The most well-known particle pollutant is DEPs. In a mouse model, DEPs functioned as adjuvant and induced ovalbumin (OVA)-specific IgE responses, infiltration of eosinophils, and goblet cell hyperplasia [5, 7]. Similar to DEP, ASD, an aerosol from central and northwestern China that is considered to affect respiratory health, has also been reported to induce allergic inflammation in the lung. Like DEPs, ASD is thought to function as an adjuvant inducing allergen (OVA)-specific responses [3, 4]. A recent study demonstrated that particulate matter 2.5 (PM2.5 is defined as having a particulate size of around or less than 2.5 μm in diameter), present in air, exacerbated allergic lung inflammation in OVA-sensitized and challenged asthmatic mice [37]. In addition, another study showed that intranasal administration of PM2.5 exacerbated allergic lung inflammation in allergy-prone NC/Nga mice [38]. These results suggested that PM2.5 may increase the risk of allergic asthma in allergy-prone children.

Many studies have shown that metal-, chemical-, and mineral-derived particulates have strong adjuvant activity. The most well-known inorganic particulate for immunologists is alum. In general, alum adjuvant preferentially activates humoral immunity to induce antigen-specific antibody responses [18–20]. However, alum is frequently used to generate mouse models of allergic diseases because it strongly induces type-2 immune responses. Similar to alum, crystalline silica is also reported to induce antigen-specific IgE and IgG1 in a mouse model [15]. A recent study demonstrated that inorganic crystalline materials consisting of heterogeneous layers of double hydroxides, such as lithium aluminum, calcium aluminum, and magnesium aluminum, have strong adjuvant activities and induce high levels of antigen-specific IgE [39]. Nickel is a representative allergen that induces contact dermatitis, and nickel oxide nanoparticles also function as type-2 immune adjuvants inducing IgE [14].

Several particulates and crystals are generated in the body and induce immune responses through their adjuvanticity. Monosodium urate (MSU) crystals are generated from saturating concentration of uric acid and are the causative agent of gout. Since uric acid is released from damaged cells, MSU crystals are thought to be DAMPs and exhibit strong type-2 adjuvant activity [12, 34, 40–42]. Recent studies demonstrated that MSU crystals play a pivotal role in house dust mite antigen-induced allergic lung inflammation [43]. Chitin is a component of the cell wall of fungi, helminth, and insects. Chitin particles, which are biopolymers of N-acetyl-D-glucosamine, can elicit type-2 immune responses through the induction of interleukin (IL)-4 from eosinophils and basophils [17].

Carbon nanotubes (CNTs) are a well-known carbon nanomaterial with a cylindrical structure that have been applied for use in drug delivery and as semiconductors. Several studies have indicated that CNTs induce lung inflammation [44, 45]. In addition, recent studies have demonstrated that subcutaneous, intranasal, or intratracheal instillation of CNTs plus OVA significantly induced OVA-specific IgE in serum and infiltration of eosinophils in the lung. As with other particulates, CNTs function as type-2 adjuvants to induce antigen-specific Th2 responses [6, 8]. It is not only cylindrical carbon structures that display adjuvant activity; carbon black, a colloidal carbon particle, also possesses adjuvant activity and particularly in ultrafine form enhanced antigen-specific IgE and induced allergic airway inflammation [46–48].

Induction of type-2 responses is one of the features of particulate adjuvant; however not all particulates induce allergic inflammation. The biocrystalline substance hemozoin is a hemin detoxification by-product of malaria parasites. Hemozoin and synthetic hemozoin (also known as β-hematin) that do not induce allergic inflammation display strong adjuvant activity [49, 50]. Recently, synthetic hemozoin was tested as a potential influenza vaccine adjuvant [51, 52]. Poly(lactide-co-glycolide) (PGLA) is a biodegradable polymeric nanoparticle that initially attracted attention as a potent delivery system [53]. However, this particle also displays adjuvant activity inducing antigen-specific IgG1 and IgG2c [53, 54]. Interestingly, PGLA stimulates innate cells such as DCs to produce IL-1α and IL-1β through PRR activation [54]. Recently, we reported that hydroxyapatite nanoparticles function as a vaccine adjuvant [55]. The hydroxyapatite particle is a less-toxic particulate adjuvant due to its characteristic of biodegradation and is a promising influenza vaccine adjuvant, similar to PGLA.

