An Unexpected Role for Uric Acid as an Inducer of T Helper 2 Cell Immunity to Inhaled Antigens and Inflammatory Mediator of Allergic Asthma

Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation. Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation. UA is an essential initiator and amplifier of allergy induced by alum or HDM UA crystal-induced Th2 cell immunity does not require inflammasome or IL-1R signaling Dendritic cells are necessary and required for Th2 cell immunity to OVA+UA Dendritic cells get activated in a pathway requiring Syk and PI3Kδ Allergic asthma is caused by an inappropriate adaptive T helper 2 (Th2) cell-mediated immune response to innocuous antigen, leading to eosinophilic airway inflammation, mucus hypersecretion, structural changes to the airway wall, and variable airway obstruction (Barnes, 2008Barnes P.J. Immunology of asthma and chronic obstructive pulmonary disease.Nat. Rev. Immunol. 2008; 8: 183-192Crossref PubMed Scopus (955) Google Scholar). The cytokines produced by allergen-specific Th2 cell-type lymphocytes are held largely responsible for orchestrating the many features of asthma. 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Allergens have a complex molecular structure and often display enzymatic activity that stimulates epithelial and innate immune cells through protease-activated receptors (Hammad and Lambrecht, 2008Hammad H. Lambrecht B.N. Dendritic cells and epithelial cells: Linking innate and adaptive immunity in asthma.Nat. Rev. Immunol. 2008; 8: 193-204Crossref PubMed Scopus (485) Google Scholar, Sokol et al., 2008Sokol C.L. Barton G.M. Farr A.G. Medzhitov R. A mechanism for the initiation of allergen-induced T helper type 2 responses.Nat. Immunol. 2008; 9: 310-318Crossref PubMed Scopus (714) Google Scholar, Tang et al., 2010Tang H. Cao W. Kasturi S.P. Ravindran R. Nakaya H.I. Kundu K. Murthy N. Kepler T.B. Malissen B. Pulendran B. The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling.Nat. 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Gioannini T.L. Weiss J.P. Karp C.L. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein.Nature. 2009; 457: 585-588Crossref PubMed Scopus (560) Google Scholar). Triggering of PAMP receptors on epithelial cells and/or basophils subsequently leads to release of innate pro-Th2 cell cytokines like thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-25, and IL-33 that instruct DCs to promote Th2 cell-type immunity in the mediastinal lymph nodes (Hammad and Lambrecht, 2008Hammad H. Lambrecht B.N. Dendritic cells and epithelial cells: Linking innate and adaptive immunity in asthma.Nat. Rev. Immunol. 2008; 8: 193-204Crossref PubMed Scopus (485) Google Scholar, Hammad et al., 2009Hammad H. Chieppa M. Perros F. Willart M.A. Germain R.N. Lambrecht B.N. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells.Nat. Med. 2009; 15: 410-416Crossref PubMed Scopus (806) Google Scholar, Hammad et al., 2010Hammad H. Plantinga M. Deswarte K. Pouliot P. Willart M.A. Kool M. Muskens F. Lambrecht B.N. Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.J. Exp. Med. 2010; 207: 2097-2111Crossref PubMed Scopus (445) Google Scholar, Rank et al., 2009Rank M.A. Kobayashi T. Kozaki H. Bartemes K.R. Squillace D.L. Kita H. IL-33-activated dendritic cells induce an atypical TH2-type response.J. Allergy Clin. Immunol. 2009; 123: 1047-1054Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, Sokol et al., 2008Sokol C.L. Barton G.M. Farr A.G. Medzhitov R. A mechanism for the initiation of allergen-induced T helper type 2 responses.Nat. Immunol. 2008; 9: 310-318Crossref PubMed Scopus (714) Google Scholar, Zhou et al., 2005Zhou B. Comeau M.R. De Smedt T. Liggitt H.D. Dahl M.E. Lewis D.B. Gyarmati D. Aye T. Campbell D.J. Ziegler S.F. Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice.Nat. Immunol. 