Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome

Abstract

Dying cells are capable of activating the innate immune system and inducing a sterile inflammatory response. Here, we show that necrotic cells are sensed by the Nlrp3 inflammasome resulting in the subsequent release of the proinflammatory cytokine IL-1β. Necrotic cells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflammasome. Nlrp3 inflammasome activation was triggered in part through ATP produced by mitochondria released from damaged cells. Neutrophilic influx into the peritoneum in response to necrotic cells in vivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dysfunction, and neutrophil influx in an in vivo renal ischemic acute tubular necrosis model. These findings suggest that the inhibition of Nlrp3 inflammasome activity can diminish the acute inflammation and damage associated with tissue injury.

Sterile inflammatory insults such as trauma or ischemia trigger a robust influx of neutrophils and monocytes to the site of tissue injury (1). Damaged cells are thought to release danger-associated molecular patterns (DAMPs) that can alert the innate immune system to the impending tissue damage. However, in the absence of an infectious etiology, the collateral damage caused by the initiation of this inflammatory response can be extremely detrimental. Hence, it is important to understand the mechanisms initiating the sterile inflammatory response to devise strategies with which to control it.

The innate immune system possesses germline-encoded pattern recognition receptors that are capable of recognizing highly conserved molecules. The best described class of these receptors are Toll-like receptors (TLRs), which predominantly sense pathogen-associated molecular patterns (PAMPs) (2). The innate immune system serves to monitor for more than just the presence of microbes; pattern recognition receptors also recognize DAMPs released by cells in response to cellular damage or stress. Although there is evidence that some DAMPs, such as high-mobility group box 1 protein (HMGB1) and heat-shock proteins, can stimulate TLR2 and TLR4, the inflammatory response to necrotic cells in vivo is predominantly independent of TLR signaling (3–6). However, mice deficient in MyD88 [which in addition to TLR signaling is also essential for signaling through the IL-1 receptor I (IL-1R)] or IL-1R had markedly impaired recruitment of neutrophils in response to necrotic cells (6).

IL-1α and IL-1β signal through the IL-1R, and both require caspase-1 for secretion of their mature forms (7). Nlrp3 (also known as Nalp3, cryopyrin, and CIAS1), a member of the NLR (nucleotide-binding domain leucine-rich repeat containing) family, along with ASC and caspase-1, form a multiprotein complex called the Nlrp3 inflammasome (8). The Nlrp3 inflammasome can activate caspase-1 in response to a number of diverse stimuli including ATP, monosodium urate crystals (MSU), and bacterial pore forming toxins resulting in the processing and secretion of the proinflammatory cytokines IL-1α, IL-1β, and IL-18 (9–12).

Here, we investigate the mechanisms of necrotic cell-induced inflammation and demonstrate that specific forms of cell death are sensed by the Nlrp3 inflammasome. We further found that viable mitochondria released from these necrotic cells were responsible for Nlrp3 inflammasome activation. Nlrp3-deficiency also had a profound effect on mortality and renal function in an in vivo model of renal ischemic acute tubular necrosis. Together our data indicate that the Nlrp3 inflammasome plays a crucial role in initiating sterile inflammatory responses to tissue injury.

Results

Inflammatory Response to Necrotic Cells Is Mediated by the Nlrp3 Inflammasome.

To examine if the Nlrp3 inflammasome was involved in the inflammatory response to cellular damage, we injected pressure-disrupted B16 cells (a murine melanoma cell line) i.p. into mice deficient in IL-1R, caspase-1, Nlrp3, or ASC (Fig. 1 A–C). Sixteen hours after challenge with pressure disrupted B16 cells, wild-type (WT) mice displayed a marked influx of neutrophils into the peritoneal cavity. This neutrophil influx was significantly diminished in mice deficient in components of the Nlrp3 inflammasome (Fig. 1 A–C). Neutrophil influx in Nlrp3- and ASC-deficient mice in response to thioglycollate remained intact demonstrating that Nlrp3- and ASC-deficiency does not lead to a global defect in neutrophil migration (Fig. S1).

Fig. 1.

Inflammation induced in vivo by pressure-disrupted necrotic cells is dependent on the Nlrp3 inflammasome. (A–C) Neutrophil influx into the peritoneum of WT, IL-1R-, caspase-1-, ASC-, and Nlrp3-deficient mice 16 h after i.p. challenge with pressure-disrupted B16 cells (B16). Control WT mice were challenged i.p. with PBS. *, P = 0.0017; **, P = 0.0021; ***, P = 0.0159; ****, P = 0.0240.

