Reciprocal regulation of IL-33 receptor-mediated inflammatory response and pulmonary fibrosis by TRAF6 and USP38.

The warning cytokine interleukin-33 receptor (IL-33R) mediates local inflammatory responses and plays crucial roles in the pathogenesis of immune diseases such as pulmonary fibrosis and rheumatoid arthritis. Whether and how IL-33R is regulated remain enigmatic. Here, we identified ubiquitin-specific protease 38 (USP38) as a negative regulator of IL-33R­mediated signaling. USP38 deficiency promotes interleukin-33 (IL-33)­induced downstream proinflammatory responses in vitro and in vivo. Usp38−/− mice are more susceptible to inflammatory damage and death and developed more serious pulmonary fibrosis after bleomycin treatment. USP38 is constitutively associated with IL-33R and deconjugates its K27-linked polyubiquitination at K511, resulting in its autophagic degradation. We further show that the E3 ubiquitin ligase tumor necrosis factor receptor­associated factor 6 (TRAF6) catalyzes K27-linked polyubiquitination of IL-33R at K511, and that deficiency of TRAF6 inhibits IL-33­mediated signaling. Our findings suggest that K27-linked polyubiquitination and deubiquitination of IL-33R by TRAF6 and USP38 reciprocally regulate IL-33R level and signaling, which represents a critical mechanism in the regulation of IL-33­triggered lung inflammatory response and pulmonary fibrosis.

The Paradigm Change of IL-33 in Vascular Biology.

In this review, we focus on the actual understanding of the role of IL-33 in vascular biology in the context of the historical development since the description of IL-33 as a member of IL-1 superfamily and the ligand for ST2 receptor in 2005. We summarize recent data on the biology, structure and signaling of this dual-function factor with both nuclear and extracellular cytokine properties. We describe cellular sources of IL-33, particularly within vascular wall, changes in its expression in different cardio-vascular conditions and mechanisms of IL-33 release. Additionally, we summarize the regulators of IL-33 expression as well as the effects of IL-33 itself in cells of the vasculature and in monocytes/macrophages in vitro combined with the consequences of IL-33 modulation in models of vascular diseases in vivo. Described in murine atherosclerosis models as well as in macrophages as an atheroprotective cytokine, extracellular IL-33 induces proinflammatory, prothrombotic and proangiogenic activation of human endothelial cells, which are processes known to be involved in the development and progression of atherosclerosis. We, therefore, discuss that IL-33 can possess both protective and harmful effects in experimental models of vascular pathologies depending on experimental conditions, type and dose of administration or method of modulation.

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

Nasal alum-adjuvanted vaccine promotes IL-33 release from alveolar epithelial cells that elicits IgA production via type 2 immune responses.

Aluminum hydroxide salts (alum) have been added to inactivated vaccines as safe and effective adjuvants to increase the effectiveness of vaccination. However, the exact cell types and immunological factors that initiate mucosal immune responses to alum adjuvants are unclear. In this study, the mechanism of action of alum adjuvant in nasal vaccination was investigated. Alum has been shown to act as a powerful and unique adjuvant when added to a nasal influenza split vaccine in mice. Alum is cytotoxic in the alveoli and stimulates the release of damage-associated molecular patterns, such as dsDNA, interleukin (IL)-1α, and IL-33. We found that Ag-specific IgA antibody (Ab) production was markedly reduced in IL-33-deficient mice. However, no decrease was observed in Ag-specific IgA Ab production with DNase I treatment, and no decrease was observed in IL-1α/ß or IL-6 production in IL-33-deficient mice. From the experimental results of primary cultured cells and immunofluorescence staining, although IL-1α was secreted by alveolar macrophage necroptosis, IL-33 release was observed in alveolar epithelial cell necroptosis but not in alveolar macrophages. Alum- or IL-33-dependent Ag uptake enhancement and elevation of OX40L expression were not observed. By stimulating the release of IL-33, alum induced Th2 immunity via IL-5 and IL-13 production in group 2 innate lymphoid cells (ILC2s) and increased MHC class II expression in antigen-presenting cells (APCs) in the lung. Our results suggest that IL-33 secretion by epithelial cell necroptosis initiates APC- and ILC2-mediated T cell activation, which is important for the enhancement of Ag-specific IgA Ab production by alum.

