American Journal of Respiratory and Critical Care Medicine

Rationale: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1β production, inflammation, and fibrosis remain poorly understood.

Objectives: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung.

Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid.

Measurements and Main Results: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1β production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor.

Conclusions: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1β production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1β production and chronic inflammatory lung pathology.

Scientific Knowledge on the Subject

Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1β production and chronic inflammatory lung pathology.

What This Study Adds to the Field

Uric acid released from injured cells exposed to bleomycin constitutes a major endogenous danger signal that activates the NALP3 inflammasome leading to IL-1β product leading to lung inflammation and fibrosis.

Recurrent episodes of acute lung injury of unknown origin lead to interstitial pulmonary fibrosis with respiratory failure, and no effective therapy is available (1). Bleomycin (BLM) causes oxidative damage and cell death of alveolar macrophages and epithelial cells, leading to experimental lung fibrosis (2). In this experimental model, we demonstrated that inflammation, repair, and fibrosis are dependent upon IL-1β production and IL-1R1/myeloid differentiation primary response gene 88 (MyD88) signaling and that the ASC adaptor molecule of the inflammasome complex is required for BLM-induced inflammation (3). However, the upstream mechanisms leading to caspase-1 activation and IL-1β secretion upon lung injury are largely unknown. Abnormal self or cell damage provide signals that alert the immune system to danger, triggering innate immunity that results in inflammation and repair (4). The mechanisms of innate immunity activation in response to tissue injury are just emerging (5). Dying cells release danger signals that alert the immune system and stimulate innate and adaptive immunity (6). The danger signals are recognized via membrane receptors, such as TLRs (79), or cytosolic receptors, such as NLRs. In particular, NALP3 (also called cryopyrin or NLRP3) is a major proinflammatory danger receptor (1014) activated by uric acid in gout arthritis (10, 15). Uric acid, a product of purine catabolism, was identified in dying cells and enhanced dendritic cell maturation and the CD8–T-cell response (16). Large amounts of uric acid are produced from injured tissue in vivo after tumor chemotherapy, leading to tumor lysis syndrome characterized by hyperuricemia (17). At high local concentration, uric acid precipitates and forms crystals that cause inflammation, as observed in clinical gout, and activate the caspase-1–containing NALP3 inflammasome, resulting in the production of active IL-1β (10). Based on the fact that cell/tissue injury and necrosis result in the production of uric acid, we hypothesized that uric acid crystals formed at the injury site might represent a key danger signal activating the inflammasome to release IL-1β, thereby causing inflammatory lung pathologies.

Some of the results of these studies have been previously reported in the form of an abstract (18).


The following mice deficient for MyD88 (19), IL-1R1 (20), IL-18R (21), Casp-1 (22), TLR4 (23), TLR2 (24), NALP3 (10), or ASC (25) were used in this study. MyD88−/−, Casp-1−/−, TLR2−/−, ASC−/− TLR4−/−, double-deficient TLR2−/−TLR4−/−, and IL-18R−/− mice were backcrossed 10 times on the wild-type C57BL/6 genetic background, except in Figure 1, where ASC−/−, backcrossed four times, were compared with their ASC+/+ littermates. IL-1R1−/− mice were backcrossed seven times, and NALP3−/− mice were directly generated on the C57BL/6 genetic background. All mice, including control C57BL/6, were bred in our animal facility at the Transgenose Institute (CNRS, Orleans, France). For experiments, adult (6–10 wk old) animals were kept in sterile, isolated, ventilated cages. All animal experiments complied with the French Government's ethical and animal experiment regulations.

Uric Acid or Allopurinol Crystals Preparation

Uric acid or allopurinol crystals were obtained by dissolving 1.68 mg of powder in 0.01 M NaOH preheated to 70°C and added as required to maintain pH between 7.1 and 7.2. The solution was filtered and incubated at room temperature under slowly and continuously agitation until crystals formed. Crystals were washed twice with ethanol 100%, dried, autoclaved, and kept sterile. The weight of dry crystals was determined under sterile conditions. The crystals were resuspended in PBS by sonication and examined under phase microscopy.

