Pollen shells and soluble factors play non-redundant roles in the development of allergic conjunctivitis in mice

Abstract

Purpose

We aimed to clarify the role of particulate allergen exposure to the conjunctiva in the development of allergic conjunctivitis.

Methods

We administered ragweed pollen suspension, pollen extract, pollen shell, particulate air pollutants, and their combinations to the mouse conjunctiva five days a week without prior sensitization. Clinical signs were scored. Histological changes, cellular infiltrations, mRNA expressions, lymph node cell recall responses, and serum immunoglobulin levels were assessed. Immune cell-depleting antibodies and ST2 knockout mice were used to investigate the cellular and molecular requirements.

Results

Pollen suspension, but not the extract or shell alone, induced robust eosinophilic conjunctivitis, accompanied by a proliferative response of epithelial cells. A combination of pollen extract and shell completely restored eosinophil accumulation. In addition, eosinophilic conjunctivitis was induced by a mixture of particulate air pollutants and pollen extract. Mechanistically, eosinophil accumulation was ameliorated by deficiency of the IL-33 receptor ST2 and abolished by depleting CD4⁺ T cells. Pollen shells, but not the extract, induced IL-33 release from conjunctival epithelial cells in vivo.

Conclusions

Our results indicate the non-redundant roles for the allergens’ particulate properties and soluble factors in the development of allergic conjunctivitis.

Introduction

Ocular allergy is a common allergic manifestation [[1], [2], [3], [4]]. Its prevalence has increased in the past decades [3,5,6] and is estimated to have reached approximately 50% in Japan [1] and 40% in the US [3]. Seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) are the most common but least severe forms, while vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) are relatively rare but severe forms that potentially lead to corneal involvement and ultimately to vision loss [1,2,4,[7], [8], [9], [10]]. The proliferative lesions such as giant papillae are observed in VKC, and in some cases of AKC [2,[7], [8], [9], [10]]. In addition, giant papillary conjunctivitis (GPC) may develop as a result of mechanical irritation such as contact lenses [2,7,11].

The ocular allergy often accompanies other allergic conditions such as allergic rhinitis, atopic eczema [1,12], asthma [13], and food allergy [2,14]. Among them, the most popular comorbidity is the allergic rhinitis; however, isolated ocular symptoms have also been documented in 6% of the general population [3]. Importantly, the sensitized allergen patterns confirmed by skin test were skewed to the weed pollens in patients with ocular symptoms, compared to the grass, mold, or indoor allergens [3]. However, it remains obscure what properties of weed allergens are essential to sensitize patients to cause solely ocular symptoms.

Epidemiological studies have been performed to elucidate the association between particulate air pollutants and conjunctivitis [15,16]. In addition, their association with allergic conjunctival diseases has been reported [1,17,18]. For instance, particulate matter (PM) exposure increases outpatient visits of allergic conjunctivitis [17]. The levels of PM10, which is a PM with an aerodynamic diameter less than 10 μm, are associated with the prevalence of VKC [1]. Furthermore, the skin prick test response to Asian dust has been found to correlate with the mean wheal diameters for pollens and fungi, implying its adjuvant effect [18]. Although the mechanistic studies on the effect of particulate air pollutants on the ocular surface are relatively limited [[19], [20], [21], [22], [23]], their biological and immunomodulatory effects on the airways have been extensively studied [24]. However, since the lower respiratory tract is accessible to only small particles with a median aerodynamic diameter under 2.5 μm, the effect of larger particles such as pollen grains onto the mucosal surface has been less characterized.

Interleukin (IL)-33 is an epithelial cell-derived cytokine, which is considered as one of the key regulators of type 2 immunity [25,26]. It is stored in the nuclei in the steady state, and actively released upon detection of external damage, potential pathogens, or allergens [[25], [26], [27], [28], [29]]. Necrotic cells also release IL-33 slowly from their chromatin [30]. Thereafter, the protease activity from various sources including allergen proteases from various pollens, fungi, mites, and cockroaches act as amplifiers of IL-33 activity [25,31,32].

IL-33 has been implicated in allergic conjunctival diseases [33]. The serum level of IL-33 was found to be elevated in the Cedar pollen allergic rhinoconjunctivitis patients [34]. In addition, local expression of IL-33 in giant papillae of AKC patients was confirmed in the epithelial cells as well as in the vascular endothelial cells [35]. Functionally, in a sensitization-elicitation type of murine allergic conjunctivitis model, exogenous IL-33 augmented eosinophil accumulation [36]. On the other hand, IL-33 deficiency, but not thymic stromal lymphopoietin (TSLP) or IL-25 deficiency, ameliorated conjunctivitis in the elicitation phase [37]. Furthermore, transgenic expression of IL-33 in epithelial cells in vivo provoked spontaneous keratoconjunctivitis resembling phenotypes of AKC, with marked increase of type 2 innate lymphoid cell (ILC2) population in the cornea [38]. Instillation of papain-soaked contact lenses to murine eyes induced IL-33 expression, leading to eosinophil accumulation dependently on TSLP, basophils, and ILC2, but not on T cells [39]. These observations suggest that IL-33 plays a role both in the innate type of conjunctival inflammation and in the elicitation phase of allergic conjunctivitis. However, the role of allergens’ particulate properties on the release of IL-33 remains unclear.

In addition to the above-mentioned observation that a substantial percentage of the population develops ocular allergic symptoms without concomitant rhinitis or other allergies [3], it has been reported that up to 60% of ocular allergy patients consulting ophthalmologists had solely ocular symptoms [40]. Furthermore, the patients with elevated levels of tear IgE, without detectable serum allergen specific IgE have been reported [[41], [42], [43]]. Thus, we hypothesized that these patients might have been sensitized to allergens through their eyes. To address this hypothesis, we chose ragweed (RW) pollens as allergens, and repeatedly applied them to the mouse conjunctiva. Intriguingly, RW pollen suspension, but not its extract, induced allergic symptoms, accompanied by robust accumulation of eosinophils. By generating virtually protein-free pollen shells, we attempted to dissect the mechanisms underlying this novel particulate pollen-induced mucosal sensitization model of allergic conjunctivitis.