Innate immunity in allergy

Abstract Innate immune system quickly responds to invasion of microbes and foreign substances through the extracellular and intracellular sensing receptors, which recognize distinctive molecular and structural patterns. The recognition of innate immune receptors leads to the induction of inflammatory and adaptive immune responses by activating downstream signaling pathways. Allergy is an immune‐related disease and results from a hypersensitive immune response to harmless substances in the environment. However, less is known about the activation of innate immunity during exposure to allergens. New insights into the innate immune system by sensors and their signaling cascades provide us with more important clues and a framework for understanding allergy disorders. In this review, we will focus on recent advances in the innate immune sensing system.


| INTRODUC TI ON
Our body is endlessly exposed to microbial agents and environmental noxious substances. These may cause serious illness, or toxicity to the body; therefore, they must be eliminated. This is mediated by the innate immune system, which is the first line of host defense against foreign invasion. Any disruption in the physical barriers that prevent pathogens from entering the body triggers pro-inflammatory responses by activating myeloid cells and dendritic cells (DCs) that are central players of the innate immune defense. Furthermore, pro-inflammatory responses induce antigen presentation shifting from an innate immune response to an acquired immune response.
B cells and T cells, in which antigen receptors are individually specialized by DNA rearrangement, mainly mediate acquired immune responses. One of the main features making the innate immune system highly specialized is the germline-encoded receptors distinguishing between self and nonself. This discrimination is mediated by membrane-bound or cytoplasmic pattern recognition receptors (PRRs). 1 The membrane-bound receptors are Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). The cytoplasmic-type receptors are retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs). These receptors directly sense various components from pathogens and distinguish conserved microbial structural features, called pathogen-associated molecular patterns (PAMPs). 2 The recognition of PAMPs leads to robust innate immune responses through the activation of these downstream signaling pathways. PRRs also recognize self-components released from the damaged cells, called damage/danger-associated molecular patterns (DAMPs), and can thus be associated with the pathogenesis of many diseases.
Allergy is an increasing problem in the health sector, with a soaring number of patients in recent years. Accumulating evidence suggests the importance of the innate immune system in the development of allergy susceptibility. Therefore, in this review, we will summarize recent advances in the involvement of PRRs in allergic diseases, and improvements to current allergy treatment modalities.

| T-HELPER RE S P ON S E AND ALLERGY
In recent years, although highly criticized, the hygiene hypothesis has been proposed to explain the increase in frequency of patients with allergy worldwide. 3 It suggests that a cleaner environment may lead to development of allergic diseases, highlighting that early exposure to microbes and parasites during childhood is essential to reduce development of susceptibility. 4 As evidence in favor of the hygiene hypothesis, it is demonstrated that maternal intranasal exposure to the nonpathogenic microbe Acinetobacter protected against the development of experimental asthma in the progeny. 5 Allergy is characterized by a T-helper 2 (Th2) hypersensitivity response with a significant increase in immunoglobulin (Ig) E. Type 2 immune responses are characterized by the expression of type 2 cytokines, such as interleukin (IL)-4, IL-5, IL-9, and IL-13. Contact with bacteria during early development may be protective by inducing T-helper 1 (Th1) cell differentiation. Intestinal microbiota also plays an important role in the prevention of allergies. 6,7 Although oral administration of ovalbumin (OVA) abrogates both Th1 and Th2 responses in specific pathogen-free mice, only Th1 responses are reduced in germ-free mice. 8 The reconstitution of the intestinal microbiota of germ-free mice suppressed the susceptibility of the Th2 responses in neonates, 9 suggesting the importance of exposure to intestinal microbiota at the neonatal stage for prevention of allergies.

| TOLL-LIK E RECEP TOR S
The TLRs were the first discovered PRR and are mammalian homologues of Drosophila Toll protein involved in innate immune response. 21,22 Studies over the past two decades have revealed important roles of TLRs in a variety of biological phenomena including inflammation, the bridging between innate and acquired immune responses, and cancer cell proliferation and survival.

