122 MC leukemias (90% of which are aleukemic) and sarcomas (30% of which progress to 40 41 123 MC leukemia) makes up <1% of patients with systemic mastocytosis, stain for CD117 and 42 43 124 tryptase, and are often associated with additional mutations in PDGFR, FIPIL1, TET2, SRSF2, 44 45 402,413 46 125 ASXL2 or K/N-RAS genes. MC cancers usually present with fever, weight loss, cytopenias, 47 48 126 bone pain and hepatosplenomegaly (c-findings). Absence of symptoms and signs of organ 49 50 127 damage (c-findings) is associated with a more stable clinical course and better prognosis.424 51 52 128 Serum MC tryptase is usually >200ng/ml. Response to radiotherapy and chemotherapy is usually 53 54 55 129 transient and the prognosis is poor with median survival of less than two years. Chemotherapy 56 57 58 59 60 Allergy Page 10 of 84

1 2 3 130 combined with midostaurin (a multi-tyrosine kinase inhibitor) and followed by hematopoietic 4 5 6 131 stem cell transplantation may be curative, but due to the rarity of these cancers definitive data on 7 8 132 the efficacy of this approach is limited. The pathogenesis and clinical disease spectrum of 9 10 133 systemic mastocytosis parallels sporadic JAK2V617F gain of function mutations known to drive 11 12 adult-onset myeloproliferative disorders.435 Systemic mastocytosis with activating KIT mutations 13 134 14 15 135 in bone marrow MCs is rare in children. 16 17 18 136 Children usually present with cutaneous rather than systemic mastocytosis (CM). Up to 19 For Peer Review 20 137 ninety percent have benign MC aggregates due to somatic KIT mutations confined to the 21 22 446,457 23 138 skin. The common D816V is only found in 34 – 42% of cases. In one study an additional 24 25 139 44% of patients had gene mutations outside exon 17.468 Seventy-five percent have multiple 26 27 140 brown skin patches. In a quarter, the rash is noticed at birth and in 90% by the age of 2 years 28 29 141 old.479-502 Patches urticate on rubbing or bathing (urticaria pigmentosa) (Figure 4). CM can also 30 31 32 142 present as one or more circumscribed reddish skin lesions (mastocytoma, 20%) or as a diffuse 33 34 143 rash (5%). In two thirds of children the disease regresses by puberty. NSAIDs, opiates, muscle 35 36 144 relaxants, insect stings and physical factors such as temperature changes with bathing or 37 38 39 145 swimming can trigger urtication and rarely hypotension. Treatment with second or third 40 41 146 generation antihistamines is usually effective. The risk of anaphylaxis after insect venom stings 42 43 147 in children with CM is currently considered very low.513,524 Prescription of an adrenaline auto- 44 45 535 46 148 injector is left to individual specialist, rather than being a mandatory requirement. 47 48 49 149 3.2 MC activation syndrome (MCAS) including -tryptasemia 50 51 52 150 Primary MCAS is characterized by excessive activation of MCs, and although its characteristics 53 54 151 are yet to be fully defined, diagnosis is based on clinical features and increased MC tryptase, 55 56 57 58 59 60 Page 11 of 84 Allergy

1 2 3 152 where mastocytosis and secondary allergic triggers have been excluded.333 Histamine and 4 5 546 6 153 prostaglandins may also be elevated, but further work is required before definitive 7 8 154 recommendations can be provided regarding use of these measurement in the diagnosis of 9 10 155 MCAS.557 11 12 13 156 -tryptase gene allele frequency varies between individuals. Twenty-nine percent of 14 15 568 16 157 people (up to 45% in white Europeans) express no α-tryptase genes. Five percent of 17 18 158 individuals are thought to have allelic TPSAB1 gene duplications inherited in an autosomal 19 For Peer Review 20 159 dominant pattern. Adults and children with TPSAB1 gene replications have serum MC tryptase 21 22 34 23 160 ≥8.0 ng/ml and clinical features of MCAS. These include flushing and angioedema, abdominal 24 25 161 pain, diarrhea and food intolerances, joint hypermobility, systemic reaction to Hymenoptera, 26 27 162 hypotensive episodes and autonomic dysfunction. Triplications and even quintuplications are 28 29 30 163 less common, associated with even higher serum MC tryptase concentration and more severe 31 32 164 disease (Table I).34,35 Serum MC tryptase is a simple screening test for this disorder. Treatment is 33 34 165 symptomatic with antihistamines and MC stabilizers. Anecdotal experience suggests that 35 36 166 imatinib is not effective. 37 38 39 40 167 4 SECONDARY MAST CELL DISORDERS (EXCLUDING IMMEDIATE IgE- 41 42 168 MEDIATED HYPERSENSITIVITY) 43 44 45 46 169 As mentioned in the Foreword, this review focuses on MC diseases other than acute IgE- 47 48 170 mediated allergic reactions. This section discusses secondary disorders of MCs associated with 49 50 171 less-well recognized, chronic features of MC dysfunction. Secondary MC dysfunction is often a 51 52 53 172 poorly recognized feature of common chronic inflammatory disorders. Some of these secondary 54 55 173 MC disorders may in the future be recognized as symptoms of primary MC disorders, for 56 57 58 59 60 Allergy Page 12 of 84

1 2 3 174 example -tryptasemia and ADGRE2-associated vibratory urticaria. There is also growing 4 5 6 175 evidence that hypoxic insults in neonates and infants may trigger MC degranulation and 7 8 176 subsequent pathology in the eyes (Retinopathy of Prematurity (ROP)), brain (Hypoxic Ischemic 9 10 177 Encephalopathy (HIE)), lungs (BronchoPulmonary Dysplasia (BPD)) and circulation (sudden- 11 12 13 178 infant death syndrome (SIDS)) (Table II). Furthermore, neovascularisation and vascular 14 15 179 remodelling may well contribute to atherosclerotic vascular disease, aneurysms and cancer 16 17 180 growth and metastases. 18 19 For Peer Review 20 181 4.1 MC dysfunction in chronic atopic and inflammatory disorders 21 22 23 24 182 MCs have a well-recognized role in the gastrointestinal defense against parasites, particularly 25 26 183 helminths.579,5860 Upon detecting foreign antigens, MCs situated close to nerves trigger enteric 27 28 184 nerves, leading to epithelial ion secretion and propulsive peristaltic activity, which help to expel 29 30 185 the pathogen.6159,602 MCs also regulate intestinal epithelial and endothelial permeability, barrier 31 32 602 33 186 function and mucin secretion. MCs release cytotoxic factors from their granules inducing 34 35 187 recruitment of other proinflammatory cells and granuloma formation, all of which promotes 36 37 188 parasite containment.613 For instance, in Trichinella spiralis (tissue-dwelling roundworm) 38 39 40 189 infections, MCs enhance intestinal epithelial permeability and Th2-cell infiltration resulting in 41 42 190 parasite expulsion.624 Fasciola hepatica (common liver fluke) invasion is accompanied by 43 44 191 increased MC infiltration and inflammation.635 MCs have also been associated with enhanced 45 46 646 47 192 responses to helminth infections in other tissues, such as in cutaneous leishmaniasis. 48 49 50 193 4.1.1 Inflammatory bowel disease 51 52 53 194 By disrupting mucosal epithelial barriers, MCs may also contribute to the pathogenesis of 54 55 195 inflammatory bowel disease (IBD).657-679 MC infiltrate and degranulate in ulcerative colitis (UC), 56 57 58 59 60 Page 13 of 84 Allergy

1 2 3 196 particularly in areas of inflammation. This is less obvious in Crohn’s disease (CD).7068-702 MCp 4 5 3 6 197 and other immune cells such as T-lymphocytes home to the gut via integrin 47. Vedolizumab, 7 8 198 a neutralizing 47 monoclonal antibody, is now licensed for the adults with IBD poorly 9 10 responsive to other therapies. It is currently not clear as to the extent to which the therapeutic 11 199 12 13 200 effects of vedolizumab are via MCs or T-lymphocytes, but it is interesting that, in keeping with 14 15 201 the in vitro data, RDBPCT suggest a greater beneficial effect in UC than CD.713-735 16 17 18 4.1.2 Asthma 19 202 For Peer Review 20 21 22 203 MCs have been implicated in the pathogenesis and severity of asthma in both children and 23 24 204 adults.746 MC stabilizers such as inhaled cromoglycate have been used to treat asthma for over 50 25 26 27 205 years, although since 1990 their use has been largely replaced by inhaled steroids and leukotriene 28 29 206 receptor antagonists. A Cochrane review of 24 studies concluded that there was insufficient 30 31 207 evidence of a beneficial effect of cromoglycate over placebo in children with moderately severe 32 33 757 34 208 asthma. 35 36 37 209 In 2001, anti-IgE monoclonal antibody therapy (omalizumab) was shown to be effective 38 39 210 in reducing exacerbations and medication use in a subset of asthmatic who had evidence of 40 41 211 clinical allergen sensitivity, suggesting a role for IgE-induced MC degranulation in acute 42 43 768,779 44 212 asthma. Omalizumab has since been licenced by the U.S. Food and Drug Administration 45 46 213 (FDA) and other licencing bodies around the world for older children and adults, having positive 47 48 214 skin or blood tests to aeroallergens, moderate to severe persistent allergic asthma not controlled 49 50 215 by inhaled corticosteroids and require frequent courses of oral steroids. These clinical findings 51 52 53 216 are supported by histological evidence of MC infiltrate the bronchial smooth muscle and the 54 55 217 alveolar parenchyma of asthmatics, particularly those with frequent exacerbations.8078-82,79 Basal 56 57 58 59 60 Allergy Page 14 of 84