2005; 6: 1047-1053Crossref PubMed Scopus (636) Google Scholar). Although contamination of allergens with PAMPs that stimulate pattern recognition receptors provides a good explanation for how immunity can be induced to harmless allergens, another could be that allergens trigger release of damage-associated molecular patterns (DAMPs) that occur intracellularly in homeostasis but are released extracellularly upon physical or metabolic stress (Matzinger, 2002Matzinger P. The danger model: A renewed sense of self.Science. 2002; 296: 301-305Crossref PubMed Scopus (3159) Google Scholar). A well-known DAMP is uric acid (UA) released from dying or stressed cells (Shi et al., 2003Shi Y. Evans J.E. Rock K.L. Molecular identification of a danger signal that alerts the immune system to dying cells.Nature. 2003; 425: 516-521Crossref PubMed Scopus (1325) Google Scholar, Vorbach et al., 2003Vorbach C. Harrison R. Capecchi M.R. Xanthine oxidoreductase is central to the evolution and function of the innate immune system.Trends Immunol. 2003; 24: 512-517Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Uric acid crystals potently trigger acute neutrophilic inflammation through stimulation of the NLRP3 inflammasome and release of interleukin-1β (Chen et al., 2006Chen C.J. Shi Y. Hearn A. Fitzgerald K. Golenbock D. Reed G. Akira S. Rock K.L. MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals.J. Clin. Invest. 2006; 116: 2262-2271Crossref PubMed Scopus (341) Google Scholar, Martinon et al., 2006Martinon F. Pétrilli V. Mayor A. Tardivel A. Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome.Nature. 2006; 440: 237-241Crossref PubMed Scopus (3334) Google Scholar). This pathway is at the heart of gouty inflammation, as supported by the absence of UA-induced neutrophilic inflammation in mice lacking the NLRP3, ASC (Pycard), IL-1R, or its downstream adaptor MyD88 and by the success of IL-1 receptor antagonist (IL-1RA) treatment for gout. We have reported recently that UA is released in the peritoneal cavity after injection of the Th2 cell adjuvant aluminum hydroxide or alum, commonly coinjected with harmless antigens intraperitoneally to induce allergic sensitization and asthma (Kool et al., 2008bKool M. Soullié T. van Nimwegen M. Willart M.A. Muskens F. Jung S. Hoogsteden H.C. Hammad H. Lambrecht B.N. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.J. Exp. Med. 2008; 205: 869-882Crossref PubMed Scopus (699) Google Scholar). In addition to inducing release of UA, alum can also directly trigger the NLRP3 inflammasome and stimulate secretion of bioactive IL-1β (Eisenbarth et al., 2008Eisenbarth S.C. Colegio O.R. O'Connor W. Sutterwala F.S. Flavell R.A. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants.Nature. 2008; 453: 1122-1126Crossref PubMed Scopus (1103) Google Scholar, Kool et al., 2008aKool M. Pétrilli V. De Smedt T. Rolaz A. Hammad H. van Nimwegen M. Bergen I.M. Castillo R. Lambrecht B.N. Tschopp J. Cutting edge: Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome.J. Immunol. 2008; 181: 3755-3759PubMed Google Scholar, Li et al., 2008Li H. Willingham S.B. Ting J.P. Re F. Cutting edge: Inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3.J. Immunol. 2008; 181: 17-21Crossref PubMed Scopus (31) Google Scholar, McKee et al., 2009McKee A.S. Munks M.W. MacLeod M.K. Fleenor C.J. Van Rooijen N. Kappler J.W. Marrack P. Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity.J. Immunol. 2009; 183: 4403-4414Crossref PubMed Scopus (275) Google Scholar). These studies have aroused interest in the UA-inflammasome-IL-1 axis as a trigger of Th2 cell development. Here, we hypothesized that uric acid is a general inducer and amplifier of Th2 cell immunity. By using models of intraperitoneal injection, as well as airway exposure of HDM allergen in mice and patients, we found that UA is present at sites of Th2 cell development, where it is necessary and sufficient for induction of Th2 cell immunity. Contrary to expectation, this Th2 cell adjuvant effect did not require triggering of the NLRP3-ASC complex, nor signaling through the IL-1R. Rather, UA induced Th2 cell immunity by triggering DC activation in a spleen tyrosine kinase (Syk)- and PI3-kinase δ-dependent manner. These findings identify UA as an unexpected initiator and amplifier of Th2 cell immunity and allergic inflammation. We have reported that UA is released in the peritoneal cavity after injection of alum (Kool et al., 2008bKool M. Soullié T. van Nimwegen M. Willart M.A. Muskens F. Jung S. Hoogsteden H.C. Hammad H. Lambrecht B.N. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.J. Exp. Med. 2008; 205: 869-882Crossref PubMed Scopus (699) Google Scholar) and is a necessary intermediate for early DC activation in vivo, whereas others argued that the immunostimulatory effects of alum did not require UA (Franchi and Núñez, 2008Franchi L. Núñez G. The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity.Eur. J. Immunol. 2008; 38: 2085-2089Crossref PubMed Scopus (331) Google Scholar). As shown in Figure 1A , the degradation of UA by uricase treatment reduced secretion of bioactive IL-1β into the peritoneal cavity, whereas production of IL-1β by DCs in vitro did not require endogenous release of UA. Therefore, the important role of UA in mediating the adjuvant activity of alum is mainly revealed in vivo. The increased UA production after injection of OVA in alum was found to be intact in Tlr4−/−, Myd88−/−, and Casp1−/− mice, excluding an important role for the TLR4 or inflammasome-IL1β axis in its induction (Figure S1 available online). Because alum is frequently used to induce allergy in animal models of asthma, we hypothesized that UA is necessary for promoting adaptive Th2 cell immunity to OVA+alum. Injection of OVA+alum into BALB/c mice led to an increased concentration of UA in the peritoneal cavity, which could be neutralized by treatment with uricase (Figure 1B). Uricase treatment during priming to OVA+alum eliminated Th2 cell-dependent bronchoalveolar lavage (BAL) fluid eosinophilia (Figure 1C), peribronchial inflammation and goblet cell hyperplasia (Figure 1D), secretion of IL-5 in the BAL fluid (Figure 1E), production of IL-4, IL-5, IL-10, and IL-13 (but not IFN-γ) in mediastinal LN (MLN) cultures restimulated with OVA antigen for 3 days ex vivo (Figure 1F), and production of serum OVA-specific IgE and IgG1 (Figure 1G). There was also a trend toward reduced production of IgG2a by uricase treatment. OVA+alum injection also led to release of extracellular ATP, another DAMP known to activate the NLRP3 inflammasome via the P2X7 receptor (Mariathasan et al., 2006Mariathasan S. Weiss D.S. Newton K. McBride J. O'Rourke K. Roose-Girma M. Lee W.P. Weinrauch Y. Monack D.M. Dixit V.M. Cryopyrin activates the inflammasome in response to toxins and ATP.Nature. 2006; 440: 228-232Crossref PubMed Scopus (2146) Google Scholar) and promote Th2 cell immunity (Idzko et al., 2007Idzko M. Hammad H. van Nimwegen M. Kool M. Willart M.A. Muskens F. Hoogsteden H.C. Luttmann W. Ferrari D. Di Virgilio F. et al.Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells.Nat. Med. 2007; 13: 913-919Crossref PubMed Scopus (445) Google Scholar), yet its neutralization or use of P2rx7−/− mice did not reveal a contribution to Th2 cell immunity (Figure S2). To address whether UA was also sufficient to induce Th2 cell immunity, we injected OVA admixed with UA crystals. Compared with control mice receiving OVA, this treatment dose dependently increased airway eosinophilia (Figures 1H and 1I), goblet cell hyperplasia (Figure 1I), IL-5 in BAL fluid (Figure 1J), and production of OVA-specific IgE, IgG1, and IgG2a (Figure 1K). Injection of OVA+UA also led to increased production of IL-4, IL-5, and IL-10, but not IFN-γ, in MLN cells (data not shown). These responses were of similar strength as those induced by injection of OVA+alum (Figures 1C–1E). Therefore, UA is necessary and sufficient for Th2 cell response development to intraperitoneal injection of harmless antigen, identifying it as a potent Th2 cell adjuvant. We next investigated the