| TLR family
Toll-like receptors include 10 and 13 family member proteins in humans and mice, respectively. The TLR family is evolutionally conserved and contains the ligand-binding domains via leucinerich repeat (LRR) motifs at the N-terminus and intracellular Toll/ IL-1 receptor (TIR) domain at the C-terminus ( Figure 1). TLRs act as the gatekeepers of host defense to various pathogens through structure-and sequence-dependent immune recognition ( Figure 1). [23][24][25][26] Most TLRs in humans and mice recognize similar PAMPs with some exceptions. TLR11, TLR12, and TLR13 have been lost in the human genome, and the Tlr10 gene is disrupted in the mouse genome.

| TLR signaling
Once activated by their ligands, individual TLRs recruit two major TIR-containing adaptor molecules, myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adaptor inducing interferon-β (IFN-β) (TRIF) (Figure 1). The engagement of all TLRs except for TLR3 induces the signaling complex, named the Myddosome, which consists of MyD88 and IL-1 receptor-associated kinases (IRAKs), leading to activation of NF-κB. 27 The ligand engagement to TLR3 or TLR4 recruits TRIF, which results in activa-

| TLR and allergy
Allergic development is believed to result from genetic backgrounds and environmental factors. Mutations in TLR family genes have been investigated using single nucleotide polymorphism (SNP) analysis and meta-genome-wide association studies (GWAS). Indeed, SNPs in the TLR4 gene are a risk factor for asthma, 28,29 indicating that genetic variations of TLR family genes are related to susceptibility to allergic diseases. Barrier epithelial cells function as an origin of allergic response to external signals from the mucous membranes of the respiratory tract, intestinal tract, or skin. Tight junction barriers are extremely sensitive to detergents. 30 LPS also increases tight junction permeability in a TLR4-dependent manner. 31 The dysregulation of the epithelial barrier may increase uptake of allergens in the pathogenesis of allergy.
Microbial-treated TLR2/3/4/7/9 knockout mother mice are no longer protected from the development of asthma in their offspring, 5 suggesting that maternal TLR signaling plays a pivotal role in the transfer of protective effects. However, the precise roles of TLRs in the development of allergic diseases are greatly influenced by many factors, such as cell types, expression level, and the nature of antigens. Indeed, TLR4 signaling leads to allergic responses. [32][33][34] This TLR4-mediated allergic reaction develops only by intranasal sensitization but not by subcutaneous or intraperitoneal sensitization, suggesting that TLR-mediated reactions influence the dose of immune-stimulatory components, as well as the route of administration and the timing of exposure. Treg cells have also been implicated in allergy development. 35 Manipulation of Th1/Th2 balance or Treg cell function by administrating TLR agonists may be promising for the treatment of allergic diseases. 36

| Allergen-specific immunotherapy for TLRs
Different TLR agonists have been assayed in clinical trials as adjuvants. 37 These were further developed in the context of allergen-specific immunotherapy (AIT) with different outcomes. Oral administration of TLR9 agonists displayed a significant benefit in the treatment of asthma and food allergy in mice. 38 Several kinds of TLR agonists have now been applied to asthmatic or allergic patients in clinical trials. 37,39 In the case of TLR9 agonists, CpG-containing nucleotides (Amb a 1-CpG vaccine) 40,41 and bacteriophage-derived virion-like particles (VLPs) packaging A-type CpG motif 42 have been shown to be effective in treating rhinitis and allergic asthma.
Intranasal administration of a TLR7 agonist (AZD8848) and TLR8 (VTX-1463) has also reduced nasal symptoms in patients with allergic rhinitis. 43

TLR4 agonist monophosphoryl A (MPL) promotes Th1
and Treg cell responses in cooperation with switching from IgE to IgG blocking antibody production. 39

| C-T YPE LEC TIN RECEP TOR S
CLRs recognize a diverse range of nonself PAMPs derived from microbes, especially fungi and house dust mites. 44

| CLRs and allergy
Dectin-1 is involved in fungal-mediated allergic inflammation mediating T-helper 17 (Th17) cell differentiation. 55,56 Genetic polymorphisms of Dectin-1, TLR3, and TLR9 are significantly associated with susceptibility to severe asthma with fungal sensitization. 57 Dectin-2 has been implicated in allergic inflammation to house dust mites with Th2 polarization. 58-60 A recent study shows that Mincle recognizes not only glycolipids but also self-derived cholesterol sulfate in skin epithelial cells and is involved in the induction of allergic skin inflammatory response. 61