Lung Inflammation Model Experimental Design

BLM sulfate (10 mg/kg) from Bellon Laboratories (Montrouge, France), uric acid, or allopurinol crystals (5–50 mg/kg) in saline or vehicle alone were administered by intranasal instillation under light ketamine-xylasine anesthesia, and bronchoalveolar lavage (BAL) and lung tissue were assayed after 6 hours (for uric acid crystal) or 24 hours (for BLM) for markers of inflammation, including cell recruitment and in particular neutrophil influx, chemokine, and cytokine levels, including KC, IL-6, and IL-1β and 14 days later for markers of tissue remodeling, such as gelatinases MMP9 and MMP2 and their inhibitor TIMP-1. Allopurinol (Sigma-Aldrich, St. Quentin Fallavier, France) was injected at 25 mg/kg subcutaneously, and uricase (Fasturtec; Sanofi Synthelabo, Paris, France) was given at 0.2 mg/kg intraperitonally or intranasally with similar efficacy. IL-1Ra (Anakinra; Amgen Thermo Shandon, Cergy Pontoise, France) was injected at 10 mg/kg subcutaneously. Optimized doses of allopurinol or uricase were tested, and repeated administrations were more effective than higher doses (data not shown).

Bronchoalveolar Lavage Fluid

After incision of the trachea, a plastic cannula was inserted, and airspaces were washed using 0.3 ml of PBS solution heated to 37°C. The rib cage was gently massaged to enable maximum cell collection. The fluid was collected by careful aspiration. This procedure was performed 10 times, and the recovery of the total lavage exceeded 95%. The samples collected were dispatched in two fractions: the first sample (1 ml corresponding to the two first lavages) was used for mediator measurement, and the second sample was used for cell determination (4 ml). The first fraction was centrifuged (600 × g for 10 min), and the supernatant was fractionated and kept at −80°C until mediator determination. The cell pellet was resuspended in 0.4 ml PBS, pooled with the second fraction, and maintained at 4°C until cell determination.

Lung Homogenization

After BAL was performed, the whole lung was removed and placed inside a microtube (Lysing matrix D; Q Bio Gene, Illkrich, France) with 1 ml of PBS. Total lung tissue extract was prepared using a Fastprep system (FP120; Q Bio Gene). The extract was centrifuged, and the supernatant was stored at −80°C before mediator measurement, myeloperoxidase (MPO), or collagen assay with Sircol Collagen Assay (France Biochem Division, Meudon, France).

MPO Activity in Lung

Lung tissue MPO activity was evaluated as described (3). In brief, the right heart ventricle was perfused with saline to flush the vascular content, and lungs were frozen at −20°C until use. Lung tissue was homogenized by polytron and centrifuged, and the supernatant was discarded. The pellets were resuspended in 1 ml PBS containing 0.5% hexadecyltrimethyl ammonium bromide (HTAB) and 5 mM ethylene-diamine tetra-acetic acid (EDTA). After centrifugation, 50 μL of supernatants were placed in test tubes with 200 μL PBS-HTAB-EDTA, 2 ml Hanks' balanced salt solution, 100 μL of o-dianisidine dihydrochloride (1.25 mg/mL), and 100 μL H2O2 0.05%. After 15 minutes of incubation at 37°C in an agitator, the reaction was stopped with 100 μL NaN3 1%. The MPO activity was determined as absorbance at 460 nm against medium.

Cell Count and Determination

Total cell count was determined in BAL fluid (BALF) using a particle counter (Z2 Coulter; Beckman Coulter, Paris, France). Differential cell counts were performed on cytospin preparations (Cytospin 3; Thermo Shandon) after staining for 4 minutes with May-Grünwald stain (MG-1L; Sigma Chemical, St. Louis, MO) and 8 minutes in 95% Giemsa stain (GS-500; Sigma Chemical). Differential cell counts were made on 100 cells using standard morphologic criteria.