1 2 3 218 serum MC tryptase can be significantly higher in chronic asthmatics (4.2 – 4.7ng/ml) than 4 5 830 6 219 healthy controls and patients with mild disease (2.5 - 3.1ng/ml).. MC tryptase is more likely to 7 8 220 be high in certain subgroups of asthmatics: older, obese patients, and non-atopic females with 9 10 221 salicylate sensitivity.84 Serum MC tryptase was also significantly higher in post-mortem lung 11 12 from individuals who died of asthma (58-120ng/ml) compared with those dying from other 13 222 14 15 223 causes.851,862 16 17 18 224 In 2017, a RDBPCT of 62 adults with severe, poorly-controlled asthma despite high dose 19 For Peer Review 20 225 inhaled or systemic corticosteroids showed that imatinib led to a significantly reduction in not 21 22 873 23 226 only serum MC tryptase but also methacholine airway responsiveness. Although the patients 24 25 227 in this trial were not selected for their allergic predisposition, they had an mean on three positive 26 27 228 skin prick test results. Overall, the current evidence indicates that MCs may well be important in 28 29 229 severe allergic asthma and that drugs inhibiting MC activity can be beneficial. 30 31 32 33 230 4.1.3 Chronic spontaneous urticaria 34 35 36 231 Chronic spontaneous urticaria (CSU) is associated with frequent, often daily urticaria for six 37 38 232 weeks or more. Symptoms often continue for many months or even years. Episodes of 39 40 233 angioedema occur in half of cases. Unlike acute urticaria, specific IgE tests are not recommended 41 42 43 234 in these patients. Symptomatic treatment of CSU is with a regular second or third generation 44 45 235 antihistamine. Where these fail omalizumab may be considered. Recent evidence suggests that 46 47 236 complete responders have significantly higher serum total IgE concentrations than non- 48 49 responders, suggesting that IgE-dependent MC degranulation may play a role in CSU.884,895 50 237 51 52 53 238 In 20% of patients, there is a clear physical trigger, such as pressure, temperature changes 54 55 239 or sun exposure.9086 CSU is usually sporadic and the underlying pathogenesis unknown, but in a 56 57 58 59 60 Page 15 of 84 Allergy

1 2 3 240 rare autosomal dominant form triggered by vibration and rubbing of the skin, activating 4 5 6 241 mutations in the dermatan sulphate-binding ADGRE2 cell surface receptor on MC promotes MC 7 8 242 degranulation with physical forces.9187 9 10 11 243 4.2 MC dysfunction, vascular remodelling and response to hypoxia 12 13 14 244 4.2.1 Cancer neovascularization and growth 15 16 17 245 Paul Ehrlich was the first to notice that MCs congregating around blood vessels of cancers and 18 19 For Peer Review 20 246 other tissues. He hypothesized that these cells helped to remodel the vasculature, promoting 21 22 247 delivery of oxygen and nutrients, leading him to coin the term mastzellen (feeding cells).8928 In 23 24 248 vascular, hematological and solid tumors, MC accumulation correlates with neovascularization, 25 26 more rapid tumor growth and metastases. In a pancreatic cancer model, MC recruitment was an 27 249 28 29 250 absolute requirement for tumor expansion. Treatment of established tumors with the MC 30 31 251 stabilizer cromoglycate triggered hypoxia and cell death.893 Cromoglycate has been used as an 32 33 252 adjuvant for the treatment of other cancers.940 Inhibitors of MC tryptase such as gabexate 34 35 36 253 mesilate and nafamostat mesylate have also been considered as adjuvant treatment of 37 38 254 tumors.951,96 2The clinical relevance of MCs and impact of manipulating MC function in cancer 39 40 255 therapy still remains unclear. 41 42 43 256 4.2.2 Retinal neovascularization in premature infants 44 45 46 47 257 Matsuda et al, 2017 discovered that MC tryptase was essential for neovascularization in murine 48 49 258 models of retinopathy of prematurity (ROP).973 MC-deficient mice did not develop retinal 50 51 259 neovascularisation (stage 3+ ROP), while retinopathy was induced after infusion of MCs or MC 52 53 260 tryptase. Intraperitoneal cromoglycate blocked retinal neovascularisation and prevented severe 54 55 56 261 ROP. Plasma MC tryptase in human preterm neonates (28-32 week’s gestation) with ROP was 57 58 59 60 Allergy Page 16 of 84

1 2 3 262 found to be 5-fold higher (median 62ng/ml) than in age-matched controls (12ng/ml). Results of 4 5 6 263 this study also allows a better understanding of the mechanism linking hypoxic insults and MC 7 8 264 degranulation. Transient Receptor Potential Ankyrin 1 (TRPA1) acts as an O2 tension sensor in 9 10 265 MCs, triggering degranulation. The development of ROP could be blocked with a specific 11 12 TRPA1 inhibitor. 13 266 14 15 16 267 4.2.3 Vascular remodelling and damage to the aorta, cerebral and coronary arteries 17 18 19 268 MCs contribute toFor the development Peer of atherosclerosis, Review as well as destabilization and 20 21 269 rupture of atherosclerotic plaques with ensuing atherothrombotic complications.984 In infarct- 22 23 270 related coronary events, the number of degranulated MCs in the adventitia backing ruptured 24 25 26 271 plaques is increased. Histamine released from the degranulated MCs may reach the media, where 27 28 272 it provokes local coronary spasm.995 There are many published case reports describing the 29 30 273 association between myocardial ischemia and acute allergic reactions (Kounis 31 32 10096,10197 33 274 syndrome). In a subgroup of patients, myocardial insufficiency may not be an 34 35 275 independent co-morbidity but rather a direct consequence of chronic allergic disease. Although 36 37 276 in the majority of patients suffering acute myocardial infarction, MC tryptase is not elevated,10298 38 39 277 it may be elevated in patients dying of acute coronary disease and of acute dissecting 40 41 10399,104 42 278 aorta. The effect of MCs on primary cardiac function is complex. On the one hand, there 43 44 279 is evidence that renin released directly by myocardial MCs can activate the renin-angiotensin 45 46 280 system and promote arrhythmias,105,106 and that chymase may exacerbate damage after acute 47 48 107 49 281 ischemia/reperfusion injuries. On the other, in murine models of myocardial infarction and 50 51 282 myocarditis, MCs have been shown to improve rather than worsen cardiac contractility.108,109 52 53 54 55 56 57 58 59 60 Page 17 of 84 Allergy

1 2 3 283 There is also evidence for the role of MCs in disrupting the integrity of blood vessels in 4 5 11001 6 284 the brain leading to saccular intracranial artery aneurysms, and also the aorta resulting in 7 8 285 abdominal aortic aneurysms. In a mouse model, MC tryptase MCP6-/- mice or treatment with 9 10 286 MC inhibitors prevented the development of aneurysms.11102-10134 MC-deficient rats showed 11 12 reduction of tPA-mediated cerebral hemorrhage compared with wild-type littermates.11405 It will 13 287 14 15 288 be interesting to study the prevalence of these vascular diseases in patients with inherently higher 16 17 289 MC tryptase concentrations, such as those with -tryptasemia. 18 19 For Peer Review 20 290 4.2.4 Hypoxic ischaemic encephalopathy 21 22 23 24 291 Animal studies have shown infiltration of MCs and histamine release in the brain after hypoxic 25 26 292 ischaemic insults in neonatal rats.11506,10716 MC stabilization with cromoglycate during the first 27 28 293 24 hours after hypoxic injury was neuroprotective, limiting neuronal loss, brain atrophy and 29 30 294 microglial activation. The results suggest that MC degranulation may exacerbate ischemia- 31 32 33 295 induced neuronal death in the preterm brain. 34 35 36 296 4.2.5 Bronchopulmonary dysplasia 37 38 297 MCs may also contribute to the development of BPD in premature neonates. Bhattacharya et al, 39 40 298 2012 used genome-wide expression profiling in lung tissue obtained at biopsy from 11 neonates 41 42 11708 43 299 with BPD and 17 age-matched controls. Expression of 159 genes was significantly different, 44 45 300 with three of the five most significantly dysregulated genes encoded MC biomarkers. 46 47 301 Immunohistochemistry demonstrated a 50-fold increase in chymase-expressing MCs in BPD 48 49 50 302 tissue biopsies when compared with controls. 51 52 303 A recent animal study of hyperoxia induced lung injury provided further evidence on the 53 54 304 potential mechanistic role of MCs.11809 Increased MC numbers and expression of MC TPSAB1, 55 56 57 58 59 60 Allergy Page 18 of 84

1 2 3 305 TPSAB2 and CPA3 genes were found in mouse lung after exposure to hyperoxia conditions 4 5 6 306 compared controls. Alveoli were disrupted in the test mice, but not in MC deficient mice or 7 8 307 controls, suggesting that MC may also disrupt lung structure and function. 9 10 308 5.2.6 Sudden infant death syndrome 11 12 13 309 Serum MC tryptase have been found to be elevated in SIDS in some studies and it has been 14 15 16 310 suggested that anaphylaxis may have a role in the pathogenesis of this syndrome in some 17 18 311 patients.1190-12112. In one study of 50 infants with SIDS, mean serum MC tryptase was 6.2ng/ml 19 For Peer Review 20 312 compared with 1.1 ng/ml in controls.12213 Forty percent of SIDS cases had MCT >10 ng/ml 21 22 23 313 compared with none of the controls. In contrast, Nishio et al, 2004, found that MC tryptase was 24 25 314 normal in 21 infants with SIDS and 14 controls.12314 Genetic testing for KIT and TPSAB1 26 27 315 mutations in these patients might provide further insights into the pathogenesis of this disease. 28 29 30 316 5 CONCLUSIONS 31 32 33 317 Despite 140 years having elapsed since Paul Ehrlich first described the MC,8928 there are still 34 35 36 318 exciting new discoveries in MC biology. In early childhood, somatic activating mutations in the 37 38 319 KIT gene of MC precursors can lead to cutaneous mastocytosis, with MC aggregates localizing 39 40 320 to the skin and usually following a benign remitting course. In contrast later in life, somatic 41 42 43 321 activating KIT gene mutations manifest in bone marrow MC precursors rather than in the skin 44 45 322 and are associated with systemic disease and a more aggressive course, particularly if additional 46 47 323 somatic oncogene mutations have accumulated as an inherent part of ageing. 48 49 50 324 Extensive MC protease gene replication over the millennia is now recognized to have 51 52 53 325 driven diversification of MC function. Indeed, gene replication is a common mechanism by 54 55 326 which all plant and animal species diversify and avoid extinction.12415,12516 Recent studies have 56 57 58 59 60 Page 19 of 84 Allergy