The cysteine protease caspase-1 is required for the processing and secretion of IL-1β and also partially for IL-1α (7, 13). To determine if necrotic cells were capable of inducing the secretion of these cytokines, LPS-primed macrophages (Mϕ) were stimulated with necrotic B16 cells derived by UV-irradiation, freeze-thawing, or pressure disruption. Pressure-disrupted B16 cells induced a robust secretion of IL-1β, however, UV-irradiated or freeze-thawed B16 cells induced substantially less IL-1β (Fig. 2A). The secretion of IL-1β was not specific to B16 cells, as pressure-disrupted HEK293 cells similarly induced IL-1β secretion from LPS-primed Mϕ (Fig. S2A). In contrast to pressure-disrupted B16 cells, which induce a robust neutrophilic influx in vivo following i.p. injection, necrotic B16 cells produced by freeze-thaw induced significantly less i.p. inflammation, which was independent of caspase-1 (Fig. 2B). Together, these findings demonstrate that specific forms of necrotic cell death are capable of inducing the secretion of IL-1β from LPS-primed Mϕ and inflammatory responses in vivo in a manner dependent on the Nlrp3 inflammasome.

Fig. 2.

Cellular injury induced by pressure-disruption is sensed by the Nlrp3 inflammasome. (A) LPS-primed WT Mϕ were stimulated with either UV-treated, freeze-thawed, or pressure-disrupted B16 cells at a ratio of 10 necrotic cells per Mϕ; culture supernatants were collected 12 h later, and IL-1β release was measured by ELISA. (B) Neutrophil influx into the peritoneum of WT and caspase-1-deficient mice 16 h after i.p. challenge with either 1 × 10⁷ pressure-disrupted B16 cells (B16) or freeze-thawed B16 cells. *, P = 0.0041. (C and D) LPS-primed WT, Nlrp3-, caspase-1-, or ASC-deficient Mϕ were stimulated with pressure-disrupted B16 cells and supernatants collected 12 h later or at the indicated time. IL-1β release was measured by ELISA. (E) Lysates from LPS-primed WT, Nlrp3-, or ASC-deficient Mϕ stimulated with pressure-disrupted B16 cell for 12 h were immunoblotted with antibodies against the p10 subunit of caspase-1, IL-1β, and GAPDH. (A, C, and D) Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two (A) and three (C and D) separate experiments.

Nlrp3-, ASC-, and caspase-1-deficient LPS-primed Mϕ displayed a marked defect in their ability to process and secrete IL-1β in response to pressure-disrupted B16 cells compared to WT Mϕ (Fig. 2 C–E). Caspase-1 activation in LPS-primed WT Mϕ stimulated with pressure-disrupted B16 cells was detected by Western blot by the appearance of the p10 cleavage product (Fig. 2E), which occurred by 3 h post-stimulation (Fig. S2B). We did not observe caspase-1 activation in response to pressure-disrupted B16 cells in either Nlrp3- or ASC-deficient LPS-primed Mϕ (Fig. 2E). In addition, there is a defect in IL-1α secretion in Nlrp3-deficient LPS-primed Mϕ stimulated with pressure-disrupted B16 cells, although the absolute amounts of IL-1α secreted were substantially lower compared to IL-1β (Fig. S2C). Thus, pressure-disrupted necrotic cells drive an inflammatory cell infiltrate in vivo and induce the production of IL-1α and IL-1β in a Nlrp3 inflammasome-dependent manner.

Complement- and Hypoxia-Mediated Cellular Damage Is Sensed by the Nlrp3 Inflammasome.

