An Inflammasome-Independent Role for Epithelial-Expressed Nlrp3 in Renal -

This information is current as Alana A. Shigeoka, James L. Mueller, Amanpreet Kambo, of September 30, 2021. John C. Mathison, Andrew J. King, Wesley F. Hall, Jean da Silva Correia, Richard J. Ulevitch, Hal M. Hoffman and Dianne B. McKay J Immunol 2010; 185:6277-6285; Prepublished online 20

October 2010; Downloaded from doi: 10.4049/jimmunol.1002330 http://www.jimmunol.org/content/185/10/6277

References This article cites 62 articles, 15 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/185/10/6277.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/186/3/1880.full.pdf

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Inflammasome-Independent Role for Epithelial-Expressed Nlrp3 in Renal Ischemia-Reperfusion Injury

Alana A. Shigeoka,* James L. Mueller,†,‡,x Amanpreet Kambo,* John C. Mathison,* Andrew J. King,{ Wesley F. Hall,‖ Jean da Silva Correia,* Richard J. Ulevitch,* Hal M. Hoffman,†,‡,x and Dianne B. McKay*

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflam- matory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules as- sociated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits -associated speck-like protein containing a recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1b and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Downloaded from components of the Nlrp3 inflammasome (Nlrp32/2 and Asc2/2 mice). To examine the role of the inflammasome in renal ischemia- reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1b, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflamma- some targets, dramatically protected from IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production. The Journal of Immunology, 2010, 185: 6277–6285. http://www.jimmunol.org/

remendous interest has evolved over the past decade in Naip, CIITA, HET-E, and telomerase-associated protein 1 domain, innate immune sensing receptors and their role in disease and several leucine-rich repeats at the C-terminal end (2). One of the T processes. Of the known innate immune receptors, the best best-described members of the NLR family is Nlrp3 (also called described are the membrane-bound TLRs; many recent discoveries cyropyrin, CIAS1, or Nalp3) (3). Nlrp3 is an essential component have highlighted their importance in human diseases. Another of the caspase 1 inflammasome, a multiprotein molecular scaffold family of intracellular innate sensors, called the nucleotide-binding necessary for caspase 1 activation (4). Upon ligand sensing, Nlrp3 oligomerization domain-like receptors (NLRs), has attracted fo- recruits the adaptor protein apoptosis-associated speck-like protein by guest on September 30, 2021 cused interest as potential therapeutic targets for diseases associ- containing a caspase recruitment domain (CARD) (Asc) through ated with the overproduction of proinflammatory cytokines. The a PYD–PYD interaction. Asc then recruits inflammatory NLRs are critical signaling molecules that contribute to formation (e.g., caspase 1) to the assembly complex through CARD–CARD of intracellular macromolecular complexes and regulate host im- interactions (5). The assembly complex brings into close proximity mune responses to a variety of stimuli, including pathogens, mol- the inflammatory caspases, leading to their activation (6). Activa- ecules released from injured tissue, and a variety of nonspecific tion of caspase 1 is required for processing and release of active molecules, including immune adjuvants (1). proinflammatory cytokines such as IL-1b and IL-18. The NLR family includes several different groups of intracellular A large variety of structurally unrelated stimuli activate Nlrp3, molecules sharing a N-terminal pyrin domain (PYD), a central such as microbial RNA and the imidazoquinoline compounds R837 and R848, muramyl dipeptide, polyinosinic-polycytidylic *Department of Immunology and Microbial Sciences, The Scripps Research In- acid, ATP, potassium efflux, asbestos, silica, aluminum hydroxide, stitute; {Division of Nephrology, Department of Medicine and ‖Department of Pa- amyloid b peptide, pore-forming toxins, cellular components re- thology, Scripps Clinic and Green Hospital, La Jolla, CA 92037; and †Department of x leased into the extracellular milieu by distressed cells (including Pediatrics, ‡Department of Medicine, and Ludwig Institute of Cancer Research, School of Medicine, University of California at San Diego, La Jolla, CA 92093 monosodium urate and calcium pyrophosphate dihydrate crystals Received for publication July 12, 2010. Accepted for publication September 8, 2010. found in gout and pseudogout), and cholesterol crystals (7–11). The This work was supported by National Institutes of Health Grant 1R01DK60151, fact that so many different stimuli trigger activation of Nlrp3, and Scripps Translational Science Institute Pilot Grant 1UL1RR025774 (to D.B.M.), subsequent inflammasome formation, suggests that Nlrp3 is a com- and a Renal Research Collaborative/Price Charities grant (to A.J.K.). mon hub for many different cellular triggers that lead to overpro- Address correspondence and reprint requests to Dr. Dianne McKay, Department of duction of proinflammatory cytokines. Immunology and Microbial Sciences, IMM-16, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: dmckay@ Several human diseases have been linked to Nlpr3. For instance, scripps.edu mutations in the Nlrp3 gene cause familial cold autoinflammatory Abbreviations used in this paper: Asc, apoptosis-associated speck-like protein con- syndrome, Muckle-Wells syndrome, and neonatal-onset multisys- taining a caspase recruitment domain; CARD, caspase recruitment domain; HEK, tem inflammatory disease, which are collectively known as cry- HEK 293 cells; hRTE, human RTE cells extracted from normal human kidneys; IRI, ischemia-reperfusion injury; LN, lymph node; NLR, nucleotide-binding oligomeri- opyrinopathies (3, 12–14). More recently, inflammation associated zation domain-like receptor; pre, preinjury; PYD, pyrin domain; RTE, renal tubular with gout has been linked to Nlrp3 and overproduction of IL-1b epithelium; sham, sham-operated; Thp1, Thp1 cells; WT, wild-type; WTLN, lymph (10). A central role for overproduction of IL-1b in the pathogenesis node cells from wild-type mice. of Nlrp3-related diseases has been identified by the successful Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 treatment of these diseases with IL-1–targeted therapies. The rIL- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002330 6278 INFLAMMASOME-INDEPENDENT ROLE FOR Nlrp3 IN RENAL IRI

