Bonini, S

Calder, VL

Eskandarpour, M

Larkin, FDP

Micera, A

Nunn, M

Weston-Davies, W

Zaher, S

Zhang, X

English

UCL Discovery

Allergy. 2021;00:1–4.   | 1wileyonlinelibrary.com/journal/allReceived: 24 June 2021  | Revised: 27 September 2021  | Accepted: 3 October 2021DOI: 10.1111/all.15128  L E T T E RAllergic eye disease: Blocking LTB4/C5 in vivo suppressed disease and Th2 & Th9 cellsTo the EditorVernal keratoconjunctivitis (VKC) affects children and can impair vision if the cornea becomes involved. Immunosuppressives (ste-roids and cyclosporin A) are required but can have side effects, and novel immunotherapeutic approaches are needed.1 The aim of this study was to investigate the contributions of LTB4 and complement C5 in a model of allergic eye disease (experimental allergy conjuncti-vitis, EAC).2 Previous studies have demonstrated that nomacopan, a bifunctional recombinant biologic derived from blood- feeding ticks, has anti- inflammatory properties by capturing LTB4 and preventing it from interacting with its two known G protein- coupled cell surface receptors (GPCR BLT1 and BLT2). Simultaneously, nomacopan inhib-ited C5 end terminal complement activation, thereby preventing for-mation of C5b- 9 and C5a.3 These two pathways have evolutionary connections as phylogenetic analysis showed that tick saliva started as an LTB4 inhibitor, and subsequently acquired the ability to inhibit C5. It has been shown in a mouse model of inflammatory arthritis that C5a production resulted in the release of LTB4 to promote fur-ther neutrophil migration to the interstitium.4EAC is a model of allergic eye disease mainly driven by effector Th2 cells and mast cells (MC). During EAC, conjunctival inflammation can be detected after 5 days, scored non- invasively2 (Figure S1A– C), with elevated levels of conjunctival IL- 9- expressing CD4+T cells and MC detected in tissues and cells expanded from conjunctival explants (Figure S1D– J). Significantly increased levels of tryptase+ conjunctival MC were observed, co- expressing intracellular IL- 9 in the sub- epithelial area within the fornix of conjunctival EAC tissues (Figure S1G).In this study, we investigated the effects of topically admin-istered nomacopan in EAC and observed a significant suppres-sion of disease in treated mice, and a decrease in IL- 9- expressing CD4+T cells (Figure 1A– C; Figure S2A). Recent studies have demonstrated that IL- 9 can be produced by Th9 and Th2 cells.5,6 To determine which CD4+T cell subset was producing IL- 9, tran-scription factor expression was investigated. IL- 9- producing Th2 cells express GATA3, but not PU- 1, whereas IL- 9- producing Th9 cells express PU.1, but not GATA3. Within the infiltrating CD4+T cells in EAC, Th9 cells (IL- 9+PU.1+) and IL- 9- expressing Th2 cells (GATA3+IL- 4+IL- 9+) cells were increased in OVA- challenged mice compared with controls (Figure S1H– J).During early stages of EAC (Days 1– 6), no effect by nomacopan was observed due to low inflammatory scores overall. However, at days 7 and 10, a significant decrease in IL- 9+CD4+ T cells (Figure 1C– F) following nomacopan treatment was observed. The levels of Th2 (IL- 4+ PU.1− GATA3+) cells and IL- 9- expressing Th2 cells were also significantly reduced (Figure S2B– D). Interestingly, although there was an overall reduction in IL- 9 expression levels, there were signif-icant changes in Th9 (IL- 9+PU.1+IL- 4−) levels only at the higher con-centration of nomacopan (Figure 1D).This in vivo model shares many features with VKC in man since there is a predominant CD4+T cell infiltration of the conjunctival tis-sues and evidence of fibrosis.2 Hence, we used VKC tissue specimens and tear fluids to compare with EAC tissues. Tears collected from VKC patients during