AIT: Allergen Immunotherapy; Dp: Dermatophagoides pteronyssinus; Df: Dermatophagoides farinae; dsRNA: double-stranded RNA; ILC2: group 2 Innate Lymphoid Cells; HDM: House Dust Mite; TLR3: Toll-Like Receptor 3

HDM is the main source of indoor aeroallergens that cause several allergic conditions including atopic dermatitis, conjunctivitis and asthma, in which patients commonly present type 2 immunopathologies characterized by the increased number of eosinophils and elevated levels of type 2 effector cytokines such as IL-4, IL-5, IL-9 and IL-13 [1,2]. Asthma, a common chronic airway disease, is estimated to affect more than 300 million people worldwide, among which most asthmatics are sensitized to HDM [3,4]. Both innate and adaptive immune responses contribute to the establishment of long-lasting type 2 inflammation caused by HDM (Figure 1). Therefore, there is a great need to better understand how HDM allergens and HDM-associated immunostimulatory components contribute to the development of type 2 immunopathologies [3,5].

HDM are microscopic insects found in many places such as mattresses, pillows, carpets and blankets. They feed on human skin flakes and prefer humid environments in order to proliferate. There are 13 HDM species, however, the most common ones are Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df) and Euroglyphus maynei (Em) [1]. Dp and Df are the main species that are known to cause respiratory allergic diseases [1,6]. Natural forms of HDM allergens are composed of both protein antigens and non-protein immunomodulatory elements. There are over 10 protein allergens in HDM including a cysteine protease (Der p1), chymotrypsin (Der p6), tropomyosin (Der p10), and chitinases (Der p15) [7-9]. Der p1 alone is highly allergenic and sufficient to initiate the production of pro-inflammatory and pro-Th2 cytokines such as IL-4, IL-5, IL-9, and IL-13 [10-12]. In addition to protein allergens, HDM also contains various microbial components such as LPS, ß-Glucan (a component of fungal spores) and various bacterial cell wall components [13-15]. Microbial products may trigger innate immune responses to orchestrate the initiation and amplification of type 2 inflammation. For example, HDM-associated LPS was previously shown to activate TLR4 in airway epithelial cells and induce the expression of thymic stromal lymphopoietin (TSLP), interleukin-25 (IL-25) and IL-33 cytokines [5,15] which are all known as primary activators of group 2 innate lymphoid cells (ILC2). HDM-LPS is also believed to directly activate TLR4 to aid the Th2 polarization [16]. Moreover, other molecules associated with HDM such as ß-Glucans and lipoproteins or lipopeptides can elicit pro-allergenic innate immune responses during HDM allergen exposures [14,15]. Activation of C type lectin receptors and TLR2 by glycan-derived molecules or lipoproteins has also been seen to lead to the infiltration of eosinophils and induction of Th2 cytokines [15-17]. Furthermore, Der p2 and chitin in HDM have also been shown to act as innate immune stimulants to induce the release of pro-inflammatory cytokines such as TNFa [15]. Taken together, HDM acts not only as an aeroallergen but also a carrier for numerous allergenic epitopes (present mainly in fecal pellets) and immunomodulatory molecules that exacerbate type 2 inflammation upon allergen exposure.

While HDM components have been known to promote type 2 inflammation and have been characterized extensively, it is somehow counterintuitive to realize that HDM allergens might contain factors that are able to suppress or reduce type 2 immune reactions as we demonstrated that HDM species (Dp) and (Df) contain immunomodulatory dsRNA > 40bp. At present, it remains unclear whether dsRNA species exist in other aeroallergens. It is known that dsRNA is immunologically active and can induce the production of type-I and type-III interferons [13], which may suppress type 2 inflammation, at least in part, through restricting ILC2 functions [18]. ILC2 cells are very important initiators and amplifiers for type 2 immune responses (more details about the role of ILC2 will be discussed later in the review).

In summary, HDM is a very complex allergen consisting of pro-allergenic proteins (e.g., Der p1) and microbial adjuvants (e.g., LPS) (Table 1). Paradoxically, new evidence indicates that HDM may also contain anti-allergenic factors such as dsRNA, which seems to be able to trigger innate immune responses to inhibit allergic inflammation.

