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Macrophage Uptake of Necrotic Cell DNA Activates the AIM2 Inflammasome to Regulate a Proinflammatory Phenotype in CKD

Takanori Komada ,1,2 Hyunjae Chung,1,2 Arthur Lau,1,2 Jaye M. Platnich,1,2 Paul L. Beck,1,2 Hallgrimur Benediktsson,2,3 Henry J. Duff,4 Craig N. Jenne,2,5 and Daniel A. Muruve1,2

1Departments of Medicine, 3Pathology and Laboratory Medicine, 4Cardiac Sciences, and 5Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada; and 2Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

ABSTRACT Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in 2(AIM2)isaninflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, 2 2 UUO induced substantially more tubular injury and IL-1b cleavage in Aim2 / or WT mice that re- 2 2 ceived WT bone marrow than in WT mice that received Aim2 / bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5–6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active -1. DNA uptake occurred in large vacuolar structures within + recruited macrophages but not resident CX3CR1 renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1b,aswellasthe formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1b levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

J Am Soc Nephrol 29: 1165–1181, 2018. doi: https://doi.org/10.1681/ASN.2017080863

Renal inflammation and fibrosis are the common include Nod-like receptor (NLR) family pyrin pathogenic pathways of progressive CKD.1 Danger- domain–containing 3 (NLRP3), NLR associated molecular patterns (DAMPs) contribute family CARD domain–containing protein to progression of CKD by driving inflammation 4 (NLRC4), and absent in melanoma 2 (AIM2).4–7 through the activation of the innate immune sys- tem.2,3 Inflammasomes are innate immune com- plexes that regulate inflammation and cell death Received August 9, 2017. Accepted December 19, 2017. through the assembly of multiprotein platforms Published online ahead of print. Publication date available at for caspase activation. Canonical inflammasomes www.jasn.org. activate caspase-1 to regulate cytokine maturation, Correspondence: Dr. Daniel A. Muruve, University of Calgary, notably IL-1b, and via gasdermin 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada. Email: D (GSDMD). Inflammasome-forming pattern rec- [email protected] ognition receptors (PRR) expressed in the kidney Copyright © 2018 by the American Society of Nephrology

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Inflammasome-forming also display noncanonical roles Significance Statement that include the activation of alternate , such as caspase-4 or caspase-8, and inflammasome (caspase)-independent func- Theinflammasomesareinnateimmunepathwaysthatcontribute tions in cellular .8–12 We and others have demon- to kidney inflammation and fibrosis. The NLRP3 inflammasome strated canonical and noncanonical roles for NLRP3 in numerous has been extensively characterized; however, the roles of other inflammasomes in kidney disease are unknown. Furthermore, models of kidney injury including the mouse model of unilateral the mechanisms that drive inflammasome activation during – ureteric obstruction (UUO).13 17 Theroleofotherinflamma- kidney injury in vivo have not been demonstrated. This paper some-forming receptors in the pathogenesis of renal injury, in- characterizes the biology of the absent in melanoma 2 (AIM2) flammation, and fibrosis has not been reported. inflammasome in the kidney and its role in tubulointerstitial in- – flammation and fibrosis. This study also unravels the mechanism AIM2 is a non-NLR cytosolic PRR that forms a caspase-1 fl in vivo fl of AIM2 in ammasome activation that occurs through activating in ammasome in response to double-stranded DNA the macrophage uptake of necrotic cell DNA. These results – (dsDNA).18 21 Most studies have examined the role of AIM2 in identify the AIM2 inflammasome as a mediator of necroin- the host response to microbial DNA.22–26 Recently, several stud- flammation in the kidney and provide additional support for ies have suggested that self-dsDNA in special circumstances can targeting the inflammasomes and their pathways in activate the AIM2 inflammasome.27,28 Nucleosomes and kidney disease. dsDNA are released during necrotic cell death in acute and chronic kidney injury,29–31 but whether these DAMPs are capa- To confirm the localization of Aim2 in the mouse kidney, ble of activating the AIM2 inflammasome in this context is un- immunofluorescence microscopy was attempted, but avail- known. Furthermore, the mechanism by which endogenous able antibodies for Aim2 were ineffective and nonspecific. DAMPs activate the AIM2 inflammasome during nonmicrobial Therefore, laser capture microdissection was used to identify injury has never been shown directly in vivo. Aim2 mRNA expression in the glomerular and tubulointer- In this study, we identify for the first time a role for the stitial compartments. The purity of dissection was con- canonical AIM2 inflammasome in kidney disease. AIM2 ex- firmed by the mRNA expression of glomerular specific pression was found in various compartments of normal and marker Nphs2 (Figure 2C). In sham controls, Aim2 mRNA diseased kidneys, including glomeruli, tubular cells, and in- expression was primarily in the glomerulus and accounted terstitial inflammatory infiltrates. In the mouse model of kid- for the majority of total kidney expression at baseline. ney injury induced by UUO, the Aim2 inflammasome played However, Aim2 expression increased 15-fold in the tubu- an essential role in regulating renal inflammation and fibrosis lointerstitial fraction after UUO (Figure 2D). Thus, Aim2 through the uptake of necrotic cell DNA in recruited proin- is expressed constitutively in the glomerulus, but signifi- flammatory macrophages. cantly induced in the tubulointerstitial compartment during kidney injury.