1R antagonist anakinra has shown remarkable therapeutic efficacy Jolla, CA). Immediately postnephrectomy, the renal pathologist removed for patients with cryopyrinopathies as well as gout (15–20). a wedge section of normal tissue (distal from the site of tumor). After Another of the disease processes linked to innate immune re- transport to the laboratory in sterile media, the renal capsule was removed and cortex dissected from the medulla. The cortex was then finely minced, ceptors is ischemic kidney injury. Ischemic kidney injury occurs placed in collagenase A (Sigma-Aldrich, St. Louis, MO), and incubated at commonly in hospitalized patients and is a complex pathophysio- 37˚C for 30 min with frequent shaking. Postincubation, the digested mixture logical process contributed to by both cell death and inflammation. was differentially sieved (200–50 mm) and washed three times with fresh Although recent attention has highlighted the important role for media. The sieved contents were then centrifuged, pelleted, and layered over 30% Percoll (Sigma-Aldrich). After spinning the Percoll mixture at TLRs in ischemic injury of the kidney (21, 22), as well as other 21,500 3 g for 30 min, four distinct bands were apparent as previously organs (23–28), less is known about the role of the NLRs in is- described (30, 31). The cells were collected from band 3 and washed three chemic organ injury (29). times with HBSS. The cells were then cultured in specialized tubular Because Nlrp3 plays a central role in immune regulation associ- epithelial cell growth media (DMEM–Ham’s-F-12 [Fischer Scientific, m m atedwith cellularinjury,weinvestigatedwhetherNlrp3playsarolein Pittsburgh, PA]; insulin [5 g/ml]; transferrin [5 g/ml]; selenium [50 nmol/l] supplements; hydrocortisone [0.05 mM; Sigma-Aldrich]; epidermal . To mimic acute kidney injury, we used a well- growth factor [10 ng/ml; Sigma-Aldrich]; tri-iodo thyronine [32 ng/ml]; and characterized murine model of ischemia-reperfusion injury (IRI). penicillin/streptomycin [1 ml]) to promote growth of tubular epithelial cells We examined the dependence of kidney injury and cytokine/chemo- but not other cell types. Within a few days, a cobblestone morphology kine secretion on the Nlrp3 inflammasome using mice with targeted characteristic of cultured renal tubular epithelium was evident. Confirma- tion of renal tubular epithelium was made by positive staining for Lotus deletions in the inflammasome components, Nlrp3 and Asc. Because tetragonolobus lectin (32) (Vector Laboratories, Burlingame, CA). inflammasome formation activates caspase 1, we also asked whether targeted deletion of caspase 1 influenced acute kidney injury. The Induction of in vivo IRI Downloaded from dependence of acute kidney injury on IL-1 was tested using an IL-1R All experimental mice were matched for age (8–12 wk), and only male mice antagonist and also using IL-1R knockout mice. The role of IL-18 were used. As previously published (21, 33), the following methods were was tested using IL-18 knockout mice. Our studies are the first to our used to induce nonlethal IRI. The mice were anesthetized with isofluorane knowledge to isolate an inflammasome-independent role of the and injected i.p. with ketamine (100 mg/kg)/xylazine (8 mg/kg) in saline. Core body temperatures were maintained between 36˚C and 37.5˚C during Nlrp3 protein in renal epithelial cells and to identify a new surgery by continuous monitoring with a rectal thermometer and automatic inflammasome-independent function in vivo for Nlrp3. heating blanket. Both kidneys were exposed with bilateral flank incisions http://www.jimmunol.org/ and ischemia induced by clamping both renal arteries with nontraumatic microvessel clamps (S&T, Neuhausen, Switzerland) for 25 min. Renal Materials and Methods veins remained unoccluded. Cessation of blood flow was documented by Mice visual inspection. After 25 min of ischemia, the clamps were released and reflow verified by visual inspection of the kidneys. All mice received 200 ml Mice were housed in the vivariums at The Scripps Research Institute and saline dripped over the open flanks during surgery to keep the tissue moist University of California at San Diego (La Jolla, CA) and approved for use by m 2/2 2/2 and 30 l saline/g body weight injected s.c. postsurgery to replenish for the Laboratory Animal Care and Use Committee. Asc and Nlrp3 mice fluid loss. were provided by Millenium Pharmaceuticals (San Diego, CA). Animals were handled according to the standards of the Association for the Assess- Measurement of renal function 2/2 ment and Accreditation of Laboratory Animal Care. C57BL/6, IL-1R , by guest on September 30, 2021 and IL-182/2 mice were obtained from The Jackson Laboratory (Bar Har- Measurement of renal function was conducted at indicated times following bor, ME). Caspase 12/2 mice were obtained from R. Ulevitch (The Scripps reperfusion. In all cases, the mice were anesthetized prior to sacrifice, and Research Institute). All mice used in these experiments had been bred onto blood was collected from the inferior vena cava into a syringe preloaded a C57BL/6 background by .10 generations. with 3.8% sodium citrate. Sham controls were also anesthetized prior to sacrifice and blood collected in an identical manner. Plasma was isolated by Detection of Asc and Nlrp3 in wild-type mice and human cells centrifugation at 4000 3 g for 10 min at 4˚C. Renal function was assessed using the Sigma Diagnostics kit (Sigma-Aldrich), running all Asc and Nlrp3 mRNA were detected in renal tubular epithelium (RTE) or samples in triplicates, and repeating measurements three times for each lymph node (LN) cells by semiquantitative PCR. To isolate and detect Asc sample. Baseline (2 wk prelaparotomy) and terminal (at the time of sac- and Nlrp3 from murine RTE, the same protocol was used as noted below rifice) plasma creatinines were measured in all animals. (isolation of human RTE), except that collagenase D was used instead of collagenase A for murine RTE. Asc and Nlrp3 were also detected in pooled Histological assessments LN cellsfromdissectedinguinalandaxillary LNs.ThefirststrandofcDNAof each sample was synthesized from 1 mg total RNA using a QuantitectRT kit To assess renal histology, kidneys were harvested 24 h after kidney IR injury (Qiagen, Valencia, CA) according to the manufacturer’s instructions. For or sham surgery, fixed in a solution of zinc formalin, and paraffin embedded. RT-PCR, 1 mg total cellular RNA was reverse transcribed and cDNA am- Sections (4 mm) were stained with H&E or periodic acid-Schiff stain. The plified. The expression of Asc and Nlrp3 mRNA in murine RTE was tissue slides were blind labeled and reviewed by the renal pathologist who detected by semiquantitative PCR with 59-GCAGCGCATCGCCTTCT- had no knowledge of the experimental groups. A histological scoring sys- ATC-39 as a forward primer and 59-GTCCATCACCAAGTAGGGATG-39 tem adapted from Kelly et al. (34) was used. The percentage of tubules in as a reverse primer for Asc mRNA and with 59-GCAGCGCATCGC- the outer medulla that showed epithelial cell was estimated and CTTCTATC-39 as a forward primer and 59-CGGTGGTTGCTAGGAGA- a numerical score assigned to represent degree of injury: 0 (no injury); 1 TGG-39 as a reverse primer for Nlrp3 mRNA. The level of GADPH mRNA in (0–10%); 2 (11–20%); 3 (21–40%); 4 (41–60%); 5 (61–75%); and 6 each sample was also determined by PCR using 59-GGTCTTACTCCTTG- (.75%) tubules injured. The scores represented the pathologist’s impres- GAGGCCATGT-39 as a forward primer and 59-GACCCCTTCATTGAC- sion of the severity of tubular injury (including loss of CTCAACTACA-39 as a reverse primer. For detection of Asc in human RTE brush border, cell blebbing, or vacuolization, and/or cell necrosis): the score the forward primer was 59-GCCGAGGAGCTCAAGAAGTT-39 and reverse ranges of 1 to 2 represented mild injury, 3 to 4 represented moderate injury, was 59-TCCTCCACCAGGTAGGACTG-39. For detection of Nlrp3 in and 5 to 6 represented severe injury. Infiltrating were counted HEK293, Thp1 and human RTE cells the forward primer was 59-AAAAG- at an original magnification of 3400. In the regions containing neutrophils, ACTCATCCGTGTGCC-39 and reverse 59-TTCCTGGCATATCACAGT- 10 fields were counted to derive the extent of infiltration per field GG-39. The GADPH housekeeping gene was detected in the human cells (0 to 1 represented little or no infiltration; 1 to 2 moderate infiltration; and by forward primer 59-CACACCATCTTCCAGGAGC-39 and reverse primer 3+ extensive infiltration). 