Although RNA sensors have been extensively studied in the context of viral infections, their roles in allergic responses remain controversial. Multiple RNA sensors have been discovered and can be classified into two types, the endosomal RNA sensor and the cytosolic RNA sensor, based on their cellular locations. The former includes TLR3, TLR7/8 and TLR13 while the latter includes RIG-I and MDA5. TLR3 binds to relatively short strands of dsRNA (>40 bp) whereas MDA5 binds to longer dsRNA sequences (>1 kb). TLR7/8 recognizes the GC-rich ssRNA while TLR13 recognizes bacterial 23S rRNA with an exquisite sequence specificity [19,20]. Finally, RIG-I binds to 5’ppp-dsRNA. Upon engagement of the corresponding RNA ligand, these RNA sensors initiate different signaling cascades that culminate in the common downstream transcriptional pathways including NF-κB and IRF3, which ultimately result in the production of pro-inflammatory cytokines (e.g., IL-1ß, TNFα) or type I interferons (IFNα/β) [16]. TLR7/8 and TLR13 recruit the adaptor protein Myd88. Stimulation of TLR3 leads to the activation and recruitment of Trif adaptor protein [16,21,22]. The cytosolic RNA sensors RIG-I and MDA5 recruit the adaptor protein MAVS [23]. Notably, TLR3, 7, 9 ligands have been tested in clinical trials for treating allergic rhinitis or asthma [24]. 
Our traditional view is that respiratory viral infections usually lead to the exacerbations of allergic diseases, however, emerging data from animal models suggests otherwise [25,26]. A certain number of viruses such as gammaherpesvirus or influenza virus have been shown to activate protective innate immune responses against the development of type 2 immunopathology [25,26]. Additionally, in the gastrointestinal system, enteric viruses were shown to activate innate RNA sensing immunity which is required for sustaining intestinal homeostasis via educating intestinal intraepithelial lymphocytes (IELs) [18,27,28]. These observations collectively suggest that RNA sensing immunity seems to play a beneficial role in host defense against both viral infections and allergic inflammation [29-32].

Emerging evidence suggest that RNA sensing immunity may play a significant role in modulating HDM-induced allergy. We have recently shown that HDM-associated dsRNA induced a protective responsive against type 2 inflammation and airway hyperresponsiveness [13]. Mechanistically, our data suggests that lung exposure to HDM-dsRNA induces the production of type I interferons through TLR3- and MAVS-mediated signaling pathways. Mice lacking both TLR3 and MAVS failed to activate interferon responses, and therefore developed much worse type 2 immunopathology upon exposure to HDM allergen [13]. Interferon responses induced by TLR3 were stronger compared to interferon responses induced by the RNA sensors upstream of MAVS, MDA5 and RIG-I [13]. This could be due to the ability of TLR3 to recognize nucleic acids sequence over 40 bp.

Human genetic studies have suggested that polymorphisms of TLR3 and TLR7/8 may be associated with asthma-related phenotypes in patients [33,34]. In prophylactic studies, dsRNA (TLR3 agonist) and ssRNA (TLR7/8 agonist) were used as immune adjuvants to prevent the onset of type 2 allergic inflammation [16]. Our data suggested the presence of dsRNA structures with >40 bp length in common HDM species (Df and Dp) [13,35,36]. Mechanistically, natural dsRNAs derived from either HDM or viruses, or a synthetic form, poly (I:C), mainly engage the TLR3-TRIF-IRF3-IFN pathway [13,36]. Unlike other HDM components that are known to be pro-allergenic, HDM dsRNA induces an anti-allergic interferon response which can efficiently suppress type 2 inflammation and airway hyperresponsiveness [13,16,37,38]. More importantly, we have shown that total RNAs derived from both DNA- and RNA- virus-infected cells were also able to suppress type 2 inflammation and airway hyperresponsiveness [39]. Thus, these findings collectively support the hygiene hypothesis, which states that early childhood microbial exposure confers protection against the development of allergic diseases [36,39].

Although we have clearly demonstrated in acute models of allergy that the dsRNA-activated interferon pathway is required for restraining the development of type 2 inflammation, it remains to be determined whether additional mechanisms are involved in negatively regulating the development of type 2 inflammation. Future studies would provide useful information for the clinical development of dsRNA as a vaccine adjuvant [40].

ILC2 are the innate counterpart of the T helper 2 lymphocytes (Th2) [41-44]. In response to alarmin cytokines such as IL-33, IL-25 and TSLP, ILC2s produce large amounts of the type 2 effector cytokines IL-5 and IL-13 [45-48], which promote the development of type 2 inflammatory responses characterized by eosinophilia, airway remodeling, mucus hypersecretion, and airway hyperresponsiveness (AHR) [45-53].

