Semaphorin 3A controls allergic and inflammatory responses in experimental allergic conjunctivitis

Abstract

Aim: To assess the efficacy of topical Semaphorin-3A (SEMA3A) in the treatment of allergic conjunctivitis.

Methods: Experimental allergic conjunctivitis (EAC) mice model induced by short ragweed pollen (SRW) in 4-week-old of BALB/c mice, mice were evaluated using haematoxylin and eosin (H&E) staining, immunofluorescence and light microscope photographs. Early phase took the samples in 24h after instillation and late phase took the samples between 4 to 14d after the start of treatment. The study use of topical SEMA3A (10 U, 100 U, 1000 U) eye drops and subconjunctival injection of SEMA3A with same concentration. For comparison, five types of allergy eyedrops were quantified using clinical characteristics.

Results: Clinical score of composite ocular symptoms of the mice treated with SEMA3A were significantly decreased both in the immediate phase and the late phase compared to those treated with commercial ophthalmic formulations and non-treatment mice. SEMA3A treatment attenuates infiltration of eosinophils entering into conjunctiva in EAC mice. The score of eosinophil infiltration in the conjunctiva of SEMA3A 1000 U-treated group were significantly lower than low-concentration of SEMA3A treated groups and non-treated group. SEMA3A treatment also suppressed T-cell proliferation in vitro and decreased serum total IgE levels in EAC mice. Moreover, Treatment of SEMA3A suppressed Th2-related cytokines (IL-5, IL-13 and IL-4) and pro-inflammatory cytokines (IFN-γ, IL-17 and TNF-α) release, but increased regulatory cytokine IL-10 concentration in the conjunctiva of EAC mice.

Conclusions: SEMA3A as a biological agent, showed the beneficial activity in ocular allergic processes with the less damage to the intraocular tissue. It is expected that SEMA3A may be contributed in patients with a more severe spectrum of refractory ocular allergic diseases including allergic conjunctivitis in the near future.

Keywords: Semaphorin 3A; cytokines; eosinophil infiltration; experimental allergic conjunctivitis; instillation; serum total IgE; subconjunctival administration.

Figure 1. Score for the symptoms of allergic conjunctivitis, was a total statistics of five items

A: All mice were scored biomicroscopically 15-30min post challenge for clinical signs [Redness, Chemosis, Discharge and Tearing]. Difference in clinical symptom suppression was confirmed in the observation period for maximum 4wk (From 3wk to 4wk had no significant changes); B: Classification of allergic conjunctivitis induced by short ragweed. After the evaluation of clinical symptoms in the eye, the severity of disease was graded by summation of scores: none, 0 points (1); minimal, 1-3 points (2); mild, > 4 points or 2 grade 2 symptoms (3); moderate, 3 or 4 grade 2 symptoms (4); severe, 5 grade 2 symptoms (5).

Figur 2. In instillation groups, allergy inhibitory effects of treatment with SEMA3A 1000 U has been evaluated and compared to commercial ophthalmic formulations

A: Instillation groups (Instill); B: Subconjunctival administration groups (SC admin). Topical SEMA3A (10 U, 100 U, 1000 U)- treatment and PBS (as non-treatment) were used in the therapy experiments. Each experimental group consisted of six mice. ^aP< 0.05; ^bP < 0.01, SEMA3A 1000 U treatment group compared with NT; C: Commercial ophthalmic formulations, which are mainly used in the allergic conjunctivitis. NT (non-treatment) used PBS as control; D: The score of the symptoms of allergic conjunctivitis in the immediate phase reaction in 24h (average value of total evaluation). Data represent means ± SD of six mice/group, ^a P < 0.05; ^bP < 0.01, SEMA3A 1000 U treatment group compared with commercial ophthalmic formulation and non-treatment groups; E: The score of the symptoms of allergic conjunctivitis in the late phase reaction on day 14 (average value of total evaluation). Data represent means±SD of six mice / group. ^aP < 0.05; ^bP < 0.01, SEMA3A 1000 U treatment group compared with commercial ophthalmic formulation and NT groups.

Figure 3. SEMA3A administration prevents development of allergic conjunctival inflammation

A: Instillation groups (Instill); B: Subconjunctival administration groups (SC admin). Each experimental group consisted of six mice. ^aP < 0.05; ^bP < 0.01, compared with NT; C: Light micrograph of conjunctival sections in mice from allergic untreated and SENA3A 1000 U treated groups. Eosinophil infiltration in the conjunctiva on day 4 and day 14 after short ragweed (SRW) challenge. C1 (Ctrl, control group): Sensitized but not challenged (the others groups are all sensitized and SRW challenged); C2, 3 (NT, Non-treatment group): PBS ocular instillation group; C4, 5 [SEMA (instill)]: SEMA3A 1000 U ocular instillation treatment group. C6, 7 [SEMA (SC admin)]: SEMA3A 1000 U subconjunctival injection treatment group. Scale bar: 40 um.

Figure 4. A significant reduction of proliferation in the in vitro inhibition of T cell proliferation was confirmed by SEMA3A administration of the concentration-dependent

In vitro, T cell proliferation inhibition of SEMA3A was confirmed on anti-Mouse CD3 plate after 48h incubation. SEMA3A 1000 U treatment group was maximum about 31 percent lower than NT group. All groups are run in triplicate. Pooled values of three repeat experiments are expressed as mean ± SE. ^aP < 0.05; ^bP < 0.01, SEMA3A 1000 U treatment group compared with the NT group or SEMA3A 100 U treatment group.

Figure 5. SEMA3A showed tissue penetration and the effect of maintaining the barrier function of the conjunctival epithelium by administration of high concentrations on day 4

Sections were stained with antibodies against SMA3A (C,H), fluorescent Alexa Fluor 546 phalloidin (B,G) for actin, and 4′ 6′-diamidino-2-phenylondole dihydrochloride (DAPI) (A,F) was used to label cell nuclei. SEMA3A 1000 U instillation mice (F-J) compared with the non-treatment mice (A-E). Scale bar: 50um.

Figure 6. SEMA3A treatment decreased serum total IgE levels and prevents the development of allergic responses, suppressed Th2-related cytokines (IL-5, IL-13 and IL-4) and pro-inflammatory cytokines (IFN-γ, IL-17 and TNF-α) release, but increased regulatory cytokine IL-10 concentration in the conjunctiva of EAC mice

Serum levels of total IgE (A), in normal mice, non-treated mice, and mice treated with SEMA3A (10 U, 100 U, 1000 U) on day 14. SEMA3A 1000 U treatment group compared with NT group and the other low-concentration of SEMA3A treated groups. Levels of IL-4 (B), IL-5 (C), IL-10 (D), IL-13 (E), IL-17 (F), TNF-α (G) and IFN-γ (H) were measured by MILLIPLEX Cytokine panel. SEMA3A 1000 U treatment group (SEMA) compared with NT group. Pooled values of two repeat experiments, each with six mice per group, data represent means ± SD. ^aP < 0.05; ^bP < 0.001.