59-CATGAGTCCTTCCACGATACC-39. The PCR products were separated on a 1% agarose gel and digital photographs taken on a transilluminator. Detection of cytokines Isolation of human RTE For cytokine/chemokine measurements, animals were sacrificed at in- dicated times after ischemic injury, a sample of blood was taken from the Human kidney tissue was obtained from discarded nephrectomies for inferior vena cava, and then kidneys harvested and snap-frozen in liquid renal cell carcinoma (IRB:08-5054, Scripps Clinic and Green Hospital, La nitrogen. The kidneys were homogenized in extraction buffer (10 mM Na The Journal of Immunology 6279 phosphate [pH 7.5], 0.1 mM amino caproic acid, 10 U/ml heparin Na salt, irradiated mice. The mice were kept in microisolator cages for 6 wk to complete protease inhibitor tablet) for 30 s and incubated overnight at 4˚C complete engraftment with donor bone marrow and were given tri- on a rotator. Supernatants were collected postcentrifugation at 10,000 3 g methoprim-sulfa–enriched water until induction of IRI. Confirmation of for 10 min at 4˚C. Cytokine and chemokine levels were measured in tissue Asc2/2 or Nlrp32/2 engraftment after bone marrow transplantation was homogenates using commercial ELISA kits (R&D Systems, Minneapolis, performed on bone marrow cells of chimeric WT mice engrafted with either MN) according to the manufacturer’s instructions. The detection limits in Nlrp32/2 bone marrow or WT bone marrow. To detect the Nlrp3 mutation, tissue were 16 pg/ml for IL-1b. Plasma concentrations of mouse IL-1b and the WT forward and reverse primers were 59-GCTGTGCCCAGGTCC- IL-18 were measured using ELISA kits (limited to 16 pg/ml for IL-1 b and 26 TAGC-39 and 59-CAGCAGCAGCCCTTTCGAG-39, respectively, and the pg/ml for IL-18). To control for the ability to produce the cytokines, wild- KO allele forward and reverse primers were 59-GCAGCGCATCGCC- type (WT) and Nlrp32/2 mice were injected i.p. with LPS from Salmonella TTCTATC-39 and 59-CGGTGGTTGCTAGGAGATGG-39, respectively. To minnesota R595 (Alexis Biochemicals, Enzo Life Sciences International, detect the Asc mutation, the WT forward and reverse primers were 59- Plymouth Meeting, PA) at a concentration of 10 mg/kg body weight. WTand GTGGACGGAGTGCTGGATG-39 and 59-GTCCATCACCAAGTAGGGA- Nlrp32/2 serum and kidneys were harvested at serial time points. Non- TG-39, respectively, and the KO allele forward and reverse primers were manipulated controls were also sacrificed to compare baseline cytokine/ 59-GCAGCGCATCGCCTTCTATC-39 and 59-GTCCATCACCAAGTAG- chemokine levels. GGATG-39, respectively. Detection of active caspase 1 and mature IL-1b Apoptosis To detect the Nlrp3 targets, caspase 1 and IL-1b, and to assess whether they To evaluate for evidence of apoptosis in renal cortical tissue after renal IRI, were activated in WT and/or Nlrp32/2 mice after IRI, whole kidneys were tissue cryosections (6 mm) were assayed with the TUNEL reaction using harvested immediately following 25 min bilateral renal artery clamping/ the TUNEL assay as provided in the fluorescein FragEL DNA Fragmen- 24 h reperfusion. The kidneys were then lysed in a buffer containing 60 tation detection kit (Calbiochem, San Diego, CA). Cryosections were fixed mM Tris, 10% glycerol, and 2% SDS and protease inhibitors. The lysis in 4% formaldehyde and permeabilized in proteinase K (in 10 mM Tris supernatants were then resolved by SDS-PAGE, transferred to nitrocellu- [pH 8]). The sections were then exposed to the TdT equilibration buffer Downloaded from lose, and immunoblotted with anti-caspase 1 Ab, anti-cleaved IL-1b Ab, or and the free 39-hydroxyl groups of DNA labeled with the fluorescein- anti–hsp-90 loading control (all Abs from Cell Signaling Technology, conjugated deoxynucleotides. Nuclear staining was identified in cell nu- Danvers, MA). The blots were then developed by the ECL system clei with DAPI and DNA breakage imaged by fluorescence microscopy. (Amersham Biosciences, Louisville, CO). Eight random nonoverlapping sections were viewed and counted under a grid at 3200 original magnification. All tissue sections were blind la- Blockade of IL-1 and IL-18 beled and viewed without knowledge of experimental groups. The effect of IL-1 blockade on renal IRI was tested in two ways. The first http://www.jimmunol.org/ employed the IL-1R antagonist anakinra (Kineret, Amgen, Thousand Oaks, Results CA). Anakinra (25 mg/kg) was injected i.p. into WT mice at two intervals Nlrp3 and Asc are expressed in murine and human kidneys [upon induction of anesthesia, and at 6 h postoperatively, following published dosing and interval recommendations (35, 36)]. To assess the potential dose/ A pathognomonic finding of renal IRI is renal tubular epithelial interval dependence, anakinra was also given over a range doses from 1–150 injury (37, 38). To evaluate the role of Nlrp3 and Asc in kidney IRI, mg/kg and a range of administration times (from 6 h presurgery to 12 h postsurgery). Higher/lower and/or more frequent anakinra dosing did not we first asked whether the Nlrp3 and Asc genes were present in show any different results from used regimen. The second method to test for renal tubular epithelial cells of murine and human kidneys. As seen the effect of IL-1 blockade was by inducing IRI in IL-1R2/2 mice using in Fig. 1, using semiquantitative PCR, both Nlrp3 and Asc were 2/2 methodology described above (see Induction of in vivo IRI). IL-18 mice well expressed in renal tubular epithelial cells in the two species. by guest on September 30, 2021 were used to test the effect of IL-18 deficiency in renal IRI. These genes were also expressed in murine lymphocytes and in Bone marrow transplantation the human monocytic cell line Thp1, but not in nontransfected 2 2 HEK cells, as previously described (1, 5, 39, 40). To evaluate the role of inflammation in kidney IR injury, Nlrp3 / versus WT bone marrow was transplanted into WT mice. Male WT mice were lethally Contribution of Nlrp3, Asc, and caspase 1 to kidney injury irradiated with two doses of 5 Gy, separated by 3 h, using a 137Cs Gam- following ischemia and reperfusion macell 40 Extractor irradiator (MDS Nordion, Ottawa, Ontario, Canada). 2 2 The next day, bone marrow was collected from WT or Nlrp3 / mice by Because Nlrp3 and Asc were highly expressed in mouse and human flushing femurs and tibia with sterile 10K media (RPMI 1640 containing L- renal tubular epithelial cells, the cells most susceptible to renal glutamine [2 mM], FBS [10%], penicillin [100 U/ml], streptomycin [100 mg/ml], 2-ME [0.05 mM], HEPES [10 mM]). The bone marrow cells were , the participation of these inflammasome component pro- then washed with sterile PBS and 5 3 106 WT or Nlrp32/2 bone teins in responses to acute kidney injury were tested in a well- marrow cells in sterile PBS were injected into the tail vein of recipient characterized model of murine kidney IRI. Using WT and mutant