involvement of endogenous UA in response to complex and relevant allergens in the airways. A single intranasal HDM administration in naive mice resulted in a rapid increase in UA concentration in BAL fluid and lung (Figure 2A ) compared with PBS. Mice sensitized and challenged with HDM developed features of allergic asthma, like BAL fluid and lung eosinophilia, lymphocytosis, goblet cell hyperplasia (Figures 2B and 2D), and airway hyperresponsiveness (AHR) to increasing doses of metacholine (Figure 2C). Intratracheal administration of uricase on day 0 at the time of sensitization neutralized the increase in UA (Figure 2A) and strongly reduced airway lymphocytosis and eosinophilia and AHR (Figures 2A–2D). To explain the mechanism of action, we evaluated the production of cytokines known to promote eosinophilic inflammation and Th2 cell immunity (Hammad and Lambrecht, 2008Hammad H. Lambrecht B.N. Dendritic cells and epithelial cells: Linking innate and adaptive immunity in asthma.Nat. Rev. Immunol. 2008; 8: 193-204Crossref PubMed Scopus (485) Google Scholar, Saenz et al., 2008Saenz S.A. Taylor B.C. Artis D. Welcome to the neighborhood: epithelial cell-derived cytokines license innate and adaptive immune responses at mucosal sites.Immunol. Rev. 2008; 226: 172-190Crossref PubMed Scopus (375) Google Scholar). As already reported, the concentration of GM-CSF, TSLP, and IL-25 (Figure 2E) were increased in the lungs of animals exposed to a single HDM injection, compared with PBS (Hammad et al., 2009Hammad H. Chieppa M. Perros F. Willart M.A. Germain R.N. Lambrecht B.N. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells.Nat. Med. 2009; 15: 410-416Crossref PubMed Scopus (806) Google Scholar). However, this innate cytokine response was severely reduced in mice treated with uricase. House dust mite-driven Th2 cell development relies on antigen presentation by inflammatory Ly6ChiCD11b+ DCs that take up antigen in the lung and migrate to the MLN (Hammad et al., 2010Hammad H. Plantinga M. Deswarte K. Pouliot P. Willart M.A. Kool M. Muskens F. Lambrecht B.N. Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.J. Exp. Med. 2010; 207: 2097-2111Crossref PubMed Scopus (445) Google Scholar). To address antigen uptake and migration of these inflammatory DCs, we injected fluorescent OVA together with HDM allergen. In mice injected with OVA+HDM, migratory DCs carrying fluorescent cargo were easily identified in the MLN. After uricase treatment, this migration was strongly reduced (Figure 2F). We next searched for the source of increased UA production. Within 2 hr after HDM administration, there was increased immunoreactivity for UA in airway epithelial cells and major histocompatibility complex II (MHCII)-positive alveolar macrophages and recruited monocytes (Figures 2G) compared to mice administered PBS. Strong UA immunoreactivity was seen along the basement membrane of HDM-exposed mice. Although UA release occurs in response to necrotic cell death, UA is also produced as a pulmonary defense mechanism to oxidative damage, through increased xanthine oxidoreductase (XOR) enzyme activity inside peroxisomes (Vorbach et al., 2003Vorbach C. Harrison R. Capecchi M.R. Xanthine oxidoreductase is central to the evolution and function of the innate immune system.Trends Immunol. 2003; 24: 512-517Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In bronchial epithelial cells and MHCII-positive cells, HDM exposure induced enhanced staining for the 70 kDa peroxisomal marker PMP70, suggestive of peroxisomal biogenesis (Figure 2H; Imanaka et al., 1999Imanaka T. Aihara K. Takano T. Yamashita A. Sato R. Suzuki Y. Yokota S. Osumi T. Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter.J. Biol. Chem. 1999; 274: 11968-11976Crossref PubMed Scopus (75) Google Scholar). The expression of XOR was increased in BAL cells of HDM-exposed mice 2 hr after instillation compared with PBS (Figure 2I). We (Hammad et al., 2009Hammad H. Chieppa M. Perros F. Willart M.A. Germain R.N. Lambrecht B.N. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells.Nat. Med. 2009; 15: 410-416Crossref PubMed Scopus (806) Google Scholar) and others (Phipps et al., 2009Phipps S. Lam C.E. Kaiko G.E. Foo S.Y. Collison A. Mattes J. Barry J. Davidson S. Oreo K. Smith L. et al.Toll/IL-1 signaling is critical for house dust mite-specific helper T cell type 2 and 17 [corrected] responses.Am. J. Respir. Crit. Care Med. 2009; 179: 883-893Crossref PubMed Scopus (133) Google Scholar, Trompette et al., 2009Trompette A. Divanovic S. Visintin A. Blanchard C. Hegde R.S. Madan R. Thorne P.S. Wills-Karp M. Gioannini T.L. Weiss J.P. Karp C.L. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein.Nature. 2009; 457: 585-588Crossref PubMed Scopus (560) Google Scholar) recently identified TLR4 signaling as a crucial early trigger for the innate pro-Th2 cell immune response to allergens. In contrast to the alum-induced increase of UA in the peritoneal lavage (Figure S1), HDM-driven production of UA was severely reduced in C57BL/6 Tlr4−/− mice (Figure 2J), suggesting that triggering of TLR4 by HDM is necessary for activation of XOR and UA production in the airways in response to allergens. To study whether our findings had any bearing on human asthma, BAL fluid was collected from 21 asthmatic subjects, 10 min and 24 hr after segmental challenge with saline or specific allergens (rye, birch, or HDM). There was no difference in the concentration of UA between saline- and allergen-challenged segments after 10 min. In contrast, 24 hr after allergen provocation, the UA concentration was markedly elevated in the BAL fluid from allergen- compared to saline-challenged segments (Figure 3A ). This increase in UA concentration correlated with the influx of eosinophils and lymphocytes into the BAL (Figures 3B and 3C). Subgroup analysis of patients showing a minor rise in UA showed that this was not correlated with the type of allergen used nor the concentration of IgE in the serum, but rather on maintenance inhaled corticosteroid (ICS) therapy up to 7 days prior to allergen challenge (Figure S3). To test whether UA neutralization would have any therapeutic potential, we again turned to the mouse model of HDM-induced asthma. Mice were first sensitized to HDM and were treated with uricase prior to each allergen challenge. HDM challenge induced a rise in UA in BAL fluid, still measurable 3 days after the last of five challenges (Figure 3D), and uricase treatment during challenge reduced airway eosinophilia (Figure 3E), metacholine-induced AHR, and IL-13 concentration in the BAL fluid (Figures 3F and 3G). Allergen challenge induced high numbers of eosinophils in the BAL fluid, which indicates that cellular death of recruited eosinophils could be a trigger for UA production. Uricase treatment had similar effects in a milder sensitization model in which only 20% of the number of eosinophils was recruited compared with the high dose sensitization model (Figure S4), arguing against the idea that UA plays a role only when there would be massive cellular recruitment and death. Although UA is the prototypical activator of the NLRP3 inflammasome and several groups (Eisenbarth et al., 2008Eisenbarth S.C. Colegio O.R. O'Connor W. Sutterwala F.S. Flavell R.A. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants.Nature. 2008; 453: 1122-1126Crossref PubMed Scopus (1103) Google Scholar, Franchi and Núñez, 2008Franchi L. Núñez G. The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity.Eur. J. Immunol. 2008; 38: 2085-2089Crossref PubMed Scopus (331) Google Scholar, Hornung et al., 2008Hornung V. Bauernfeind F. Halle A. Samstad E.O. Kono H. Rock K.L. Fitzgerald K.A. Latz E. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization.Nat. Immunol. 2008; 9: 847-856Crossref PubMed Scopus (1914) Google Scholar, Kool et al., 2008aKool M. Pétrilli V. De Smedt T. Rolaz A. Hammad H. van Nimwegen M. Bergen I.M. Castillo R. Lambrecht B.N. Tschopp J. Cutting edge: Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome.J. Immunol. 