RESULTS Effects of Aim2 on Kidney Injury Induced by UUO Given the significant upregulation of AIM2 in the tubuloin- AIM2 Expression in Mouse and Human Kidney Disease terstitial compartment of diseased kidneys in mice and To understand the biology of AIM2 in the kidney, AIM2 ex- humans, the specific role of Aim2 in UUO-induced tubuloin- pression was first determined in normal and diseased human terstitial injury was examined in WTand Aim22/2 littermate kidney tissue by immunofluorescence (Figure 1A). In normal mice. Because NLRP3 also regulates kidney inflammation, ap- kidney, AIM2 was expressed primarily in glomeruli, with optosis, and fibrosis,16,32,33 double deficient Nlrp32/2 some sporadic expression in tubules. Few AIM2-positive Aim22/2 mice were generated (Figure 3A). Gross pathology leukocytes were observed in the normal renal interstitium. revealed similar distention of the renal pelvis and ureter in In biopsy samples from patients with CKD due to diabetes WT, Aim22/2,andNlrp32/2Aim22/2mice (Supplemental or hypertension, a significant increase of AIM2-positive Figure 1A). Obstructed kidneys from Aim22/2 and Nlrp32/2 leukocytes was present in chronic inflammatory tubulointer- Aim22/2 mice showed significantly less histopathologic stitial lesions. AIM2 expression increased in tubular epithelial injury compared with WT (Figure 3, B and C). Expression of cells in diseased kidney sections, but remained unchanged in the tubular injury marker, kidney injury molecule–1(KIM-1), the glomeruli (Figure 1B). and caspase-3 was also decreased in Aim22/2 and Nlrp32/2 Next, Aim2 expressionwas determined in mouse kidneys at Aim22/2kidneys (Figure 3, D–F, Supplemental Figure 1, B and C). baseline and after injury induced by UUO. Aim2 mRNA Consistent with these findings, Aim22/2 and Nlrp32/2 expression progressively increased over time after UUO (Fig- Aim22/2 mice demonstrated less renal fibrosis by picrosirius red ure 2A). Aim2 protein also increased in obstructed kidneys staining, fibronectin, a-smooth muscle actin (aSMA), and type I compared with sham-operated wild-type (WT) mice 7 days and type III collagen expression (Figure 4, Supplemental Figure 1, D after UUO, but not in the Aim22/2 kidneys (Figure 2B). and E). The effect of Aim2 deficiency on kidney injury was evident Thus, Aim2 is induced in the kidney during UUO, 14 days post-UUO because Aim22/2 and Aim2 2/2Nlrp32/2 suggesting a role in the pathophysiology of kidney injury. both demonstrated less tubular injury and fibrosis at this

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Figure 1. AIM2 is expressed constitutively in glomeruli, and up-regulated in tubules and infiltrating leukocytes in human kidney diseases. AIM2 and CD45 immunofluorescence microscopy in kidney sections from patients with diabetic nephropathy (n=3) and hypertensive nephrosclerosis (n=3). Healthy control sections were obtained from nondiseased margins of nephrectomy samples (n=3). (A) Representative images are shown. AIM2 expression (red) in glomerular cells is indicated by white arrows, tubular epithelial cells