1 2 3 327 highlighted how common gene variability is in chymotrypsin-related serine proteases, 4 5 6 328 particularly in TPSAB1. The gene is completely absent in a third to a half of the population, 7 8 329 while duplication occur in approximately 5%. TPSAB1 gene plasticity may have provided 9 10 330 communities living in the tropics with a survival advantage after snake envenomation, and may 11 12 explain the absence of TPSAB1 from a higher proportion of Europeans where the risk of being 13 331 14 15 332 bitten poisonous snake is less. Excessive TPSAB1 gene duplication causes raised basal MC 16 17 333 tryptase activity and a propensity to multi-system disease. Until now, the underlying diagnosis of 18 19 334 many of these patients withFor nonspecific Peer symptoms Review may have gone unrecognized. Measurement 20 21 22 335 of basal serum MC tryptase offers a simple screening test, and if ≥8.0ng/ml a possible 23 24 336 explanation for their disease. 25 26 27 337 There is no doubt that MC infiltrate and degranulate in the tissues of patients with IBD 28 29 338 and asthma, but the relevance of this to clinical disease remains unclear. There are however clues 30 31 32 339 that as with eosinophilia, which is now deemed clinically relevant and effective suppressed with 33 34 340 mepolizumab in a subset of severe asthmatics,12617 dampening down MC activity using imatimab 35 36 341 in patients with refractory asthma is showing some promise. 37 38 39 342 Finally, studies of animal models of common neonatal diseases of the lungs, eye and 40 41 42 343 brain suggests that MC may well have an important role in their pathogenesis. Although it is 43 44 344 important to remain cautious when translating finding from animals into human, these results 45 46 345 should provide the impetus to look for new solutions to these diseases with high healthcare 47 48 12718 49 346 burdens. Furthermore, results from these studies suggest an important role of changes in O2 50 51 347 tension in triggering MC degranulation. 52 53 54 55 56 57 58 59 60 Allergy Page 20 of 84

1 2 3 348 140 years on, MCs remain central to allergic and inflammatory diseases, well beyond 4 5 6 349 IgE-dependent degranulation and immediate hypersensitivity. The challenge for scientists and 7 8 350 clinicians alike is to look beyond the current dogma if old problems are to be solved with new 9 10 351 therapies. 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 21 of 84 Allergy

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1 2 3 536 84. Hinks TS, Zhou X, Staples KJ et al. Innate and adaptive T cells in asthmatic patients: 4 5 6 537 Relationship to severity and disease mechanisms. J Allergy Clin Immunol 2015;136:323-33. 7 8 9 538 815. Scarpelli MP, Keller S, Tran L, et al. Postmortem serum levels of IgE and mast cell tryptase 10 11 539 in fatal asthma. Forensic Sci Int 2016;269;113-8. 12 13 14 540 826. Elliot JG, Abramson MJ, Drummer OH, et al. Time to death and mast cell degranulation in 15 16 541 fatal asthma. Respirology 2009;14,808-13. 17 18 19 For Peer Review 20 542 837. Cahill KN, Katz HR, Cui J, et al. KIT Inhibition by Imatinib in Patients with Severe 21 22 543 Refractory Asthma. N Engl J Med 2017;376;1911-20. 23 24 25 544 848. Zhao ZT, Ji CM, Yu WJ, et al. Omalizumab for the treatment 26 27 545 of chronic spontaneous urticaria: A meta-analysis of randomized clinical trials. J Allergy Clin 28 29 30 546 Immunol 2016;137:1742-1750.e4. 31 32 33 547 859. Weller K, Ohanyan T, Hawro T, et al. Total IgE levels are linked to the response of chronic 34 35 548 spontaneous urticaria patients to omalizumab. Allergy 2018, Aug 4. 36 37 38 549 8906. Saini SS. Chronic spontaneous urticaria: etiology and pathogenesis. Immunol Allergy Clin 39 40 North Am 2014;34:33-52. 41 550 42 43 551 8791. Boyden SE, Desai A, Cruse G, et al. Vibratory urticaria associated with a missense variant 44 45 552 in ADGRE2. N Engl J Med 2016;374:656-63. 46 47 48 553 8928. Blank U, Falcone FH, Nilsson G. The history of mast cell and basophil research - some 49 50 51 554 lessons learnt from the last century. Allergy 2013;68:1093-101. 52 53 54 555 8993. Soucek L, Lawlor ER, Soto D, et al. Mast cells are required for angiogenesis and 55 56 556 macroscopic expansion of Myc-induced pancreatic islet tumors. Nat Med 2007;13:1211-8. 57 58 59 60 Page 31 of 84 Allergy

1 2 3 557 904. Wroblewski M, Bauer R, Cubas Córdova M, et al. Mast cells decrease efficacy of anti- 4 5 6 558 angiogenic therapy by secreting matrix-degrading granzyme B. Nat Commun 2017;8:269. 7 8 9 559 915. Ribatti D, Ranieri G. Tryptase, a novel angiogenic factor stored in mast cell granules. Exp 10 11 560 Cell Res 2015;332:157-62. 12 13 14 561 926. Ribatti D. Mast cells as therapeutic target in cancer. Eur J Pharmacol 2016;778:152-7. 15 16 17 562 937. Matsuda K, Okamoto N, Kondo M, et al. Mast cell hyperactivity underpins the development 18 19 For Peer Review 20 563 of oxygen-induced retinopathy. J Clin Invest 2017;127;3987-4000. 21 22 23 564 948. Kovanen PT, Bot I. Mast cells in atherosclerotic cardiovascular disease - Activators and 24 25 565 actions. Eur J Pharmacol 2017;816:37-46. 26 27 28 566 959. Laine P, Kaartinen M, Penttilä A, et al. Association between myocardial infarction and 29 30 567 the mast cells in the adventitia of the infarct-related coronary artery. Circulation 1999;99:361-9. 31 32 33 34 568 91006. Fassio F, Almerigogna F. Kounis syndrome (allergic acute coronary syndrome): different 35 36 569 views in allergologic and cardiologic literature. Intern Emerg Med 2012;7:489-95. 37 38 39 570 97101. Abdelghany M, Subedi R, Shah S, et al. Kounis syndrome: a review article on 40 41 571 epidemiology, diagnostic findings, management and complications of allergic acute coronary 42 43 44 572 syndrome. Int J Cardiol 2017;232:1–4. 45 46 47 573 10298. Kervinen H, Kaartinen M, Mäkynen H, et al. Serum tryptase levels in acute coronary 48 49 574 syndromes. Int J Cardiol 2005;104:138-43. 50 51 52 575 10399. Palmiere C, Comment L, Vilarino R, et al. Measurement of β-tryptase in postmortem 53 54 serum in cardiac deaths. J Forensic Leg Med 2014;23:12-8. 55 576 56 57 58 59 60 Allergy Page 32 of 84

1 2 3 577 1040. Xiao N, Li DR, Wang Q, et al. Postmortem Serum Tryptase Levels with Special Regard to 4 5 6 578 Acute Cardiac Deaths. J Forensic Sci 2017;62:1336-1338. 7 8 9 579 105. Silver RB, Reid AC, Mackins CJ, et al. Mast cells: a unique source of renin. Proc Natl Acad 10 11 580 Sci USA 2004;101:13607-12. 12 13 14 581 106. Koda K, Salazar-Rodriguez M, Corti F, et al. Aldehyde dehydrogenase activation prevents 15 16 582 reperfusion arrhythmias by inhibiting local renin release from cardiac mast cells. Circulation 17 18 19 583 2010;122:771-81. For Peer Review 20 21 22 584 107. Zheng J, Wei CC, Hase N, et al. Chymase mediates injury and mitochondrial damage in 23 24 585 cardiomyocytes during acute ischemia/reperfusion in the dog. PLoS One 2014;9:e94732. 25 26 27 586 108. Ngkelo A, Richart A, Kirk JA, et al. Mast cells regulate myofilament calcium sensitization 28 29 30 587 and heart function after myocardial infarction. J Exp Med 2016;213:1353-74. 31 32 33 588 109. Mina Y, Rinkevich-Shop S, Konen E, et al. Mast cell inhibition attenuates myocardial 34 35 589 damage, adverse remodeling, and dysfunction during fulminant myocarditis in the rat. J 36 37 590 Cardiovasc Pharmacol Ther 2013;18:152-61. 38 39 40 11001. Ollikainen E, Tulamo R, Lehti S, et al. Mast cells, neovascularization, and 41 591 42 43 592 microhemorrhages are associated with saccular intracranial artery aneurysm wall remodelling. J 44 45 593 Neuropathol Exp Neurol 2014;73:855-64. 46 47 48 594 10112. Mäyränpää MI, Kovanen PT. Mast cells: important players in the orchestrated 49 50 51 595 pathogenesis of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2011;31:734-40. 52 53 54 596 11203. Zhang J, Sun J, Lindholt JS, et al. Mast cell tryptase deficiency attenuates mouse 55 56 597 abdominal aortic aneurysm formation. Circ Res 2011;108:1316-27. 57 58 59 60 Page 33 of 84 Allergy

1 2 3 598 11304. Sun J, Zhang J, Lindholt JS, et al. Critical role of mast cell chymase in mouse abdominal 4 5 6 599 aortic aneurysm formation. Circulation 2009;120:973-82. 7 8 9 600 11405. Strbian D, Karjalainen-Lindsberg ML, Kovanen PT, et al. Mast cell stabilization reduces 10 11 601 hemorrhage formation and mortality after administration of thrombolytics in experimental 12 13 602 ischemic stroke. Circulation 2007;116:411-8. 14 15 16 603 11506. Biran V, Cochois V, Karroubi A, et al. Stroke Induces Histamine Accumulation and 17 18 19 604 Mast Cell Degranulation inFor the Neonatal Peer Rat Brain. Review Brain Pathol 2008;18;1-9. 20 21 22 605 11607. Jin Y, Silverman A, Vannucci S. Mast Cells Are Early Responders After Hypoxia- 23 24 606 Ischemia in Immature Rat Brain. Stroke 2009;40;3107-12. 25 26 27 607 11708. Bhattacharya S, Go D, Krenitsky DL, et al. Genome-Wide Transcriptional Profiling 28 29 30 608 Reveals Connective Tissue Mast Cell Accumulation in Bronchopulmonary Dysplasia. Am J 31 32 609 Respir Crit Care Med 2012;186;349–58. 33 34 35 610 11809. Veerappan A, Thompson M, Savage AR, et al. Mast cells and exosomes in hyperoxia- 36 37 611 induced neonatal lung disease. Am J Physiol Lung Cell Mol Physiol 2016;310;1218–32. 38 39 40 1190. Buckley M, Variend S, Walls A. Elevated serum concentrations of beta-tryptase, but not 41 612 42 43 613 alpha-tryptase, in Sudden Infant Death Syndrome (SIDS). An investigation of anaphylactic 44 45 614 mechanisms. Clin Exp Allergy 2001;31;1696-1704. 46 47 48 615 12011. Gold Y, Goldberg A, Sivan Y. Hyper-releasability of mast cells in family members of 49 50 51 616 infants with sudden infant death syndrome and apparent life-threatening events. J Pediatr 52 53 617 2000;136;460-5. 54 55 56 57 58 59 60 Allergy Page 34 of 84

