To the Editor,

Vernal keratoconjunctivitis (VKC) affects children and can impair vision if the cornea becomes involved. Immunosuppressives (steroids 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 conjunctivitis, 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 inhibited C5 end terminal complement activation, thereby preventing formation 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 further neutrophil migration to the interstitium.⁴

EAC 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 administered nomacopan in EAC and observed a significant suppression 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, transcription 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 significant changes in Th9 (IL-9⁺PU.1⁺IL-4⁻) levels only at the higher concentration 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 tissues 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 examined 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 significantly 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 receptors) 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 expression 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.

We 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 allergic eye disease due to its ability to down-regulate LTB4/C5 pathways in EAC.

致编辑，

春季角结膜炎 (VKC) 会影响儿童，如果角膜受累，可能会损害视力。需要使用免疫抑制剂（类固醇和环孢菌素 A），但可能会产生副作用，因此需要新的免疫治疗方法。¹本研究的目的是调查 LTB4 和补体 C5 在过敏性眼病（实验性过敏性结膜炎，EAC）模型中的作用。²先前的研究表明，nomacopan 是一种源自吸血蜱的双功能重组生物制剂，通过捕获 LTB4 并阻止其与其两种已知的 G 蛋白偶联细胞表面受体（GPCR BLT1 和 BLT2）相互作用，具有抗炎特性。同时，nomacopan 抑制 C5 末端补体激活，从而阻止 C5b-9 和 C5a 的形成。³这两种途径具有进化联系，因为系统发育分析表明，蜱唾液最初是一种 LTB4 抑制剂，随后获得了抑制 C5 的能力。已经在炎症性关节炎的小鼠模型中显示，C5a 的产生导致 LTB4 的释放，以促进中性粒细胞进一步迁移到间质。⁴

EAC 是一种过敏性眼病模型，主要由效应 Th2 细胞和肥大细胞 (MC) 驱动。在 EAC 期间，结膜炎症可在 5 天后检测到，无创评分为² （图 S1A-C），在结膜外植体扩增的组织和细胞中检测到结膜表达 IL-9 的 CD4 ⁺ T 细胞和 MC 水平升高（图 S1D-J)。^{观察到类胰蛋白酶+}结膜 MC水平显着增加，在结膜 EAC 组织穹窿内的上皮下区域共表达细胞内 IL-9（图 S1G）。

在这项研究中，我们研究了在 EAC 中局部给药的 nomacopan 的作用，并观察到治疗小鼠的疾病显着抑制，以及表达 IL-9 的 CD4 ⁺ T 细胞减少（图 1A-C；图 S2A）。最近的研究表明，IL-9 可以由 Th9 和 Th2 细胞产生。^{5, 6}为了确定哪个 CD4 ⁺ T 细胞亚群产生 IL-9，研究了转录因子的表达。产生 IL-9 的 Th2 细胞表达 GATA3，但不表达 PU-1，而产生 IL-9 的 Th9 细胞表达 PU.1，但不表达 GATA3。在 EAC 中浸润的 CD4 ⁺ T 细胞内，Th9 细胞 (IL-9 ⁺ PU.1 ⁺ ) 和表达 IL-9 的 Th2 细胞 (GATA3 ⁺ IL-4 ⁺与对照相比，OVA 攻击的小鼠中IL-9 ⁺ ) 细胞增加（图 S1H-J）。

在 EAC 的早期阶段（第 1-6 天），由于总体炎症评分较低，未观察到 nomacopan 的影响。然而，在第 7 天和第 10 天，观察到在 nomacopan 治疗后IL-9 ⁺ CD4 ^{+ T 细胞（图 1C-F）显着减少。}Th2（IL-4 ⁺ PU.1 ^- GATA3 ⁺）细胞和表达 IL-9 的 Th2 细胞的水平也显着降低（图 S2B-D）。有趣的是，虽然 IL-9 表达水平总体降低，但 Th9（IL-9 ⁺ PU.1 ⁺ IL-4 ^-）水平仅在较高浓度的 nomacopan 时发生显着变化（图 1D）。

这种体内模型与人体中的 VKC 有许多共同特征，因为结膜组织中存在主要的 CD4 ⁺ T 细胞浸润和纤维化的证据。²因此，我们使用 VKC 组织标本和泪液与 EAC 组织进行比较。与非活动性 VKC（受控；图 2A）相比，在活动性疾病期间从 VKC 患者收集的眼泪显示出显着更高水平的 IL-9（临床上不受控制）。检查 VKC 结膜组织切片中 CD4、IL-9、IL-9 受体 (IL-9R) 和类胰蛋白酶的表达（图 2B-E）。 与对照相比，所有活性 VKC 标本 ( n = 7) 中表达 IL-9 的 CD4 ⁺ T 细胞和 MC 均显着上调。我们观察到 CD4 ⁺T 细胞与 IL-9 共定位在 VKC 切片的上皮下和基质区域（图 2B）和 IL-9R 表达（图 2C）。浸润性 MC（类胰蛋白酶⁺）和其他细胞类型也在基质区域内表达 IL-9R（图 2D），表达 IL-9R 的 MC 的频率显着高于表达 IL-9R 的 CD4 ⁺ T 细胞（图 2E）。

为了进一步研究 nomacopan 靶向的那些配体的受体是否在 EAC 期间表达，研究了 C5aR 和 BLT1（LTB4 受体）的结膜组织表达和定位（图 S2E）以及 VKC 和健康组织对照（图 2F，G ）。BLT1 和 C5aR 的相对表达通过计数至少 5 个非重叠区域中的阳性染色细胞进行分析 (ImageJ)。有趣的是，基质区域内浸润免疫细胞的增加与 EAC 中 BLT1（平均值 ± SD；34.98 ± 15）和 C5aR（56.67 ± 12）的表达增加相关，并且在 VKC 中也观察到相当的水平：BLT1（40.48 ± 10 ); C5aR (44.90 ± 20)。很少有细胞同时表达这两种受体，而很少有任何 BLT1 ⁺或 C5aR1 ⁺在健康小鼠和人体组织中检测到的细胞，表明这些受体仅在疾病期间表达，可能通过浸润 CD4 ⁺ T 细胞和 MC。

我们得出结论，IL-9 在 VKC 和 EAC 期间上调。Nomacopan 显着抑制 EAC 严重程度，伴随着产生 IL-9 的 Th2 细胞、Th2 细胞和较小程度的 Th9 细胞减少，这表明分泌 IL-9 的 CD4 ⁺ T 细胞在过敏性眼中具有关键的促炎作用疾病。

我们的研究结果支持 nomacopan 作为过敏性眼病的潜在治疗方法，因为它能够下调 EAC 中的 LTB4/C5 通路。