J Am Soc Nephrol 29: 1165–1181, 2018 AIM2 Inflammasome and Kidney Injury 1167 BASIC RESEARCH www.jasn.org time point (Supplemental Figure 2). Nlrp32/2Aim22/2 consistent with a noncanonical role for parenchymal AIM2 mice displayed a nonsignificant trend toward lower caspase- in kidney injury as previously reported for Nlrp3.33 In this 3 activation, KIM-1, and aSMA expression compared with regard, primary tubular epithelial cells isolated from Aim22/2 mice after UUO, but pathologic tubulointerstitial Aim22/2 mice exhibited less Smad2 phosphorylation injury and fibrosis were generally similar between Aim22/2 and aSMA expression in response to TGFb (Supplemental and Nlrp32/2Aim22/2 miceat7or14days.Nlrp32/2 Figure 4C). mice displayed slightly more tubular injury, but comparable fibrosis compared with Aim22/2 mice (Figures 3, B–F, and 4, Characterization of Leukocytes Responsible for AIM2 Supplemental Figures 1 and 2). Together, these data suggest Inflammasome Activation in UUO Kidney that AIM2 and NLRP3 both contribute to the pathogenesis of Flow cytometry was used to characterize leukocyte subsets chronic kidney injury but redundant mechanisms exist be- responsible for inflammasome activation in UUO. Total re- yond these -forming . cruited CD45+F4/80+ macrophages and CD45+F4/80+ + + CX3CR1 CCR2 inflammatory macrophages were sup- Aim2 Inflammasome Activation and Inflammation pressed in both Aim22/2 and Nlrp32/2Aim22/2 mice during Kidney Injury at 7 days after UUO (Figure 7, A–C). Activated caspase-1, Immunohistochemistry for the pan-leukocyte marker CD45 detected using the caspase-1–FLICA (FAM-YVAD-fmk) and the macrophage marker F4/80 revealed an increase in in- probe, was increased 7 days post-UUO in CD45+ renal flammatory cells after UUO that was attenuated in Aim22/2 leukocytes (Figure 7D). Caspase-1–FLICA+ leukocytes 2 mice (Figure 5, A–C). Aim22/2 mice also displayed less IL- were mainly Ly6G ,and.50% expressed F4/80+CD11b+ 1b, IL-18, and caspase-1 cleavage at 7 days post-UUO (Figure Ly6C+ consistent with proinflammatory macrophages re- 5, D and E; images of uncropped immunoblots: Supplemental cruited from the circulation34,35 (Figure 7D). These CD45+ Figure 3). Similarly, the expression of the chemokine Ccl2 F4/80+CD11b+Ly6C+ macrophages were significantly re- (MCP-1) was diminished in Aim22/2 and Nlrp32/2 duced in UUO kidneys from Nlrp32/2Aim22/2 mice at Aim22/2 mice compared with controls (Figure 5F). The de- 3 and 7 days (Supplemental Figure 5). At 14 days post-UUO, gree of IL-18 processing and caspase-1 cleavage was less in inflammatory macrophage populations were generally lower Nlrp32/2Aim22/2 mice, but not universal for all inflamma- compared with 7 days but did not differ between WT, tory markers, again suggesting that Aim2 and Nlrp3 both con- Aim22/2,andAim22/2Nlrp32/2 mice (Supplemental tribute to renal inflammation but redundant pathways exist Figure 6). These results indicated that after UUO, Nlrp3 (Figure 5, D and E). and Aim2 influence the early recruitment of inflammatory The AIM2 inflammasome is activated predominantly in macrophages to the kidney, a major reservoir of inflamma- macrophages.19 To further determine whether Aim2 function some activity. in kidney injury was canonical or noncanonical, the contri- bution of hematopoietic cells in inflammasome activation was Uptake of Necrotic Cell DNA in Macrophages assessed in WT to WT, WT to Aim22/2, Aim22/2 to WT, Macrophages exhibit multiple diverse functions including the and Aim22/2 to Aim22/2 bone marrow chimera (BMT) clearance of dying cells.36 We speculated that macrophage up- mice undergoing UUO. Histologic analysis demonstrated less take of DNA from necrotic cells drove Aim2-dependent in- 2 2 tubular injury and KIM-1 expression in BMTAim2 / to WT flammation during kidney injury. To address this question, 2 2 2 2 2 2 and BMTAim2 / to Aim2 / but not BMTWT to Aim2 / mice intravital microscopy was used to examine leukocyte behavior at 7 days post-UUO (Figure 6, A–D). IL-1b cleavage was re- in LysM(gfp/gfp) mice undergoing UUO. LysM(gfp/gfp) reporter 2 2 duced in BMTAim2 / to WT compared with BMTWT to WT or mice express the GFP transgene primarily in circulating neu- 2 2 BMTWT to Aim2 / mice, suggesting that Aim2 inflammasome trophils and monocytes/macrophages.37 The membrane im- activation was driven by leukocytes recruited to the kidney permeable dye Sytox orange was injected intravenously at the (Figure 6, E and F). The reduction in injury and cytokine time of imaging to label DNA and visualize necrotic cells. At 2 2 maturation in BMTAim2 / to WT was not associated with 5–6 days after UUO, a significant influx of GFP+ leukocytes less fibrosis (Supplemental Figure 4, A and B). On the into the kidney occurred with the majority of tubular cells 2 2 contrary, fibrosis was significantly diminished in BMTWT to Aim2 / showing positive Sytox labeling and signs of (Figure 2 2 2 2 and BMTAim2 / to Aim2 / mice despite severe tubular injury, 8A). Furthermore, GFP+ leukocytes crawled along and within

indicated by red arrows, and CD45+ leukocytes (green) indicated by yellow arrows. Sc, sclerotic glomerulus. Scale bars=50 mm. (B) Renal compartment–specific quantitative analysis of AIM2 expression. Data are expressed as mean6SD, and analyzed using ANOVA with Dunnet’s post hoc test or nonparametric test. *P,0.05, **P,0.01. DMN, diabetic nephropathy; HTN, hypertensive nephrosclerosis; Pt, patient.