Although the complement system plays an important role in protecting the host against infectious diseases and aiding in the repair of damaged tissue, unregulated or inappropriate activation of the complement cascade can in itself lead to tissue injury (14). To determine if complement-mediated damage induced an inflammatory response similar to pressure-disrupted necrotic cells, we challenged Mϕ with complement-lysed splenocytes. Complement-lysed splenocytes did induce the secretion of IL-1β from LPS-primed WT Mϕ (Fig. 3A). Heat inactivation of serum used for complement lysis abrogated the ability of the splenocytes to induce IL-1β secretion (Fig. 3B), suggesting that cellular damage was required for this process. Nlrp3-, ASC-, and caspase-1-deficient Mϕ failed to secrete IL-1β in response to complement-lysed splenocytes (Fig. 3A). In contrast, Mϕ deficient in Nlrc4, which is required for caspase-1 activation in response to infection with type III and type IV secretion system carrying Gram-negative bacteria (15, 16), were capable of secreting IL-1β in response to complement-lysed splenocytes (Fig. 3A). Similar to primary splenocytes, complement-mediated lysis of a hybridoma cell line was also capable of inducing the activation of caspase-1 and the secretion of IL-1β from LPS-primed Mϕ in a Nlrp3-dependent manner (Fig. 3 C–D and Fig. S2D). Therefore, complement-mediated damage to host cells can be sensed by the Nlrp3 inflammasome. These findings also demonstrate a communication between these two evolutionary ancient components of the innate immune system.

Fig. 3.

Complement- and hypoxia-induced cellular injury is sensed by the Nlrp3 inflammasome. (A) LPS-primed WT, caspase-1-, ASC-, Nlrp3-, or Nlrc4-deficient Mϕ were stimulated with complement-lysed splenocytes at a ratio of 25 splenocytes per Mϕ; supernatants were collected 12 h later, and IL-1β release assessed by ELISA. (B–D) Splenocytes or the hybridoma cell line 6F10 were either left untreated or opsonized with anti-MHC class II Aβ(b) IgG2a antibody followed by incubation with human serum (serum) or heat-inactivated human serum (HI-serum). LPS-primed WT or Nlrp3−/− Mϕ were stimulated with complement-damaged cells at a ratio of 25 splenocytes per Mϕ, 20 6F10 cells per Mϕ, or as indicated; supernatants were collected 12 h later, and IL-1β release assessed by ELISA. *, P = 0.0003. (E) LPS-primed WT or Nlrp3−/− Mϕ were stimulated with B16 cells grown under normoxic (control) or hypoxic conditions at a ratio of five cells per Mϕ; supernatants were collected 12 h later, and IL-1β release assessed by ELISA. **, P = 0.0013. (F) WT Mϕ were primed with either biglycan (8 μg/mL) or hyaluronic acid (25 μg/mL) for 12 h followed by stimulation with pressure-disrupted B16 cell at a ratio of 10 necrotic cells per Mϕ; supernatants were collected 12 h later, and IL-1β release measured by ELISA. (A–F) Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two (C–F) and three (A and B) separate experiments.

To assess if hypoxia-mediated cellular damage was capable of activating the Nlrp3 inflammasome, Mϕ were exposed to B16 cells grown under hypoxic conditions. LPS-primed Mϕ exposed to hypoxic B16 cells, but not B16 cells grown under normoxic conditions, secreted IL-1β in a Nlrp3-dependent manner (Fig. 3E). These results suggest that in addition to pressure-disruption and complement-mediated damage, the Nlrp3 inflammasome is capable of sensing cellular damage induced by hypoxic conditions.

Extracellular Matrix Molecules Can Prime the Nlrp3 Inflammasome for Activation.

Activation of the Nlrp3 inflammasome is a two-step process requiring a priming step (signal 1) followed by an activation step (signal 2). Signal 1 serves two functions: in addition to stimulating the production of pro-IL-1β it is also a prerequisite for inflammasome activation. LPS priming of Mϕ allowed for pressure-disrupted B16 cells to activate caspase-1 and induce the secretion of IL-1β as unprimed Mϕ failed to activate caspase-1 and secrete IL-1β (Fig. S3 A and B). Importantly, endogenous molecules can also serve a priming role for Nlrp3 inflammasome activation. Biglycan and hyaluronic acid, components of the extracellular matrix, were capable of priming Mϕ for Nlrp3 inflammasome activation in response to pressure-disrupted B16 cells (Fig. 3F). Hence, extracellular matrix components that accumulate in nonphysiological sites or amounts can function as signal 1, suggesting that Nlrp3 inflammasome activation can occur in vivo in sterile settings without microbes being present to provide signal 1.

Mitochondria Activate the Nlrp3 Inflammasome.

