Exogenous interleukin-10 alleviates allergic inflammation but inhibits local interleukin-10 expression in a mouse allergic rhinitis model

Allergic rhinitis (AR) is one of the most prevalent airway diseases worldwide, which exerts a heavy burden on patients due to its impact on physical, social and emotional functioning, as well as the cost of treatment. Despite the increasing worldwide prevalence of AR, treatments for AR remain limited in effect [1]. Thus, it is greatly important to develop novel therapies with higher efficacy for AR patients.

AR is characterized by a disturbance of T-cell subsets and an accumulation of eosinophils and mast cells. CD4⁺ T-cell subsets are central regulators of immune responses and allergic inflammatory diseases. After encountering specific antigen, they become activated, proliferate, and differentiate into specific Type 1 helper T (Th1), Th2, Th17, or regulatory T (Treg) cells distinguished by cytokine profile, surface phenotype, and specific transcription factor expression. AR has long been considered as an allergic response involving predominantly Th2 cells, with a relative insufficiency of Th1 cells [2]. However, in recent years, the classic paradigm of Th1/Th2 cell-mediated immunity has been evolved to include novel subsets of Th cell, such as Th17 cells and Treg cells. Exaggerated Th17 and impaired Treg responses have been implicated in the development of AR and have been associated with corticosteroid resistance [2–7]. These T-cell subsets regulate inflammatory responses mainly by secreting specific cytokines such as interferon-γ (IFN-γ), IL-5, IL-17, IL-10, and transforming growth factor-β1 (TGF-β1), as well as other non-specific cytokines such as tumor-necrosis factor-α (TNF-α) [3–6, 8, 9].

IL-10 is a multifunctional cytokine secreted by a variety of cells, including T cells, monocytes, macrophages, dendritic cells, and endothelial cells. IL-10 has diverse effects on most hemopoietic cells. Its crucial role is to limit and ultimately terminate immune and inflammatory responses [10]. In the sensitization phase of allergic responses, IL-10 can down-regulate major histocompatibility complex class II and costimulatory molecule expression on dendritic cells and induce anergic immune responses possibly through up-regulating cytotoxic T lymphocyte-associated molecule-4 (CTLA)-4 expression. In the effector phase, IL-10 directly inhibits active CD4⁺ T cells proliferation and their function by crosslinking IL-10R on the surface of activated CD4⁺ T cells, which engages the Jak family signalling pathway of tyrosine kinases Jak1 and Tyk2 [11]. Because of its inhibitory properties, IL-10 has been suggested as a potential therapy for systemic and localized autoimmune and allergic inflammatory diseases, including inflammatory bowel disease, allergic asthma, rheumatoid arthritis, and experimental autoimmune encephalomyelitis [10, 12, 13]. It has been reported that endogenous IL-10 levels in the nasal endothelium are decreased and negatively correlated with AR development and severity in AR patients [14, 15], which makes us hypothesize that IL-10 may alleviate allergic inflammation in AR. Here, we investigated the effect of intranasal exogenous IL-10 administration on the allergic inflammation of AR in a mouse model induced by ovalbumin (OVA).

In this study, we demonstrated that rmIL-10 administration during challenge could alleviate allergic inflammatory response in AR mice, which was evidenced by the decreased infiltration of eosinophils and mast cells, and reduced Th2 and Th17 responses. In contrast, interestingly, rmIL-10 inhibited the local IL-10 expression in the nasal mucosa.

AR is characterized by the accumulation of eosinophils and mast cells. Th cells and their secreted cytokines play a central role in regulating the migration, apoptosis, and function of eosinophils and mast cells in AR. Th2 cells secrete IL- 4, IL-5, IL-10, and IL-13, which mount humoral immunity by inducing IgE production by B cells and by activating mast cells and eosinophils, while Th1 cells mediate cellular immunity by secreting IFN-γ. It has been considered that the function of Th1 and Th2 cells is inhibited by each other [2]. In line with a previous finding, we found that T-bet expression was not significantly changed in AR mice compared with the controls [16]. However, IFN-γ expression levels in the NLF and nasal mucosa were slightly increased in AR mice compared with the controls, and were down-regulated with rmIL-10 treatment during challenge. The inconsistent expression between IFN-γ and T-bet was not very surprising. It has been reported that IFN-γ is secreted by a variety of cells, including Th1 cells, macrophages, and epithelial cells, but T-bet is only expressed by Th1 cells. We also showed that Th2 responses (IL-5, IL-10, and GATA-3 expression) were markedly down-regulated with rmIL-10 treatment, suggesting that IL-10 can suppress Th2 reactions [12, 13].

IL-17-producing Th17 cells, induced in vitro by IL-6, IL-23, and TGF-β1, and regulated by transcription factor ROR-c, have multiple functions in different inflammatory airway diseases. Th17 cells have not only been indicated in control of the mucosal pathogen, but also are involved in the pathogenesis of Th1-mediated inflammatory diseases [3, 4]. Recently, Th17 cells have been associated with Th2 predominated allergic diseases. Increased expression of Th17 responses has been found in asthma, AR, and nasal polyps [5, 17–19]. Th17 cells have been demonstrated to promote the development and augment the severity of allergic inflammatory airway diseases by secreting pro-inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, and IL-17 [5, 17, 18]. Interestingly, increased IL-17 expression is associated with resistance to corticosteroid in AR patients [20]. Consistent with previous reports [5, 7], we showed that the expression levels of IL-17, ROR-c, and TGF-β1 were significantly increased in AR mice compared with the control mice. Moreover, we found that the increased expression of Th17 responses could be significantly down-regulated by IL-10 treatment in AR mice. A probable underlying mechanism may be that IL-10 combines IL-10R on the surface of activated Th17 cells and inhibits proliferation and function of Th17 cells [11]. It has also been reported that an intermediate subset of T cells, Foxp3⁺ IL-17⁺ T cells, exist in the nasal mucosa of patients with AR, which can be converted to Foxp3⁺ Treg under the influence of IL-10 [21]. In this study, we found an inhibition of ROR-c expression without an increase of Foxp3⁺ expression. Therefore, we could not preclude the possibility that rmIL-10 may down-regulate Th17 responses by promoting the conversion of Foxp3⁺ IL-17⁺ T cells to Foxp3⁺ Treg [21]. However, this needs further investigation in future.