IL-33/ST2 Signaling Regulates Synaptic Plasticity and Homeostasis in the Adult Hippocampal Circuitry.

In response to neuronal activity changes, the adult hippocampal circuits undergo continuous synaptic remodeling, which is essential for information processing, learning, and memory encoding. Glial cells, including astrocytes and microglia, actively regulate hippocampal synaptic plasticity by coordinating the neuronal activity-induced synaptic changes at the circuit level. Emerging evidence suggests that the crosstalk between neurons and glia in the adult hippocampus is region specific and that the mechanisms controlling this process are critically dependent on secreted factors. Interleukin-33 (IL-33), a cytokine of the IL-1 family, is a key factor that modulates such glia-driven neuromodulations in two distinct hippocampal circuits. The activation of IL-33 and its receptor complex is important for maintaining the excitatory synaptic activity in the cornu ammonis 1 subregion and the remodeling of dentate gyrus synapses through activity-dependent astrocyte-synapse and microglia-synapse interactions, respectively. Meanwhile, the dysregulation of this signaling is implicated in multiple neurological disorders, especially Alzheimer's disease. Further investigations of how IL-33/ST2 signaling is regulated in a region-specific manner as well as its diverse functions in glia-synapse communications in the adult hippocampal circuitry will provide insights into the nature of hippocampal synaptic plasticity and homeostasis in health and disease.

Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption.

Regulatory T cells (Tregs) suppress immune responses and maintain immunological self-tolerance and homeostasis. We currently investigated relationships between skin barrier condition and Treg behavior using skin barrier-disrupted mice. Skin barrier disruption was induced by repeated topical application of 4% sodium dodecyl sulfate (SDS) on mice. The number of CD4+ forkhead box protein P3 (Foxp3)+ Tregs was higher in 4% SDS-treated skins than in controls. This increasing was correlated with the degree of acanthosis. The numbers of interleukin (IL)-10+ and transforming growth factor (TGF)-ß+ Tregs also increased in 4% SDS-treated skins. Localization of IL-33 in keratinocytes shifted from nucleus to cytoplasm after skin barrier disruption. Notably, IL-33 promoted the migration of Tregs in chemotaxis assay. The skin infiltration of Tregs was cancelled in IL-33 neutralizing antibody-treated mice and IL-33 knockout mice. Thus, keratinocyte-derived IL-33 may induce Treg migration into barrier-disrupted skin to control the phase transition between healthy and inflammatory conditions.

The chemerin-CMKLR1 axis limits thermogenesis by controlling a beige adipocyte/IL-33/type 2 innate immunity circuit.

IL-33-associated type 2 innate immunity has been shown to support beige fat formation and thermogenesis in subcutaneous inguinal white adipose tissue (iWAT), but little is known about how it is regulated in iWAT. Chemerin, as a newly identified adipokine, is clinically associated with obesity and metabolic disorders. We here show that cold exposure specifically reduces chemerin and its receptor chemerin chemokine-like receptor 1 (CMKLR1) expression in iWAT. Lack of chemerin or adipocytic CMKLR1 enhances cold-induced thermogenic beige fat via potentiating type 2 innate immune responses. Mechanistically, we identify adipocytes, particularly beige adipocytes, as the main source for cold-induced IL-33, which is restricted by the chemerin-CMKLR1 axis via dampening cAMP-PKA signaling, thereby interrupting a feed-forward circuit between beige adipocytes and type 2 innate immunity that is required for cold-induced beige fat and thermogenesis. Moreover, specific deletion of adipocytic IL-33 inhibits cold-induced beige fat and type 2 innate immune responses. Last, genetic blockade of adipocytic CMKLR1 protects against diet-induced obesity and enhances the metabolic benefits of cold stimulation in preestablished obese mice. Thus, our study identifies the chemerin-CMKLR1 axis as a physiological negative regulator of thermogenic beige fat via interrupting adipose-immune communication and suggests targeting adipose CMKLR1 as a potential therapeutic strategy for obesity-related metabolic disorders.

Inflammasomes and Fibrosis.