To determine what cellular component from necrotic cells was capable of inducing the activation of the Nlrp3 inflammasome, we performed subcellular fractionation of the pressure-disrupted B16 cells. Cytosolic, nuclear, and plasma membrane fractions all failed to induce the secretion of IL-1β from LPS-primed Mϕ (Fig. 4A). Surprisingly, mitochondria isolated from pressure-disrupted B16 cells stimulated the secretion of IL-1β from LPS-primed WT Mϕ (Fig. 4A). This was not unique to B16 cells, as mitochondria isolated from HEK293 cells were similarly capable of inducing the secretion of IL-1β from LPS-primed Mϕ (Fig. S4A). Purity of the mitochondrial fraction was confirmed by Western blot analysis using antibodies against Cox4 (Fig. S4B). Unprimed Mϕ failed to secrete IL-1β in response to mitochondria, demonstrating that a separate priming step was required for mitochondria to activate caspase-1 (Fig. S3 A and B). Similar to results obtained using pressure-disrupted cells, we observed that LPS-primed Mϕ from Nlrp3-, ASC-, and caspase-1-deficient mice failed to secrete IL-1β in response to mitochondria (Fig. 4B). LPS-primed Mϕ from Nlrc4-deficient mice had an intact response to mitochondria and secreted IL-1β (Fig. 4B), suggesting that Mϕ that encounter extracellular mitochondria specifically activate the Nlrp3 inflammasome. Stimulation of LPS-primed WT Mϕ, but not Nlrp3- or ASC-deficient Mϕ, with mitochondria resulted in activation of caspase-1 as detected by Western blot (Fig. 4C). Mitochondria injected i.p. into WT mice resulted in a marked inflammatory response as determined by neutrophilic influx; however, Nlrp3-deficient mice had a significantly diminished inflammatory response in vivo compared to WT mice (Fig. 4D).

Fig. 4.

Mitochondria released from necrotic cells stimulate Nlrp3 inflammasome activation. (A) LPS-primed WT Mϕ were stimulated with pressure-disrupted B16 cells or nuclei, plasma membrane, cytosol, and mitochondrial fractions derived from B16 cells at a ratio of five cell equivalents per Mϕ; supernatants were collected 12 h later, and IL-1β release was measured by ELISA. (B) LPS-primed WT, caspase-1-, ASC-, Nlrp3-, or Nlrc4-deficient Mϕ were left untreated or stimulated with mitochondria (100 μg/mL); supernatants were collected 12 h later, and IL-1β release was assessed by ELISA. (C) Lysates from LPS-primed WT, Nlrp3-, or ASC-deficient Mϕ stimulated with mitochondria for 12 h were immunoblotted with antibodies against the p10 subunit of caspase-1 and GAPDH. (D) Neutrophil influx into the peritoneum of WT and Nlrp3-deficient mice 16 h after i.p. challenge with mitochondria isolated from 1 × 10⁷ B16 cells. Control WT mice were challenged i.p. with homogenization buffer. *, P = 0.0317. (E) Mitochondria were treated with rotenone (10 μM), myxothiazol (10 μM), apyrase (1 U) for 20 min or heat-treated at 65 °C for 20 min, washed, and ATP content quantified. (F) LPS-primed WT Mϕ were stimulated with mitochondria (100 μg/mL) that had been treated as described; supernatants were collected 12 h later, and IL-1β release was measured by ELISA. (G) LPS-primed WT or P2X7R-deficient Mϕ were stimulated with ATP (5 mM), silica (50 μg/cm²), pressure-disrupted B16 cells, or mitochondria; supernatants were collected 12 h later, and IL-1β release was measured by ELISA. **, P = 0.0041; ***, P = 0.0009. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two (A, E, and F) and three (B and G) separate experiments.

Pretreatment of mitochondria with rotenone or myxothiazol, inhibitors of mitochondrial complex I and III, respectively, apyrase, an ATPase, or heat treatment resulted in the loss of ATP content of the mitochondria (Fig. 4E) as well as diminished their ability to induce Mϕ secretion of IL-1β (Fig. 4F), suggesting that mitochondria must be actively respiring to induce the activation of the Nlrp3 inflammasome. To confirm that residual rotenone, myxothiazol, and apyrase were not affecting Mϕ function, silica, an activator of the Nlrp3 inflammasome, was added in combination with untreated and treated mitochondria; no inhibition in silica-induced IL-1β was observed (Fig. S4C). Consistent with these findings, pretreatment of pressure-disrupted necrotic B16 cells with rotenone, myxothiazol, apyrase, or heat treatment also resulted in a diminished ability to induce Mϕ secretion of IL-1β (Fig. S4D). Thus, mitochondria activate the Nlrp3 inflammasome, and this activation can be blocked by inhibitors of cellular respiration.