Mediator Measurements

IL-1β, KC, IL-6, and TIMP-1 levels in BALF or lung homogenate were determined using ELISA assay kits according to the manufacturer's instructions (Mouse DuoSet; R&D Systems, Minneapolis, MN). An IL-1β ELISA assay kit (mouse IL-1β/IL-1F2) specific for natural and recombinant mouse IL-1β exhibited no cross-reactivity or interference with recombinant mouse IL-1α, IL-1ra, IL-1RI/Fc chimera, or IL-1RII/Fc chimera (mouse IL-1–specific polyclonal goat IgG and monoclonal rat IgG1, clone #30,311).

Uric Acid Measurement

Uric acid concentration was determined in BAL samples and lung homogenates using an Amplex Red Uric Acid/Uricase Assay Kit (Molecular Probes, Eugene, OR). Briefly, uricase catalyzes the conversion of uric acid to allantoin, hydrogen peroxide (H2O2), and carbon dioxide. In the presence of horseradish peroxidase, H2O2 reacts stochiometrically with Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin, which is measured spectrophotometrically.

Zymographic Analysis of MMPs

MMP-2 and MMP-9 levels were determined by gelatin zymography. Briefly, nonreduced supernatant samples of BALF (15 μl) and standards (161–0305; Bio-Rad, Hercules, CA) were loaded onto 7% polyacrylamide gels (wt/vol) incorporating 0.1% (wt/vol) gelatin substrate. The MMP in the gelatinolytic bands were evaluated using recombinant murine Pro–MMP-9 (100 kD) and recombinant murine Pro–MMP-2 (72 kD) as references. Proteins were subjected to electrophoresis at 20 to 30 mA for 3 hours. The gel was washed twice in 2.5% Triton (vol/vol), rinsed three times quickly with distillated water, and placed three times for 20 minutes in distillated water. Each wash was performed under gentle stirring. Gels were incubated overnight at 37°C in 50 mM Tris buffer containing 5 mM CaCl2 and 2 μM ZnCL2. Finally, gels were stained in Coomassie Blue and then destained progressively until bands of lysis (enzyme activity) in the gels showed up as regions of negative staining. The areas of lysis in the gels were analyzed using a densitometric analyzer (Bioprofil; Vilbert Lournet, Marne la vallée, France), images were taken, and band densities were measured. After treatment with BLM, we observed an increase in the activity of Pro–MMP-9 (100 kD), Pro-MMP-2 (72 kD), and active MMP-2 (65 kD). Only these MMP were quantified.

Total Lung Collagen Measurements

Aliquots of lung homogenate (50 μl) were assayed for lung collagen levels and compared with a standard curve prepared from bovine skin using the Sircol collagen dye binding assay according to the manufacturer's instructions (Biocolor Ltd, Carrickfergus, UK).

α-I Collagen mRNA Measurement

Quantification of α-I collagen was described previously (26). Briefly, frozen lung samples were ground to a fine powder and homogenized in 2 ml of Trizol reagent (Invitrogen Life Technology, Paisley, UK). After vigorous shaking, chloroform was added, and the samples were centrifuged at 12,000 × g for 20 minutes. Total RNA was precipitated with isopropanol and dissolved in RNAse-free water. RNAs were reverse-transcribed into cDNA using SuperScriptTMII (Invitrogen Life Technology). Real-time quantitative PCR was performed by fluorescent dye SYBR Green methodology using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7000 apparatus (Perkin-Elmer, Foster City, CA). The relative quantification of the steady-state of the target mRNA levels was calculated by an active reference, GAPDH.


After BAL and lung perfusion, the large lobe was fixed in 4% buffered formaldhehyde for standard microscopic analysis. Sections (3-μm) were stained with hematoxylin and eosin or Chromotrope Aniline Blue as described previously (3). The severity of the morphologic changes (infiltration by neutrophils and mononuclear cells, destruction and thickening of the alveolar septae, and fibrosis) were assessed semiquantitatively using a numeric fibrotic scale (Ashcroft score) (27). The mean score was considered the fibrotic score (0–8) by two independent observers (I.C., B.R.).