Thus, not all but most of the particulates described above function as adjuvants to enhance antigen (allergen)-specific type-2 immune responses. Therefore, it has been hypothesized that particulate adjuvants causing allergy evoke similar and unique signals in innate cells to preferentially activate type-2 immune responses.

TLR and MyD88 signaling is one of the major signaling pathways for the activation of innate cells such as macrophages and DCs. Schnare et al. investigated the effect of the particulate adjuvant alum on IgG and IgE responses in MyD88-deficient mice [56]. MyD88-deficient mice naturally display excess quantities of total IgE, because T cells from knockout (KO) mice release increased amounts of IL-13. However, antigen-specific IgE responses were comparable between wild-type (WT) and KO mice that were immunized with alum plus OVA by footpad injection [56]. Furthermore, Gavin et al. also investigated alum adjuvanticity using MyD88 and Toll/IL-1 receptor domain-coding adaptor inducing IFN-γ (TRIF) double-KO mice, which completely lack TLR signaling. Similarly to MyD88 deficiency, the antibody responses were comparable between WT and double-KO mice immunized with trinitrophenol-hemocyanin plus alum by intraperitoneal injection [57]. However, Matsushita et al. demonstrated that B-cell-specific MyD88-deficient mice displayed reduced levels of serum antigen-specific IgE and IgG1 after the immunization with alum plus OVA through direct administration to the airway [58]. Thus, the requirement for MyD88 signaling might be dependent on the administration route and the specific cell type.

Several studies also demonstrated the requirement of MyD88 signaling in ambient air pollution particle-induced allergic responses. Becker et al. showed that microbial components on ambient particulate matter stimulate alveolar macrophage to produce inflammatory cytokines in a TLR4-dependent manner [59]. In addition, Ichinose et al. reported that microbial materials, in particular TLR2 ligands such as β-glucan and peptidoglycan, are involved in ASD-induced lung eosinophilia. ASD, to which LPS and β-glucan adhere, induced eosinophil infiltration in the lung; however this was reduced after heating of the ASD to exclude microbial components [60, 61]. In fact, TLR ligands are known to induce allergic lung inflammation [62], suggesting that ambient particulate adjuvant activates type-2 immune responses, in part, through TLR and MyD88 signaling promoted by microbial components adhered to the particulate.

Natural and synthetic hemozoin crystals that are not particulate adjuvants causing allergy also seem to activate MyD88 signaling which is required for adjuvant activity. The underlying mechanisms of the adjuvant activity of hemozoin remain to be investigated [49].