Mitochondrial Activation of the Nlrp3 Inflammasome in Vitro Partially Requires P2X7R.

Given its association with cellular damage, it was possible that liberated uric acid (monosodium urate; MSU) from pressure-disrupted B16 cells was responsible for Nlrp3 inflammasome activation (11, 17). We thus challenged LPS-primed Mϕ with pressure-disrupted B16 cells or mitochondria that had been pretreated with uricase. As expected, uricase treatment of MSU abolished its ability to induce the secretion of IL-1β (Fig. S5A) (18). However, uricase treatment of pressure-disrupted B16 cells or mitochondria did not inhibit their ability to induce IL-1β secretion (Fig. S5A), suggesting that pressure-disrupted necrotic cells and mitochondria activate the Nlrp3 inflammasome in a manner that is independent of MSU.

To determine if necrotic cells and mitochondria require internalization to activate the Nlrp3 inflammasome in a manner similar to MSU, silica, and alum (11, 18–23), LPS-primed Mϕ were pretreated with cytochalasin B or D. As expected, cytochalasin B and D inhibited silica-induced IL-1β secretion (Fig. S5B) (23, 24); however, cytochalasin B or D did not markedly inhibit pressure-disrupted B16 cells or mitochondria from inducing IL-1β secretion from LPS-primed WT Mϕ (Fig. S5B). Hence, endocytosis of pressure-disrupted necrotic cells or mitochondria is not required for Nlrp3 inflammasome activation.

ATP, acting via the purinergic P2X7 receptor, can activate the Nlrp3 inflammasome without a requirement for endocytosis (10, 12, 25). To determine if signaling through the P2X7 receptor was required for the inflammatory response to injured cells, we used Mϕ from P2X7R-deficient mice. As expected P2X7R-deficient LPS-primed Mϕ failed to secrete IL-1β in response to ATP, however, IL-1β secretion in response to silica remained intact (Fig. 4G). P2X7R-deficient LPS-primed Mϕ had a marked defect in their ability to secrete IL-1β in response to either pressure-disrupted B16 cells, mitochondria, or complement-lysed 6F10 cells (Fig. 4G and Fig. S5C); suggesting that ATP released from disrupted cells or generated by actively respiring mitochondria is capable of activating the Nlrp3 inflammasome. However, while IL-1β secretion from P2X7R-deficient LPS-primed Mϕ was diminished in response to necrotic cells or mitochondria, it was not abrogated (Fig. 4G and Fig. S5C), suggesting that additional pathways leading to Nlrp3 inflammasome activation and IL-1β secretion exist. Consistent with these in vitro findings, in vivo i.p. challenge of P2X7R−/− mice with pressure-disrupted B16 cells did not result in a significant decrease in neutrophilic influx into the peritoneal cavity in comparison to WT mice (Fig. S5D).

Nlrp3- and ASC-Deficient Mice Are Protected from Renal Ischemia-Reperfusion Injury.

The events that occur during ischemia-reperfusion (I/R) injury are complex and involve altered renal morphology and hemodynamics, death of epithelial and endothelial cells, and exuberant inflammation (26). It is known that mice deficient in the components of the membrane attack complex are protected from renal I/R injury, suggesting cell lysis by complement is a key initiator of the inflammatory response (27). To explore the mechanism by which necrotic injury is translated in vivo into inflammation, mice were subjected to bilateral renal artery occlusion and killed one day after reperfusion. This revealed that nonlethal renal I/R injury resulted in a significant upregulation of Nlrp3 gene expression (Fig. 5A), which was accompanied by pronounced acute tubular necrosis (Fig. S6) that was similar between WT and Nlrp3-deficient animals (Fig. 5B). There was also increased expression of the extracellular matrix molecules biglycan and hyaluronic acid following renal I/R injury (Fig. S7 A and B), consistent with previous findings (28). Importantly, Nlrp3 deficiency protected animals from lethal renal ischemic injury; Nlrp3-deficient mice had a 86% survival following renal acute tubular necrosis at 5.5 days post-ischemia compared with 8% survival in WT mice (P < 0.0001) (Fig. 5C). Nlrp3-deficient mice were moreover functionally protected against renal dysfunction as reflected by significantly lower plasma urea and creatinine levels as compared to WT animals 24 h after nonlethal renal ischemic injury (Fig. 5 D and E). This protection was associated with reduced numbers of neutrophils infiltrating the renal interstitium on day 1 compared to WT mice (Fig. 5 F and G). Consistent with this, the level of neutrophil chemoattractant KC and total IL-1β in the kidneys of Nlrp3-deficient mice after ischemic acute tubular necrosis was reduced compared to WT mice (Fig. 5 H and I).