Statistical Analysis

Statistical evaluation of differences between the experimental groups was determined by Mann Whitney test using Prism software. P values < 0.05 were considered statistically significant.

BLM Activates the Inflammasome NALP3 Leading to IL-1β Production and Inflammation in Lung

Intranasal administration of a single dose of BLM induces a rapid inflammation of the airways within 24 hours, followed by tissue remodeling and lung fibrosis within 14 days. Because we showed that BLM-induced lung injury causes an inflammation dependent of IL-1R1 and IL-1β (3), we investigated the upstream mechanisms leading to IL-1β release and in particular the role of the inflammasome, a cytosolic multiprotein complex composed of receptors, adaptors, and cysteine proteases, which cleaves pro–IL-1β into IL-1β (28). Twenty-four hours after BLM administration, neutrophil recruitment in BALF and lung (Figures 1A and 1B), production of the neutrophil attracting proinflammatory chemokine KC, and the inflammatory mediator IL-6 (Figures 1C and 1D), and, of the profibrotic mediators, IL-1β (Pro–IL-1β plus mature IL-1β) and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) (Figures 1E and 1F) were significantly reduced in NALP3- and ASC-deficient mice. BLM-induced inflammation was reduced in caspase-1–deficient mice (Figure 2A) or in wild-type mice treated with the inhibitor of caspase-1 z-YVAD-fmk with reduced neutrophil recruitment in BALF (Figure 2B), production of IL-1β (Figure 2C), KC (Figure 2D), and TIMP-1 (Figure 2E) in the lung. The difference in IL-1β measured in lung of wild-type versus Casp-1–deficient mice or wild-type mice treated with the inhibitor of caspase-1 z-YVAD-fmk after BLM likely represents the maturation of IL-1β by the caspase-1 protease. Therefore, the data indicate that the NALP3 inflammasome is activated upon BLM lung injury, resulting in enhanced production of IL-1β and subsequent inflammation.

NALP3 Inflammasome Is Critical for BLM-Mediated Late Inflammation and Tissue Remodeling

To evaluate the involvement of NALP3 inflammasome in late inflammation and tissue remodeling in lung inflammation induced by BLM, we analyzed cell recruitment and gelatinase activities 14 days after a single intranasal BLM administration by the measurement of matrix metalloproteinase 9 (MMP-9, or gelatinase A) and matrix metalloproteinase 2 (MMP-2, or gelatinase B) by zymography of the BALF. BLM promoted lymphocyte recruitment into the BALF of wild-type mice, which was markedly decreased in NALP3 and Casp-1–deficient mice (Figure 3A). MMP-9 was shown to be largely produced by neutrophils, and its activity was associated with neutrophil recruitment, whereas MMP-2 was produced by fibroblasts and associated with fibrosis (29). Fourteen days after BLM administration, Pro–MMP-9 (100 kD) and Pro–MMP-2 (71 kD) activities were measured after activation, and active MMP-2 (65 kD) activity were up-regulated in the BALF of wild-type mice but were significantly reduced in NALP3 and Casp-1–deficient mice (Figure 3B). Because the balance of TIMPs and MMPs is an important factor in the fibrotic process, we analyzed the late production of TIMP-1, a hallmark for the evolution to fibrosis (26), and showed that TIMP-1 was up-regulated at 14 days in lung homogenates of wild-type mice but decreased in NALP3 and Casp-1–deficient mice (Figure 3C). Enhancement of total pulmonary collagen content upon BLM administration was attenuated in NALP3 and Casp-1–deficient mice (Figure 3D). Therefore, late inflammation and repair processes depend on the NALP3 inflammasome.