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Figure 2. AIM2 is induced in the mouse kidney during UUO. (A) Aim2 mRNA expression by real-time RT-PCR analysis (n=7 for each) in UUO kidneys harvested at indicated time points (d, day). Relative Aim2 expression of contralateral (cntrl) and obstructed (UUO) kidneys to sham kidneysamplesisshown.(B)Immunoblotting for AIM2 in sham-operated andobstructedkidneysfromWTandAim22/2 mice at 7 days. Bone marrow–derived macrophages (BMDMs) are used as controls. b-actin was used as the loading control. (C) Laser capture microdissection (LCM) of mouse kidney tissue to isolate glomeruli. Fresh-frozen tissue sections were stained with HistoGene to visualize the structures. mRNA level of Nphs2 in total kidney section (tot), glomeruli (glom), and tubules (tub) was analyzed with real-time RT-PCR (n=2–3foreach).(D)Aim2 mRNA level in total kidney section (tot), glomeruli (glom), and tubules (tub) was analyzed by real-time RT-PCR (n=3 for each). Data are ex- pressed as mean6SDandanalyzedwitht test. Sham versus obstructed, **P,0.01. Sham glom versus sham tubules, **P,0.01. obst, ob- structed UUO kidney; RIPA, radioimmunoprecipitation assay buffer.

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Figure 3. AIM2 contributes UUO-induced mouse kidney injury. Mice were euthanized at 7 days after UUO induction and sham op- eration. The kidneys of WT, Nlrp32/2, Aim22/2,andNlrp32/2Aim22/2 were obtained. (A) Kidney tissue protein was extracted with

1170 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1165–1181, 2018 www.jasn.org BASIC RESEARCH injured tubules and necrotic cells (Figure 8B, Supplemental inhibitor YVAD-fmk (Figure 9C). In mouse bone marrow– Movie 1). GFP+ leukocytes formed large phagocytic vacuoles derived macrophages (BMDM), necrotic primary mouse consistent with the uptake of cellular debris in the tubuloin- tubular epithelial cells also induced DNaseI-dependent IL- terstitium. Three-dimensional reconstruction revealed the 1b maturation in WT but not Aim22/2 or Aim22/2Nlrp32/2 presence of Sytox orange–positive DNA within GFP+ leuko- macrophages (Figure 9D). Finally, confocal microscopy cytes and their vacuoles, confirming that infiltrating leuko- confirmed inflammasome assembly and the formation of cytes were actively taking up DNA during injury in vivo AIM2 and ASC-positive specks adjacent to internalized ne- (Figure 8C, Supplemental Movie 2). The phagocytosis of crotic HTEC DNA in THP-1 macrophages (Figure 9, E DNA was restricted to LysM-GFP+ cells recruited to the kid- and F). ney. Studies in Cx3cr1(gfp/+) reporter mice that express GFP in resident renal macrophages/dendritic cells38 demon- Cell Death and Inflammasome Activation in strated an increase in cell number over 6 days of UUO but Macrophages little to no movement, and no Sytox orange positivity or The induction of the AIM2 inflammasome by DNA released evidence of DNA uptake (Supplemental Figure 7, Supplemental from necrotic cells is consistent with necroinflammation, a Movie 3). DAMP-driven process observed during kidney injury.39,40 To To confirm inflammasome activation in leukocytes re- evaluate whether other forms of necrotic cell death, including cruited to the injured kidney, LysM(gfp/gfp) mice underwent pyroptosis and necroptosis,39,40 could also activate the UUO and were administered caspase-1–FLICA to probe for inflammasome in macrophages, additional coincubation activated caspase-1. The majority of LysM-GFP+ leukocytes studies were performed. Because HTECs do not assemble in- recruited to the kidney were CD45+F4/80+CD11b+Ly6C+ flammasomes or undergo pyroptosis in response to conven- 2 Ly6G inflammatory macrophages (Supplemental Figure 8, tional agonists,32 pyroptotic and necroptotic THP-1 cells were – A–C). CD45+GFP+Ly6G macrophages sorted by flow cytom- first employed as stimuli in coincubation experiments. Unlike – etry but not CD45+GFP leukocytes (likely resident phago- necrotic cells, both necroptotic and pyroptotic THP-1 cells cytes and other nonmyeloid leukocytes) expressed Aim2 contained activated caspase-1 and IL-1b before incubation mRNA by real-time RT-PCR, confirmingthesecellsasa with viable cells (Figure 10, A and B). Necroptosis is known source of Aim2 expression in the UUO kidney (Supplemental to activate the Nlrp3 inflammasome,41 andpyroptosisisan Figure 8D). LysM-GFP+ macrophages were also caspase-1– inflammasome-dependent effector mechanism.39 Necrotic FLICA positive, consistent with inflammasome activation in cells efficiently activated IL-1b and caspase-1 in viable THP- these cells (Figure 8, D and E). Caspase-1–FLICA–positive 1 cells (Figure 10, A and B). Pyroptotic THP-1 cells also cells were not significantly increased in non-GFP leukocyte increased IL-1b and caspase-1 activation above baseline; populations, confirming that canonical inflammasome acti- however, necroptotic THP-1 cells were less effective at induc- vation occurs primarily in proinflammatory macrophages ing the inflammasome in viable THP-1 cells (Figure 10, A and during kidney injury. B). Necrotic HTECs activated the inflammasome in THP-1 To confirm that AIM2 inflammasome activation in mac- cells as determined by caspase-1 and IL-1b secretion, as well rophages in vivo resulted from phagocytosis of necrotic cell as a small amount of GSDMD cleavage, a marker of pyroptosis DNA, Sytox orange–labeled necrotic cell debris was gener- (Figure 10, B–D). Similar to the necroptotic THP-1 cells, nec- ated from primary human tubular epithelial cells (HTECs). roptotic HTECs had a modest stimulatory effect on THP-1 Differentiated human monocytic THP-1 cells treated with cells (Figure 10, B and D) that was DNA-dependent in part, necrotic cell debris ingested DNA as demonstrated by fluo- because pretreatment of cell debris with DNaseI significantly rescence microscopy (Figure 9A). Necrotic cell debris, but attenuated IL-1b secretion (Figure 10D). Together, these data not live cells or apoptotic cell debris, induced IL-1b matura- support the premise that the AIM2 inflammasome contrib- tion, effects that were reversed by DNaseI (Figure 9, B and C). utes to necroinflammation through the sensing of DNA re- Gel electrophoresis confirmed the elimination of necrotic cell leased from dying cells. Indeed, the inflammatory response to DNA by DNaseI within 2 hours (Supplemental Figure 9). The UUO was attenuated at 7 days in WTmice administered daily activation of IL-1b was also reversed by the caspase-1 DNaseI (Supplemental Figure 10).