Fig. 5.

Nlrp3-deficiency protects animals against mortality, renal dysfunction, and impairs an inflammatory response during renal ischemic acute tubular necrosis. (A) Relative Nlrp3 mRNA levels were measured in kidneys of WT mice 1 day after nonlethal renal I/R or sham operation. (B) Tubular necrosis score of WT mice (n = 8) and Nlrp3-deficient mice (n = 8) after nonlethal renal I/R injury using PAS-D-stained renal tissue sections. (C) Survival of Nlrp3-deficient mice (n = 14) compared to WT mice (n = 13) after lethal renal ischemia. (D and E) Renal dysfunction of Nlrp3-deficient mice (n = 8) compared to WT mice (n = 8) as reflected by increased levels of urea (D) and creatinine (E) in plasma after nonlethal renal acute tubular necrosis. (F and G) Neutrophil influx in kidneys from WT and Nlrp3-deficient mice 1 day after renal I/R or sham operation as assessed by immunohistochemistry (F) and counted in at least 10 randomly selected high-power fields in the outer medulla (G). Original magnification of pictures, ×400. (H and I) Total KC and IL-1β levels in kidneys from Nlrp3-deficient and WT mice subjected for 1 day to renal I/R injury. *, P < 0.05.

ASC gene expression was significantly upregulated 5 days after nonlethal renal I/R injury, while there was no difference after 1 day (Fig. S8A). The amount of acute tubular necrosis was similar between WT and ASC-deficient animals at both time points (Fig. S8B). ASC deficiency protected animals from lethal renal ischemic injury, although the difference was less pronounced than in Nlrp3-deficient mice. ASC-deficient mice had a 38% survival following renal acute tubular necrosis at 5.5 days post-ischemia, while none of the WT mice survived (P = 0.001) (Fig. S8C). ASC-deficient mice also displayed a more preserved renal function 5 days after renal I/R injury as compared to WT (Fig. S8 D and E). In line with the Nlrp3-deficient mice, protection of the ASC-deficient mice correlated with reduced neutrophil influx and renal KC and IL-1β levels (Fig. S8 F–H). The difference seen between Nlrp3-deficient and ASC-deficient mice in survival and early renal dysfunction following renal ischemic injury suggests that Nlrp3 may play additional roles independently of ASC and, by extension, caspase-1. Together these data demonstrate that the Nlrp3 inflammasome induces an exaggerated acute inflammatory response during ischemic acute tubular necrosis and as a consequence contributes to I/R-induced renal dysfunction and lethality.

Discussion

We postulate that specific forms of cellular injury result in the release of viable mitochondria into the extracellular space, triggering the activation of the Nlrp3 inflammasome, in part through the release of ATP. Extracellular ATP is quickly degraded by exonucleases, therefore release of intact ATP-producing mitochondria provides an effective mechanism to alert the immune system to cellular damage. Triggering of the Nlrp3 inflammasome results in activation of caspase-1 and the processing and secretion of IL-1β, which can recruit neutrophils to sites of infection and injury, and also possibly lead to host tissue damage (Fig. S9).

It is interesting that specific modes of cell death, such as pressure disruption, complement lysis and hypoxia, were sensed by the Nlrp3 inflammasome, whereas cell death induced by freeze-thaw and UV irradiation failed to induce a robust secretion of IL-1β (Fig. 2A). We speculate that freeze-thaw or UV irradiation may either result in damaged nonviable mitochondria or that mitochondria remain within the killed cell, inaccessible to the extracellular space and hence fail to activate the Nlrp3 inflammasome. A recent study by Li and colleagues also demonstrated that necrosis induced by 7-bromoindirubin-3′-oxime was capable of activating the Nlrp3 inflammasome (29). These findings highlight the heterogeneity of cell death even within the necrotic subset.