FIGURE 1. Nlrp3 and Asc in murine and human cells. The figure shows three panels depicting the Nlrp3 (left panel), Asc (middle panel), or house- keeping GADPH (right panel) genes. Each panel illustrates gene expression detected by semiquantitative PCR in the RTE of Nlrp32/2 mice (Nlrp32/2 mRTE), wild-type (WTmRTE) mice, lymph node cells from WT mice (WTLN), nontransfected HEK 293 cells (HEK), Thp1 cells (Thp1), or human RTE cells extracted from normal human kidneys (hRTE). The results are representative of four mice per group and five different human kidneys extracted for renal tubular cells with identical results. 6280 INFLAMMASOME-INDEPENDENT ROLE FOR Nlrp3 IN RENAL IRI

Fig. 2 shows plasma creatinine levels 2 wk preinjury for WT mice and 24 h postinjury for each indicated group of mice. Pre- injury and sham creatinines are listed in the figure legend for Asc2/2, caspase 12/2, and Nlrp32/2 mice; no differences were observed between any of the groups. As seen in Fig. 2, there were no significant differences in postinjury creatinines between WT and Asc2/2 mice (p , 0.05) or between WT versus caspase 12/2 mice (p , 0.05). However, an extremely significant reduction in serum creatinine was noted in IR-treated Nlrp32/2 mice (p , 0.001), suggesting greater renal protection was afforded by a de- ficiency of Nlrp3 than by a deficiency in the protein Asc, which collaborates with Nlrp3 to form the inflammasome. Confirming that Nlrp3 can also signal independently of the inflammasome, there was no protection from renal IRI by deletion of caspase 1, FIGURE 2. Kidney IRI in WT, NLR, and caspase 1-deficient mice. WT, 2/2 2/2 2/2 the downstream signaling target of this inflammasome. Asc , caspase 1 , or Nlrp3 mice were subjected to 25 min is- Acute tubular necrosis in each group of mice was evaluated by chemia/24 h reperfusion and plasma obtained at 24 h to detect creatinine 2 2 2 2 2 2 a pathologist blinded to the experimental groups (Fig. 3A). Using from injured WT, Asc / , caspase 1 / , and Nlrp3 / mice (n = 6 mice/ group). Preinjury creatinines were obtained 2 wk earlier on each of the a modified standard scoring system (21), necrosis was scored 24 h experimental mice: WT is shown in the figure (pre); Asc2/2 (0.42 6 0.01); after the initial ischemic insult (Fig. 3A). The means of each group Downloaded from caspase 12/2 (0.47 6 0.01); and Nlrp32/2 (0.48 6 0.04). Creatinines were are shown in the figure legend. As seen in the top panel of Fig. 3A, 2 2 2/2 also obtained from sham-operated mice: WT (0.4 6 0.1); Asc / (0.3 6 there was no difference in necrosis between WT, caspase 1 2 2 0.1); caspase 12/2 (0.4 6 0.2); and Nlrp32/2 (0.3 6 0.2). Error bars (p . 0.05), and Asc / mice (p . 0.05), but a significant re- represent SD of creatinines, and statistical significance was determined duction in tubular necrosis was seen between WT mice and mice with a one-way ANOVA with Bonferroni’s multiple comparison test. The deficient in Nlrp3 (p , 0.01). A representative photomicrograph is data represent one of five identical experiments with the same results. shown for each of the groups below Fig. 3A. Neutrophil infiltration http://www.jimmunol.org/ was also assessed in each group, in a blinded manner, by the mice, bred for .10 generations to the same genetic background pathologist (Fig. 3B). There were no significant differences in 2/2 (C57BL/6), nonlethal renal IRI was induced in WT, Asc , and neutrophil infiltration noted between any of the groups. Nlrp32/2 mice using our previously well-characterized model b 2/2 (21). Upon ligand recognition, Nlrp3 and Asc form a multimo- IL-18 and IL-1 secretion in WT and Nlrp3 mice after lecular complex that is critical for triggering the activation of cas- kidney IRI pase 1 (41). Therefore, we tested whether a deficiency in caspase 1 Protection from kidney injury following IRI was most prominent in 2 2 influenced kidney IRI using caspase 1 / mice. mice lacking the innate sensor molecule Nlrp3. As such, postinjury by guest on September 30, 2021

FIGURE 3. Histological renal injury after bilateral renal artery clamping. A shows blinded scoring of tu- bular necrosis from WT (n = 6), caspase 12/2 (n = 6), Asc2/2 (n = 6), Nlrp32/2 (n = 6), and sham-operated (n = 3) mice 24 h after bilateral renal artery clamping (25 min ischemia/24 h reperfusion). The top panel shows the blinded necrosis score (see Materials and Methods for details), and the bottom panel shows a representative micrograph of each group of mice (periodic acid-Schiff, 3200 original magnification). Means of necrosis scores were as follows: WT (5.0 6 1.1); caspase 12/2 (4.7 6 1.0); Asc2/2 (3.7 6 2.2); and Nlrp32/2 (2.3 6 0.8). There were no statistical differ- ences between WT versus caspase 12/2 (p . 0.50) or WT versus Asc2/2 (p . 0.50), but significant differ- ences in histological injury were observed between WT versus Nlrp32/2 (p , 0.01) mice. B shows blinded scores of neutrophil infiltration on the same sections (see Materials and Methods). Means of neutrophil scores were: WT (2.1 6 0.8); caspase 12/2 (1.8 6 1.3); Asc2/2 (1.6 6 1.1); and Nlrp32/2 (1.4 6 0.7). No statistical differences in neutrophil infiltration were seen between the groups: WT versus caspase 12/2 (p . 0.05), versus Asc2/2 (p . 0.05), or versus Nlrp32/2 (p . 0.05). Statistical significance was determined by a one-way ANOVA with Bonferroni’s multiple com- parison test. The Journal of Immunology 6281

IL-18 and IL-1b secretion was compared and contrasted between IL-1R antagonist prior to renal artery clamping. IL-1–targeted WT and Nlrp32/2 mice (Fig. 4). Fig. 4A shows there was signifi- therapy is highly useful for treatment of several disorders asso- cantly less IL-18 and IL-1b produced after kidney IRI in the ciated with mutations in Nlrp3 (10, 15–20). Shown in Fig. 5A, Nlrp32/2 than WT mice. In the bottom panels of Fig. 4A,LPS there were no significant differences in creatinine levels after renal treatment indicated that, as expected, mice deficient in Nlrp3 were IRI in unmanipulated WT mice compared with WT mice treated unable to secrete the inflammasome-dependent cytokines IL-18 with the IL-1Ra anakinra (p = 0.13). A wide range of anakinra or IL-1b. Fig. 4B shows that mature IL-1b and activated caspase 1 doses were compared and contrasted (see Materials and Methods), was detected in WT kidneys but not in Nlrp32/2 kidneys subjected and there was no renal protection afforded by any of the tested to renal IRI. The fact that Nlrp32/2 mice were not able to activate doses of anakinra (data not shown). In Fig. 5B, a slight, but caspase 1 or produce mature IL-1b following IRI confirms that nonsignificant, decrease in renal injury in IL-1R2/2 mice was Nlrp3 is necessary for inflammasome-mediated proinflammatory noted compared with unmanipulated WT mice (p . 0.05), but no cytokine production in vivo. But, as seen below in Fig. 5, the renal differences were seen in histology score or neutrophil infiltrates protection conferred by the deficiency in Nlrp3 is independent of its (data not shown). In Fig. 5C, there were no differences in crea- role in inflammasome-induced proinflammatory cytokine secretion. tinines of WT mice versus IL-182/2 mice (p . 0.05) treated with bilateral renal artery clamping, consistent with prior reports of the IL-18 and IL-1R blockade and kidney IRI ineffectiveness of IL-18 deficiency on renal IRI (42). b Because Nlrp3-deficient mice did not produce IL-18 or IL-1 , the 2/2 2/2 experiments shown in Fig. 5 were performed to determine whether Inflammation in WT, Asc , and Nlrp3 mice after renal IRI protection afforded by the absence of Nlrp3 was mediated through To determine whether the absence of Nlrp3 protected kidneys Downloaded from these cytokines. In Fig. 5A, WT mice were pretreated with an from IRI by abrogating hematopoietic cell-mediated inflammation, http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. Cytokine secretion after ischemia reper- fusion or injection of LPS. A shows WT or Nlrp32/2 mice (n = 6/group) subjected to 25 min ischemia and indicated times of reperfusion. At the indicated time points, plasma (for IL-18) or kidney homogenates (for IL-1 b) was assayed for the cytokines (top panels, IRI). Bottom panels show plasma cytokine secretion over time after LPS injection in WT (d) or Nlrp32/2 (:) mice (n = 6/group). The data are representative of three experiments with the same results. Error bars repre- sent standard deviations. B shows IL-1b and caspase 1 in WT versus Nlrp32/2 kidneys from sham-operated (sham) or IRI mice. The top panel shows mature IL-1b (mature IL-1b identified by the p17 fragment). The middle panel shows caspase 1 (active subunit p20), and the lower panel shows hsp-90 for a loading control. This represents one of three experiments with identical results. 6282 INFLAMMASOME-INDEPENDENT ROLE FOR Nlrp3 IN RENAL IRI

that in addition to the protection from necrosis, the absence of Nlrp3 also protected the kidney from renal tubular apoptosis in our model.