RIPA buffer. NLRP3 protein expression was analyzed by immunoblotting. b-actin was used as the loading control. (B) Quantitative analysis of tubular injury score was performed (n=4, 10, 6, 9) on (C) periodic acid–Schiff (PAS)–stained sections from sham-operated and obstructed kidneys in WT, Aim22/2,andNlrp32/2Aim22/2 mice at 7 days. (D) KIM-1 immunofluorescence (green) in paraffin-embedded kidney sections to identify tubular injury. Nuclei were costained with DAPI (blue). Representative photographs are shown. (E) Quantitative analysis of KIM-1 immunohistochemistry (n=4 for each). (F) Renal Havcr1 (KIM-1) mRNA expression assessed by real-time PCR (n=3, 9, 5, 8). Scale bars=50 mm. Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. *P,0.05, **P,0.01. d, day; DAPI,49,6-diamidino-2-phenylindole; IHC, immunohistochemistry; obst, obstructed UUO kidney; RIPA, radioimmunoprecipitation assay lysis buffer.

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Figure 4. AIM2 contributes to tubulointerstitial fibrosis in mice undergoing UUO. (A) Representative picrosirius red staining of the sham-operated and obstructed (Ob) kidneys of WT, Aim22/2, Nlrp32/2,andNlrp32/2Aim22/2 mice at 7 days after UUO. Collagen bundles appear red/yellow/green under polarized light. (B) Quantitative analysis of fibrosis (picrosirius red) was performed (n=4,8,7,6, 9). (C) Immunofluorescence probing for collagen type I (red) on fresh-frozen sections of the kidneys from WT, Aim22/2,andNlrp32/2 Aim22/2 mice at 7 days after UUO. Nuclei were contained with DAPI (blue). Representative photographs are shown. (C) Quantitative analysis of immunostaining for collagen type I was performed. (D) Renal expression of collagen type I and type III was assessed by immunoblotting. b-actin was used as the loading control. (E) Quantitative analysis of immunostaining for collagen type I was performed (n=4–10). Scale bars=50 mm. Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. *P,0.05, **P,0.01. d, day; DAPI,49,6-diamidino-2-phenylindole; PR, picrosirius red.

DISCUSSION infiltrates during kidney disease. AIM2 contributes to UUO-induced kidney inflammation and injury primarily In this study, we characterize for the firsttimeAIM2expressionin through canonical inflammasome function in recruited mac- the kidney and its role in kidney injury. Our findings show rophages triggered by the uptake of necrotic cell DNA. that AIM2 is constitutively expressed in the glomerulus, but Efferocytosis is the process of phagocytic clearance of dead upregulated in the tubular epithelium and inflammatory or dying cells. Engulfment of apoptotic or programmed