BLM-induced Inflammation Is Mediated by Uric Acid

We then asked about the danger signals triggering NALP3 inflammasome activation leading to IL-1β maturation and lung inflammation upon BLM lung injury. NALP3 inflammasome activation is triggered by many microbial or danger stimuli (3036). In particular, NALP3 is a major proinflammatory danger receptor activated by uric acid in the gout arthritis model (10). Because uric acid was identified as a principal endogenous danger signal released from injured cells, we hypothesized that uric acid can be important in the induction of immunity after lung injury (16). We first assessed whether uric acid is released upon BLM-induced lung injury in mice. Uric acid production was enhanced in the BALF (Figure 4A) and in the lung (Figure 4B) 24 hours after intranasal BLM administration. We next assayed whether modulation of lung uric acid levels in mice airway can influence BLM-induced inflammation and remodeling. We demonstrated that subcutaneous administration of the xanthine oxidase inhibitor allopurinol, which impairs uric acid synthesis, prevented uric acid increases in the lung upon BLM administration (Figure 4B). Allopurinol greatly inhibited BLM-induced acute inflammation, resulting in reduced total cell (Figure 4C) and neutrophil recruitment in the BALF (Figure 4D) and reduced production of KC in the lung (Figure 4E). The profibrotic cytokine IL-1β (Figure 4F) and TIMP-1, which are involved in repair processes and are characteristic of the evolution to fibrosis, were diminished in the lung (Figure 4G) and in the BALF (data not shown) upon allopurinol treatment. We administered mice with uricase used to treat hyperuricemia in tumor lysis syndrome associated with cancer chemotherapy (17). Intraperitoneal or intranasal administration of uricase, which rapidly degrades uric acid into soluble allantoin, also reduced BLM-induced lung uric acid increase (Figure 5A), neutrophil influx (Figure 5B), and pulmonary IL-1β production (Figure 5C). Therefore, BLM-induced lung inflammation and remodeling are largely mediated by uric acid, which represents a major danger signal likely released from dying pulmonary cells upon injury and a new target to control inflammation upon lung injury.

BLM-induced Repair and Fibrosis Are Mediated by Uric Acid

We extended our investigation to test whether the inhibition of uric acid synthesis reduces BLM-induced repair and fibrosis. Subcutaneous administration of allopurinol reduced BLM-induced late lymphocyte and neutrophil recruitment in the BALF at Day 8 (Figure 6A). Gelatinase activities were assessed by the measurement of MMP-9 (gelatinase A) and MMP-2 (gelatinase B) by zymography. Pro–MMP-9 and active MMP-2 activities, which were up-regulated in BALF 14 days after BLM administration, were significantly reduced by allopurinol or uricase treatment (Figure 6B). We then analyzed the late production of TIMP-1 and showed that TIMP-1 was up-regulated at 8 days in the lung homogenates (Figure 6C) and BALF (data not shown) of BLM-treated mice but inhibited by allopurinol administration. Pulmonary α-I collagen mRNA content was increased 14 days after BLM administration but was inhibited by uricase or allopurinol treatment (Figure 6D). Lung sections showed that BLM-induced alveolar wall destruction, collagen deposition, and lung fibrosis at 14 days were significantly reduced when uric acid synthesis was inhibited or after uricase administration (Figure 6E). The fibrosis induced by BLM was assessed semiquantitatively. Fibrosis with thickening of alveolar septae and inflammation were significantly reduced in mice treated with uricase or allopurinol in comparison to B6 mice (Figure 6F). Therefore, not only inflammation, but also repair processes and fibrosis depend on the danger signal uric acid, bridging early events to late pathology development.

Exogenous Uric Acid Causes Acute Lung Inflammation and Remodeling

Uric acid released from the lung upon BLM injury (see Figure 4A) might trigger NALP3 inflammasome activation. To validate this point, we asked whether exogenous uric acid administration in the airways causes similar lung inflammation. Upon intranasal administration, uric acid crystals were found engulfed by alveolar macrophages (Figure 7A). They induced dose-dependent cell recruitment in the BALF with macrophages and neutrophils and few lymphocytes (Figure 7B), whereas the chemically and structurally similar allopurinol crystals caused only little neutrophil recruitment into the BALF (Figure 7C). The inflammation was transient, reaching a maximum at 6 hours, decreasing at Day 1, and being resolved at Day 14 (Figure 7D). Uric acid crystals dose-dependently induced pulmonary TIMP-1, a marker of incipient fibrosis (Figure 7E), which returned to basal levels at Day 14 (data not shown), as reported after exogenous IL-1β, whereas BLM administration induced a long-lasting production of TIMP-1 (3). Rapid degradation of uric acid occurs in mice due to their functional uricase, in contrast to humans (37), and repeated uric acid crystal administration may be required to develop lung fibrosis. Thus, local administration of uric acid crystals triggers inflammation and repair in the lung, similar to BLM.