Discussion Renal IRI is a common clinical problem complicated by substantial morbidity and mortality; thus, tremendous interest has evolved in putative triggers of the renal injury. The pathognomonic features of renal IRI include hypoxia-induced tubular epithelial damage fol- lowed by postreperfusion inflammation. Multiple cell types and cell signals contribute to the syndrome, but tubular epithelial apoptosis and necrosis are predominant findings, particularly in the ischemic phases of injury. Many recent studies have pointed to innate pattern recognition receptors as important triggers of tissue injury induced by ischemia (21, 29). Because apoptosis and inflammation are essential com- ponents of kidney IRI, our study focused on a recently identified intracellular innate immune receptor, Nlrp3, known to play a role

in apoptosis and inflammatory responses to cellular injury. Downloaded from Within the cytoplasm, Nlrp3 forms a complex with Asc and procaspase 1 called the Nlrp3 inflammasome. Recent studies have suggested that inflammasome components may signal death path- FIGURE 5. IL-1 and IL-18 blockade and kidney IRI. A shows WT, WT ways independent from the inflammasome (44). We investigated 2/2 mice treated with anakinra (per Materials and Methods), or IL-1R mice the dependence of Nlrp3 on the intact inflammasome in vivo by that were treated with 25 min ischemia/24 h reperfusion followed by plasma testing the inflammasome components, Asc and Nlrp3, as well as http://www.jimmunol.org/ creatinine determination at 24 h. Sham mice underwent laparotomy without the downstream targets caspase 1, IL-1b, and IL-18 in our well- bilateral renal artery clamping. These data represent six mice per group. There were no significant differences between WT versus anakinra (p . characterized ischemic kidney injury model. 0.05) and WT versus IL-1R2/2 (p . 0.05) groups. Error bars represent Examining murine and human renal tubular epithelial cells, we SDs. B shows blinded necrosis scores of the IR-injured mice. The means of found that Nlrp3 and Asc were expressed in both species and, necrosis score were: WT (4.2 6 0.8), anakinra (4.5 6 0.8), and IL-1R2/2 importantly, within tissue highly susceptible to renal IRI/renal (4.0 6 0.6). There were no statistical differences in necrosis scores: WT tubular epithelium. These data are consistent with reports of in- 2 2 versus anakinra (p . 0.05) versus IL-1R / (p . 0.05). The means of nate immune receptor expression in many different cell types and in neutrophil infiltration were: WT (2.3 6 0.8), anakinra (1.7 6 0.5), IL- diverse species (45–52), suggesting these phylogenetically con- 2/2

6 . by guest on September 30, 2021 1R (2.0 0.6); statistical differences: WT versus anakinra (p 0.05) served proteins are involved in the response to endogenous stress. 2/2 . versus IL-1R (p 0.05). Statistical significance was determined by To determine whether Nlrp3 plays an important role in tissue a one-way ANOVA with Bonferroni’s multiple comparison test. responses to renal ischemia, we compared and contrasted injury responses in WT and inflammasome-deficient mice using a well- WT mice were transplanted with bone marrow from WT, Asc2/2, characterized model of renal IRI. Interestingly, despite the pres- or Nlrp32/2 mice, and renal IRI was induced (Fig. 6). As shown ence of both Nlrp3 and Asc in the cells most susceptible to IRI, only in Fig. 6A, there were no differences in creatinine levels after IRI the Nlrp3 deficiency offered protection from tissue injury. Nlrp3 is between the WT mice transplanted with WT versus Asc2/2 (p . well known to respond to a wide variety of structurally different 0.05) or Nlrp32/2 (p . 0.05) bone marrow. Confirming that there ligands, positioning it as a key sensor for cellular injury (53). Our 2/2 2/2 2/2 were no differences in tubular necrosis or inflammation between data showing that Nlrp3 mice, but not Asc or capsase 1 the two groups, blinded necrosis scores (Fig. 6B) and neutrophil mice, were protected suggest that Nlrp3 might be uncoupled from counts (Fig. 6C) showed no differences between the WT and Asc, and from the inflammasome, in renal tubular epithelium. 2/2 chimeric mice (Fig. 6B). Fig. 6D confirms that the WT bone Consistent with our data, Nlrp3 mice have been found to be marrow was indeed replaced by the mutant marrow in the chi- protected from renal IRI in another model, but these investigators 2/2 meric mice. Irradiated mutant mice were noted to have severe also found that Asc mice were protected (54). The mice used weight loss, and therefore, reverse bone marrow transplants were in the study were backcrossed onto C57BL/6 mice for only four not performed until further studies could determine the cause of generations, and therefore, it is possible that the founder strain the irradiation-induced weight loss in the mutant mice. might account for the variance in findings. We have carefully confirmed that our mice are on a C57BL/6 background by absence Apoptosis and renal IRI of rejection of skin grafts from the mutant mice on the WT mice. An early histological finding of kidney IRI is apoptosis of renal Because the absence of Nlrp3, but not Asc or caspase 1, pro- tubular cells; necrosis follows longer ischemic intervals (43). To tected from renal injury, our data strongly suggest an inflam- determine whether Nlrp3 contributed to renal tubular apoptosis in masome-independent role for Nlrp3. The lack of protection from our IRI model, kidney sections were examined for evidence of injury we observed in caspase 1-knockout mice is supported by apoptosis in situ by the TUNEL assay. We have previously shown studies of others using a similar model of renal ischemia (55). that maximal apoptosis occurs at 4 h postinjury in this model Activation of the Nlrp3 inflammasome is known to cause caspase (29) and so focused on this interval, comparing results in WT ver- 1 activation and cleavage of pro–IL-1b and pro–IL-18 into their 2 2 2 2 sus Asc / or Nlrp3 / kidneys. As shown in Fig. 7, there were active, mature forms (4, 6). Consistent with this, we found that 2 2 significant differences in apoptosis between WT and Nlrp3 / a deficiency in Nlrp3 blocked IL-1b production, as well as IL-18 mice (p , 0.001), whereas there were no differences between IRI- production. But, blockade of IL-1 and IL-18 (with either anakinra 2 2 treated WT and Asc / kidneys (p . 0.05). These data suggest or with IL-1R2/2 or IL-182/2 mice) had no significant effect The Journal of Immunology 6283 Downloaded from

FIGURE 6. Bone marrow chimeric mice subjected to IRI. A shows WT mice that were irradiated and injected with WT (n = 6), Asc2/2 (n = 6), or Nlrp32/2 (n = 6) bone marrow 6 wk earlier. Postengraftment, the three groups of mice were treated with 25 min bilateral renal artery clamping followed by 24 h reperfusion. Precreatinine values were obtained 2 wk preinjury (pre). Sham creatinines were obtained from mice that underwent laparotomy without http://www.jimmunol.org/ undergoing bilateral renal artery clamping (n = 3). Error bars represent SDs. B shows the necrosis score of the injured animals. The means of scores were: WT (4.3 6 0.8), Asc2/2 (4.0 6 1.1), Nlrp32/2 (4.3 6 0.8); and statistical differences: Wt versus Asc2/2 (p . 0.05) versus Nlrp32/2 (p . 0.05). C shows the scores of neutrophil infiltration: WT (2.8 6 0.4), Asc2/2 (2.5 6 0.5), Nlrp32/2 (2.3 6 0.8); and the statistical differences were: WT versus Asc2/2 (p . 0.05) and WT versus Nlrp32/2 (p . 0.05). Statistical significance was obtained by a one-way ANOVAwith Bonferroni’s multiple comparison test. D shows that chimeric bone marrow was engrafted in the indicated experimental groups. The control panels show the Nlrp3 knockout allele (indicating the Nlrp3 gene) was present in the Nlrp32/2 mice, but not in the WT or the Asc2/2 mice (controls). The WT mice transplanted with the Nlrp32/2 bone marrow show that the Nlrp3 knockout allele was highly expressed (bone marrow chimeras, Nlrp32/2 → WT). Likewise, the Asc knockout allele was expressed in Asc2/2 controls but not in the WT controls and also highly expressed in the mice transplanted with Asc2/2 bone marrow (bone marrow chimeras, Asc2/2 →