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Figure 5. AIM2 contributes to renal inflammation and inflammasome activation during UUO. (A) Immunohistochemistry probing for CD45 and F4/80 in the kidneys of WT, Aim22/2,andNlrp32/2Aim22/2 mice at 7 days post-UUO. Representative photographs are shown. Scale bars=50 mm. (B and C) Quantitative analyses of CD45 and F4/80 immunohistochemistry (n=4 for each). (D) Renal ex- pression of inflammasome-dependent cytokines pro–IL-1b,IL-1b (p17), pro–IL-18, IL-18 (p18), pro–caspase-1, and caspase-1 (p20) was assessed by immunoblotting. b-actin was used as the loading control. (E) Quantitative analyses of IL-1b (p17)/Pro–IL-1b, IL-18 (p-18)/ Pro–IL-18, and casp-1(p20)/pro–casp-1 were performed (n=2, 4, 4, 4 for each). (F) The expression of monocyte chemotactic protein–1 (MCP-1) was analyzed using Luminex assay (n=3–6). Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. *P,0.05, **P,0.01. d, day; IHC, immunohistochemistry. necrotic cells is generally an anti-inflammatory event.42–44 On the activating confirmed in vitro.27,28 We provide the the other hand, leakage of intracellular contents from uncon- first direct evidence in vivo that the uptake of necrotic cell trolled necrosis induces a proinflammatory response referred DNA underlies the mechanism of Aim2 inflammasome acti- to as necroinflammation.39,40,45 Many potential DAMPs re- vation during sterile injury, positioning Aim2 as a key player in leased from necrotic cells can trigger an immune response necroinflammation. including DNA. Several studies have suggested a role for the Our study characterizes AIM2 expression in the human AIM2 inflammasome in nonmicrobial disease, with DNA as kidney and provides evidence that the AIM2 inflammasome

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2 2 Figure 6. Aim2 in bone marrow–derived cells contributes to tubular injury and inflammation in UUO. BMTWT to WT,BMTAim2 / to WT, 2 2 2 2 2 2 BMTWT to Aim2 / ,andBMTAim2 / to Aim2 / bone marrow chimera mice were generated and underwent UUO. The kidneys were harvested after 7 days (n=7, 7, 6, 5). (A) Representative photographs of periodic acid–Schiff (PAS) staining are shown. (B) Quantitative analysis of tubular injury score. (C) Immunofluorescence probing for KIM-1 (green) and E-cadherin (white) to identify tubular injury. Nuclei were contained with DAPI (blue). Representative photographs are shown. (D) Quantitative analysis of KIM-1 immunofluores- cence. (E) Renal expression of pro–IL-1b and IL-1b (p17) was assessed by immunoblotting. b-actin was used as the loading control. (F) Quantitative analysis of IL-1b (p17)/Pro–IL-1b (n=3 for each). Scale bars=50 mm. Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. *P,0.05, **P,0.01. DAPI,49,6-diamidino-2-phenylindole; E-Cad, E-cadherin; exp, exposure; IB, immunoblotting; IHC, immunohistochemistry. contributes to renal inflammation and fibrosis in mice. the tubular epithelium during injury and human kidney disease. Although this study specifically addresses the contribution of ca- The observation of less fibrosis in Aim22/2 mice reconstituted nonical AIM2 inflammasome in the leukocyte compartment and with WT bone marrow, and the resistance of tubular epithelial renal inflammation, data also suggest noncanonical roles for AIM2 cells to TGFb, identify a noncanonical role for AIM2 in fibrosis. in kidney disease. First, AIM2 is constitutively expressed in glo- These results mirror previous findings with NLRP3 and its non- merular podocytes. Studies from our group indicate a noncanon- canonical functions in the kidney described by our group and icalfunction forAIM2in podocyte proliferation anddifferentiation others.46,47 AIM2 is known to play a noncanonic role in the reg- (manuscript in review). Second, AIM2 expression was induced in ulation of cell proliferation and , the exact mechanism of

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Figure 7. Inflammatory macrophages are responsible for inflammasome activation and are reduced in Aim22/2 and Nlrp32/2 Aim22/2 mice. (A) Flow cytometric analysis of kidney leukocytes from WT, Aim22/2,andNlrp32/2Aim22/2 mice after sham operation or UUO induction. (A) Representative flow cytometry plots of mouse kidney inflammatory monocytes/macrophages 7 days after UUO. (B) Quantitative analysis of CD45+F4/80+ (n=4–6 for each group, n=1 for each sham). (C) Quantitative analysis of in- + + + + flammatory macrophages defined by CD45 F4/80 CX3CR1 CCR2 (n=4–6 for each group, n=1 for each sham). (D) Populations of casp-1–FLICA–positive leukocytes were identified with the markers CD45, F4/80, CD11b, Ly6C, and Ly6G. Note that .46% of casp-1– – FLICA–positive leukocytes were F4/80+CD11b+Ly6C+Ly6G macrophages. Data are expressed as mean6SD and analyzed with AN- OVA with Tukey’s post hoc test. *P,0.05, **P,0.01. d, day; FSC, forward scatter; SSC, side scatter. which remains incompletely understood.48,49 Dissecting the non- Finally, we generated double Nlrp32/2Aim22/2 mice canonical role for AIM2 in renal epithelial cells will require further to examine the relative contribution of NLRP3 and AIM2 in investigation. renal injury. We and others have demonstrated canonical