Unlike other effector CD4⁺ T cells, Treg cells have been implicated in peripheral tolerance as an inhibitor of immune responses. Treg cells are classified into natural Treg (nTreg) generated in the thymus and induced Treg (iTreg) cells induced by IL-10 or TGF-β1 in the periphery. Both of them are required for the full expression of tolerance but have a division of labour in immune homeostasis. nTreg cells migrate preferentially to lymphoid organs, prevent the development of autoimmune disease, and can be recruited and activated early during an immune response to control its magnitude, whereas iTreg cells are induced upon repeated antigen stimulation, act later to reduce the activation of T cells directed against environmental antigens and to restore and maintain tolerance [22–25]. One of the iTreg cells is Type 1 Regulatory T (Tr1) cells that are induced by IL-10. A few studies have demonstrated that iTreg cells also express Foxp3 upon activation. However, Foxp3 expression on Tr1 cells is low and transient or never reaches the high expression levels that characterize Foxp3⁺ Treg. Furthermore, Foxp3 is not required for Tr1 cell induction or function since suppressive Tr1 cells can be generated or isolated from peripheral blood of patients with Foxp3-mutations, even in those patients with complete deletion of Foxp3 [26, 27]. Therefore, Tr1 cells may not express Foxp3 and their immunosuppressive effects are partly inhibited by anti-IL-10 neutralizing antibody [27]. Tr1 cells, but not nTreg cells, were decreased in the peripheral blood of patients with AR [25, 28]. In the present study, total Foxp3 mRNA expression was slightly increased in AR mice compared with the controls, reflecting a possible increased recruitment of nTreg to nasal mucosa or activation of T cells in nasal mucosa. IL-10 treatment may induce accumulation of Tr1 in nasal mucosa, however, the limited amount of murine nasal mucosa does not allow the flow cytometric analysis of Tr1 in nasal mucosa based on the co-expression of CD49b and lymphocyte activation gene 3 on Tr1 [26].

We found that IL-10 protein levels in NLF, and IL-10 mRNA expression level and IL-10-positive cell number in the nasal mucosa were significantly increased in AR mice compared with the control mice. Immunohistochemistry showed that IL-10-immunoreactivity was mainly in epithelial cells and inflammatory cells in LP. Previous studies have demonstrated that IL-10-positive cells are mainly T and B lymphocytes, monocytes, macrophages, and mast cells in LP in allergic responses [10, 29]. In this study, we found that rmIL-10 treatment further increased IL-10 protein levels in NLF. In order to determine if increased IL-10 protein levels in NLF came from the exogenous rmIL-10 or induced local IL-10 expression in nasal mucosa, we investigated the IL-10 mRNA and protein expression in the nasal mucosa and found that IL-10 expression in nasal mucosa was markedly decreased with rmIL-10 treatment. The results indicate that exogenous IL-10 treatment can inhibit the local IL-10 expression in the nasal mucosa and the increased IL-10 protein levels in NLF are derived from exogenous rmIL-10.

In the study, local treatment with rmIL-10 significantly alleviated Th2 and Th17 responses in the nasal mucosa. The increased expression of IL-17 has been shown to be negatively associated with the sensitivity of AR patients to topical corticosteroid treatment [20]. IL-10 can upregulate glucocorticoid-receptor expression of asthmatic CD4⁺ T cells with glucocorticoid-resistant [29]. Thus, IL-10 may have potential benefits for the treatment of AR patients with corticosteroid resistance. It is well accepted that Th1 and Treg cells play an important role in constraining allergic reactions. Their under-expression or dysfunction is closely related to the development of AR. Our study suggests that IL-10 administration at current dose may not cause significant suppression to the immunity against allergic airway responses since it had not apparently inhibitory effect on the expression of Th1 and Treg cells.

Although glucocorticoids have been used for the treatment of inflammatory and allergic airway diseases for a long time, their side effects have been concerned for many years, such as growth inhibition, osteoporosis, and gastric ulcer [30–32]. Based on its anti-inflammation function, intranasally delivered IL-10 has the potential to be an alternative drug for glucocorticoids and therefore to avoid side effects of glucocorticoids.

Our current study has several limitations. One is that given the limited amount of mouse nasal tissues, we could not perform the flow cytometric analysis of Th subsets at cellular level. Second, our AR mouse model was established by OVA sensitization and challenge. It may be better to induce AR with relevant allergens found in human AR, such as pollens and dust mites. Third, we administrated exogenous IL-10 during challenge instead of after the establishment of AR. Therefore, the therapeutic effect of IL-10 on established AR needs to be studied in the future research.

In conclusion, the study demonstrates that rmIL-10 administration during challenge down-regulates allergic reactions in the context of AR, most likely through inhibiting Th2 and Th17 responses without impairment of Th1 and Treg responses. Exogenous IL-10 can also inhibit local IL-10 expression in the nasal mucosa in the context of AR. Given its anti-inflammatory and immunosuppressive functions, IL-10 may have a promising future as an inhibitor of AR. However, this needs further studies in future.