The inflammasome is a type of intracellular PRR that is categorized into the NOD-like receptors. There are four classes of inflammasome, NLRP1, NLRP3, NLRC4 (IPAF), and AIM2, of which NLRP3 is the best characterized [24]. Upon activation, NLRP3 forms a multiprotein complex with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. This complex promotes the secretion of the pro-inflammatory cytokines IL-1β and IL-18 through the action of caspase-1 [24]. Initially, it was demonstrated that the NLRP3 inflammasome is activated by invasive infection and induces inflammatory responses [24]. Later in 2008, several reports demonstrated that particulates or crystals, such as silica, alum, and asbestos, stimulate macrophages and DCs to produce IL-1β and IL-18 through the activation of the NLRP3 inflammasome [63–65]. The NLRP3 inflammasome has been reported to be involved in type-2 immune responses, and in addition, alum-induced type-2 responses, especially IgG1 and IgE responses, are significantly reduced in NLRP3-, ASC-, and caspase-1-deficient mice. Interestingly, most particulate adjuvants activate the NLRP3 inflammasome in macrophages and DCs to produce mature IL-1β. For example, in addition to alum and silica, MSU acts as activator of the NLRP3 inflammasome [66]. Ambient particle pollutants, such as DEP, ASD, and PM2.5, are reported to stimulate innate cells to produce IL-1β and IL-18, implicating the importance of inflammasome activation [38, 67, 68]. In addition, PGLA and hydroxyapatite nanoparticles that do not induce allergic responses function as a vaccine adjuvant and induce IL-1β through the NLRP3 inflammasome [54, 55]. However, other reports have shown that the NLRP3 inflammasome is dispensable for adjuvant activity of particulates. In addition, it has been reported that the NLRP3 inflammasome is not required for antigen-specific antibody production induced by sensitization with alum plus antigens [69–71]. These contradictory reports on the role of the NLRP3 inflammasome indicate that further investigation is required to determine the importance of the inflammasome in particulate adjuvanticity. Inflammasome activation induced by particulates seems to be important for the induction of acute inflammation in the lung. Furthermore, several studies have shown that most inflammasome activators also induce IL-1α release from DCs, and particulate adjuvants such as alum, silica, MSU, and TiO₂ stimulate DCs to release IL-1α, a process that is partially dependent on the NLRP3 inflammasome [72, 73]. Another study also reported that IL-1α release was partially reduced in response to PGLA and polystyrene microparticles in DCs from NLRP3-deficient mice [54]. The detailed mechanism of inflammasome-dependent IL-1α release by particulate adjuvant is still unclear. Interestingly, a recent study demonstrated that IL-1α and IL-1β control inflammation at different stages, i.e., initiation and maintenance [74]. Thus, particulate adjuvants may regulate a many-sided immune response through NLRP3 inflammasome activation during inflammation. Recent reports have demonstrated a novel mechanism of inflammasome to perpetuate inflammatory responses [75, 76]. After the activation of inflammasome, ASC assembles into a large protein aggregate with NLRP3 and caspase-1. The inflammasome particles, or “specks,” are released during pyroptotic cell death and taken up by surrounding phagocyte to lead to inflammation through the release of IL-1β. Thus, ASC speck acts as danger signals to spread inflammatory responses.

Uric acid is an intracellular purine catabolite that is released from dying or stressed cells. At saturated concentrations of uric acid, MSU crystals are formed. MSU crystals are considered as DAMPs that regulate immune responses at the site of inflammation, such as those that occur during gout. In 2003, Shi et al. demonstrated that uric acid and its crystals induce DC maturation and activation [77]. Interestingly, MSU crystals are reported to preferentially induce type-2 immune responses through their adjuvant activity. Kool et al. reported that levels of uric acid are increased in the peritoneal cavity after i.p. injection of alum. In addition, released uric acid seems to induce antigen-specific T-cell responses because administration of uricase significantly reduced the activation of T cells [12, 40]. Some particulates, such as alum and silica, are known to induce cell death. Furthermore, it is reported that ambient particle pollutants, PM2.5 and DEP, and metal-based nanoparticles also showed cytotoxic activity on macrophages and monocytes through the production of reactive oxygen species [78–80]. Given that particulates induce cell death, uric acid and MSU crystals released from dying cells by particulates might contribute to allergic inflammation. In fact, a recent study showed that allergic lung inflammation induced by the administration of airborne allergen to the airway is a uric acid- and IL-33-dependent mechanism and treatment with an inhibitor of uric acid synthesis attenuated the onset of asthma [43]. Kool et al. demonstrated that uric acid-primed inflammatory monocytes and DCs participate in T-cell activation through IL-1 and MyD88 signaling [40]. In addition, since uric acid (and its crystal form) is known to induce IL-1β through the activation of the NLRP3 inflammasome as described above, this adjuvant effect was considered to be dependent on IL-1β released by the activated inflammasome. However, it was reported that the NLRP3 inflammasome, IL-1, and MyD88 signaling are dispensable for the adjuvanticity of uric acid induced by alum [12, 14]. In addition, it was shown that spleen tyrosine kinase (Syk) and PI3-kinase δ in inflammatory monocytes and DCs participate in type-2 immune responses induced by uric acid [12]. So far, several papers have shown the importance of Syk in activation of macrophages and DCs. The underlying mechanisms of Syk activation by particulates and the role of Syk in type-2 immune responses are interesting issues that require further investigation [12, 14, 81]. Recent studies have reported the importance of Syk in immune responses induced by dead cells, demonstrating that Clec9a, one of the C-type lectin receptors (CLRs) in PRRs, senses necrotic cells and induces DC activation through the Syk-dependent signaling pathway [82, 83]. Several CLRs are coupled with Syk and transduce activating signals into cells [27]. Clec9a recognizes F-actin expressed on necrotic cells [82, 83]. Thus, Syk may play an important role in signal transduction for factors from dying cells.