Uric Acid–induced Acute Lung Inflammation Is Dependent upon Inflammasome and MyD88/IL-1R1

We investigated whether the NALP3 inflammasome was involved in the lung inflammation triggered by uric acid crystals. Acute lung neutrophil recruitment (Figures 8A and 8C) and IL-1β production (Figures 8B and 8D) induced 6 hours after exogenous uric acid crystals administration were significantly reduced in mice deficient for the inflammasome NALP3 receptor or ASC adaptor in comparison to wild-type mice. Moreover, the inflammatory response to uric acid crystals was drastically reduced in MyD88 and IL-1R1–deficient mice (Figure 8E) or after IL-1 neutralization by IL-1Ra administration (Figure 8F), as evidenced by reduced neutrophil influx in BALF. The inflammatory mediators IL-6 (Figure 8G) and KC (Figure 8H) and the fibrotic mediator TIMP-1 (Figure 8I) induced by uric acid crystals were reduced in the lung of MyD88 and IL-1R1–deficient mice. IL-1β was significantly decreased in lungs from MyD88-deficient mice but not from IL-1R1–deficient mice (Figure 8J), suggesting that other receptors using the common MyD88 adaptor, such as TLR or IL-18R, may be involved. Previous works proposed that uric acid crystals activate TLR2 and TLR4 receptors (38), whereas others showed that these receptors are not involved in uric acid crystals–induced inflammation (11). We show here that mice deficient for TLR2 or TLR4 developed inflammation in response to uric acid crystals (Figures 9A and 9B), as did IL-18R–deficient mice (Figures 9C and 9D). However, mice deficient for both TLR2 and TLR4 displayed an attenuated inflammatory response (Figures 9A and 9B), showing that the combined action of TLR2 and TLR4 may be required for optimal inflammation in response to uric acid crystals. Therefore, uric acid crystals-induced–inflammation is likely TLR2/TLR4 dependent but is also IL-18R independent and activates the NALP3 inflammasome and signals via IL-1R1/MyD88.