| Allergen-specific immunotherapy for CLRs
Recent findings have shown that allergoids conjugated to nonoxidized mannan from Saccharomyces cerevisiae are next-generation vaccines targeting DCs through CLRs. These vaccines are candidates for AIT of allergic diseases as they promote the generation of Treg cells by mechanisms partially depending on programmed death-ligand 1 (PD-1) and IL-10 in both humans and mice. 62,63 Phase II clinical trials for grass-pollen and house dust mite allergens are currently ongoing. 64

| RLR family
Tlr3 -deficient cells showed normal type I IFN production toward viral infection, 65 suggesting that additional mechanisms were hidden in the RNA sensing. As the cytosolic dsRNA sensor sensing both RNA helicases, RIG-I (DDX58) 66 and melanoma differentiation-associated gene 5 (MDA5, IFIH1) 67 were identified. 68 The

| RLRs and allergy
Early viral infections in children are associated with further allergic sensitization and asthma persistence. Similarly, viral infections in asthma patients (both allergic and nonallergic) are also associated with asthma exacerbations. In the development of respiratory disease, innate immune mechanisms are involved in virus-infected airway epithelial cells. 80 Loss-of-function mutations in the IFIH1 gene increase susceptibility to severe respiratory infection caused by human rhinovirus in children. 81,82 A meta-phenome-wide association study also revealed a novel association of an IFIH1 allele mutation to increased risk for asthma. 83 In an experimental model, poly(I:C) and rhinovirus-derived dsRNA exacerbated asthma. [84][85][86] Taken together, RLRs play a nonredundant and critical role in the development and progression of asthma. IFN production via the TBK1/IRF-3 pathway (Figure 3). 96,97 cGAMP senses viral-derived DNA as well as host-derived DNA. 98,99 cGAS is involved in DNA damage-induced inflammatory signaling in cancer cells. 100,101 The cGAS-STING-mediated DNA sensing system also contributes to the induction of apoptosis, control of ER stress response, and autophagy.

| NOD -LIK E RECEP TOR S
A third group of PRRs are NLRs. 102

NOD1 is widely expressed by a variety of cell types, whereas NOD2
expression is limited to certain cell types such as hematopoietic cells 105 and Paneth cells in the intestine. 106

| NODs and allergy
NOD1 and NOD2 promote Th1 and Th17 adaptive immunity by inducing the secretion of TNF and IL-1 107,108 in addition to Th2 immune response, 109 suggesting that signaling through these receptors may be central to susceptibility and exacerbation of allergies. Although inhalation of NOD1 and NOD2 ligands induces Th2 response, NOD2 appears to have more potent activity than NOD1 in Th2-driven allergic inflammation. It is shown that NOD2 displays this function by promot- Polymorphisms of NOD1 and NOD2 are highly associated with inflammation development in the respiratory airways, 111 childhood asthma, 112 and atopic diseases. [113][114][115] It has been thought that these phenotypes arise from a defect in NOD sensing fragments of bacterial peptidoglycan. However, a recent study shows that NODs also participate in sensing infection with viruses and parasites by inducing ER stress-induced inflammation, 116 and further research is needed to elucidate the role of NODs in allergic diseases.

| Canonical inflammasome
Caspases are a group of cysteine-aspartic acid proteases, of which 12 and 10 family members have been identified in humans and mice, respectively. Caspases play an essential role in programmed cell death and are involved in either apoptosis or inflammation along with pyroptosis 122 (Section 8). In the steady state, the caspase family is present in inactive forms called zymogens, which require activation. Inflammasome mediates the activation of caspase-1 through CARD-CARD interactions. Caspase-1, caspase-4 (human only), caspase-5 (human only), caspase-11 (mouse only), and caspase-12 are known to be involved in the inflammatory pathway, and those inflammatory caspase genes are clustered in the human and mouse genome. 123 Inflammasomes activate caspase-1, resulting in production of pro-inflammatory cytokines IL-1β and IL-18 upon activation by various signals 124 ( Figure 5). Thus, "canonical" inflammasome relies on the activation of caspase-1.