The recognition mechanisms of MSU crystals have also been investigated. Ng et al. demonstrated that MSU crystals interact with the lipid raft on DCs in a receptor-independent manner. This interaction causes lipid sorting and transduces a signal into cells that then leads to recruitment and activation of Syk [81]. Flach et al. reported that alum also binds to the lipid raft on DCs and then activates Syk and PI3-kinase, similarly to MSU crystals. Then DCs activate T cells through the interaction with intracellular adhesion molecule (ICAM)-1 and leukocyte function-associated antigen (LFA)-1 [84].

Previously, we reported the unique function of particulates in eliciting type-2 immune responses. We focused on the particulates alum and silica and found that these particulates stimulated macrophages and DCs to produce the lipid mediator prostaglandin (PG), similar to the activation of the NLRP3 inflammasome [14]. PGE₂, a well-characterized pro-inflammatory lipid mediator, is an arachidonic acid metabolite that is mainly produced by myeloid-lineage cells such as DCs and macrophages [85]. PGE₂ performs various functions in the regulation of immune responses, one of which is the suppression of type-1 immune responses by inhibiting the production of type-1 cytokines such as IFN-γ and IL-12 [86–88].

Alum and silica stimulate LPS-primed macrophages and DCs to activate the NLRP3 inflammasome and release IL-1β and IL-18; in addition, we also observed PGE₂ release from particulate-activated macrophages and DCs. PGE₂ release was shown to be independent of the NLRP3 inflammasome and IL-1 signaling because inflammasome-deficient macrophages, such as NLRP3 KO, ASC KO, or caspase-1 KO macrophages, still produced normal levels of PGE₂ in response to alum and silica. Interestingly, PGE₂ production in response to alum and silica was mediated by the activation of Syk, which is an important molecule for the activation of DCs by MSU crystals. To clarify the role of PGE₂ in immune responses in vivo, we used PGE synthase (PTGES)-deficient mice [89]. Macrophages from PTGES-deficient mice did not produce PGE₂ in response to alum or silica. In addition, PTGES-deficient mice displayed reduced levels of antigen-specific IgE after immunization with alum + OVA or silica + OVA, indicating that particulate-induced PGE₂ regulates IgE production in vivo. Previous studies have shown that PGE₂ facilitates IgE production from B cells stimulated with IL-4, through the intracellular accumulation of cAMP [90, 91]. Furthermore, a recent study demonstrated that the elevated levels of PGE₂ in the gut are involved in allergic airway inflammation by the alteration of macrophage phenotypes toward type-2 macrophage (M2) [92].

We also found that many particulates such as MSU crystals, PGLA, nickel oxide, amorphous silica, and CNTs also stimulate macrophages to produce inflammasome-dependent IL-1β and inflammasome-independent PGE₂. In addition, increased amounts of PGE₂ release were observed in dying cells, indicating that, as with uric acid release, PGE₂ release is an indicator of cell death and might function as a DAMP (Kuroda et al. unpublished data). These results suggest that PGE₂ release in response to particulates is one of the markers of the adjuvanticity of particulates and may be a potent inducer of allergic inflammation.