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Figure 8. LysM-GFP+ macrophages engulf necrotic DNA and activate caspase-1 during UUO. LysM(gfp/gfp) mice underwent UUO and obstructed kidneys were imaged with two-photon intravital microscopy at 6 days. Sytox orange dye was injected intravenously before

1176 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1165–1181, 2018 www.jasn.org BASIC RESEARCH and noncanonical roles for NLRP3 in kidney disease. obtained from the nondiseased margins of human nephrectomy sam- 13–17,32,33 Furthermore, recent papers have shown a link ples. between AIM2 and NLRP3 inflammasomes in response to microbial .50,51 Hence, we expected less injury, in- Cell Culture flammation, or fibrosis in Nlrp32/2Aim22/2 after UUO. Human monocytic leukemia THP-1 cells were differentiated with Although Nlrp32/2Aim22/2 mice generally showed a 100 nM phorbol-12-myristate-13 acetate for 48 hours before ex- trendtowardlessinjury,inflammation, and fibrosis, no periments. Primary human and mouse tubular epithelial cells were dramatic phenotypic differences were observed compared prepared as previously reported.54,55 HTEC debris was generated with Aim22/2 mice. These data suggest that both AIM2 by incubation with 500 mM H2O2 to induce necrosis for 3 and NLRP3 contribute to renal injury. Ongoing injury and hours.56–58 Cells were stained with Sytox orange (Thermo- inflammation in Nlrp32/2Aim22/2 mice also identifies Fisher), washed, and centrifuged at 20,000 3 g for 5 minutes. redundancy in inflammasome and caspase-1–activating Necrotic cells were completely disrupted using a QIAshredder ho- pathways in the kidney. These could include the NLRC4 mogenizer (Qiagen). DNase inactivation reagent (Ambion) was and the noncanonical caspase-11 (caspase-4 in humans) added to cell debris with or without DNaseI treatment at 37°C inflammasomes, both of which are expressed in the for 12 hours. kidney.4,7,32 In conclusion, we demonstrate a role for AIM2 in the pathogenesis of kidney injury, inflammation, and fibrosis. Kidney Intravital Microscopy UUO was induced in LysM(gfp/gfp) or CX CR1(gfp/+) mice. The kid- Together, these data provide further insight into the role of 3 ney was exposed with lateral incisionandextendedontheheated inflammasomes in the pathogenesis of kidney disease and imaging platform. Intravenous Sytox orange and Hoechst 33258 identify additional potential therapeutic targets. were administered to visualize DNA of necrotic and viable cells, respectively. Capillaries were visualized with Qtracker 655 (Thermo-Fisher). Imaging was performed by using a Leica multi- CONCISE METHODS photon confocal microscope equipped with 253 and 603 objec- tives and a MaiTai pulsed infrared laser (Leica). Image processing Mouse Studies used the LASX software. Nlrp32/252 and -trap Aim2/224 mice on the C57Bl/6 back- ground were used to generate Nlrp3+/+Aim+/+(WT), Nlrp32/2 Aim2+/+, Nlrp3+/+Aim22/2,andNlrp32/2Aim22/2 litter- mate mice. Eight to 12-week-old male mice underwent left ACKNOWLEDGMENTS UUO or sham operation as described previously.7,53 For chimera studies, bone marrow cells were isolated from male WT and The authors thank Sharon A. Clark for her technical support and Aim22/2 miceandadministeredtoirradiatedrecipientsasde- Dr. May Ho for her critical review of the manuscript. scribed previously.16 This work was supported by Operating and Team grants from the Canadian Institutes for Health Research (CIHR). Research was also support by the Canadian National Transplantation Research Program Human Biopsy Samples and the CIHR Inflammation in Chronic Disease Signature Initiative. Human biopsy samples of diabetic nephropathy (n=3) and hyperten- D.A.M. holds a Tier II Canada Research Chair. T.K. holds a fellowship sive nephrosclerosis (n=3) without any findings of primary glomer- from the Manpei Suzuki Diabetes Foundation, Japan. A.L. holds a ular diseases were used. Histologically normal kidney tissue was Postdoctoral Scholarship Award from Alberta Innovates Health So-