Fibrosis is the final common pathway of inflammatory diseases in various organs. The inflammasomes play an important role in the progression of fibrosis as innate immune receptors. There are four main members of the inflammasomes, such as NOD-like receptor protein 1 (NLRP1), NOD-like receptor protein 3 (NLRP3), NOD-like receptor C4 (NLRC4), and absent in melanoma 2 (AIM2), among which NLRP3 inflammasome is the most studied. NLRP3 inflammasome is typically composed of NLRP3, ASC and pro-caspase-1. The activation of inflammasome involves both "classical" and "non-classical" pathways and the former pathway is better understood. The "classical" activation pathway of inflammasome is that the backbone protein is activated by endogenous/exogenous stimulation, leading to inflammasome assembly. After the formation of "classic" inflammasome, pro-caspase-1 could self-activate. Caspase-1 cleaves cytokine precursors into mature cytokines, which are secreted extracellularly. At present, the "non-classical" activation pathway of inflammasome has not formed a unified model for activation process. This article reviews the role of NLRP1, NLRP3, NLRC4, AIM2 inflammasome, Caspase-1, IL-1ß, IL-18 and IL-33 in the fibrogenesis.

Nuclear IL-33 Plays an Important Role in the Suppression of FLG, LOR, Keratin 1, and Keratin 10 by IL-4 and IL-13 in Human Keratinocytes.

IL-33 is a chromatin-associated multifunctional cytokine implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. The previous reports show that IL-33 is highly detected in the nucleus of epidermal keratinocytes in AD lesions compared with that in unaffected or normal skin. However, it is unclear whether intracellular IL-33 directly contributes to the pathogenesis of AD. T helper type 2 cytokines IL-4 and IL-13 that are elevated in AD lesions suppress keratinocyte differentiation to impair skin barrier function. We investigated whether intracellular IL-33 is involved in IL-4 and IL-13 function. In monolayer culture and living skin equivalent analyses, IL-4 and IL-13 increased the expression of full-length IL-33 in the nucleus of keratinocytes by activating the MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase signaling pathway, which is necessary for the inhibition of differentiation markers FLG, LOR, keratin 1, and keratin 10. The nuclear IL-33 functions as a transcription cofactor of signal transducer and activator of transcription 3, increasing the binding of phosphorylated signal transducer and activator of transcription 3 to FLG promoter, thereby inhibiting its transcription, and it inhibits the expression of transcription factor RUNX1 by signal transducer and activator of transcription 3 and signal transducer and activator of transcription 6, thereby downregulating LOR, keratin 1, and keratin 10. Thus, the elevated nuclear IL-33 in the epidermis of AD lesions may be involved in the pathogenesis of AD by inhibiting keratinocyte differentiation and skin barrier function.

Mechanical Ventilation with Moderate Tidal Volume Exacerbates Extrapulmonary Sepsis-Induced Lung Injury via IL33-WISP1 Signaling Pathway.

ABSTRACT: IL-33 and WNT1-inducible secreted protein (WISP1) play central roles in acute lung injury (ALI) induced by mechanical ventilation with moderate tidal volume (MTV) in the setting of sepsis. Here, we sought to determine the inter-relationship between IL-33 and WISP1 and the associated signaling pathways in this process.We used a two-hit model of cecal ligation puncture (CLP) followed by MTV ventilation (4 h 10 mL/kg) in wild-type, IL-33-/- or ST2-/- mice or wild-type mice treated with intratracheal antibodies to WISP1. Macrophages (Raw 264.7 and alveolar macrophages from wild-type or ST2-/- mice) were used to identify specific signaling components.CLP + MTV resulted in ALI that was partially sensitive to genetic ablation of IL-33 or ST2 or antibody neutralization of WISP1. Genetic ablation of IL-33 or ST2 significantly prevented ALI after CLP + MTV and reduced levels of WISP1 in the circulation and bronchoalveolar lung fluid. rIL-33 increased WISP1 in alveolar macrophages in an ST2, PI3K/AKT, and ERK dependent manner. This WISP1 upregulation and WNT ß-catenin activation were sensitive to inhibition of the ß-catenin/TCF/CBP/P300 nuclear pathway.We show that IL-33 drives WISP1 upregulation and ALI during MTV in CLP sepsis. The identification of this relationship and the associated signaling pathways reveals a number of possible therapeutic targets to prevent ALI in ventilated sepsis patients.

