Discussion
Humulus pollen is an important allergen that causes asthma in East Asia. In this study, we showed that, compared with IP sensitization, SC sensitization provides a murine model that better recapitulates the typical clinical characteristics of allergic asthma. Using this model, we successfully developed a rapid SCIT model for HSE-induced asthma. This SCIT model elucidated the mechanism of desensitization treatment, and provided a rationale for the use of HSE in clinical SCIT.
Compared with animals in the IP.HSE group, animals in the SC.HSE group showed significantly higher levels of allergen-specific IgE and significantly higher quantity and proportion of eosinophils in BALF. These findings are consistent with
the typical characteristics of allergic asthma patients[16]. In our study, mice sensitized via the IP route showed an elevated number of eosinophils, but no change in their proportion. Following airway stimulation by atomized pollen, the number of inflammatory cells is globally elevated; thus, enhancement of absolute eosinophil number does not necessarily reflect the occurrence of a typical type I hypersensitivity reaction.
Allergic asthma is characterized by elevated expression of Th2 cytokines (i.e., IL-4 and IL-13) and reduced expression of Th1 cytokines (i.e., IFN-γ)[20]. IL-4 is a representative Th2 cytokine that can stimulate the proliferation of B and T cells; it enhances IgE synthesis[21] and induces eosinophil infiltration into the airway[22]. In this study, IL-4 levels in the BALF and SHS were significantly higher in mice in the SC.HSE group, compared with mice in the SC.PBS and IP.HSE groups. This may explain why mice in the SC.HSE group showed higher HSE-specific IgE levels and higher proportions of BALF eosinophils, while mice in the IP.HSE group did not. IL-13 has been proposed to induce inflammatory cell infiltration into the airway, as well as mucus secretion and AHR[23]. Compared with the respective control groups, BALF IL-13 levels were significantly higher among mice in both SC.HSE and IP.HSE groups. Thus, IL-13 may have promoted inflammatory cell infiltration, mucus secretion, and AHR in these animals, although IL-4 levels were not elevated in the IP.HSE group. However, IFN-γ levels in BALF and SHS of mice in the SC.HSE and IP.HSE groups only showed a slight downward trend, but no significant reduction.
The SC and IP sensitization routes may produce different immune responses because of the presence of different local antigen-presenting cell populations at the site of antigen contact. In the skin, dendritic cells, which vary in quantity and phenotype, are reportedly found in atopic individuals in a manner that favors type 2 immune responses[24]. In addition, mast cells , which can produce Th2 cytokines and amplify allergic response, are widely distributed in the dermis and act as sentinels at sites of initial antigen exposure[25]. In contrast, in the peritoneal cavity, macrophages are found in large numbers and tend to favor the induction of Th1/Th0 immune responses[26].
Our group previously found that the approximately 10-kDa component is a major allergen in H. scandens pollen among Chinese individuals; we also identified the complete amino acid sequence of this protein (GenBank: ADB97919.1). Except for this 10-kDa component ,Park et al.[27] reported that three protein components (13 kDa,74 kDa, and 80 kDa) from Humulus pollen extract were bound to IgE, among sera from more than 50% of the patients. Sun et al.[28] reported that 30.8% of patient serum samples exhibited binding interactions with 100.5-kDa proteins in Humulus pollen. The immunoblotting experiments in the present study showed that the IgE antibodies of sensitized mice were mainly bound to relatively large fragments (48–100 kDa), and rarely bound to the 10-kDa band. This finding is consistent with the study work of U.S. Eitzer et al.[29] who found that mice sensitized with whole protein of timothy-grass pollen did not produce IgE antibodies against PHLP1, the major allergen of timothy in humans. These findings suggested that mice and humans may be sensitive by somewhat different proteins.
Clinically, SCIT typically requires SC injection twice per week for 3–5 years. Considering differences in the life cycles of mice and humans, we used a rapid desensitization treatment method (eight treatments every other day) and effectively alleviated pulmonary inflammation and AHR in asthmatic mice. Our results were similar to those of a study in which mice received weekly treatments for a continuous 8-week period[30]. This short-term and rapid desensitization method saves time and reduces the cost of mouse maintenance.
Despite the short treatment duration, allergen-specific IgE was significantly lower in the treatment group than in the vehicle group; anti-IgE therapy has been effective for some allergic diseases[31]. In addition, SCIT has been shown to significantly enhance allergen-specific IgG2a levels. Elevated levels of allergen-specific IgG4 in humans (similar to IgG2a in mice) are accompanied by improvement of clinical symptoms[32]. Therefore, the therapeutic effect of SCIT in our model may have been achieved by inhibiting production of allergen-specific IgE and enhancing production of allergen-specific IgG2a in serum, as in previous studies[33].
SCIT corrected the imbalance of Th1/Th2 immune responses in asthmatic mice and converted the immune response to a Th1 type. This may be the underlying mechanism of desensitization treatment. IL-10 is a pleiotropic cytokine that has both pro- and anti-inflammatory effects during immune responses[34]. In our study, IL-10 levels were significantly increased in asthmatic mice and significantly decreased after SCIT, indicating that this cytokine may play a role in exerting pro-inflammatory effects.
There were some limitations in this study. Changes in the systemic immune response were examined by analysis of SHS because of limited laboratory conditions; the sensitivity of this method is relatively low, we will try to co-culture spleen cells with specific allergens in vitro to observe changes in the systemic immune response in the further studies.
In conclusion, we successfully developed a better murine asthma model for Humulus pollen allergy through SC sensitization and atomization challenge. Using this model, short-term and rapid SCIT improved symptoms and pathophysiology in asthmatic mice. The murine models of asthma and SCIT for Humulus pollen allergy developed in this study may be valuable tools for studying the mechanisms of asthma and SCIT for airborne allergens, as well as for designing more efficacious therapies for allergic asthma.