1 2 3 618 12112. Edston E, Gidlund E, Wickman M, et al. Increased mast cell tryptase in sudden infant 4 5 6 619 death-anaphylaxis, hypoxia or artefact? Clin Exp Allergy 1999;29;1648-54. 7 8 9 620 12213. Platt MS, Yunginger JW, Sekula-Perlman A, et al. Involvement of mast cells in sudden 10 11 621 infant death syndrome. J Allergy Clin Immunol 1994;94;250-6. 12 13 14 622 12314. Nishio H, Suzuki K. Serum tryptase levels in sudden infant death syndrome in forensic 15 16 623 autopsy cases. Forensic Sci Int 2004;139;57-60. 17 18 19 For Peer Review 20 624 12415. Proulx SR. Multiple routes to subfunctionalization and gene duplicate specialization. 21 22 625 Genetics 2012;190:737-51. 23 24 626 12516. Taylor JS, Raes J. Duplication and divergence: the evolution of new genes and old ideas. 25 26 Annu Rev Genet 2004;38:615-43. 27 627 28 29 628 12617. Ortega HG, Liu MC, Pavord ID, et al. Mepolizumab treatment in patients with severe 30 31 629 eosinophilic asthma. N Engl J Med 2014;371:1198-207. 32 33 34 630 12718. Bischoff S. Role of mast cells in allergic and non-allergic immune responses: comparison 35 36 37 631 of human and murine data. Nat Rev Immunol 2007;7;93-104. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 84 Allergy

1 2 3 632 Figure Legends 4 5 6 633 Figure 1 Differentiation of mast cells from bone marrow precursors. Hematopoietic stem 7 8 9 634 cells (HSC) pass through a number of stages of differentiation in the bone marrow (multipotent 10 11 635 progenitor (MMP), common myeloid progenitor (CMP), granulocyte/monocyte progenitor 12 13 636 (GMP), basophil/mast cell progenitor (BMCP)) before reaching the mast cell progenitor (MCp). 14 15 16 637 MCp are proliferating agranular MC precursors that enter the blood stream and home to tissues, 17 18 638 where they further differentiate into mature nonproliferating granule positive mast cells (MC). 19 For Peer Review 20 21 639 Figure 2 Homing of mast cell progenitors (MCp) from the bone marrow and peripheral 22 23 640 blood into tissues. Homing of MCp depends on the expression of specific adhesion molecules 24 25 26 641 and chemokines. In the gut, integrins 4 7 on MCp bind to Mucosal vascular Addressin Cell 27 28 642 Adhesion Molecule 1 (MAdCAM1) on blood vessel endothelium, while in the lung these 29 30 643 integrins bind Vascular Cell Adhesion Protein 1 VCAM-1. In the skin, MCp are attracted by 31 32 33 644 prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and chemokine (C-C motif) ligand 2 (CCL2) 34 35 645 also known as monocyte chemoattractant protein 1 (MCP1) to prostaglandin EP3 receptor and 36 37 646 CCR2. Percentages of MCT and MCTC in tissues is based in information from reference 15. 38 39 40 647 Figure 3 Spectrum of primary mast cell diseases. Infants (i) sporadic activating KIT 41 42 43 648 mutations (cutaneous mastocytosis), (ii) dominantly inherited TPSAB1 gene duplication (- 44 45 649 tryptasemia), Older children and adults sporadic activating KIT mutations ± other activating 46 47 mutations (systemic mastocytosis) 48 650 49 50 51 651 Figure 4 Examples of clinical features and histology in mast cell disorders. A. Cutaneous 52 53 652 mastocytosis, B. Dariers sign – urtication of skin lesions after rubbing, C. Excessive mast cell 54 55 653 influx (stained with CD117) within the gastrointestinal tract in a patient with -tryptasemia. 56 57 58 59 60 Allergy Page 36 of 84

1 2 3 654 Table I Primary mast cell disorders 4 5 Mastocytosis 6 Overview clonal MC proliferation, rare, sporadic, activating gene mutations 7 Subtypes systemic (bone marrow cutaneous (usually without bone 8 involvement) marrow involvement) 9 10 more common in children than adults 11 common in adults, rare in children diffuse, multicentric (urticaria 12 pigmentosa), solitary (mastocytoma) 13 Histology multifocal, dense infiltrates of MC 14 (≥15 mast cells in aggregates) in 15 bone marrow and/or other 16 extracutaneous organs 17 MC may have atypical morphology 18 e.g. spindle-shape, CD2/CD25 19 expressionFor or in Peer more aggressive Review 20 forms an undifferentiated phenotype 21 Clinical course ranges from indolent to malignant indolent, starts in infancy, usually 22 (acute or chronic leukemia) regresses before puberty 23 Clinical features flushing flushing, urticaria, angioedema, 24 abdominal pain, diarrhoea, muscle non-cutaneous symptoms less 25 aches and pains, fractures, common 26 27 neurological and psychiatric 28 symptoms, hypotension/anaphylaxis 29 with malignancy: fevers, weight 30 loss, hepatosplenomegaly 31 Genetics sporadic D816V activating c-kit sporadic D816V activating c-kit 32 mutation in 90%, may have mutation in 20 – 40%, other activating 33 additional mutations in PDGFRA/B, c-kit mutations in 60% 34 TET2, N-RAS 35 MC tryptase >20 ng/ml <20 ng/ml 36 Potential therapies second or third generation second or third generation 37 antihistamines, ranitidine, mast cell antihistamines usually suffice 38 stabilisers (cromoglycate, ketotifen), 39 corticosteroids, imatinib only if no 40 D816V mutation, midostaurin 41 (protein kinase inhibitor), 42 chemotherapy for leukemia 43 44 45 Mast cell activation syndromes 46 Overview excessive mast cell activity without clonal proliferation or obvious secondary 47 triggers, -tryptasemia is a common autosomal dominant condition occurring 48 in a subset of patients 49 Subtypes -tryptasemia (up to 5% of general idiopathic (no evidence of 50 population) mastocytosis, alpha-tryptasemia or 51 secondary cause) 52 Histology may have increased number of MC, no MC aggregates 53 particularly in GI tract, but do not 54 form aggregates, may be associated 55 with increased eosinophils 56 57 58 59 60 Page 37 of 84 Allergy

1 2 3 Clinical course variable, may depend of degree of similar to alpha-tryptasemia 4 gene replication, other genetic or 5 environmental cofactors 6 Clinical features flushing, urticaria, angioedema similar to alpha-tryptasemia 7 8 abdominal pain, diarrhoea, but have 9 mucous and blood 10 muscle aches and pains, headaches 11 hypermobile joints 12 POTS 13 Neurological and psychiatric 14 symptoms 15 assess parents and siblings 16 Genetics allelic TPSAB1 gene replication no defined genetic cause 17 usually autosomal dominant 18 Basal MC tryptase ≥8.0ng/ml no consensus regarding MC tryptase, 19 For Peer Reviewhistamine or prostaglandin 20 concentrations 21 Potential therapies secondary or third generation as for alpha-tryptasemia 22 antihistamines, ranitidine, mast cell 23 stabilisers (cromoglycate/ketotifen), 24 dietary manipulation, imatinib 25 26 typically ineffective 27 28 655 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 38 of 84

1 2 3 656 Table II Neonatal and paediatric disorders associated with secondary MC dysfunction 4 5 tissue MC pathology increased MC inhibitor MC inhibitor 6 infiltration & serum blocks blocks pathology 7 degranulation MC activity 8 tryptase 9 Disorders associated with chronic atopic & inflammatory disorders 10 11 helminth infections ++ promotes NT NT NT 12 peristalsis & 13 epithelium ion 14 secretion 15 expulsion, 16 granuloma  17 containment 18 inflammatory ++ unknown NT NT ?vedolizumab 19 bowel disease For Peer Review 20 21 22 asthma ++ asthma flares cromoglycate imatinib 23 acute death imatinib 24 25 Disorders associated with hypoxic insults or vascular remodelling 26 27 possible TRPA1 oxygen sensor triggered MC degranulation and inflammation 28 retinopathy of -- neo- ++ cromoglycate cromoglycate 29 prematurity vascularisation nafamostat nafamostat (tryptase 30 stage 3+ ROP (tryptase inhibitor) 31 inhibitor) 32 hypoxic-ischemic ++ neuronal death NT cromoglycate cromoglycate 33 encephalopathy 34 35 36 bronchopulmonary ++ disrupts NT NT ?ketotifen 37 dysplasia alveoli 38 39 40 sudden-infant death -- anaphylaxis ++ NT 41 NT 42 syndrome 43 44 45 atherosclerotic ++ ischemia heart ++ cromoglycate cromoglycate 46 vascular disease disease, 47 Kounis 48 syndrome 49 vascular aneurysms ++ abdominal ++ cromoglycate cromoglycate 50 (brain, aorta) aortic 51 aneurysms, 52 cerebral 53 aneurysms 54 neovascularisation ++ more -- cromoglycate cromoglycate, 55 of tumors aggressive 56 57 58 59 60 Page 39 of 84 Allergy

1 2 3 growth and gabexate, mesylate, 4 metastasis nafamostat 5 6 7 657 NT not tested 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 40 of 84