Present knowledge suggests that tissue injury may cause sterile inflammation (15, 39), and several mediators such as uric acid, ATP, high mobility group box1 protein, heat shock protein 70, lysophosphatidic acid (40), and others have been identified (41). These danger signals may be produced after lung damage, triggering inflammation, remodeling, and fibrosis. Here, using lung injury induced by BLM, we present evidence that uric acid is locally released and activates the NALP3 inflammasome, resulting in IL-1β production. We show that BLM-induced lung injury resulting in IL-1β production and subsequent inflammation is dependent on the inflammasome NALP3 and ASC. The partial decrease in IL-1β measured in lung of NALP3-deficient versus wild-type mice after BLM administration may represent a direct defect in the maturation of IL-1β by the NALP3 inflammasome or some indirect effects. Indeed, production of Pro–IL-1β, known to be independent on NALP3 inflammasome, maturation in IL-1β (which depends on inflammasome activation) and secretion of mature IL-1β are separate processes that regulate the production of such a powerful inflammatory cytokine. Because NALP3 and ASC were recently implicated in the formation of other multiprotein complexes than inflammasomes leading to a caspase-independent cell death called pyronecrosis (42), we verified the implication of caspase-1 in the BLM-induced inflammation and confirmed the role of the NALP3 inflammasome in this pathology. Nevertheless, pyronecrosis may also be involved, leading to pulmonary tissue cell death and amplifying the inflammatory response by the release of inflammatory intracellular contents of dying cells, as shown in familial autoinflammatory diseases associated with mutations in NALP3 (43). Moreover, late inflammation and repair processes depend on the NALP3 inflammasome. We demonstrate that lung injury results in the local accumulation of uric acid, which acts as an endogenous danger signal, probably activating the NALP3 inflammation and leading to IL-1β–dependent inflammation. Recently, uric acid was shown to be the endogenous danger signal released after necrosis induced by the adjuvant alum used for vaccination (44). Nevertheless, these results were not confirmed in another study of the same group (45). Indeed, the adjuvant alum was shown to activate directly NALP3 inflammasome, but it is possible that uric acid may be released upon alum-induced cell death, amplifying the activation of NALP3 inflammasome (33, 45). Here we show that regulation of uric acid levels by xanthine oxidase inhibition with allopurinol or degradation of uric acid by uricase abrogate inflammation and reduce remodeling and fibrosis upon BLM-induced lung injury. Although allopurinol is known to decrease the uric acid levels, it may also reduce oxygen species, which play a role in BLM-induced inflammation and fibrosis (46). Nevertheless, treatment with uricase confirms that reducing local uric acid levels attenuates inflammation and remodeling. Inflammation, but also repair processes and fibrosis, depend on the danger signal uric acid, bridging early events to further pathology development. Our data provide evidence that the NALP3 inflammasome is activated by uric acid, as shown previously in the gout arthritis model (10), although it is not excluded that other danger signals may be involved in the pathology, such as extracellular ATP, which also activates NALP3. However, the fact that uricase and allopurinol inhibit BLM-induced inflammation and fibrosis represents compelling evidence that uric acid plays a critical role in BLM-induced lung pathology.

Exogenous uric acid crystals have been shown to activate the NALP3 inflammasome, leading to IL-1β–dependent inflammation in the peritoneal cavity (10, 11). Here we demonstrate that exogenous uric acid crystals given by the airways cause NALP3 inflammasome activation, the production of IL-1β, IL-1R1/MyD88–dependent lung inflammation, and TIMP-1 expression, a hallmark for the evolution to fibrosis (26). IL-18R, TLR2, and TLR4 are dispensable for lung inflammation to exogenous uric acid crystals. Nevertheless, we observed that the combined action of TLR2 and TLR4 is required for optimal inflammation in response to uric acid crystals, as shown in lung inflammation caused by airway administration of dying cells (47). This requirement for either TLR2 or TLR4 was also shown in response to peritoneal administration of dying cell (15), which probably releases uric acid (16). TLR2 and TLR4 may be involved in the generation of the Pro–IL-1β upon uric acid crystals stimulation and maturated after uric acid crystal-mediated activation of the NALP3 inflammasome (10). TLR2 and TLR4 via CD14 may also play a role in uric acid crystal uptake by alveolar macrophages (48). Nevertheless, a recent publication showed that dendritic cell activation by uric acid crystals is receptor independent but depends on direct membrane binding of crystals, which leads to cell-surface lipid sorting, Syk kinase activation, and topological membranes changes resembling to phagocytosis (49). Thus, the role of TLR2 and TLR4 in inflammatory response to uric acid crystal is unclear and may depend on the cell type, on endogenous or exogenous origin of uric acid crystals, and on different experimental conditions. Our data raise the possibility that regulating uric acid production at the level of synthesis or metabolism might be useful in limiting chronic lung inflammation, repair, and fibrosis. Indeed, allopurinol is used to treat gout, and uricase is an alternative therapy of acute gout arthritis (50) or hyperuricemic syndromes (17). The model presented in Figure 10 summarizes our data. BLM-induced injury of lung cells (probably epithelial cells) induces activation of the NALP3 inflammasome, leading to lung IL-1β production, inflammation, and remodeling. BLM-induced cell injury results in the release of uric acid, which represents a major danger/stress signal likely generated from dying pulmonary cells upon injury. Local increase probably induces uric acid crystallization. The xanthine oxidase inhibitor allopurinol, which impairs uric acid synthesis or uricase (which rapidly degrades uric acid into soluble allantoin), prevents uric acid release in the lung upon BLM and decreases IL-1β production, inflammation, remodeling, and fibrosis, suggesting that uric acid crystals activate the NALP3 inflammasome, leading to the processing and maturation of Pro–IL-1β into biologically active IL-1β. Administration of exogenous uric acid crystals induces pulmonary inflammation and remodeling that are typical of the evolution toward fibrosis with TIMP-1 accumulation. IL-1β production, inflammation, and remodeling upon administration of uric acid crystals are dependent on the NALP3 inflammasome. TLR2 and TLR4 double deficiency impairs IL-1β production and cellular influx upon administration of uric acid crystals and may be involved in crystal-induced production of Pro–IL-1β or in uric acid crystals uptake by alveolar macrophages and/or resident cells.