2008; 181: 3755-3759PubMed Google Scholar, McKee et al., 2009McKee A.S. Munks M.W. MacLeod M.K. Fleenor C.J. Van Rooijen N. Kappler J.W. Marrack P. Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity.J. Immunol. 2009; 183: 4403-4414Crossref PubMed Scopus (275) Google Scholar) have shown that alum directly triggers NLRP3 in vitro, there is considerable controversy whether the inflammasome-IL-1β pathway is necessary for adjuvanticity in vivo (Franchi and Núñez, 2008Franchi L. Núñez G. The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity.Eur. J. Immunol. 2008; 38: 2085-2089Crossref PubMed Scopus (331) Google Scholar, Williams et al., 2010Williams A. Flavell R.A. Eisenbarth S.C. The role of NOD-like receptors in shaping adaptive immunity.Curr. Opin. Immunol. 2010; 22: 34-40Crossref PubMed Scopus (63) Google Scholar). We therefore studied the involvement of this pathway in UA-driven Th2 cell immunity in vivo. Wild-type C57BL/6 mice immunized with OVA+UA showed a marked increase in eosinophilia and lymphocytosis in BAL (Figures 4A–4E ), and this response was not affected in Nlrp3−/− or Pycard−/− mice, lacking essential components of the inflammasome complex (Martinon et al., 2006Martinon F. Pétrilli V. Mayor A. Tardivel A. Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome.Nature. 2006; 440: 237-241Crossref PubMed Scopus (3334) Google Scholar). Because there might be inflammasome-independent pathways to generate IL-1β, we also immunized mice lacking the IL-1R or mice lacking the critical IL-1R signaling intermediate MyD88. It has also been suggested that UA crystals signal via TLR4, although this could be due to contaminating endotoxin. Strikingly, Il1r−/−, Tlr4−/−, and Myd88−/− mice all developed normal Th2 cell immunity to OVA+UA (Figures 4C–4E). We also studied whether development of Th2 cell immunity and asthma in response to OVA+alum relied on Nlrp3. Contrary to a recent report (Eisenbarth et al., 2008Eisenbarth S.C. Colegio O.R. O'Connor W. Sutterwala F.S. Flavell R.A. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants.Nature. 2008; 453: 1122-1126Crossref PubMed Scopus (1103) Google Scholar), we observed that Th2 cell-dependent asthmatic airway inflammation developed normally in Nlrp3−/− mice immunized with OVA+alum (Figure S5). To address whether HDM-driven asthma was dependent on these pathways, Nlrp3−/−, Pycard−/−, and P2rx7−/− mice were sensitized and challenged with HDM via the airways but showed no reduction in Th2 cell-dependent eosinophilia (Figure S6). As already reported, Myd88−/− and Tlr4−/− mice had reduced HDM-driven airway inflammation (data not shown; Hammad et al., 2009Hammad H. Chieppa M. Perros F. Willart M.A. Germain R.N. Lambrecht B.N. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells.Nat. Med. 2009; 15: 410-416Crossref PubMed Scopus (806) Google Scholar, Hammad et al., 2010Hammad H. Plantinga M. Deswarte K. Pouliot P. Willart M.A. Kool M. Muskens F. Lambrecht B.N. Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.J. Exp. Med. 2010; 207: 2097-2111Crossref PubMed Scopus (445) Google Scholar). In conclusion, the prototypical Th2 cell stimuli alum and HDM, like UA crystals, do not rely on the NLRP3 inflammasome to induce Th2 cell-adaptive immunity leading to allergic airway inflammation. Twenty-four hours after injection of OVA-Alexa Fluor 647+UA, there was a strong increase in the number of inflammatory Ly6ChiCD11b+ monocytes and inflammatory Ly6C+CD11b+ DCs that had ingested OVA in the peritoneal cavity compared with mice injected with OVA-AF647 alone (Figure 5A ), and these recruited cells had higher expression of the costimulatory molecule CD86 (Figure 5B). Although recruitment and activation of DCs suggests a functional role for these cells in the UA effect, we still wanted to address whether DCs are really necessary for mediating Th2 cell immunity, because recent work sugges