WT). by guest on September 30, 2021 on renal injury responses, as previously reported (56). Others have generally involve direct induction of cell death pathways in tubular reported that blockade of IL-18 provides either minimal (57) or epithelia (58), glomerular injury, renal endothelial injury and ex- no (42) protection from renal IRI. Therefore, Nlrp3 does not pression of adhesion molecules (59), and leukocyte recruitment likely contribute to renal injury primarily through inflammasome- and activation (60). Nlrp3 has been detected in the cytosol of var- mediated IL-1b or IL-18 production. Our data suggest that Nlrp3, ious immune cells, such as T cells, macrophages, and dendritic rather than Asc, drives responses to tissue injury in this model cells (49), and we thought it possible that Nlrp3 might contribute and suggests activation of Nlrp3 can induce inflammasome- to renal injury through inflammation initiated by hematopoietic- independent responses in renal epithelial cells. derived immune cells. However, WT mice engrafted with Nlrp32/2 The pathogenesis of kidney IR is complex, and to date, many bone marrow were not protected from injury. Although Nlrp32/2 causative mechanisms have been proposed. These mechanisms mice do not produce IL-1b or IL-18 (neutrophil chemotaxins),

FIGURE 7. Apoptosis of RTE cells after renal IRI. WT versus Nlrp32/2, Asc2/2,or sham mice were treated with 25 min of bi- lateral renal artery clamping followed by 4 h of reperfusion. The mice were sacrificed and their kidneys removed and placed into for- malin for paraffin embedding and TUNEL assay. A shows representative images of TUNEL and DAPI (Nuclei) staining 4 h postreperfusion. Original magnification 3200. B shows results of blinded counting of TUNEL-positive cells, 4 h postreperfusion, in nine random, nonoverlapping sections (n = 3 mice/group). Data are expressed as means 6 SD. Statistical significance was determined by a one-way ANOVA with Bonferroni’s multiple comparison test. 6284 INFLAMMASOME-INDEPENDENT ROLE FOR Nlrp3 IN RENAL IRI neutrophils are still recruited to the injured tissue. Therefore, future recruitment domain is a regulator of procaspase-1 activation. J. Immunol. 171: 6154–6163. studies will need to be conducted to completely exclude any role 6. Martinon, F., K. Burns, and J. Tschopp. 2002. The inflammasome: a molecular for Nlrp3 in activation of specific innate and adaptive inflammatory platform triggering activation of inflammatory caspases and processing of proIL- mediators to ischemic renal tissue in our model. A major role, beta. Mol. Cell 10: 417–426. 7. Kanneganti, T. D., M. Body-Malapel, A. Amer, J. H. Park, J. Whitfield, though, for Nlrp3-mediated inflammation does not appear to be L. Franchi, Z. F. Taraporewala, D. Miller, J. T. Patton, N. Inohara, and G. Nu´n˜ez. relevant from these data. 2006. Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to The most susceptible region of the kidney to ischemia appears to viral infection and double-stranded RNA. J. Biol. Chem. 281: 36560–36568. 8. Kanneganti, T. D., N. Ozo¨ren, M. Body-Malapel, A. Amer, J. H. Park, be the renal tubular epithelium located in the outer stripe of the L. Franchi, J. Whitfield, W. Barchet, M. Colonna, P. Vandenabeele, et al. 2006. medulla (37, 38). It is thought that ischemia-related reduction in Bacterial RNA and small antiviral compounds activate caspase-1 through cry- microvascular blood flow to this region of the kidney coupled with opyrin/Nalp3. Nature 440: 233–236. 9. Pe´trilli, V., C. Dostert, D. A. Muruve, and J. Tschopp. 2007. The inflammasome: a limited capacity of the proximal tubule cells to undergo anaer- a danger sensing complex triggering innate immunity. Curr. Opin. Immunol. 19: obic metabolism makes this region particularly susceptible. In 615–622. 10. Martinon, F., V. Pe´trilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Gout- fact, the most prominent histologic findings are the loss of the associated uric acid crystals activate the NALP3 inflammasome. Nature 440: periodic acid-Schiff–positive brush border of the proximal tubular 237–241. epithelium and scattered single-cell necrosis or desquamation of 11. Chu, J., L. M. Thomas, S. C. Watkins, L. Franchi, G. Nu´n˜ez, and R. D. Salter. 2009. Cholesterol-dependent cytolysins induce rapid release of mature IL-1beta proximal tubular cells with intact basement membranes (61). In- from murine macrophages in a NLRP3 inflammasome and cathepsin B- terestingly, those proximal tubular epithelial cells that do not dependent manner. J. Leukoc. Biol. 86: 1227–1238. undergo apoptosis/necrosis (e.g., the surviving proximal tubular 12. Feldmann, J., A. M. Prieur, P. Quartier, P. Berquin, S. Certain, E. Cortis, D. Teillac-Hamel, A. Fischer, and G. de Saint Basile. 2002. Chronic infantile epithelial cells) divide and repopulate the tubular epithelium, neurological cutaneous and articular syndrome is caused by mutations in CIAS1, Downloaded from thereby making kidney IRI a reversible disorder, unless all of the a gene highly expressed in polymorphonuclear cells and chondrocytes. Am. J. epithelium is destroyed by the ischemia (62). Significantly less Hum. Genet. 71: 198–203. 2/2 13. Aksentijevich, I., M. Nowak, M. Mallah, J. J. Chae, W. T. Watford, tubular necrosis was observed in Nlrp3 mice than in WT, S. R. Hofmann, L. Stein, R. Russo, D. Goldsmith, P. Dent, et al. 2002. De novo 2 2 2 2 Asc / , or caspase 1 / mice following renal IRI, consistent with CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in the known role for Nlrp3 as a mediator of necrosis in monocytes patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. and macrophages (63). Interestingly, significantly less renal tu- Arthritis Rheum. 46: 3340–3348. http://www.jimmunol.org/ bular apoptosis was also seen in the Nlrp3-deficient mice sub- 14. Hull, K. M., N. Shoham, J. J. Chae, I. Aksentijevich, and D. L. Kastner. 2003. The expanding spectrum of systemic autoinflammatory disorders and their jected to renal IRI compared with renal IRI treated WT or Asc- rheumatic manifestations. Curr. Opin. Rheumatol. 15: 61–69. deficient mice. These data suggest that Nlrp3 might contribute to 15. Hawkins, P. N., H. J. Lachmann, and M. F. McDermott. 2003. -1- renal injury following ischemia/reperfusion by inducing apoptosis receptor antagonist in the Muckle-Wells syndrome. N. Engl. J. Med. 348: 2583– 2584. within a few hours of the ischemic insult. Future studies are on- 16. Yamazaki, T., J. Masumoto, K. Agematsu, N. Sawai, S. Kobayashi, going in our laboratory to identify Nlrp3-dependent signaling T. Shigemura, K. Yasui, and K. Koike. 2008. Anakinra improves sensory deaf- events that contribute to renal tubular necrosis and apoptosis ness in a Japanese patient with Muckle-Wells syndrome, possibly by inhibiting the cryopyrin inflammasome. Arthritis Rheum. 58: 864–868. following IRI. 17. Mirault, T., D. Launay, L. Cuisset, E. Hachulla, M. Lambert, V. Queyrel, Our data point to a role for Nlrp3 in the kidney that is in- T. Quemeneur, S. Morell-Dubois, and P. Y. Hatron. 2006. Recovery from by guest on September 30, 2021 dependent of the inflammasome. Future studies will need to focus deafness in a patient with Muckle-Wells syndrome treated with anakinra. Ar- thritis Rheum. 54: 1697–1700. on additional direct injury mechanisms that might be playing 18. Rynne, M., C. Maclean, A. Bybee, M. F. McDermott, and P. Emery. 2006. a role in Nlrp3-medated protection. Our study is the first to our Hearing improvement in a patient with variant Muckle-Wells syndrome in re- sponse to interleukin 1 receptor antagonism. Ann. Rheum. Dis. 65: 533–534. knowledge to show that in renal epithelium Nlrp3 can signal in- 19. Hoffman, H. M., S. Rosengren, D. L. Boyle, J. Y. Cho, J. Nayar, J. L. Mueller, jury responses independent of the inflammasome and independent J. P. Anderson, A. A. Wanderer, and G. S. Firestein. 2004. Prevention of cold- of proinflammatory cytokines/chemokines. associated acute inflammation in familial cold autoinflammatory syndrome by interleukin-1 receptor antagonist. Lancet 364: 1779–1785. 20. Granel, B., J. Serratrice, P. Disdier, and P. J. Weiller. 2005. Dramatic improve- Acknowledgments ment with anakinra in a case of chronic infantile neurological cutaneous and We thank Susan Abel for assistance with the acquisition of human tissue articular (CINCA) syndrome. Rheumatology (Oxford) 44: 689–690. 21. Shigeoka, A. A., T. D. Holscher, A. J. King, F. W. Hall, W. B. Kiosses, for extraction of renal tubular epithelium and Terri Thinnes for technical P. S. Tobias, N. Mackman, and D. B. McKay. 2007. TLR2 is constitutively help with the caspase 1-deficient mice. We also thank John Bertin, Anthony expressed within the kidney and participates in ischemic renal injury through Coyle, and Ethan Grant, formerly of Millenium Pharmaceuticals, for pro- both MyD88-dependent and -independent pathways. J. Immunol. 178: 6252– viding the Nrlp3 and Asc knockout mice and Matthew McGeough and 6258. 22. Shirali, A. C., and D. R. Goldstein. 2008. Tracking the toll of kidney disease. J. Carla Pena for mouse technical support. Am. Soc. Nephrol. 19: 1444–1450. 23. Frantz, S., R. A. Kelly, and T. Bourcier. 2001. Role of TLR-2 in the activation of Disclosures nuclear factor kappaB by in cardiac myocytes. J. Biol. Chem. 276: 5197–5203. The authors have no financial conflicts of interest. 24. Shen, X. D., B. Ke, Y. Zhai, F. Gao, R. W. Busuttil, G. Cheng, and J. W. Kupiec- Weglinski. 2005. Toll-like receptor and heme oxygenase-1 signaling in hepatic ischemia/reperfusion injury. Am. J. Transplant. 5: 1793–1800. References 25. Chen, L. W., W. J. Chang, P. H. Chen, W. C. Liu, and C. M. Hsu. 2008. TLR 1. Geddes, K., J. G. Magalha˜es, and S. E. Girardin. 2009. Unleashing the thera- ligand decreases mesenteric ischemia and reperfusion injury-induced gut dam- peutic potential of NOD-like receptors. Nat. Rev. Drug Discov. 8: 465–479. age through TNF-alpha signaling. Shock 30: 563–570. 2. Fritz, J. H., R. L. Ferrero, D. J. Philpott, and S. E. Girardin. 2006. Nod-like 26. Kilic, U., E. Kilic, C. M. Matter, C. L. Bassetti, and D. M. Hermann. 2008. TLR- proteins in immunity, inflammation and disease. Nat. Immunol. 7: 1250–1257. 4 deficiency protects against focal cerebral ischemia and axotomy-induced 3. Hoffman, H. M., J. L. Mueller, D. H. Broide, A. A. Wanderer, and neurodegeneration. Neurobiol. Dis. 31: 33–40. 27. Victoni, T., F. R. Coelho, A. L. Soares, A. de Freitas, T. Secher, R. Guabiraba, R. D. Kolodner. 2001. Mutation of a new gene encoding a putative pyrin-like F. Erard, R. M. de Oliveira-Filho, B. B. Vargaftig, G. Lauvaux, et al. 2010. Local protein causes familial cold autoinflammatory syndrome and Muckle-Wells and remote tissue injury upon intestinal ischemia and reperfusion depends on the syndrome. Nat. Genet. 29: 301–305. TLR/MyD88 signaling pathway. Med. Microbiol. Immunol. (Berl.) 199: 35–42. 4. Agostini, L., F. Martinon, K. Burns, M. F. McDermott, P. N. Hawkins, and 28. Hua, F., T. Ha, J. Ma, X. Gao, J. Kelley, D. L. Williams, I. W. Browder, J. Tschopp. 2004. NALP3 forms an IL-1beta-processing inflammasome with R. L. Kao, and C. Li. 2005. Blocking the MyD88-dependent pathway protects increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20: the myocardium from ischemia/reperfusion injury in rat hearts. Biochem. Bio- 319–325. phys. Res. Commun. 338: 1118–1125. 5. Stehlik, C., S. H. Lee, A. Dorfleutner, A. Stassinopoulos, J. Sagara, and 29. Shigeoka, A. A., A. Kambo, J. C. Mathison, A. J. King, W. F. Hall, J. da Silva J. C. Reed. 2003. Apoptosis-associated speck-like protein containing a caspase Correia, R. J. Ulevitch, and D. B. McKay. 2010. Nod1 and nod2 are expressed in The Journal of Immunology 6285