Sepsis Induces Deregulation of IL-13 Production and PD-1 Expression in Lung Group 2 Innate Lymphoid Cells.

ABSTRACT: Deregulation of the immune system in sepsis plays the central role in the pathogenesis of multiple organ failure including septic lung injury. Group 2 innate lymphoid cells (ILC2s) have emerged as a new player in regulating immune homeostasis in the lung; however, the role of ILC2s in lung injury in sepsis remains poorly understood. Here, we investigated temporal changes in stimulatory and inhibitory receptor expression and intracellular type 2 cytokine expression of ILC2s in the lung using a cecal ligation and puncture mouse sepsis model. We found that IL-13 production by ILC2s, which were predominately composed of the resident natural ILC2 subset rather than the migratory inflammatory ILC2 subset, was reduced in the lungs of sepsis mice on day 1 and gradually restored through day 7. Although the expression levels of ST2 and inducible T-cell costimulator (stimulatory receptors) were high, IL-13 production by ILC2s was reduced while showing high programmed cell death 1 (PD-1) (inhibitory receptor) expression. Furthermore, using IL-33 knockout mice, we have shown that IL-33 regulates the capacity of ILC2s to produce IL-13, possibly through the modulation of ST2 and PD-1 expression and signaling in the septic lung. To the best of our knowledge, this is the first report showing differential costimulatory/inhibitory receptor expression on ILC2s in a septic lung in the context of an IL-33/IL-13 pathway-mediated type 2 immune response in the progression and resolution of inflammation. Our present findings contribute to a better understanding of the underlying immunological mechanism of ILC2s and may fill the critical knowledge gap regarding immune homeostasis in the lung that hampers the development of new therapeutic strategies for sepsis-induced acute lung injury.

Interleukin 33 Triggers Early Eosinophil-Dependent Events Leading to Metaplasia in a Chronic Model of Gastritis-Prone Mice.

BACKGROUND & AIMS: Interleukin (IL)33/IL1F11 is an important mediator for the development of type 2 T-helper cell (Th2)-driven inflammatory disorders and has also been implicated in the pathogenesis of gastrointestinal (GI)-related cancers, including gastric carcinoma. We therefore sought to mechanistically determine IL33's potential role as a critical factor linking chronic inflammation and gastric carcinogenesis using gastritis-prone SAMP1/YitFc (SAMP) mice. METHODS: SAMP and (parental control) AKR mice were assessed for baseline gastritis and progression to metaplasia. Expression/localization of IL33 and its receptor, ST2/IL1R4, were characterized in corpus tissues, and activation and neutralization studies were both performed targeting the IL33/ST2 axis. Dissection of immune pathways leading to metaplasia was evaluated, including eosinophil depletion studies using anti-IL5/anti-CCR3 treatment. RESULTS: Progressive gastritis and, ultimately, intestinalized spasmolytic polypeptide-expressing metaplasia (SPEM) was detected in SAMP stomachs, which was absent in AKR but could be moderately induced with exogenous, recombinant IL33. Robust peripheral (bone marrow) expansion of eosinophils and local recruitment of both eosinophils and IL33-expressing M2 macrophages into corpus tissues were evident in SAMP. Interestingly, IL33 blockade did not affect bone marrow-derived expansion and local infiltration of eosinophils, but markedly decreased M2 macrophages and SPEM features, while eosinophil depletion caused a significant reduction in both local IL33-producing M2 macrophages and SPEM in SAMP. CONCLUSIONS: IL33 promotes metaplasia and the sequelae of eosinophil-dependent downstream infiltration of IL33-producing M2 macrophages leading to intestinalized SPEM in SAMP, suggesting that IL33 represents a critical link between chronic gastritis and intestinalizing metaplasia that may serve as a potential therapeutic target for preneoplastic conditions of the GI tract.

IL-33 Mediates Lung Inflammation by the IL-6-Type Cytokine Oncostatin M.

