Background
Interaction of antigen-presenting cells (APCs) and T cells is crucial in both the initiation and challenge phases of allergic asthma and, therefore, has possibility as a target of anti-asthmatic drugs. Optimal T cell activation and differentiation require not only interaction between the T cell receptor (TCR) and antigen-MHC complexes but also interaction between costimulatory ligands on APCs and their putative receptors on T cells. One of the best-characterized costimulatory molecules is CD28, which binds to two costimulatory ligands, B7-1 (CD80) and B7-2 (CD86), on APCs. CD28 is constitutively expressed on both CD4
+ and CD8
+T cells. By contrast, CD80/86 expression on dendritic cells and B cells is upregulated after antigen pulse in the process of maturating into APCs. Despite sharing the same receptor, CD80 and CD86 appear to mediate different mechanisms. CD80 can be more potent than CD86 in inducing antitumor responses, while CD86 preferentially induces Th2-driven allergic responses [
1],[
2]. It is generally accepted that during APC/T cell interaction, the B7-CD28 pathway is indispensable for the activation and differentiation of naïve T cells. However, it remains controversial whether this pathway has a pivotal role in the reactivation of primed T cells in the effector phase.
It is well known that the dendritic cell (DC) is the most powerful APC for inducing allergic immune responses
in vivo. The DC network beneath the epithelium of the conducting airways is ideally positioned to perform a surveillance role for inhaled antigens (Ag). By depleting DCs before the inhaled Ag challenge, all the salient features of asthma were diminished, and the effector cytokine secretion was profoundly reduced [
3]. Moreover, recent studies suggest that the expression of CD86, but not CD80, on airway DCs is upregulated during the effector phase. Ongoing allergic inflammation induces a specific shift in airway DCs from a CD86-low to a CD86-high phenotype in periphery [
4]. Given that lung DCs maturate and upregulate the expression of CD86 following the allergen challenge [
5], it is important to know whether the upregulation of CD86 has a role in the development of asthmatic responses.
RNA interference is an endogenous cellular mechanism in which short interfering RNAs (siRNAs) elicit the sequence-specific degradation of a complementary mRNA target. Today, siRNA-mediated gene silencing has become more powerful, more specific, and much less toxic than low-molecular-weight chemical inhibitors or blocking mAbs for laboratory investigations, particularly
in vitro. It is also characterized by a low-cost and transient effect. Several studies have demonstrated the therapeutic effects of synthetic siRNA in allergen-induced asthma models [
6]-[
8]. These studies targeted STAT6, TRAIL, and PAI-1, and the siRNAs were delivered by intratracheal administration. Although low transfection efficacy might be a potential hurdle in RNA interference, it is expected that DCs can be good targets because of their location and their prominent particle uptake ability, including the uptake of short-based nucleotides. Here, we examined the effects of the downregulation of CD86 by intratracheal administration of siRNA on Th2 cytokine production in the effector phase
in vitro and on asthma phenotypes
in vivo.
Discussion
The present study demonstrated that CD86 siRNA treatment attenuated the upregulation of CD86 on maturing BMDCs. CD86 siRNA-treated BMDCs were poor stimulators of effector Th2 cells, shown by reduced productions of IL-4, IL-5, and IL-13, but not of IFN-γ. In vivo, intratracheal administration of CD86 siRNA during OVA challenge reduced the production of IL-5, IL-13, and TARC release and ameliorated airway eosinophilia, airway hyperresponsiveness, and elevation of OVA-specific IgE. These results clearly show that CD86 has a pivotal role in the reactivation of Th2 cells in the effector phase of allergic reactions. In addition, CD86 siRNA treatment did not stimulate systemic production of IL-6 or IFN-β, suggesting that CD86 may become a promising target for the treatment of allergic asthma.
The effector phase of allergic reactions has long been explained by the initiative interaction between antigen-bearing DCs and a memory phenotype of Th2 cells in the draining lymph nodes (DLNs). Thus, airway DCs capture the inhaled allergens, mature with upregulation of costimulatory molecules, including CD86, and then traffic into the DLNs, where they meet with antigen-specific memory Th2 cells. The interaction between those cells leads to clonal expansion and recirculation of effectory Th2 cells, leading to recruitment and activation of effector Th2 cells in the airway mucosa. Although this scenario might be a rationale to interrupt CD86 to CD28 interaction for the treatment of allergic responses, recent investigations have provided a novel concept regarding roles of airway DCs in the activation of Th2 in the airway mucosa. Airway DCs could present antigen directly to memory Th2 cells in the airway mucosa of OVA-sensitized and -challenged mice. Of note, this antigen-presenting activity was accompanied by upregulation of CD86 but not CD80 [
16]. Another study demonstrated that allergic lungs specifically retained antigen-bearing DCs within the airway-adjacent region without evidently altering DLN trafficking [
17]. This local presentation pathway may be a more feasible target of interruption via intra-airway remedies than is the conventional presentation pathway in the DLNs. A previous study reported that intraperitoneal administration of anti-CD86 mAb during OVA challenge failed to attenuate the subsequent allergic responses in OVA-sensitized mice [
18]. The failure might be explained by insufficient distribution of anti-CD86 mAb into the airway mucosa.