1 2 3 107 of the MC growth factor receptor KIT gene in bone marrow MC progenitors.30 The clinical 4 5 36 6 108 course ranges from indolent to malignant (acute or chronic MC leukemia, MC sarcoma). 7 8 109 Adults with Hymenoptera venom anaphylaxis should also be investigated for systemic 9 10 110 mastocytosis, even if baseline MC tryptase is normal and there are no skin signs.37 The reported 11 12 prevalence of underlying clonal mast cell disease in venom allergy varies greatly from 7% to 13 111 14 15 112 94%, but is higher in patients suffering from hypotension and syncope,38,39 and also in adults 16 17 113 suffering from anaphylactic reactions after discontinuation of immunotherapy. The latter 18 19 114 observation suggests that Forthese patients Peer may require Review life-long venom immunotherapy.40 Although 20 21 22 115 bone marrow examination is conventionally not recommended for patients where their MC 23 24 116 tryptase mast cell tryptase is less than 20ng/ml (or 11.5ng/ml in those with severe anaphylaxis or 25 26 117 other features suggestive of mastocytosis), normal MC tryptase does not exclude the diagnosis. 27 28 29 118 KIT D816V mutations has been detected in the blood of 92% of adults with Hymenoptera 30 31 119 venom-induced anaphylaxis, reducing the need for more invasive tests in some patients.41 32 33 34 120 MC leukemias (90% of which are aleukemic) and sarcomas (30% of which progress to 35 36 121 MC leukemia) makes up <1% of patients with systemic mastocytosis, stain for CD117 and 37 38 39 122 tryptase, and are often associated with additional mutations in PDGFR, FIPIL1, TET2, SRSF2, 40 41 123 ASXL2 or K/N-RAS genes.42,43 MC cancers usually present with fever, weight loss, cytopenias, 42 43 124 bone pain and hepatosplenomegaly (c-findings). Absence of symptoms and signs of organ 44 45 44 46 125 damage (c-findings) is associated with a more stable clinical course and better prognosis. 47 48 126 Serum MC tryptase is usually >200ng/ml. Response to radiotherapy and chemotherapy is usually 49 50 127 transient and the prognosis is poor with median survival of less than two years. Chemotherapy 51 52 128 combined with midostaurin (a multi-tyrosine kinase inhibitor) and followed by hematopoietic 53 54 55 129 stem cell transplantation may be curative, but due to the rarity of these cancers definitive data on 56 57 58 59 60 Page 47 of 84 Allergy

1 2 3 130 the efficacy of this approach is limited. The pathogenesis and clinical disease spectrum of 4 5 6 131 systemic mastocytosis parallels sporadic JAK2V617F gain of function mutations known to drive 7 8 132 adult-onset myeloproliferative disorders.45 Systemic mastocytosis with activating KIT mutations 9 10 133 in bone marrow MCs is rare in children. 11 12 13 134 Children usually present with cutaneous rather than systemic mastocytosis (CM). Up to 14 15 16 135 ninety percent have benign MC aggregates due to somatic KIT mutations confined to the 17 18 136 skin.46,47 The common D816V is only found in 34 – 42% of cases. In one study an additional 19 For Peer Review 20 137 44% of patients had gene mutations outside exon 17.48 Seventy-five percent have multiple brown 21 22 49-52 23 138 skin patches. In a quarter, the rash is noticed at birth and in 90% by the age of 2 years old. 24 25 139 Patches urticate on rubbing or bathing (urticaria pigmentosa) (Figure 4). CM can also present as 26 27 140 one or more circumscribed reddish skin lesions (mastocytoma, 20%) or as a diffuse rash (5%). In 28 29 141 two thirds of children the disease regresses by puberty. NSAIDs, opiates, muscle relaxants, insect 30 31 32 142 stings and physical factors such as temperature changes with bathing or swimming can trigger 33 34 143 urtication and rarely hypotension. Treatment with second or third generation antihistamines is 35 36 144 usually effective. The risk of anaphylaxis after insect venom stings in children with CM is 37 38 53,54 39 145 currently considered very low. Prescription of an adrenaline auto-injector is left to individual 40 41 146 specialist, rather than being a mandatory requirement.55 42 43 44 147 3.2 MC activation syndrome (MCAS) including -tryptasemia 45 46 47 148 Primary MCAS is characterized by excessive activation of MCs, and although its characteristics 48 49 50 149 are yet to be fully defined, diagnosis is based on clinical features and increased MC tryptase, 51 52 150 where mastocytosis and secondary allergic triggers have been excluded.33 Histamine and 53 54 151 prostaglandins may also be elevated,56 but further work is required before definitive 55 56 57 58 59 60 Allergy Page 48 of 84

1 2 3 152 recommendations can be provided regarding use of these measurement in the diagnosis of 4 5 57 6 153 MCAS. 7 8 9 154 -tryptase gene allele frequency varies between individuals. Twenty-nine percent of 10 11 155 people (up to 45% in white Europeans) express no α-tryptase genes.58 Five percent of individuals 12 13 156 are thought to have allelic TPSAB1 gene duplications inherited in an autosomal dominant pattern. 14 15 16 157 Adults and children with TPSAB1 gene replications have serum MC tryptase ≥8.0 ng/ml and 17 18 158 clinical features of MCAS.34 These include flushing and angioedema, abdominal pain, diarrhea 19 For Peer Review 20 159 and food intolerances, joint hypermobility, systemic reaction to Hymenoptera, hypotensive 21 22 23 160 episodes and autonomic dysfunction. Triplications and even quintuplications are less common, 24 25 161 associated with even higher serum MC tryptase concentration and more severe disease (Table 26 27 162 I).34,35 Serum MC tryptase is a simple screening test for this disorder. Treatment is symptomatic 28 29 30 163 with antihistamines and MC stabilizers. Anecdotal experience suggests that imatinib is not 31 32 164 effective. 33 34 35 165 4 SECONDARY MAST CELL DISORDERS (EXCLUDING IMMEDIATE IgE- 36 37 38 166 MEDIATED HYPERSENSITIVITY) 39 40 41 167 As mentioned in the Foreword, this review focuses on MC diseases other than acute IgE- 42 43 168 mediated allergic reactions. This section discusses secondary disorders of MCs associated with 44 45 46 169 less-well recognized, chronic features of MC dysfunction. Secondary MC dysfunction is often a 47 48 170 poorly recognized feature of common chronic inflammatory disorders. Some of these secondary 49 50 171 MC disorders may in the future be recognized as symptoms of primary MC disorders, for 51 52 53 172 example -tryptasemia and ADGRE2-associated vibratory urticaria. There is also growing 54 55 173 evidence that hypoxic insults in neonates and infants may trigger MC degranulation and 56 57 58 59 60 Page 49 of 84 Allergy

1 2 3 174 subsequent pathology in the eyes (Retinopathy of Prematurity (ROP)), brain (Hypoxic Ischemic 4 5 6 175 Encephalopathy (HIE)), lungs (BronchoPulmonary Dysplasia (BPD)) and circulation (sudden- 7 8 176 infant death syndrome (SIDS)) (Table II). Furthermore, neovascularisation and vascular 9 10 177 remodelling may well contribute to atherosclerotic vascular disease, aneurysms and cancer 11 12 growth and metastases. 13 178 14 15 16 179 4.1 MC dysfunction in chronic atopic and inflammatory disorders 17 18 19 180 MCs have a well-recognizedFor role in Peer the gastrointestinal Review defense against parasites, particularly 20 21 181 helminths.59,60 Upon detecting foreign antigens, MCs situated close to nerves trigger enteric 22 23 182 nerves, leading to epithelial ion secretion and propulsive peristaltic activity, which help to expel 24 25 61,62 26 183 the pathogen. MCs also regulate intestinal epithelial and endothelial permeability, barrier 27 28 184 function and mucin secretion.62 MCs release cytotoxic factors from their granules inducing 29 30 185 recruitment of other proinflammatory cells and granuloma formation, all of which promotes 31 32 63 33 186 parasite containment. For instance, in Trichinella spiralis (tissue-dwelling roundworm) 34 35 187 infections, MCs enhance intestinal epithelial permeability and Th2-cell infiltration resulting in 36 37 188 parasite expulsion.64 Fasciola hepatica (common liver fluke) invasion is accompanied by 38 39 189 increased MC infiltration and inflammation.65 MCs have also been associated with enhanced 40 41 66 42 190 responses to helminth infections in other tissues, such as in cutaneous leishmaniasis. 43 44 45 191 4.1.1 Inflammatory bowel disease 46 47 48 192 By disrupting mucosal epithelial barriers, MCs may also contribute to the pathogenesis of 49 50 193 inflammatory bowel disease (IBD).67-69 MC infiltrate and degranulate in ulcerative colitis (UC), 51 52 70-72 53 194 particularly in areas of inflammation. This is less obvious in Crohn’s disease (CD). MCp and 54 55 195 other immune cells such as T-lymphocytes home to the gut via integrin 47.3 Vedolizumab, a 56 57 58 59 60 Allergy Page 50 of 84

1 2 3 196 neutralizing 47 monoclonal antibody, is now licensed for the adults with IBD poorly 4 5 6 197 responsive to other therapies. It is currently not clear as to the extent to which the therapeutic 7 8 198 effects of vedolizumab are via MCs or T-lymphocytes, but it is interesting that, in keeping with 9 10 199 the in vitro data, RDBPCT suggest a greater beneficial effect in UC than CD.73-75 11 12 13 14 200 4.1.2 Asthma 15 16 17 201 MCs have been implicated in the pathogenesis and severity of asthma in both children and 18 19 76 For Peer Review 20 202 adults. MC stabilizers such as inhaled cromoglycate have been used to treat asthma for over 50 21 22 203 years, although since 1990 their use has been largely replaced by inhaled steroids and leukotriene 23 24 204 receptor antagonists. A Cochrane review of 24 studies concluded that there was insufficient 25 26 205 evidence of a beneficial effect of cromoglycate over placebo in children with moderately severe 27 28 77 29 206 asthma. 30 31 32 207 In 2001, anti-IgE monoclonal antibody therapy (omalizumab) was shown to be effective 33 34 208 in reducing exacerbations and medication use in a subset of asthmatic who had evidence of 35 36 37 209 clinical allergen sensitivity, suggesting a role for IgE-induced MC degranulation in acute 38 39 210 asthma.78,79 Omalizumab has since been licenced by the U.S. Food and Drug Administration 40 41 211 (FDA) and other licencing bodies around the world for older children and adults, having positive 42 43 212 skin or blood tests to aeroallergens, moderate to severe persistent allergic asthma not controlled 44 45 46 213 by inhaled corticosteroids and require frequent courses of oral steroids. These clinical findings 47 48 214 are supported by histological evidence of MC infiltrate the bronchial smooth muscle and the 49 50 215 alveolar parenchyma of asthmatics, particularly those with frequent exacerbations.80-82 Basal 51 52 53 216 serum MC tryptase can be significantly higher in chronic asthmatics (4.2 – 4.7ng/ml) than 54 55 217 healthy controls and patients with mild disease (2.5 - 3.1ng/ml).83 MC tryptase is more likely to 56 57 58 59 60 Page 51 of 84 Allergy