imaging to visualize DNA from necrotic cells. (A) Representative photographs of intravital microscopy 6 days after sham operation or UUO induction. Necrotic cell DNA was stained with Sytox orange (red). Nuclei in live cells were visualized by injection of Hoechst 33258 (cyan). An increase in crawling LysM-GFP–positive neutrophils/monocytes/macrophages (green) is observed. Scale bars=15 mm. (B) Time-lapse imaging of LysM-GFP+ cells around a necrotic tubule of an obstructed kidney at 6 days. Necrotic cell DNA was stained with Sytox (red). Images are individual frames from a continuous movie (Supplemental Material). Scale bars=5 mm. (C) Three-dimensional images of LysM- positive macrophages in the UUO kidney. Sectional views show necrotic cell DNA (red) and endocytic/phagocytic vacuoles within the cell (denoted by arrows). (D and E) Flow cytometric analysis for casp-1–FLICA in kidney LysM-GFP+ macrophages isolated from LysM(gfp/gfp) mice 7 days after sham operation (sham) or UUO induction (UUO). Contralateral kidneys (cntrl) of UUO were also analyzed. (D) Repre- sentative histograms showing casp-1–FLICA expression in LysM-GFP+ cells 7 days after UUO. (E) Quantitative analysis of casp-1–FLICA– – positive cells in LysM-GFP+ or LysM-GFP populations (n=3 for each). Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. **P,0.01. d, day; GFP, green fluorescent protein.

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Figure 9. Necrotic cell DNA activates the AIM2 inflammasome in vitro. (A) Exogenous necrotic DNA from HTECs prestained with Sytox orange was treated on human THP-1 macrophages. Necrotic DNA was taken up in the 2 hours after treatment. was stained with cholera-toxin subunit B (CTB, green). Nuclei were contained with DAPI (blue). Representative photographs are shown. Scale bars=3 mm. (B) Immunoblotting showing IL-1b processing in THP-1 after 3 hours of treatment with live HTECs, necrotic debris, apoptotic debris, or poly(dA:dT) (2 mg/ml). (C) ELISA for IL-1b in the supernatants of THP-1 cells treated with necrotic cell debris in the presence or absence of DNaseI or 20 mM YVAD-fmk (n=4). (D) BMDMs from WT, Aim22/2,andNlrp32/2Aim22/2 mice were primed with LPS followed by the treatment with necrotic cell debris or DNase I–treated cell debris. WT BMDMs treated with necrotic debris together with YVAD or Z-VAD were used as negative controls. The levels of IL-1b in the supernatants were measured by ELISA (n=4). (E) Immunofluorescence microscopy costaining for ASC (green) in THP-1 macrophages treated with exogenous necrotic cell DNA (red)

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Figure 10. Debris from various types of cell death induces inflammasome activation in macrophages. Human macrophages were cocultured with THP-1 cells or HTECs induced to undergo necrotic, pyroptotic, or necroptotic cell death with H2O2, ATP, or TNFa/Smac mimetics/zVAD, respectively. (A) ELISA for IL-1b in the supernatants of THP-1 cells treated with debris from necrotic, pyroptotic, and necroptic THP-1 cells (n=4). (B) Representative immunoblotting for caspase-1 and IL-1b in the su- pernatants from THP-1 cells incubated with debris from necrotic, pyroptotic, and necroptotic cell debris. Cell debris from pyroptotic and necroptic THP-1, but not HTEC, contains activated inflammasome products including IL-1b and caspase-1. (C) Immunoblotting for gasdermin d (GSDMD) in THP-1 cells stimulated with debris from necrotic or apoptotic HTECs. b-actin was used as the loading control. (D) ELISA for IL-1b in the supernatants of THP-1 cells treated with necrotic or necroptotic HTEC debris pre-treated with DNase I (n=4). Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. *P,0.05, **P,0.01. Apo, apoptotic; exp, exposure; necrop, necroptotic; NT, nontreatment; poly(dA-dT), poly (deoxyadenylic-deoxytymidylic) acid sodium salt; pyrop, pyroptotic; sup, supernatant. lutions. J.M.P.and A.L. were also supported by Beverly Phillips Trainee Flow Cytometry Facility, the Live Cell Imaging Facility, and the Bi- Awards from the Snyder Institute for Chronic Disease, University of obank for the Molecular Classification of Kidney Disease at the Snyder Calgary. Infrastructure and technical support was provided by the Institute for Chronic Diseases.

or poly(dA:dT). (F) Immunofluorescence microscopy costaining for AIM2 (green) in THP-1 macrophages treated with exogenous necrotic cell DNA (red) or poly(dA:dT). Scale bars=3 mm. Data are expressed as mean6SD, and analyzed using ANOVA with Tukey’s post hoc test. † †† *P,0.05, **P,0.01; P,0.05, P,0.01 with Dunnett’s post hoc test versus corresponding WT group. Apo, apoptotic; ASC, apoptosis- associated speck-like protein containing a caspase recruitment domain; 3D, three-dimensional; DAPI, 49,6-diamidino-2-phenylindole; Exo, exogenous; lys, cell lysate; MTEC, mouse kidney tubular epithelial cells; Nec, necrotic; NT, nontreatment, poly(dA-dT), poly (deoxyadenylic-deoxytymidylic) acid sodium salt; sup, supernatant; YVAD, Ac-Tyr-Val-Ala-Asp-Chloromethylketone; zVAD, carbobenzoxy- valyl-alanyl-aspartyl-(O-methyl)- fluoromethylketone.

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