Our colleagues have already showed that topical application of cream-emulsified CD86 siRNA ameliorated the clinical manifestations of murine contact hypersensitivity and atopic dermatitis-like disease [
9]. It also afford collateral evidence that CD86 has a role in effector phase. But another study reported that intratracheal injection of OVA-pulsed DCs from CD80/CD86 double-deficient mice in OVA-sensitized mice led to the reactivation of Th2 effector responses the same as OVA-pulsed DCs from wild-type mice [
19]. siRNA theoretically causes ubiquitous gene silencing, leaving a possibility that CD86 siRNA affects not only airway DCs but also other cell types. CD86 on alveolar macrophages, eosinophils, and B cells was reported to play a role in the development of allergic airway reactions [
20],[
21]. CD86 on B cells stimulates CD28 on T cells and transduces positive signals into B cells that increase IgG
1 and IgE production. This pathway may be particularly important for memory B cells in which CD86 is upregulated [
22],[
23]. The reduction of OVA-specific IgE in the serum of CD86 siRNA-treated mice in the present study is highly consistent with this pathway.
TARC is the CC chemokine that selectively attracts Th2 lymphocytes toward APCs [
24]
. A previous study showed that TARC expression was highly concentrated in purified lung DCs [
25]. In the present study, the mechanism remains unclear as to why the concentration of TARC in BAL fluid was reduced by CD86 siRNA treatment. We confirmed that CD86 siRNA treatment
per se did not alter the ability to produce TARC in BMDCs
in vitro (unpublished observation). A possible explanation is that CD86 siRNA treatment reduces recruitment of DCs into the airways by currently unknown mechanism(s), which results in the reduction of TARC in the lungs. The quantitative and qualitative assessments of airway DCs await further investigations.
Contrasting with the effects on lines of asthma phenotypes, CD86 siRNA treatment failed to ameliorate the goblet cell hyperplasia and MUC5AC gene expression, cardinal features of airway remodeling in asthma. A majority of asthma phenotypes, including airway eosinophilia, airway hyperresponsiveness, and airway remodeling, are attributable to the pluripotent effects of IL-13 and its downstream molecules. Those IL-13-mediated phenotypes vary in sensitivity to therapeutic interventions. Airway eosinophilia induced by intratracheal IL-13 was feasibly suppressed by systemic treatment with glucocorticosteroid, while airway hyperresponsiveness and remodeling were resistant to glucocorticosteroid [
12]. Similar results were obtained from our recent study that examined the effect of an inhibitor of Janus kinase (JAK), a kinase family mediating multiple cytokine signalings, on OVA-sensitized/challenged mice [
14]. In the present study, however, treatment with CD86 siRNA abolished the OVA-challenge-induced elevation of IL-13 in BAL fluid, which makes it difficult to explain the differences in the effects of siRNA via variant sensitivities of IL-13-mediated asthma phenotypes to therapeutic interventions. IL-13-independent mechanisms of airway remodeling must still be elucidated.
To our knowledge, this study is the first report targeting airway DCs for treatment with siRNA. The efficient delivery of siRNA to the target cells has been a challenge for therapeutic application of siRNA since a majority of tissue-constructing cells hardly intake a sufficient amount of siRNA for gene silencing. Given the significant effect of naked siRNA against CD86 in the present study, vigorous phagocytic activity of DCs would be meaningful. Additional carefully designed studies are required to improve pharmacokinetics and facilitate cellular uptake of siRNA [
26].
CD86 shares its ligand, CD28, with CD80. In our colleagues’ study, freshly isolated murine CD4
+T cells were incubated with murine mastocytoma P815 cells transfectants expressing a similar level of either CD80 or CD86 in the presence of anti-CD3 mAb [
27]. Both CD80 and CD86 costimulated the proliferation of CD4
+T cells at comparable time-kinetics and magnitude, but CD86 alone was able to costimulate IL-4 production in CD4
+T cells. Regarding
in vivo models, a previous study showed that intranasal administration of anti-CD86 mAb markedly reduced AHR, IgE production, and airway eosinophilia in OVA-sensitized/challenged mice whereas the treatment with anti-CD80 reduced airway eosinophilia alone [
28]. On the other hand, another study supported a role for both CD80- and CD86-mediated costimulation in allergen-induced AHR, IgE production, and airway eosinophilia [
29]. In the present study, we selected CD86 as a target based on the study indicating that T cell activation during the late-phase airway allergic response is associated with tracheal DC upregulation of CD86 but not CD80 [
16]. Concomitant silencing of CD80 and CD86 is subject to future investigation.
The limitation of this animal study around interpretation and translatability to humans may be the choice of OVA as the allergen compared an allergen more relevant to humans, and which has intrinsic activity, such as house dust mite or fungal allergens. Another limitation of this study to the translatability to human asthma may be the question of steroids. Inhaled steroid therapy is used as the mainstay for the treatment of asthma. If steroids effectively down-regulate CD86, the benefit of CD86 siRNA would be minimal. A previous study showed that glucocorticoids inhibited the maturation of human DCs induced by LPS. Thus, glucocorticoids down-regulated the expression of CD86 following LPS stimulation
in vitro[
30]. On the other hand, glucocorticoid insensitivity in some Th2 clones was reversed by blockade of CD86 to CD28 signaling
in vitro (Dr. Mori A, National Sagamihara Hospital, Japan, unpublished observation). The relevance of CD86-targeted approach in various asthma phenotypes awaits further investigations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
This work was supported by JSPS KAKENHI Grant Number 21590967, Grant-in-aid from the Ministry of Health, Labor and Welfare (MHLW), Japan, and by the National Institute of Biomedical Innovation, Japan. All authors declare no conflict of interests.
Authors’ contributions
YA-T and KM designed all the experiments, conducted the experiments, compiled the results, conducted the statistical analysis, and wrote the initial drafts of the manuscript. SF helped conceive the study. KK and KT aided with in vitro experiments. TN aided with mouse model experiments and histological analysis. TO and MA designed and provided siRNA and supervised the work. HI and YN advised the designing of experiments and helped with writing the manuscript. All authors read and approved the final manuscript.