human and murine renal tubular epithelial cells and participate in renal ischemia 46. Lev-Sagie, A., D. Prus, I. M. Linhares, Y. Lavy, W. J. Ledger, and S. S. Witkin. reperfusion injury. J. Immunol. 184: 2297–2304. 2009. Polymorphism in a gene coding for the inflammasome component NALP3 30. Detrisac, C. J., M. A. Sens, A. J. Garvin, S. S. Spicer, and D. A. Sens. 1984. and recurrent vulvovaginal candidiasis in women with vulvar vestibulitis syn- Tissue culture of human kidney epithelial cells of proximal tubule origin. Kidney drome. Am. J. Obstet. Gynecol. 200: 303.e1–303.e6. Int. 25: 383–390. 47. Lee, P., D. J. Lee, C. Chan, S. W. Chen, I. Ch’en, and C. Jamora. 2009. Dynamic 31. McLaren, J., P. Whiting, J. Simpson, and G. Hawksworth. 1995. Isolation and expression of epidermal caspase 8 simulates a wound healing response. Nature characterisation of human proximal tubular cells derived from kidney cortical 458: 519–523. segments. Hum. Exp. Toxicol. 14: 916–922. 48. McCall, S. H., M. Sahraei, A. B. Young, C. S. Worley, J. A. Duncan, J. P. Ting, 32. Nadasdy, T., Z. Laszik, K. E. Blick, D. L. Johnson, K. Burst-Singer, C. Nast, and I. Marriott. 2008. Osteoblasts express NLRP3, a nucleotide-binding domain A. H. Cohen, J. Ormos, and F. G. Silva. 1995. Human acute tubular necrosis: and leucine-rich repeat region containing receptor implicated in bacterially a lectin and immunohistochemical study. Hum. Pathol. 26: 230–239. induced cell death. J. Bone Miner. Res. 23: 30–40. 33. Frank, R. D., G. Schabbauer, T. Holscher, Y. Sato, M. Tencati, R. Pawlinski, and 49. Kummer, J. A., R. Broekhuizen, H. Everett, L. Agostini, L. Kuijk, F. Martinon, N. Mackman. 2005. The synthetic pentasaccharide fondaparinux reduces coa- R. van Bruggen, and J. Tschopp. 2007. Inflammasome components NALP 1 and 3 gulation, inflammation and neutrophil accumulation in kidney ischemia- show distinct but separate expression profiles in human tissues suggesting a site- reperfusion injury. J. Thromb. Haemost. 3: 531–540. specific role in the inflammatory response. J. Histochem. Cytochem. 55: 443–452. 34. Kelly, K. J., W. W. Williams, Jr., R. B. Colvin, S. M. Meehan, T. A. Springer, 50. Gonza´lez-Benı´tez, J. F., M. A. Jua´rez-Verdayes, S. Rodrı´guez-Martı´nez, J. C. Gutierrez-Ramos, and J. V. Bonventre. 1996. Intercellular adhesion M. E. Cancino-Diaz, F. Garcı´a-Va´zquez, and J. C. Cancino-Diaz. 2008. The molecule-1-deficient mice are protected against ischemic renal injury. J. Clin. NALP3/Cryopyrin-inflammasome complex is expressed in LPS-induced ocular Invest. 97: 1056–1063. inflammation. Mediators Inflamm. 2008: 614345. 35. Abbate, A., F. N. Salloum, E. Vecile, A. Das, N. N. Hoke, S. Straino, 51. Rosengren, S., H. M. Hoffman, W. Bugbee, and D. L. Boyle. 2005. Expression G. G. Biondi-Zoccai, J. E. Houser, I. Z. Qureshi, E. D. Ownby, et al. 2008. and regulation of cryopyrin and related proteins in rheumatoid arthritis syno- Anakinra, a recombinant human interleukin-1 receptor antagonist, inhibits ap- vium. Ann. Rheum. Dis. 64: 708–714. optosis in experimental acute myocardial . Circulation 117: 2670– 52. Anderson, J. P., J. L. Mueller, S. Rosengren, D. L. Boyle, P. Schaner, 2683. S. B. Cannon, C. S. Goodyear, and H. M. Hoffman. 2004. Structural, expression, 36. Salloum, F. N., V. Chau, A. Varma, N. N. Hoke, S. Toldo, G. G. Biondi-Zoccai, and evolutionary analysis of mouse CIAS1. Gene 338: 25–34. F. Crea, G. W. Vetrovec, and A. Abbate. 2009. Anakinra in experimental acute 53. Tschopp, J., and K. Schroder. 2010. NLRP3 inflammasome activation: The Downloaded from myocardial infarction—does dosage or duration of treatment matter? Car- convergence of multiple signalling pathways on ROS production? Nat. Rev. diovasc. Drugs Ther. 23: 129–135. Immunol. 10: 210–215. 37. Donohoe, J. F., M. A. Venkatachalam, D. B. Bernard, and N. G. Levinsky. 1978. 54. Iyer, S. S., W. P. Pulskens, J. J. Sadler, L. M. Butter, G. J. Teske, T. K. Ulland, Tubular leakage and obstruction after renal ischemia: structural-functional cor- S. C. Eisenbarth, S. Florquin, R. A. Flavell, J. C. Leemans, and F. S. Sutterwala. relations. Kidney Int. 13: 208–222. 2009. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 38. Reimer, K. A., C. E. Ganote, and R. B. Jennings. 1972. Alterations in renal inflammasome. Proc. Natl. Acad. Sci. USA 106: 20388–20393. cortex following ischemic injury. 3. Ultrastructure of proximal tubules after is- 55. Daemen, M. A., G. Denecker, C. van’t Veer, T. G. Wolfs, P. Vandenabeele, and