I/R injury occurs as blood flow is restored to ischemic tissue resulting in a profound inflammatory response. It is a significant cause of the pathology associated with clinical conditions such as myocardial infarction, cerebral ischemia, and in the operative management of trauma. Renal I/R injury is a major cause of acute and end-stage renal failure and is associated with increased acute renal transplant rejection and delayed allograft function (30–34). Although the pathophysiology of renal I/R injury is complex, it is clear that necrotic cellular injury mediated by I/R and complement activation play a major role in initiating subsequent inflammatory responses. Our studies show that although the initial necrotic insult following I/R injury is the same in WT, Nlrp3-, and ASC-deficient mice, the ensuing inflammatory response to the necrotic cells is markedly reduced in mice deficient in components of the Nlrp3 inflammasome. Limiting this Nlrp3 inflammasome-driven inflammatory response is crucial to preventing further organ damage and in preserving renal function. An important role for IL-1β in the pathogenesis of renal I/R injury is also supported by in vivo studies demonstrating that the lack of functional IL-1R, IL-1α/β, or the treatment with IL-1 receptor antagonist (IL-1Ra) impairs the inflammatory response and subsequently accelerates renal recovery (35) or reduces renal tissue destruction (36, 37) after renal I/R injury. We have also recently shown that Nlrp3-deficient mice are protected from acetaminophen-induced hepatotoxicity (38). Our current findings provide mechanistic insight into this process, suggesting that necrotic hepatocytes themselves may be responsible for triggering the inflammatory response through the Nlrp3 inflammasome.

It has been unclear what the initial priming step for Nlrp3 inflammasome activation is in vivo, but our findings suggest it may be through stimulation by endogenous DAMPs that are released concomitantly with cellular injury. Both biglycan and hyaluronic acid were capable of priming Mϕ for Nlrp3 inflammasome activation in response to pressure-disrupted necrotic cells in vitro. Biglycan and hyaluronic acid were also found to be expressed at higher levels in tissue following renal I/R injury, suggesting that they may be responsible for priming the Nlrp3 inflammasome for activation in vivo.

The sterile inflammatory response to injured cells is thought to contribute to the pathogenesis of not only ischemic diseases, such as renal-, myocardial-, and cerebral-ischemia, but also to inflammation associated with traumatic injury and chemotherapeutic-induced tumor death. It is clear this inflammatory response is an independent source of profound morbidity and mortality. Blockade of an excessive and prolonged sterile inflammatory response with specific Nlrp3 inflammasome antagonists could represent an approach to treat or prevent the adverse outcomes associated with tissue injury.

Materials and Methods

Mice.

The generation of Nlrp3-, ASC-, caspase-1-, and Nlrc4-deficient mice has been described (7, 12, 39). IL-1R-deficient and P2X7R-deficient mice (25) were purchased from Jackson Laboratories. Caspase-1-, IL-1R-, and P2X7R-deficient mice were backcrossed onto the C57BL/6 genetic background for 10, five, and seven generations, respectively. ASC- and Nlrp3-deficient mice were backcrossed onto the C57BL/6 genetic background for nine generations. Nlrc4-deficient mice were backcrossed onto the C57BL/6 genetic background for six generations. Age- and sex-matched C57BL/6 mice purchased from NCI were used as WT controls. All protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Iowa and the University of Amsterdam.

In Vivo Peritonitis.

Mice were injected i.p. with either 1 × 10⁷ pressure-disrupted B16 cells or freeze-thaw-treated B16 cells in 500 μL PBS, 1 mL 3% thioglycollate, or PBS alone. Sixteen hours later, animals were euthanized and peritoneal lavage was performed. The number of neutrophils (Ly-6G + 7/4+) in the lavage was assessed by flow cytometry as described in ref. 6.

Ischemic Acute Tubular Necrosis.

Ischemic acute tubular necrosis was induced as described in ref. 40. Briefly, renal arteries of mice were clamped for 30 min (nonlethal) or 45 min (lethal) using microaneurysm clamps through a midline abdominal incision under general anesthesia. After surgery, all mice received a s.c. injection of 50 μg/kg buprenorphin (Temgesic; Shering-Plough) for analgesic purposes and were allowed to recover from surgery for 12 h at 28 °C in a ventilated stove and were killed 1 day after surgery. Sham-operated mice (n = 6 per group) underwent the same procedure without clamping. At the time of sacrifice, blood was collected by heart puncture in heparin-containing tubes and stored at −80 °C, and kidneys were harvested for further analysis. Total RNA was extracted from renal tissue sections with TRIzol reagent (Invitrogen) and converted to cDNA.

Detailed information about materials and methods is in the SI Text.