1 2 3 218 be high in certain subgroups of asthmatics: older, obese patients, and non-atopic females with 4 5 84 6 219 salicylate sensitivity. Serum MC tryptase was also significantly higher in post-mortem lung 7 8 220 from individuals who died of asthma (58-120ng/ml) compared with those dying from other 9 10 221 causes.85,86 11 12 13 222 In 2017, a RDBPCT of 62 adults with severe, poorly-controlled asthma despite high dose 14 15 16 223 inhaled or systemic corticosteroids showed that imatinib led to a significantly reduction in not 17 18 224 only serum MC tryptase but also methacholine airway responsiveness.87 Although the patients in 19 For Peer Review 20 225 this trial were not selected for their allergic predisposition, they had an mean on three positive 21 22 23 226 skin prick test results. Overall, the current evidence indicates that MCs may well be important in 24 25 227 severe allergic asthma and that drugs inhibiting MC activity can be beneficial. 26 27 28 228 4.1.3 Chronic spontaneous urticaria 29 30 31 229 Chronic spontaneous urticaria (CSU) is associated with frequent, often daily urticaria for six 32 33 230 weeks or more. Symptoms often continue for many months or even years. Episodes of 34 35 36 231 angioedema occur in half of cases. Unlike acute urticaria, specific IgE tests are not recommended 37 38 232 in these patients. Symptomatic treatment of CSU is with a regular second or third generation 39 40 233 antihistamine. Where these fail omalizumab may be considered. Recent evidence suggests that 41 42 43 234 complete responders have significantly higher serum total IgE concentrations than non- 44 45 235 responders, suggesting that IgE-dependent MC degranulation may play a role in CSU.88,89 46 47 48 236 In 20% of patients, there is a clear physical trigger, such as pressure, temperature changes 49 50 237 or sun exposure.90 CSU is usually sporadic and the underlying pathogenesis unknown, but in a 51 52 53 238 rare autosomal dominant form triggered by vibration and rubbing of the skin, activating 54 55 56 57 58 59 60 Allergy Page 52 of 84

1 2 3 239 mutations in the dermatan sulphate-binding ADGRE2 cell surface receptor on MC promotes MC 4 5 91 6 240 degranulation with physical forces. 7 8 9 241 4.2 MC dysfunction, vascular remodelling and response to hypoxia 10 11 12 242 4.2.1 Cancer neovascularization and growth 13 14 15 243 Paul Ehrlich was the first to notice that MCs congregating around blood vessels of cancers and 16 17 244 other tissues. He hypothesized that these cells helped to remodel the vasculature, promoting 18 19 For Peer Review 92 20 245 delivery of oxygen and nutrients, leading him to coin the term mastzellen (feeding cells). In 21 22 246 vascular, hematological and solid tumors, MC accumulation correlates with neovascularization, 23 24 247 more rapid tumor growth and metastases. In a pancreatic cancer model, MC recruitment was an 25 26 absolute requirement for tumor expansion. Treatment of established tumors with the MC 27 248 28 29 249 stabilizer cromoglycate triggered hypoxia and cell death.93 Cromoglycate has been used as an 30 31 250 adjuvant for the treatment of other cancers.94 Inhibitors of MC tryptase such as gabexate mesilate 32 33 251 and nafamostat mesylate have also been considered as adjuvant treatment of tumors.95,96 The 34 35 36 252 clinical relevance of MCs and impact of manipulating MC function in cancer therapy still 37 38 253 remains unclear. 39 40 41 254 4.2.2 Retinal neovascularization in premature infants 42 43 44 255 Matsuda et al, 2017 discovered that MC tryptase was essential for neovascularization in murine 45 46 97 47 256 models of retinopathy of prematurity (ROP). MC-deficient mice did not develop retinal 48 49 257 neovascularisation (stage 3+ ROP), while retinopathy was induced after infusion of MCs or MC 50 51 258 tryptase. Intraperitoneal cromoglycate blocked retinal neovascularisation and prevented severe 52 53 259 ROP. Plasma MC tryptase in human preterm neonates (28-32 week’s gestation) with ROP was 54 55 56 260 found to be 5-fold higher (median 62ng/ml) than in age-matched controls (12ng/ml). Results of 57 58 59 60 Page 53 of 84 Allergy

1 2 3 261 this study also allows a better understanding of the mechanism linking hypoxic insults and MC 4 5 6 262 degranulation. Transient Receptor Potential Ankyrin 1 (TRPA1) acts as an O2 tension sensor in 7 8 263 MCs, triggering degranulation. The development of ROP could be blocked with a specific 9 10 264 TRPA1 inhibitor. 11 12 13 265 4.2.3 Vascular remodelling and damage to the aorta, cerebral and coronary arteries 14 15 16 266 MCs contribute to the development of atherosclerosis, as well as destabilization and 17 18 98 19 267 rupture of atherosclerotic Forplaques withPeer ensuing atherothromboticReview complications. In infarct- 20 21 268 related coronary events, the number of degranulated MCs in the adventitia backing ruptured 22 23 269 plaques is increased. Histamine released from the degranulated MCs may reach the media, where 24 25 99 26 270 it provokes local coronary spasm. There are many published case reports describing the 27 28 271 association between myocardial ischemia and acute allergic reactions (Kounis syndrome).100,101 29 30 272 In a subgroup of patients, myocardial insufficiency may not be an independent co-morbidity but 31 32 33 273 rather a direct consequence of chronic allergic disease. Although in the majority of patients 34 35 274 suffering acute myocardial infarction, MC tryptase is not elevated,102 it may be elevated in 36 37 275 patients dying of acute coronary disease and of acute dissecting aorta.103,104 The effect of MCs on 38 39 276 primary cardiac function is complex. On the one hand, there is evidence that renin released 40 41 42 277 directly by myocardial MCs can activate the renin-angiotensin system and promote 43 44 278 arrhythmias,105,106 and that chymase may exacerbate damage after acute ischemia/reperfusion 45 46 279 injuries.107 On the other, in murine models of myocardial infarction and myocarditis, MCs have 47 48 108,109 49 280 been shown to improve rather than worsen cardiac contractility. 50 51 52 281 There is also evidence for the role of MCs in disrupting the integrity of blood vessels in 53 54 282 the brain leading to saccular intracranial artery aneurysms,110 and also the aorta resulting in 55 56 57 58 59 60 Allergy Page 54 of 84

1 2 3 283 abdominal aortic aneurysms. In a mouse model, MC tryptase MCP6-/- mice or treatment with 4 5 111-113 6 284 MC inhibitors prevented the development of aneurysms. MC-deficient rats showed 7 8 285 reduction of tPA-mediated cerebral hemorrhage compared with wild-type littermates.114 It will 9 10 286 be interesting to study the prevalence of these vascular diseases in patients with inherently higher 11 12 13 287 MC tryptase concentrations, such as those with -tryptasemia. 14 15 16 288 4.2.4 Hypoxic ischaemic encephalopathy 17 18 19 289 Animal studies have shownFor infiltration Peer of MCs andReview histamine release in the brain after hypoxic 20 21 290 ischaemic insults in neonatal rats.115,116 MC stabilization with cromoglycate during the first 24 22 23 24 291 hours after hypoxic injury was neuroprotective, limiting neuronal loss, brain atrophy and 25 26 292 microglial activation. The results suggest that MC degranulation may exacerbate ischemia- 27 28 293 induced neuronal death in the preterm brain. 29 30 31 294 4.2.5 Bronchopulmonary dysplasia 32 33 34 295 MCs may also contribute to the development of BPD in premature neonates. Bhattacharya et al, 35 36 296 2012 used genome-wide expression profiling in lung tissue obtained at biopsy from 11 neonates 37 38 297 with BPD and 17 age-matched controls.117 Expression of 159 genes was significantly different, 39 40 298 with three of the five most significantly dysregulated genes encoded MC biomarkers. 41 42 43 299 Immunohistochemistry demonstrated a 50-fold increase in chymase-expressing MCs in BPD 44 45 300 tissue biopsies when compared with controls. 46 47 301 A recent animal study of hyperoxia induced lung injury provided further evidence on the 48 49 118 50 302 potential mechanistic role of MCs. Increased MC numbers and expression of MC TPSAB1, 51 52 303 TPSAB2 and CPA3 genes were found in mouse lung after exposure to hyperoxia conditions 53 54 55 56 57 58 59 60 Page 55 of 84 Allergy

1 2 3 304 compared controls. Alveoli were disrupted in the test mice, but not in MC deficient mice or 4 5 6 305 controls, suggesting that MC may also disrupt lung structure and function. 7 8 306 5.2.6 Sudden infant death syndrome 9 10 11 307 Serum MC tryptase have been found to be elevated in SIDS in some studies and it has been 12 13 308 suggested that anaphylaxis may have a role in the pathogenesis of this syndrome in some 14 15 119-121 16 309 patients. . In one study of 50 infants with SIDS, mean serum MC tryptase was 6.2ng/ml 17 18 310 compared with 1.1 ng/ml in controls.122 Forty percent of SIDS cases had MCT >10 ng/ml 19 For Peer Review 20 311 compared with none of the controls. In contrast, Nishio et al, 2004, found that MC tryptase was 21 22 123 23 312 normal in 21 infants with SIDS and 14 controls. Genetic testing for KIT and TPSAB1 24 25 313 mutations in these patients might provide further insights into the pathogenesis of this disease. 26 27 28 314 5 CONCLUSIONS 29 30 31 315 Despite 140 years having elapsed since Paul Ehrlich first described the MC,92 there are still 32 33 316 exciting new discoveries in MC biology. In early childhood, somatic activating mutations in the 34 35 36 317 KIT gene of MC precursors can lead to cutaneous mastocytosis, with MC aggregates localizing 37 38 318 to the skin and usually following a benign remitting course. In contrast later in life, somatic 39 40 319 activating KIT gene mutations manifest in bone marrow MC precursors rather than in the skin 41 42 43 320 and are associated with systemic disease and a more aggressive course, particularly if additional 44 45 321 somatic oncogene mutations have accumulated as an inherent part of ageing. 46 47 48 322 Extensive MC protease gene replication over the millennia is now recognized to have 49 50 323 driven diversification of MC function. Indeed, gene replication is a common mechanism by 51 52 124,125 53 324 which all plant and animal species diversify and avoid extinction. Recent studies have 54 55 325 highlighted how common gene variability is in chymotrypsin-related serine proteases, 56 57 58 59 60 Allergy Page 56 of 84