The interleukin-1 family member IL-33 participates in both innate and adaptive T helper-2 immune cell responses in models of lung disease. The IL-6-type cytokine Oncostatin M (OSM) elevates lung inflammation, Th2-skewed cytokines, alternatively activated (M2) macrophages, and eosinophils in C57Bl/6 mice in vivo. Since OSM induces IL-33 expression, we here test the IL-33 function in OSM-mediated lung inflammation using IL-33-/- mice. Adenoviral OSM (AdOSM) markedly induced IL-33 mRNA and protein levels in wild-type animals while IL-33 was undetectable in IL-33-/- animals. AdOSM treatment showed recruitment of neutrophils, eosinophils, and elevated inflammatory chemokines (KC, eotaxin-1, MIP1a, and MIP1b), Th2 cytokines (IL-4/IL-5), and arginase-1 (M2 macrophage marker) whereas these responses were markedly diminished in IL-33-/- mice. AdOSM-induced IL-33 was unaffected by IL-6-/- deficiency. AdOSM also induced IL-33R+ ILC2 cells in the lung, while IL-6 (AdIL-6) overexpression did not. Flow-sorted ILC2 responded in vitro to IL-33 (but not OSM or IL-6 stimulation). Matrix remodelling genes col3A1, MMP-13, and TIMP-1 were also decreased in IL-33-/- mice. In vitro, IL-33 upregulated expression of OSM in the RAW264.7 macrophage cell line and in bone marrow-derived macrophages. Taken together, IL-33 is a critical mediator of OSM-driven, Th2-skewed, and M2-like responses in mouse lung inflammation and contributes in part through activation of ILC2 cells.

Critical role of IL-33, but not IL-25 or TSLP, in silica crystal-mediated exacerbation of allergic airway eosinophilia.

Silica crystals (silica), which are a major mineral component of volcanic ash and desert dust, contribute to the pathogenesis of pulmonary disorders such as asthma and fibrosis. Although administration of silica or sand dust to rodents exacerbates development of ovalbumin-induced or house dust mite-induced asthma-like airway inflammation, the detailed mechanisms remain unclear. Here, using murine models, we found that silica can induce IL-33 expression in pulmonary epithelial cells. IL-33, but not IL-25 or TSLP, and type 2 cytokines such as IL-5 and IL-13 were critically involved in silica's exacerbation of OVA-induced airway eosinophilia in mice. Innate lymphoid cells (ILCs), but not T, B or NKT cells, were also involved in the setting. Moreover, a scavenger receptor that recognized silica was important for silica's exacerbating effect. These observations suggest that IL-33 induced in epithelial cells by silica activates ILCs to produce IL-5 and/or IL-13, contributing to silica's exacerbation of OVA-induced airway eosinophilia in mice. Our findings provide new insight into the underlying mechanisms of exacerbation of pulmonary disorders such as asthma following inhalation of silica-containing materials such as volcanic ash and desert dust.

The IL-33/ST2 pathway suppresses murine colon cancer growth and metastasis by upregulating CD40 L signaling.

Interleukin (IL)-33 is a member of the IL-1 family, participating in both helper T1 (Th1)- and Th2-type immune responses, but its ambiguous effects on tumor growth and related immune mechanisms remain unclear. Here, we report that recombinant mouse IL-33 (mIL-33) significantly inhibited colon cancer growth and metastasis to lung and liver in a murine CT26 or MC38 tumor-cell engraftment model. This effect could be associated with CD4+ T cells and CD40 L signaling, as depletion of CD4+ T cells or blocking CD40 L signaling in vivo partly abolished the antitumor function of IL-33. In addition, IL-33 treatment upregulated CD40 L expression on tumor-infiltrating lymphocytes, and promoted the activation of CD4+ T, CD8+ T and natural killer cells via CD40 L signaling. Furthermore, IL-33 was sufficient to induce the ST2 expression on CD4+ T cells, but not on CD8+ T and natural killer cells, indicating that IL-33 acted on CD4+ T cells via a positive-feedback loop. Our findings shed new light on the IL-33-mediated antitumor effects and mechanisms of Th1 action, and also suggest that IL-33 may serve as an activator to boost anticancer immune responses in singular or combinatory therapies.

Hepatitis B virus X protein (HBx) promotes ST2 expression by GATA2 in liver cells.