| NLRP3 inflammasome
NLRP3 inflammasomes can respond to a variety of substances including crystals such as urate, cholesterol, asbestos, silica, aggregated amyloid-β, and islet amyloid polypeptide. 125 As a direct interaction of NLRP3 with these substances has not been detected, NLRP3 inflammasome activation is considered to occur via mitochondrial and lysosomal damage. Reactive oxygen species (ROS) also trigger NLRP3 inflammasomes priming. 126 Extracellular ATPs released from activated or necrotic cells activate P2X purinoceptor 7 (P2X7, P2RX7) receptor 127 and induce caspase-1 activation, leading to IL-1β production via formation of an NLRP3 inflammasome. 128 IL-1β is critically involved in Th17 cell differentiation, production of Th17-associated cytokines, and neutrophilic inflammation. 129 In addition to the Th2 response, a Th17 response is associated with asthma, notably severe glucocorticoid-resistant asthma. 130 From various studies of human diseases and mouse models, the elevation of IL-1β and IL-18 is associated with the development of allergic diseases, such as asthma, dermatitis, rhinitis, and conjunctivitis. 131 All these events are connected with inflammasome activation and suggest participation of the inflammasome in the development and progression of asthma. Th2 and Th17 inflammatory pathways are mutually regulated in asthmatic patient samples. 132 Type II cytokine suppression promotes Th17 responses, indicating that combined therapy targeting both Th2 and Th17 responses may benefit asthmatic patients.

| AIM2 inflammasome
Cytoplasmic dsDNA sensor AIM2 is a member of IFI20X/IFI16 family. 133 In normal conditions, the N-terminal PYD of AIM2 F I G U R E 5 Inflammasome. NLRP3 forms a complex with ASC through their PYD binding each other and recruiting the CARD domain of pro-caspase-1. After assembly, this multimeric complex is called the inflammasome (upper box). In the canonical inflammasome assembly, ligand-sensing NLRs (NLRP1, NLRP3, NLRP6, NAIP/NLRC4, and AIM2) (blue balloons) form multisubunit disk-like structures comprising an inner ring and outer ring. Pro-caspase-1 is located in the central portion (black balloon). Activated caspase-1 processes IL-1β/IL-18 and triggers proteolytic cleavage of GSDMD (brown boxes). In the noncanonical inflammasomes, caspase-11 and caspase-8 participate in the cytoplasmic LPS sensing pathway and Dectin-1-CBM signaling pathway, respectively (pink boxes). Activated caspase-11 cleaves GSDMD similarly to caspase-1 (red arrow). GSDMD-derived NTD induces pore formation for pyroptosis and stimulates the NLRP3 inflammasome pathway (black arrows). Activated caspase-8 with FADD is capable of cleaving pro-IL-1β/pro-IL-18 (red arrow). Caspase-8 may positively regulate NLRP3 inflammasome pathway (red arrow). AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing NLRC4 is implicated in the exacerbation of psoriatic lesions, 152 and NLRP4 mutation is associated with exacerbation of asthma in smoking adults. 153 Polymorphisms of NACHT-LRR in NLRP12 and the promoter regions of NLRC4 and NLRP1 are associated with atopic dermatitis. 113 The elusive other inflammasomes remain of outstanding interest.

| Caspase-8
Caspase-8 is known to play a central role in apoptosis as initiator and apical activator. Caspase-8 has two death effecter domains (DEDs) in its N-terminus and is structurally different to CARD-containing inflammatory caspases. Interestingly, caspase-8 has been shown to form a noncanonical inflammasome in response to fungal and mycobacterial infection by Dectin-1. 164 Fungal PAMPs activate Dectin-1 signaling to induce a noncanonical caspase-8-ASC with CBM complex in a caspase-1-independent manner. 165 Caspase-8 assumes its inflammatory roles by inducing IL-1β activation 165,166 (Figure 5). Furthermore, caspase-8 mediates both canonical and noncanonical NLRP3 inflammasome priming and activation with a death domain (DD)-containing adaptor protein Fas-associated protein with DD (FADD). 167 Caspase-8mediated IL-1 signaling promotes Th2 responses in allergic airway inflammation, 168 implicating its therapeutic potential for asthma.