1 2 3 326 particularly in TPSAB1. The gene is completely absent in a third to a half of the population, 4 5 6 327 while duplication occur in approximately 5%. TPSAB1 gene plasticity may have provided 7 8 328 communities living in the tropics with a survival advantage after snake envenomation, and may 9 10 329 explain the absence of TPSAB1 from a higher proportion of Europeans where the risk of being 11 12 bitten poisonous snake is less. Excessive TPSAB1 gene duplication causes raised basal MC 13 330 14 15 331 tryptase activity and a propensity to multi-system disease. Until now, the underlying diagnosis of 16 17 332 many of these patients with nonspecific symptoms may have gone unrecognized. Measurement 18 19 333 of basal serum MC tryptaseFor offers aPeer simple screening Review test, and if ≥8.0ng/ml a possible 20 21 22 334 explanation for their disease. 23 24 25 335 There is no doubt that MC infiltrate and degranulate in the tissues of patients with IBD 26 27 336 and asthma, but the relevance of this to clinical disease remains unclear. There are however clues 28 29 337 that as with eosinophilia, which is now deemed clinically relevant and effective suppressed with 30 31 126 32 338 mepolizumab in a subset of severe asthmatics, dampening down MC activity using imatimab 33 34 339 in patients with refractory asthma is showing some promise. 35 36 37 340 Finally, studies of animal models of common neonatal diseases of the lungs, eye and 38 39 341 brain suggests that MC may well have an important role in their pathogenesis. Although it is 40 41 42 342 important to remain cautious when translating finding from animals into human, these results 43 44 343 should provide the impetus to look for new solutions to these diseases with high healthcare 45 46 344 burdens.127 Furthermore, results from these studies suggest an important role of changes in O 47 2 48 49 345 tension in triggering MC degranulation. 50 51 52 346 140 years on, MCs remain central to allergic and inflammatory diseases, well beyond 53 54 347 IgE-dependent degranulation and immediate hypersensitivity. The challenge for scientists and 55 56 57 58 59 60 Page 57 of 84 Allergy

1 2 3 348 clinicians alike is to look beyond the current dogma if old problems are to be solved with new 4 5 6 349 therapies. 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 58 of 84

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1 2 3 555 94. Wroblewski M, Bauer R, Cubas Córdova M, et al. Mast cells decrease efficacy of anti- 4 5 6 556 angiogenic therapy by secreting matrix-degrading granzyme B. Nat Commun 2017;8:269. 7 8 9 557 95. Ribatti D, Ranieri G. Tryptase, a novel angiogenic factor stored in mast cell granules. Exp 10 11 558 Cell Res 2015;332:157-62. 12 13 14 559 96. Ribatti D. Mast cells as therapeutic target in cancer. Eur J Pharmacol 2016;778:152-7. 15 16 17 560 97. Matsuda K, Okamoto N, Kondo M, et al. Mast cell hyperactivity underpins the development 18 19 For Peer Review 20 561 of oxygen-induced retinopathy. J Clin Invest 2017;127;3987-4000. 21 22 23 562 98. Kovanen PT, Bot I. Mast cells in atherosclerotic cardiovascular disease - Activators and 24 25 563 actions. Eur J Pharmacol 2017;816:37-46. 26 27 28 564 99. Laine P, Kaartinen M, Penttilä A, et al. Association between myocardial infarction and 29 30 565 the mast cells in the adventitia of the infarct-related coronary artery. Circulation 1999;99:361-9. 31 32 33 34 566 100. Fassio F, Almerigogna F. Kounis syndrome (allergic acute coronary syndrome): different 35 36 567 views in allergologic and cardiologic literature. Intern Emerg Med 2012;7:489-95. 37 38 39 568 101. Abdelghany M, Subedi R, Shah S, et al. Kounis syndrome: a review article on 40 41 569 epidemiology, diagnostic findings, management and complications of allergic acute coronary 42 43 44 570 syndrome. Int J Cardiol 2017;232:1–4. 45 46 47 571 102. Kervinen H, Kaartinen M, Mäkynen H, et al. Serum tryptase levels in acute coronary 48 49 572 syndromes. Int J Cardiol 2005;104:138-43. 50 51 52 573 103. Palmiere C, Comment L, Vilarino R, et al. Measurement of β-tryptase in postmortem serum 53 54 in cardiac deaths. J Forensic Leg Med 2014;23:12-8. 55 574 56 57 58 59 60 Page 69 of 84 Allergy

1 2 3 575 104. Xiao N, Li DR, Wang Q, et al. Postmortem Serum Tryptase Levels with Special Regard to 4 5 6 576 Acute Cardiac Deaths. J Forensic Sci 2017;62:1336-1338. 7 8 9 577 105. Silver RB, Reid AC, Mackins CJ, et al. Mast cells: a unique source of renin. Proc Natl Acad 10 11 578 Sci USA 2004;101:13607-12. 12 13 14 579 106. Koda K, Salazar-Rodriguez M, Corti F, et al. Aldehyde dehydrogenase activation prevents 15 16 580 reperfusion arrhythmias by inhibiting local renin release from cardiac mast cells. Circulation 17 18 19 581 2010;122:771-81. For Peer Review 20 21 22 582 107. Zheng J, Wei CC, Hase N, et al. Chymase mediates injury and mitochondrial damage in 23 24 583 cardiomyocytes during acute ischemia/reperfusion in the dog. PLoS One 2014;9:e94732. 25 26 27 584 108. Ngkelo A, Richart A, Kirk JA, et al. Mast cells regulate myofilament calcium sensitization 28 29 30 585 and heart function after myocardial infarction. J Exp Med 2016;213:1353-74. 31 32 33 586 109. Mina Y, Rinkevich-Shop S, Konen E, et al. Mast cell inhibition attenuates myocardial 34 35 587 damage, adverse remodeling, and dysfunction during fulminant myocarditis in the rat. J 36 37 588 Cardiovasc Pharmacol Ther 2013;18:152-61. 38 39 40 110. Ollikainen E, Tulamo R, Lehti S, et al. Mast cells, neovascularization, and 41 589 42 43 590 microhemorrhages are associated with saccular intracranial artery aneurysm wall remodelling. J 44 45 591 Neuropathol Exp Neurol 2014;73:855-64. 46 47 48 592 111. Mäyränpää MI, Kovanen PT. Mast cells: important players in the orchestrated pathogenesis 49 50 51 593 of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2011;31:734-40. 52 53 54 594 112. Zhang J, Sun J, Lindholt JS, et al. Mast cell tryptase deficiency attenuates mouse abdominal 55 56 595 aortic aneurysm formation. Circ Res 2011;108:1316-27. 57 58 59 60 Allergy Page 70 of 84

1 2 3 596 113. Sun J, Zhang J, Lindholt JS, et al. Critical role of mast cell chymase in mouse abdominal 4 5 6 597 aortic aneurysm formation. Circulation 2009;120:973-82. 7 8 9 598 114. Strbian D, Karjalainen-Lindsberg ML, Kovanen PT, et al. Mast cell stabilization reduces 10 11 599 hemorrhage formation and mortality after administration of thrombolytics in experimental 12 13 600 ischemic stroke. Circulation 2007;116:411-8. 14 15 16 601 115. Biran V, Cochois V, Karroubi A, et al. Stroke Induces Histamine Accumulation and Mast 17 18 19 602 Cell Degranulation in the ForNeonatal PeerRat Brain. Brain Review Pathol 2008;18;1-9. 20 21 22 603 116. Jin Y, Silverman A, Vannucci S. Mast Cells Are Early Responders After Hypoxia-Ischemia 23 24 604 in Immature Rat Brain. Stroke 2009;40;3107-12. 25 26 27 605 117. Bhattacharya S, Go D, Krenitsky DL, et al. Genome-Wide Transcriptional Profiling Reveals 28 29 30 606 Connective Tissue Mast Cell Accumulation in Bronchopulmonary Dysplasia. Am J Respir Crit 31 32 607 Care Med 2012;186;349–58. 33 34 35 608 118. Veerappan A, Thompson M, Savage AR, et al. Mast cells and exosomes in hyperoxia- 36 37 609 induced neonatal lung disease. Am J Physiol Lung Cell Mol Physiol 2016;310;1218–32. 38 39 40 119. Buckley M, Variend S, Walls A. Elevated serum concentrations of beta-tryptase, but not 41 610 42 43 611 alpha-tryptase, in Sudden Infant Death Syndrome (SIDS). An investigation of anaphylactic 44 45 612 mechanisms. Clin Exp Allergy 2001;31;1696-1704. 46 47 48 613 120. Gold Y, Goldberg A, Sivan Y. Hyper-releasability of mast cells in family members of 49 50 51 614 infants with sudden infant death syndrome and apparent life-threatening events. J Pediatr 52 53 615 2000;136;460-5. 54 55 56 57 58 59 60 Page 71 of 84 Allergy

1 2 3 616 121. Edston E, Gidlund E, Wickman M, et al. Increased mast cell tryptase in sudden infant 4 5 6 617 death-anaphylaxis, hypoxia or artefact? Clin Exp Allergy 1999;29;1648-54. 7 8 9 618 122. Platt MS, Yunginger JW, Sekula-Perlman A, et al. Involvement of mast cells in sudden 10 11 619 infant death syndrome. J Allergy Clin Immunol 1994;94;250-6. 12 13 14 620 123. Nishio H, Suzuki K. Serum tryptase levels in sudden infant death syndrome in forensic 15 16 621 autopsy cases. Forensic Sci Int 2004;139;57-60. 17 18 19 For Peer Review 20 622 124. Proulx SR. Multiple routes to subfunctionalization and gene duplicate specialization. 21 22 623 Genetics 2012;190:737-51. 23 24 624 125. Taylor JS, Raes J. Duplication and divergence: the evolution of new genes and old ideas. 25 26 Annu Rev Genet 2004;38:615-43. 27 625 28 29 626 126. Ortega HG, Liu MC, Pavord ID, et al. Mepolizumab treatment in patients with severe 30 31 627 eosinophilic asthma. N Engl J Med 2014;371:1198-207. 32 33 34 628 127. Bischoff S. Role of mast cells in allergic and non-allergic immune responses: comparison of 35 36 37 629 human and murine data. Nat Rev Immunol 2007;7;93-104. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 72 of 84