active disease showed a significantly higher level of IL- 9 (clinically uncontrolled) as compared with inactive VKC (controlled; Figure 2A). VKC conjunctival tissue sections were exam-ined for expression of CD4, IL- 9, IL- 9 receptor (IL- 9R), and tryptase (Figure 2B– E). IL- 9- expressing CD4+T cells and MC were both signifi-cantly up- regulated in all active VKC specimens (n = 7) as compared with controls. We observed CD4+T cells co- localized with IL- 9 in the sub- epithelial and stromal areas of VKC sections (Figure 2B) and IL- 9R expression (Figure 2C). Infiltrating MC (Tryptase+) and other cell types also expressed IL- 9R (Figure 2D) within stromal areas, with the frequency of IL- 9R- expressing MC significantly higher than IL- 9R- expressing CD4+T cells (Figure 2E).To further investigate whether the receptors for those ligands targeted by nomacopan were expressed during EAC, conjunctival tissue expression and localization of C5aR and BLT1 (LTB4 recep-tors) were investigated (Figure S2E) as well as in VKC and healthy tissue controls (Figure 2F,G). Relative expression of BLT1 and C5aR was analysed by counting positively stained cells in at least 5 non- overlapping areas (ImageJ). Interestingly, increases in infiltrating immune cells within the stromal areas correlated with increased ex-pression of BLT1 (means ± SD; 34.98 ± 15) and C5aR (56.67 ± 12) in EAC, and comparable levels were also observed in VKC: BLT1 (40.48 ± 10); C5aR (44.90 ± 20). Very few cells co- expressed both receptors while only rarely were any BLT1+ or C5aR1+ cells detected in healthy mouse and human tissues, suggesting that these receptors are exclusively expressed during disease, potentially by infiltrating CD4+T cells and MC.© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.2  |    LETTERF I G U R E  1  Topical nomacopan attenuated conjunctivitis in EAC and decreased IL- 9 in CD4+T cells. (A, B) Clinical scores of eyes were compared with saline- treated controls. (C−F) Conjunctival explant cells stained for T- cell markers. Flow cytometry plots represent staining for: (C) IL- 9, (D) IL- 9 and PU.1 (Th9 cells) and a summary graph of percentages for IL- 9+PU.1+ (Th9) or IL- 9+PU.1− (Th2) cells. (E, F) Representative plots were gated for Th2 cells (IL- 4+GATA3+PU.1−) in treatment groups and a summary graph. Four independent experiments, ANOVA and unpaired t- tests; mean ± SD (n = 6– 8 mice per group). *p < .05, **p < .01. Nom = nomacopan, CsA = Cyclosporine A(A) Day 7 of treatment (C)(E)(D)(F)EAC-salineTh9Th2EAC-Nom-0.25Th9Th2EAC-Nom-0.5Th9Th2% IL-4+ GATA3+in CD4+********IL-43ATAGctrl-saline EAC-saline EAC-Nom-0.25 EAC-Nom-0.55.803.822.5587.8v68.0 7.0022.42.60 12.92.411.9882.7 1.9089.82.84 5.44erocs yrotammalfniDay 10 of treatmentIL-9PU.1% IL9+expressing cells  2460(B) Day 10 of treatmentinflammatory score *******24602030401001020300ctrl-salineTh9Th2OVA challengectrl saline Nom-0.25 Nom-0.5 CsA3210CD4+cell number x105************OVA challenge********OVA challenge******OVA challenge OVA challenge***    | 3LETTERF I G U R E  2  (A) IL- 9 levels in tear fluids, demonstrating an increase in clinically uncontrolled (i.e. active) VKC as compared with those with clinically controlled disease; (B) Histology/immunofluorescence staining of VKC conjunctival sections for CD4 and IL- 9; (C) CD4 and IL- 9R; (D) Tryptase and IL- 9R. (E) Scatterplots based on immunoreactivity and intensity for IL- 9R, tryptase or IL- 9R and CD4 as percentages of the immunostained areas. Representative summary graph of one of 7 biopsies. Values were added from 3 fields. (F, G) Representative staining for C5aR1, BLT1, CD4 on healthy, 1 of 3 (F) and VKC 1 of 7 (G) conjunctival tissues. Mean ± SD, ANOVA, **p < .01,**p < .001. E = epithelial layer; S = stroma: G = gland (Lacrimal)IL-9 (pg)nietorp latot gm/**IL-9RTyptaseIL-9RCD4100μm100μm**Area intensity % 200150100500BLT1C5aRCD4=whiteS SE EGG50μmSE50μmBLT1C5aRCD4=whiteIL-9CD4100μmHealthy VKCSE3002001000(B)(D)(A)(E)(F) (G)(C)4  |    LETTERWe conclude that IL- 9 was up- regulated during VKC and EAC. Nomacopan significantly suppressed EAC severity, accompanied by a decrease in IL- 9- producing Th2 cells, Th2 cells and to a lesser extent, Th9 cells, suggesting a key pro- inflammatory role for IL- 9- secreting CD4+T cells in allergic eye disease.Our findings support nomacopan as a potential treatment for al-lergic eye disease due to its ability to down- regulate LTB4/C5 path-ways in EAC.FUNDING INFORMATIONThis research was supported in part by a research collaborative grant from Akari Therapeutics (V.C.), the National Institute for Health Research (NIHR) Moorfields Biomedical Research Centre and NIHR Moorfields Clinical Research Facility, based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. M.N. and W.W- D. are employees of Akari Therapeutics.ACKNOWLEDG EMENTWe would like to thank our support staff, in particular in the BSU, for their assistance.CONFLIC T OF INTERE S TNo authors have any Conflicts of Interest with the exception of Drs. Nunn and Weston- Davies, who are both employees of Akari Therapeutics Inc.Malihe Eskandarpour1Xiaozhe Zhang1Alessandra Micera2Sarah Zaher3,4Frank D. P. Larkin3,4Miles Nunn5Stefano Bonini6Wynne Weston- Davies5Virginia L. Calder1,41Ocular Immunology Group, UCL Institute of Ophthalmology, London, UK2UOSD Research Laboratories, IRCCS- fondazione Bietti, University Campus Bio- Medico, Rome, Italy3NIHR Moorfields Clinical Research Facility, Moorfields Eye Hospital, London, UK4NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust &, UCL Institute of Ophthalmology, London, UK5Akari Therapeutics plc, London, UK6Ophthalmology Operative Complex Unit, University Campus Bio- Medico, Rome, ItalyCorrespondenceVirginia L. Calder, UCL Institute of Ophthalmology, 11/43 Bath Street, London EC1V 9EL, UK.Email: v.calder@ucl.ac.ukORCIDMalihe Eskandarpour  https://orcid.org/0000-0002-6380-5389 Virginia L. Calder  https://orcid.org/0000-0003-1422-2926 R E FE R E N C E S 1. Leonardi A, Lazzarini D, Motterle L, et al. Vernal keratoconjunctivitis- like disease in adults. Am J Ophthalmol. 2013;155(5):796- 803. doi:10.1016/j.ajo.2012.11.018 2. Reyes NJ, Mathew R, Saban DR. Induction and characteriza-tion of the allergic eye disease mouse model. Methods Mol Biol. 2018;1799:49- 57. doi:10.1007/978- 1- 4939- 7896- 0_5 3. Roversi P, Ryffel B, Togbe D, et al. Bifunctional lipocalin ameliorates murine immune complex- induced acute lung injury. J Biol Chem. 2013;288(26):18789- 18802. doi:10.1074/jbc.M112.420331 4. Sidik CD, Miyabe Y, Sezin T, Luster AD. The critical role of C5a as an initiator of neutrophil- mediated autoimmune inflammation of the joint and skin. Semin Immunol. 2018;37:21- 29. doi:10.1016/j.smim.2018.03.002 5. Micosse C, von Meyenn L, Steck O, et al. Human "TH9" cells are a subpopulation of PPAR- gamma(+) TH2 cells. Sci Immunol. 2019;4(31):1- 13. 10.1126/sciim munol.aat5943 6. Chang HC, Sehra S, Goswami R, et al. The transcription factor PU.1 is required for the development of IL- 9- producing T cells and al-lergic inflammation. Nat Immunol. 2010;11(6):527- 534. https://doi.org/10.1038/ni.1867SUPPORTING INFORMATIONAdditional supporting information may be found in the online ver-sion of the article at the publisher’s website.

Allergic eye disease: Blocking LTB4/C5 in vivo suppressed disease and Th2 & Th9 cells

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Allergic eye disease: Blocking LTB4/C5 in vivo suppressed disease and Th2 & Th9 cells

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