Our findings provide insight into the molecular mechanisms linking tissue injury, inflammation, and lung fibrosis. Mechanisms of inflammation and lung fibrosis resulting in pneumoconiosis after inhalation of airborne pollutants, such as asbestos or silica, have been partially elucidated recently (31, 32, 51). In these very different lung pathologies with known origin and focal fibrosis, NALP3 inflammasome activation and IL-1β production are essential for inflammation and fibrosis. The role of reactive oxygen species (ROS), generated by NADPH oxidase upon particle phagocytosis in NALP3 inflammasome activation, was proposed, along with other possible mechanisms that may activate NALP3 inflammasome (31, 35, 52, 53). Indeed, ROS generation has been implicated in the activation of NALP3 inflammasome in response to uric acid crystals using ROS inhibitors (31, 52). Thus, in our model, we cannot exclude that allopurinol or uricase, in decreasing uric acid levels, act by reducing oxidative stress. Uric acid may indirectly activate the NALP3 inflammasome by induction of ROS. Nevertheless, the role of ROS in NALP3 activation after administration of uric acid crystals was contested in experiments with mice lacking a subunit of the phagosomal NADPH oxidase (35). Future studies need to address this question further.

Our findings support a crucial role of the NALP3 inflammasome in interstitial pulmonary fibrosis from unknown origin. We propose another mechanism whereby uric acid formation may play a pivotal role in NALP3 activation upon tissue damage–associated idiopathic lung fibrosis. Therefore, local accumulation of uric acid may act as an endogenous danger signal that activates the NALP3 inflammasome with the production of IL-1β, causing lung inflammation, repair, and fibrosis. Others danger signals released in response to unknown toxic agents are probably involved in NLR-dependent production of mature IL-1β or in NLR-dependent induction of cell death, depending on the cause of lung injury and on the type of cell death, triggering the innate immune response. Because the development of idiopathic lung fibrosis depends on unknown environmental factors associated with genetic predispositions, mutations in NALP3 or NALP12 similar to those that cause hereditary periodic fever syndromes may also predispose patients to developing idiopathic lung fibrosis (54).

In conclusion, the identification of a critical role of uric acid as the driving force of NALP3 inflammasome complex activation with the production of IL-1β represents a novel and promising therapeutic target to control inflammation and fibrosis by reducing uric acid levels with xanthine oxidase inhibitors such as allopurinol or uricase, which deserve to be tested in interstitial pulmonary fibrosis.

The authors thank Virginie Vasseur, Isabelle Maillet (UMR6218 IEM, Orleans), and Isabelle Guenon (INSERM U620, Université de Rennes 1) for their skillful assistance and la Fondation de la Recherche Médicale (France) and MRC South Africa for support. The authors thank Drs. V.M. Dixit (Genetech, San Francisco) for the gift of ASC−/− mice and Dr. S. Akira (Osaka University, Japan) for TLR and MyD88−/− mice. The authors are grateful to François Erard for scientific discussion.

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Correspondence and requests for reprints should be addressed to Isabelle Couillin, M.D., Molecular Immunology and Embryology, UMR6218, 3B rue de la Férollerie, 45071 Orleans, France. E-mail:


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