chemia or autolysis. Lab. Invest. 26: 347–363. W. A. Buurman. 2001. Activated caspase-1 is not a central mediator of in- http://www.jimmunol.org/ 39. Aggarwal, B. B. 2004. Nuclear factor-kappaB: the enemy within. Cancer Cell 6: flammation in the course of ischemia-reperfusion. Transplantation 71: 778–784. 203–208. 56. Haq, M., J. Norman, S. R. Saba, G. Ramirez, and H. Rabb. 1998. Role of IL-1 in 40. Hasegawa, M., R. Imamura, K. Motani, T. Nishiuchi, N. Matsumoto, renal ischemic reperfusion injury. J. Am. Soc. Nephrol. 9: 614–619. T. Kinoshita, and T. Suda. 2009. Mechanism and repertoire of ASC-mediated 57. Wu, H., M. L. Craft, P. Wang, K. R. Wyburn, G. Chen, J. Ma, B. Hambly, and gene expression. J. Immunol. 182: 7655–7662. S. J. Chadban. 2008. IL-18 contributes to renal damage after ischemia-reper- 41. Yu, H. B., and B. B. Finlay. 2008. The caspase-1 inflammasome: a pilot of innate fusion. J. Am. Soc. Nephrol. 19: 2331–2341. immune responses. Cell Host Microbe 4: 198–208. 58. Kaushal, G. P., A. G. Basnakian, and S. V. Shah. 2004. Apoptotic pathways in 42. Daemen, M. A., C. van’t Veer, T. G. Wolfs, and W. A. Buurman. 1999. Ischemia/ ischemic acute renal failure. Kidney Int. 66: 500–506. reperfusion-induced IFN-gamma up-regulation: involvement of IL-12 and IL-18. 59. Molitoris, B. A., and T. A. Sutton. 2004. Endothelial injury and dysfunction: role J. Immunol. 162: 5506–5510. in the extension phase of acute renal failure. Kidney Int. 66: 496–499. 43. Perl, M., C. S. Chung, and A. Ayala. 2005. Apoptosis. Crit. Care Med. 33(12 60. Bonventre, J. V., and A. Zuk. 2004. Ischemic acute renal failure: an in-

Suppl): S526–S529. flammatory disease? Kidney Int. 66: 480–485. by guest on September 30, 2021 44. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee, 61. Olsen, S. 1989. Acute Tubular Necrosis and Toxic Renal Injury. J.B. Lippincott, M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation of Philadelphia. the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218. 62. Bonventre, J. V. 2003. Dedifferentiation and proliferation of surviving 45. Bostanci, N., G. Emingil, B. Saygan, O. Turkoglu, G. Atilla, M. A. Curtis, and epithelial cells in acute renal failure. J. Am. Soc. Nephrol. 14(Suppl 1): S55–S61. G. N. Belibasakis. 2009. Expression and regulation of the NALP3 inflammasome 63. Ting, J. P., S. B. Willingham, and D. T. Bergstralh. 2008. NLRs at the in- complex in periodontal diseases. Clin. Exp. Immunol. 157: 415–422. tersection of cell death and immunity. Nat. Rev. Immunol. 8: 372–379. The Journal of Immunology

Corrections

Shigeoka, A. A., J. L. Mueller, A. Kambo, J. C. Mathison, A. J. King, W. F. Hall, J. da Silva Correia, R. J. Ulevitch, H. M. Hoffman, and D. B. McKay. 2010. An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury. J. Immunol. 185: 6277–6285.

In regards to Ref. 54 (Iyer, S. S., W. P. Pulskens, J. J. Sadler, L. M. Butter, G. J. Teske, T. K. Ulland, S. C. Eisenbarth, S. Florquin, R. A. Flavell, J. C. Leemans, and F. S. Sutterwala. 2009. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome. Proc. Natl. Acad. Sci. USA 106: 20388–20393), an error occurred in the Discussion section on p. 6282 of the article, which states that “The mice used in the study were backcrossed onto C57BL/6 mice for only four generations…”. This was a mistake; the mice used in Ref. 54 were backcrossed for nine generations. We apologize for this error and regret any inconvenience. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1090136

Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00