At present, most studies on the relationship between hepatitis B virus (HBV) and IL-33/ST2 axis focus on clinical detection, but the underlying molecular mechanisms of HBx and IL-33/ST2 axis regulation and Th cell function regulation have not been explored. In this study, serum samples of patients with chronic hepatitis B (CHB) and HBV-related liver cancer (HBV-HCC), and healthy controls, as well as the supernatant solutions of HL7702-WT, HL7702-NC, and HL7702-HBx cells were collected to detect the content of soluble ST2 (sST2). The contents of Th1 cytokines (TNF-α and TNF-γ) and Th2 cytokines (IL-6 and IL-10) in the supernatant of different co-culture groups were detected. The effects of GATA2 on ST2 promoter transcription were investigated by upregulation or interference with GATA2 expression, dual-luciferase reporting, and ChIP experiments. The combined detection of sST2 and FIB-4 was beneficial to the non-invasive diagnosis of liver fibrosis. HBx promotes sST2 expression in liver cells, upregulates Th2 cell function, and inhibits Th1 cell function through IL-33/ST2 axis. HBx interacts with GATA2 to influence the activity of ST2 promoter. Serum sST2 detection is an invaluable indicator for the assessment of the progress of HBV infectious diseases, and the IL-33/ST2 axis plays an important role in changing the cellular immune function caused by HBV infection.

Enhanced Thermal Sensitivity of TRPV3 in Keratinocytes Underlies Heat-Induced Pruritogen Release and Pruritus in Atopic Dermatitis.

Itch in atopic dermatitis (AD) is aggravated under warm conditions. Transient receptor potential vanilloid (TRPV) 3, a member of the thermosensitive transient receptor potential channels, is activated by innocuous heat and is abundantly expressed in keratinocytes. The potential role of TRPV3 in itch is illustrated in TRPV3 channelopathies of humans and mice. However, the role of TRPV3 in heat-induced itch in AD and the underlying mechanisms are unclear. Here we showed that keratinocytes isolated from patients with AD exhibit enhanced expression and heat sensitivity with hyperactive channel function of TRPV3. Heat stimulus induced enhanced secretion of thymic stromal lymphopoietin, nerve growth factor, and prostaglandin E2 by keratinocytes from patients with AD through TRPV3 activation. TRPV3 agonists increased thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 production in human keratinocytes and induced scratching behavior upon intradermal injection in mice. TRPV3 was upregulated in the skin of MC903-induced AD mouse model. Heat stimulation to MC903-treated mice increased scratching behavior and produced higher levels of thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 from the epidermis, which were attenuated by pharmacologic inhibition of TRPV3. Moreover, neutralization of thymic stromal lymphopoietin reduced heat-evoked scratching in MC903-challenged mice. These results suggest that TRPV3 is a potential therapeutic target for heat-induced itch in AD.

IL-33 Is Essential for Adjuvant Effect of Hydroxypropyl-ß-Cyclodexrin on the Protective Intranasal Influenza Vaccination.

Vaccine adjuvants are traditionally used to augment and modulate the immunogenicity of vaccines, although in many cases it is unclear which specific molecules contribute to their stimulatory activity. We previously reported that both subcutaneous and intranasal administration of hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a pharmaceutical excipient widely used to improve solubility, can act as an effective adjuvant for an influenza vaccine. However, the mechanisms by which mucosal immune pathway is critical for the intranasal adjuvant activity of HP-ß-CD have not been fully delineated. Here, we show that intranasally administered HP-ß-CD elicits a temporary release of IL-33 from alveolar epithelial type 2 cells in the lung; notably, IL-33 expression in these cells is not stimulated following the use of other vaccine adjuvants. The experiments using gene deficient mice suggested that IL-33/ST2 signaling is solely responsible for the adjuvant effect of HP-ß-CD when it is administered intranasally. In contrast, the subcutaneous injection of HP-ß-CD and the intranasal administration of alum, as a damage-associated molecular patterns (DAMPs)-inducing adjuvant, or cholera toxin, as a mucosal adjuvant, enhanced humoral immunity in an IL-33-independent manner, suggesting that the IL-33/ST2 pathway is unique to the adjuvanticity of intranasally administered HP-ß-CD. Furthermore, the release of IL-33 was involved in the protective immunity against influenza virus infection which is induced by the intranasal administration of HP-ß-CD-adjuvanted influenza split vaccine. In conclusion, our results suggest that an understanding of administration route- and tissue-specific immune responses is crucial for the design of unique vaccine adjuvants.