Damage of airway epithelial layers by proteases allergens often results in the release of cytokines like IL-25, IL-33 and TSLP, which potently activate ILC2 cells [54]. The function of ILC2 must be tightly regulated in order to prevent the onset of overzealous type 2 inflammation. Several mechanisms have been proposed for which ILC2 is regulated in allergic diseases [55]. Targeting ILC2 has shown some promising therapeutic effects in treating asthma and allergic rhinitis [55]. One of these strategies is to directly suppress ILC2 numbers and functions by interferons induced by activating TLRs [56]. Our lab has recently shown that a synthetic dsRNA analogue and natural dsRNA derived from DNA and RNA viruses can attenuate IL-33- and a fungal allergen-induced type 2 inflammation by blocking the activation of ILC2 via the TLR3-IFN signaling axis [36]. Our data suggest that dsRNA species induce the release of type I interferons in the lungs; this is sufficient to reduce airway inflammation as mice lacking interferon receptor had exacerbated lung inflammation [39]. Phenotypically, dsRNA-induced suppression of ILC2 function resulted in the decreased number of eosinophils by ~6 folds and reduced lung resistance in mice [36]. In addition to interferon-dependent suppression of ILC2, a number of studies demonstrated several other mechanisms to target these cells, for instance, prostaglandin E₂, IL-27, regulatory T cells or their associated cytokines were all shown to suppress ILC2 activation, proliferation and functions, respectively [57-59].

HDM allergen not only polarize to type 2 allergic responses by acting on epithelial cells but also contain proteases that could direct DCs, via ILC2-derived IL-13, to the lymph nodes where they present antigens to Th2 cells, hence, leading to establishment of adaptive Th2 responses [49]. Given the critical role of ILC2 in amplifying and exacerbating type 2 inflammation, it is necessary to explore new methods for targeting these cells.

Although we have clearly showed that dsRNA seizes the proliferation and expansion of ILC2 in acute models of allergic inflammation (Figure 2) [36], it has not been determined whether viral or HDM-dsRNA inhibition of ILC2 could extend to chronic models of allergic inflammation. Notably, DCs play very important role at early stages of sensitization by allergens. DCs take up allergen, then migrate to adjacent lymph nodes where they present allergen peptides to MHCII-restricted T cells, thus, leading to polarized Th2 responses. Given that dsRNA can be used to promote antigen-specific Th1-polarized responses, it would be interesting to evaluate whether DCs could be targeted by dsRNA to manipulate allergen sensitization process [60].

Allergen immunotherapy (AIT) is an effective approach to treat allergic diseases. Although not fully defined, AIT is thought to suppress Th2 responses [61]. Treating HDM-sensitized patients with both forms of immunotherapy, subcutaneous and sublingual (SCIT and SLIT), has been shown to effectively reduce the disease severity [62]. However, the process of immunotherapy is complicated and often takes many years to reach satisfying therapeutic effects. In order to shorten the treatment period, immune adjuvants that stimulate innate immune sensors such as TLR4 and TLR9 have been used clinically [63].

HDM extracts used in AIT contain not only protein allergens, but also other microbial components, of which some could be pro-allergenic while others like TLR3 agonist (dsRNA) are anti-allergic [13]. In fact, commercial HDM extracts from 10 different manufacturers induces variable IgE reactivity profiles in patients [64]. To minimize the element of variability in AIT, it would be important to clearly define the immune stimulatory nature of HDM-associated components.

TLR agonists targeting TLR2, TLR3, TLR4, TLR7/8, TLR9 seem to be effective in reducing eosinophilic inflammation by inducing the production of interferons and Th1 cytokines such as IL-12 in animal models and in clinical trials [24,56,65,66]. The hygiene hypothesis states that microbial exposures early in life may confer a protective immunity against the development of allergy in healthy individuals. Accumulating evidence from experimental and epidemiological studies in recent years supports this notion [25,67]. Mechanistically, interferon responses induced by TLR agonists have been shown to play an important role in ameliorating type 2 inflammation in animal models of asthma and allergy [17]. Other protective pathways beyond interferons should be explored in the future studies. Besides dsRNA, ssRNA can be generated during microbial infections and it would be important to evaluate whether ssRNA species derived from viruses or bacteria could lead to the activation of TLR7/8, and therefore regulation of allergen-induced type 2 inflammation.

HDM allergens cause various allergic diseases including allergic rhinitis, eczema, conjunctivitis and asthma [68]. Over 50% of asthmatic patients are sensitized to HDM. Immunotherapy with HDM extract has already been utilized to desensitize allergic patients [64,69]. We recently identified that the medical-grade HDM extracts endogenously contain long dsRNA species that potently induce innate immune responses in mouse lungs [13], which might contribute to some immunological effects during the HDM immunotherapy in patients. Besides immunotherapy, other forms of treatments are impermanent and not suitable for all groups of patients, hence, identifying immunomodulatory RNA within HDM allergen could provide an alternative therapeutic approach to control the development of airway inflammation induced by HDM or other related allergens in clinical settings.

H.H.A. is supported by the Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Al Jouf University, Sakaka, Saudi Arabia. X.D.L. is supported by the UT Health San Antonio School of Medicine Startup Fund and the Max and Minnei Voelcker Fund