| Inhibition of inflammasome activation
Inflammasomes are multiple protein complexes; therefore, abnormal assembly causes hyperinflammatory conditions, as in the case of skin inflammation in NLRP1 germline mutation. 169 Some pathogens are able to selectively inhibit the activation of the caspase-11-mediated noncanonical NLRP3 inflammasome. 170 However, the regulatory mechanisms of inflammasomes are not fully understood and need further characterization. Interestingly, a recent study shows that TAK1 restricts the NLRP3 inflammasome to regulate cell homeostasis and death in myeloid cells. 171 TAK deficiency promotes spontaneous NLRP3 inflammasome activation. TAK1 inhibits the activation of DD-containing receptor-interacting serine/threonineprotein kinase 1 (RIPK1). Activated RIPK1 induces the caspase-8-FADD pathway. TAK1 inactivation induces RIPK1 activation, leading to the caspase-8-dependent pathway, 172 indicating that RIPK1 plays a role upstream of caspase-8. This machinery is also associated with neuroinflammation, aging, and infection. 173 Hence, the mechanism requires further investigation to understand the implications of inhibiting inflammasome activation.

| Pyroptosis
Pyroptosis is one form of cell death and is morphologically different to apoptosis and necrosis. Apoptosis is an immunologically silent death mode while necrosis and pyroptosis are pro-inflammatory death modes tightly associated with inflammation. 174 Caspase-1 is a key player of pyroptosis in cell death (canonical inflammasome).
As described above, caspase-11 also contributes to the central mechanism of pyroptosis ( Figure 5). 155 Pyroptotic cells release their entire cellular contents including nuclear and mitochondrial DNA.
Pyroptosis preferentially occurs in macrophages, monocytes, and DCs. Neutrophil cell death is called NETosis (neutrophil extracellular traps) and releases chromatin components to the extracellular space. 175,176 It is found that NETosis-derived dsDNA mediates allergic asthma exacerbations during rhinovirus infection, 177,178 suggesting dsDNA acts as an adjuvant to boost type II-mediated allergic inflammation.

| GSDMB and allergy
Importantly, the genome locus of GSDMB and orosomucoid 1-like 3 (ORMDL3) on chromosome 17q21 is strongly associated with childhood-onset asthma. 190,191 GSDMB is highly expressed in airway epithelial cells. 192 GSDMB-mediated pyroptosis in epithelial cells may be involved in the pathogenesis of asthma. Furthermore, GSDMB transgenic mice assume asthma symptoms in the absence of airway inflammation, 192 implicating that the induction of GSDMB triggers asthma.

| ALPK1-TIFA-NF-κ B A XIS
Besides LPS itself, ADP-β-D-manno-heptose (ADP-Hep) and Dglycero-β-D-manno-heptose 1,7-bisphosphate (HBP), [193][194][195] intermediate products of the LPS biosynthetic pathway, activate the NF-κB signaling pathway ( Figure 6). ADP-Hep is more potent than HBP. TRAF-interacting protein with forkhead-associated domain (TIFA) was originally identified as a TRAF2-binding protein that is involved in the NF-κB pathway. 196 Both ADP-Hep and HBP sensing trigger TIFA oligomerization. 193 Recent studies have shown that TIFA oligomerization can be induced by ADP-Hep or ADP-heptose 7-P that is converted from HBP by host adenylyltransferase enzymes of the nicotinamide mononucleotide adenylyltransferase (NMNAT) family ( Figure 6). 197 Alpha-kinase 1 (ALPK1), a member of the atypical kinase family alpha kinases, is necessary for phosphorylation-dependent formation of TIFA oligomerization. 194,195 These sugar molecules directly bind the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The role of ADP-Hep as a PAMP was further confirmed on comparison with synthetic HBP. 198 This ADP-heptose sensing system stimulates host innate immune responses.

| CON CLUS ION
Over 20 years, we have witnessed a remarkable advance in understanding the mechanism of pathogen recognition by the innate immune system. New players involved in the innate immune system continue to be reported. However, despite enormous efforts, our knowledge of how the innate immune system is involved in the development of allergic diseases is still limited, and feasible target molecules or pathways are yet to be discovered. It is necessary to determine how pathogen recognition molecules and subsequent signaling pathways are involved in the development of allergic diseases.

Kazuhiko Maeda has research collaboration with Otsuka
Pharmaceutical Co., Ltd. Shizuo Akira has research support from Chugai Pharmaceutical Co., Ltd. The terms of this arrangement have been reviewed and approved by Osaka University in accordance with its policy on objectivity in research.