1 2 3 630 Figure Legends 4 5 6 631 Figure 1 Differentiation of mast cells from bone marrow precursors. Hematopoietic stem 7 8 9 632 cells (HSC) pass through a number of stages of differentiation in the bone marrow (multipotent 10 11 633 progenitor (MMP), common myeloid progenitor (CMP), granulocyte/monocyte progenitor 12 13 634 (GMP), basophil/mast cell progenitor (BMCP)) before reaching the mast cell progenitor (MCp). 14 15 16 635 MCp are proliferating agranular MC precursors that enter the blood stream and home to tissues, 17 18 636 where they further differentiate into mature nonproliferating granule positive mast cells (MC). 19 For Peer Review 20 21 637 Figure 2 Homing of mast cell progenitors (MCp) from the bone marrow and peripheral 22 23 638 blood into tissues. Homing of MCp depends on the expression of specific adhesion molecules 24 25 26 639 and chemokines. In the gut, integrins 4 7 on MCp bind to Mucosal vascular Addressin Cell 27 28 640 Adhesion Molecule 1 (MAdCAM1) on blood vessel endothelium, while in the lung these 29 30 641 integrins bind Vascular Cell Adhesion Protein 1 VCAM-1. In the skin, MCp are attracted by 31 32 33 642 prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and chemokine (C-C motif) ligand 2 (CCL2) 34 35 643 also known as monocyte chemoattractant protein 1 (MCP1) to prostaglandin EP3 receptor and 36 37 644 CCR2. Percentages of MCT and MCTC in tissues is based in information from reference 15. 38 39 40 645 Figure 3 Spectrum of primary mast cell diseases. Infants (i) sporadic activating KIT 41 42 43 646 mutations (cutaneous mastocytosis), (ii) dominantly inherited TPSAB1 gene duplication (- 44 45 647 tryptasemia), Older children and adults sporadic activating KIT mutations ± other activating 46 47 mutations (systemic mastocytosis) 48 648 49 50 51 649 Figure 4 Examples of clinical features and histology in mast cell disorders. A. Cutaneous 52 53 650 mastocytosis, B. Dariers sign – urtication of skin lesions after rubbing, C. Excessive mast cell 54 55 651 influx (stained with CD117) within the gastrointestinal tract in a patient with -tryptasemia. 56 57 58 59 60 Page 73 of 84 Allergy

1 2 3 652 Table I Primary mast cell disorders 4 5 Mastocytosis 6 Overview clonal MC proliferation, rare, sporadic, activating gene mutations 7 Subtypes systemic (bone marrow cutaneous (usually without bone 8 involvement) marrow involvement) 9 10 more common in children than adults 11 common in adults, rare in children diffuse, multicentric (urticaria 12 pigmentosa), solitary (mastocytoma) 13 Histology multifocal, dense infiltrates of MC 14 (≥15 mast cells in aggregates) in 15 bone marrow and/or other 16 extracutaneous organs 17 MC may have atypical morphology 18 e.g. spindle-shape, CD2/CD25 19 expressionFor or in Peer more aggressive Review 20 forms an undifferentiated phenotype 21 Clinical course ranges from indolent to malignant indolent, starts in infancy, usually 22 (acute or chronic leukemia) regresses before puberty 23 Clinical features flushing flushing, urticaria, angioedema, 24 abdominal pain, diarrhoea, muscle non-cutaneous symptoms less 25 aches and pains, fractures, common 26 27 neurological and psychiatric 28 symptoms, hypotension/anaphylaxis 29 with malignancy: fevers, weight 30 loss, hepatosplenomegaly 31 Genetics sporadic D816V activating c-kit sporadic D816V activating c-kit 32 mutation in 90%, may have mutation in 20 – 40%, other activating 33 additional mutations in PDGFRA/B, c-kit mutations in 60% 34 TET2, N-RAS 35 MC tryptase >20 ng/ml <20 ng/ml 36 Potential therapies second or third generation second or third generation 37 antihistamines, ranitidine, mast cell antihistamines usually suffice 38 stabilisers (cromoglycate, ketotifen), 39 corticosteroids, imatinib only if no 40 D816V mutation, midostaurin 41 (protein kinase inhibitor), 42 chemotherapy for leukemia 43 44 45 Mast cell activation syndromes 46 Overview excessive mast cell activity without clonal proliferation or obvious secondary 47 triggers, -tryptasemia is a common autosomal dominant condition occurring 48 in a subset of patients 49 Subtypes -tryptasemia (up to 5% of general idiopathic (no evidence of 50 population) mastocytosis, alpha-tryptasemia or 51 secondary cause) 52 Histology may have increased number of MC, no MC aggregates 53 particularly in GI tract, but do not 54 form aggregates, may be associated 55 with increased eosinophils 56 57 58 59 60 Allergy Page 74 of 84

1 2 3 Clinical course variable, may depend of degree of similar to alpha-tryptasemia 4 gene replication, other genetic or 5 environmental cofactors 6 Clinical features flushing, urticaria, angioedema similar to alpha-tryptasemia 7 8 abdominal pain, diarrhoea, but have 9 mucous and blood 10 muscle aches and pains, headaches 11 hypermobile joints 12 POTS 13 Neurological and psychiatric 14 symptoms 15 assess parents and siblings 16 Genetics allelic TPSAB1 gene replication no defined genetic cause 17 usually autosomal dominant 18 Basal MC tryptase ≥8.0ng/ml no consensus regarding MC tryptase, 19 For Peer Reviewhistamine or prostaglandin 20 concentrations 21 Potential therapies secondary or third generation as for alpha-tryptasemia 22 antihistamines, ranitidine, mast cell 23 stabilisers (cromoglycate/ketotifen), 24 dietary manipulation, imatinib 25 26 typically ineffective 27 28 653 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 75 of 84 Allergy

1 2 3 654 Table II Neonatal and paediatric disorders associated with secondary MC dysfunction 4 5 tissue MC pathology increased MC inhibitor MC inhibitor 6 infiltration & serum blocks blocks pathology 7 degranulation MC activity 8 tryptase 9 Disorders associated with chronic atopic & inflammatory disorders 10 11 helminth infections ++ promotes NT NT NT 12 peristalsis & 13 epithelium ion 14 secretion 15 expulsion, 16 granuloma  17 containment 18 inflammatory ++ unknown NT NT ?vedolizumab 19 bowel disease For Peer Review 20 21 22 asthma ++ asthma flares cromoglycate imatinib 23 acute death imatinib 24 25 Disorders associated with hypoxic insults or vascular remodelling 26 27 possible TRPA1 oxygen sensor triggered MC degranulation and inflammation 28 retinopathy of -- neo- ++ cromoglycate cromoglycate 29 prematurity vascularisation nafamostat nafamostat (tryptase 30 stage 3+ ROP (tryptase inhibitor) 31 inhibitor) 32 hypoxic-ischemic ++ neuronal death NT cromoglycate cromoglycate 33 encephalopathy 34 35 36 bronchopulmonary ++ disrupts NT NT ?ketotifen 37 dysplasia alveoli 38 39 40 sudden-infant death -- anaphylaxis ++ NT 41 NT 42 syndrome 43 44 45 atherosclerotic ++ ischemia heart ++ cromoglycate cromoglycate 46 vascular disease disease, 47 Kounis 48 syndrome 49 vascular aneurysms ++ abdominal ++ cromoglycate cromoglycate 50 (brain, aorta) aortic 51 aneurysms, 52 cerebral 53 aneurysms 54 neovascularisation ++ more -- cromoglycate cromoglycate, 55 of tumors aggressive 56 57 58 59 60 Allergy Page 76 of 84

1 2 3 growth and gabexate, mesylate, 4 metastasis nafamostat 5 6 7 655 NT not tested 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 77 of 84 Allergy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 Figure 1 Differentiation of mast cells from bone marrow precursors. Hematopoietic stem cells (HSC) pass 32 through a number of stages of differentiation in the bone marrow (multipotent progenitor (MMP), common 33 myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP), basophil/mast cell progenitor (BMCP)) before reaching the mast cell progenitor (MCp). MCp are proliferating agranular MC precursors that enter the 34 blood stream and home to tissues, where they further differentiate into mature nonproliferating granule 35 positive mast cells (MC). 36 37 254x190mm (96 x 96 DPI) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 78 of 84

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 Figure 2 Homing of mast cell progenitors (MCp) from the bone marrow and peripheral blood into tissues. 32 Homing of MCp depends on the expression of specific adhesion molecules and chemokines. In the gut, 33 integrins 4 7 on MCp bind to Mucosal vascular Addressin Cell Adhesion Molecule 1 (MAdCAM1) on blood vessel endothelium, while in the lung these integrins bind Vascular Cell Adhesion Protein 1 VCAM-1. In the 34 skin, MCp are attracted by prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and chemokine (C-C motif) 35 ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1) to prostaglandin EP3 receptor 36 and CCR2. Percentages of MCT and MCTC in tissues is based in information from reference 15. 37 38 254x190mm (96 x 96 DPI) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 79 of 84 Allergy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 Figure 3 Spectrum of primary mast cell diseases. Infants (i) sporadic activating KIT mutations (cutaneous 32 mastocytosis), (ii) dominantly inherited TPSAB1 gene duplication (alpha-tryptasemia), Older children and 33 adults sporadic activating KIT mutations ± other activating mutations (systemic mastocytosis) 34 254x190mm (96 x 96 DPI) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Allergy Page 80 of 84

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 Figure 4 Examples of clinical features and histology in mast cell disorders. A. Cutaneous mastocytosis, B. 32 Dariers sign – urtication of skin lesions after rubbing, C. Excessive mast cell influx (stained with CD117) 33 within the gastrointestinal tract in a patient with alpha-tryptasemia. 34 254x190mm (96 x 96 DPI) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 81 of 84 Allergy