As described above, DAMP release in response to a particulate seems to be required for the induction of immune responses [28]. Recent studies have shown that a chemical agent that has cytotoxic activity acts as an adjuvant to induce IgE production, suggesting that particulate-induced DAMPs may participate in allergic inflammation [93, 94]. DNA released from host cells appears to be a DAMP. Marichal et al. demonstrated that DNA from damaged host cells is involved in the adjuvanticity of alum [16]. This study showed that host DNA is released from damaged phagocytes such as macrophage and neutrophils at the site of alum injection. The released DNA functions as an adjuvant and induces antigen-specific IgG and IgE responses. Interestingly, treatment with DNase I at the injection site of alum + OVA significantly reduced OVA-specific antibody responses. In addition, purified genomic DNA mixed with antigen mimicked the adjuvant activity of alum and induced antigen-specific antibody responses. This finding indicates that DNA released from host cells is a critical factor for the activation of innate and adaptive immune responses. TANK-binding kinase 1(TBK-1) and interferon regulatory factor 3 (IRF-3) are reported to be important signaling molecules downstream of specific receptors for DNA recognition in innate cells [95]. This study demonstrated the attenuated adjuvant activities of alum or host DNA in TBK1- or IRF3-deficient mice, with IgE responses being markedly reduced. In addition, IRF3 participates in the recruitment of inflammatory monocytes and DCs, cells responsible for the induction of type-2 immune responses, suggesting that IRF3 regulates the type-2 adjuvant activity of alum through the signaling pathway for host DNA recognition during an innate immune response. The IRF3-mediated activation of inflammatory DCs was controlled by IL-12p80 release, a p40 homodimer. Furthermore, treatment of IL-12p80 neutralizing antibody partially reduced IgE responses in mice immunized with alum + OVA. Given that many particulates with adjuvant activity are known to induce cell death, the adjuvant activity of particulates is considered to be closely linked to cell death and consequently host DNA release. CpG oligodeoxynucleotide is a well-known DNA-based adjuvant that is recognized by TLR9, but it is reported to be a strong inducer of type-1 immunity unlike the DNA released from dying cells [96, 97], suggesting that different types of DNA induce type-2 immune responses through signal transduction pathways other than TLR9 signaling.

A recent study demonstrated that DNA released from damaged cells directly stimulates naive CD4⁺ T cells to induce Th2 differentiation through an unknown DNA sensor and might be involved in allergic inflammation [98]. The underlying mechanisms for Th2 differentiation were mediated by the downregulation of T-bet and the upregulation of GATA-3 expression. Furthermore, a recently identified DNA sensor, cyclic GMP-AMP synthase (cGAS), might be important for type-2 immunity [99]. Activated cGAS induces generation of cyclic AMP-GMP (cGAMP), and this cyclic nucleotide functions as the ligand for the ER-resident adaptor molecules, stimulator of interferon genes (STING). Activated STING is known to regulate the TBK1-IRF3 signaling pathway to induce type-1 IFN production [100, 101]. Our previous studies demonstrated that cGAMP and synthetic STING ligand function as type-2 adjuvants and induce antigen-specific Th2 responses [102, 103]. Interestingly, STING ligand-induced type-2 responses were IRF3 and type-1 IFN dependent and were completely abolished in IRF3- and type-1 IFN receptor-deficient mice [102, 103]. Given that IRF3 and TBK1 are involved in host DNA-dependent adjuvant activity by alum, these signaling molecules may be potent therapeutic targets of particulate-induced allergic inflammation. Thus, host DNA and DNA-sensing molecule and its signal transducers appear to augment allergic inflammation through both known and unknown mechanisms.

It is not only DNA that is released from damaged cells; uric acid and its crystals are also released. We also found that higher levels of PGE₂ were released during cell death (unpublished data). Taken together, it is evident that many different types of DAMPs released from dying cells induced by particulates promote immune responses and exacerbate allergic inflammation.