Inflammasome-Independent and Atypical Processing of IL-1Β

Inflammasome-Independent and Atypical Processing of IL-1 β Contributes to Acid Aspiration−Induced Acute Lung Injury

This information is current as Yoshiko Mizushina, Tadayoshi Karasawa, Kenichi Aizawa, of September 23, 2021. Hiroaki Kimura, Sachiko Watanabe, Ryo Kamata, Takanori Komada, Naoko Mato, Tadashi Kasahara, Shinichiro Koyama, Masashi Bando, Koichi Hagiwara and Masafumi Takahashi

J Immunol published online 20 May 2019 Downloaded from http://www.jimmunol.org/content/early/2019/05/17/jimmun ol.1900168 http://www.jimmunol.org/

Why The JI? Submit online.

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

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *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

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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 20, 2019, doi:10.4049/jimmunol.1900168 The Journal of Immunology

Inﬂammasome-Independent and Atypical Processing of IL-1b Contributes to Acid Aspiration–Induced Acute Lung Injury

Yoshiko Mizushina,*,†,‡ Tadayoshi Karasawa,* Kenichi Aizawa,x Hiroaki Kimura,* Sachiko Watanabe,* Ryo Kamata,* Takanori Komada,* Naoko Mato,† Tadashi Kasahara,* Shinichiro Koyama,‡ Masashi Bando,† Koichi Hagiwara,† and Masafumi Takahashi*

Inflammation plays a pivotal role in the pathophysiology of gastric aspiration–induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome–driven IL-1b production in a mouse model of acid aspiration–induced inflammation and ALI. Acid aspiration–induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1b2/2 mice, but not NLRP32/2 mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro–IL-1b into its 18-kDa mature form (p18–IL-1b), which was different from the caspase-1–processed 17-kDa form (p17–IL-1b), in human THP-1 macrophages and primary murine macrophages. Deficiency of Downloaded from NLRP3 and caspase-1 had no effect on acidic stress–produced IL-1b. The production of IL-1b by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1b production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro–IL-1b,we identified D109 as a novel cleavage site of pro–IL-1b in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress–produced IL-1b was confirmed by its ability to promote http://www.jimmunol.org/ p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1b production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI. The Journal of Immunology, 2019, 203: 000–000.

astric aspiration pneumonitis is defined as an acute lung role in the progression of acid aspiration–induced ALI (1, 2). Be- injury (ALI) following the inhalation of gastric contents cause gastric fluid is highly acidic, it is considered that gastric as- G and is associated with significant morbidity and mor- piration triggers sterile inflammation and injury in the lung.

tality (1, 2). Aspiration pneumonitis commonly occurs as a com- Recent evidence indicates that sterile inflammation is mediated by guest on September 23, 2021 plication of general anesthesia and in patients with altered levels of through the nucleotide-binding oligomerization domain-like re- consciousness due to trauma, cerebral vascular ischemia, or meta- ceptor (NLR) family pyrin domain containing 3 (NLRP3) inflam- bolic encephalopathies. The inhalation of highly acidic gastric fluid masome, an intracellular, large, multiple-protein complex that and/or particulate food matter promotes chemical pneumonitis and regulates the processing of a potent proinflammatory cytokine IL-1b the development of ALI, which can frequently be complicated by (5, 6). NLRP3 inflammasome contains NLRP3, an adaptor mole- subsequent bacterial pneumonia. Previous studies have demon- cule apoptosis-associated, speck-like protein containing a caspase strated that aspirated acid induces dysfunction of alveolar epithelial recruitment domain (ASC), and cysteine protease caspase-1, which fluid transport, followed by alveolar epithelial cell injury and neu- induces caspase-1 activation. Because caspase-1 is known to be an trophil infiltration in the lung (3, 4). However, the mechanisms that IL-1b–converting enzyme (7), its activation processes pro–IL-1b underline acid aspiration–induced ALI are unclear, because acid into its mature form and induces IL-1b production, thereby lead- itself is rapidly neutralized by proteins and bicarbonate systems (4). ing to an inflammatory response and tissue injury. Indeed, we re- Recent investigations have shown that inflammation plays a pivotal cently demonstrated that NLRP3 inflammasome was involved in the

*Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical performed liquid chromatography–tandem mass spectrometry and analyzed the data. University, Shimotsuke, Tochigi 329-0498, Japan; †Division of Pulmonary Medicine, N.M., T. Kasahara, S.K., M.B., and K.H. discussed the data and provided scientific Department of Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, advice. Y.M. and M.T. wrote the manuscript. Japan; ‡Department of Pulmonary Medicine, Jichi Medical University, Saitama Medical x Address correspondence and reprint requests to Prof. Masafumi Takahashi, Division of Center, Saitama 330-8503, Japan; and Division of Clinical Pharmacology, Department Inflammation Research, Center for Molecular Medicine, Jichi Medical University, 3311-1 of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail address: [email protected] ORCIDs: 0000-0002-7441-0382 (H.K.); 0000-0002-6110-9603 (S.W.); 0000-0001- Abbreviations used in this article: AEBSF, 4-(2-aminoethyl)benzenesulfonyl 6970-9657 (R.K.); 0000-0003-3360-3185 (T. Komada); 0000-0001-7687- fluoride hydrochloride; ALI, acute lung injury; BALF, bronchoalveolar lavage 5477 (T. Kasahara); 0000-0003-2716-7532 (M.T.). fluid; CGI, cathepsin G inhibitor I; HCl, hydrochloric acid; 4-HNE, 4-hydroxy- Received for publication February 8, 2019. Accepted for publication April 17, 2019. 2-nonenal; IL-1Ra, IL-1R antagonist; NLR, nucleotide-binding oligomerization domain-like receptor; NLRP3, NLR family pyrin domain containing 3; ROS, This work was supported by grants from the Japan Society for the Promotion of Science reactive oxygen species; sgRNA, single-guide RNA; UBSS, unbuffered balanced through Grants-in-Aid for Scientific Research 16H07151 (to Y.M.) and 18K08112 (to M.T.), salt solution; WT, wild-type. the Private University Research Branding Project (to M.T.), the Agency for Medical Research and Development-Core Research for Evolutional Science and Technology Ó (18gm0610012h0105 to M.T.), the Takeda Science Foundation (to M.T.), and a Jichi Medical Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 University Graduate Student Start-Up Award and Student Research Award (to Y.M.). Y.M. and M.T. designed the study concept and experiments. Y.M., T. Karasawa, H.K., S.W., R.K., and T. Komada performed the experiments and analyzed the data. K.A.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900168 2 IL-1b PROCESSING IN ACID-INDUCED INFLAMMATION pathogenesis of sterile inflammation-related diseases (8–12). In using a Takara TP960 PCR Thermal Cycler Dice Real Time System terms of acidic stress, a mild acidic environment (pH 6–6.5) (Takara Bio, Shiga, Japan) to detect mRNA. The primers (antisense and 9 frequently occurs at sites of inflammation and ischemia (13, 14). sense, respectively) were as follows: Il1b:5-TGAAGTTGACGGACCC- CAAA-39 and 59-TGATGTGCTGCTGTGAGATT-39, Ccl2:59-GGCTCAG- Several studies have suggested that an inflammatory response is CCAGATGCAGTTAAC-39 and 59-GCCTACTCATTGGGATCATCTTG-39, influenced under mild acidic conditions. However, the role of Cxcl1:59-GCTGGGATTCACCTCAAGAA-39 and 59-TCTCCGTTACTTG- NLRP3 inflammasome under such mild acidic conditions re- GGGACAC-39,18SrRNA(Rn18s): 59-GTAACCCGTTGAACCCCATT-39 9 9 9 mains controversial. For instance, Rajamaki et al. (15) recently and 5 -CCATCCAATCGGTAGTAGCG-3 , CCL2:5-CAGCCAGATGCA- ATCAATGCC-39and 59-TGGAATCCTGAACCCACTTCT-39,CXCL1:59-GGAAC- demonstrated that mild acidic stress triggered NLRP3 inflam- AGAAGAGGAAAGAGAGAC-39 and 59-TAGGACAGTGTGCAGGTAGA-39, + masome activation and IL-1b production through K efflux in and 18S rRNA (RNA18S5): 59-GTAACCCGTTGAACCCCATT-39 and human macrophages. In contrast, Takenouchi et al. (16) found 59-CCATCCAATCGGTAGTAGCG-39. Expression levels were quantified that mild acidic conditions modified P2X7 receptor–dependent using a standard curve and were normalized to the content of 18S IL-1b production in cultured murine microglial cells, indepen- rRNA. Each normalized value was expressed as a ratio to the value for WT mice after intratracheal aspiration of PBS. dent of caspase-1. Similarly, Edye et al. (17) reported that mild acidic conditions promoted danger signal-induced IL-1b pro- Western blot analysis duction in a caspase-1–independent manner in human monocytic Cell lysates were prepared using radioimmunoprecipitation assay buffer THP-1 cells and murine glial cells. Intriguingly, the latter two (20 mM Tris, 2.5 mM EDTA, 1% Triton X, 10% glycerol, 1% deoxycholic reports suggested that acidic stress could alter pro–IL-1b pro- acid, 0.1% SDS, 50 mM NaF, and 10 mM Na4P2O7 3 10 H2O), and sub- cessing site(s) and produce the 20-kDa mature form (p20–IL-1b) jected to SDS-PAGE. The proteins were electrophoretically transferred to a PVDF membrane. The membrane was blocked with 2% casein or 3% BSA rather than the 17-kDa, caspase-1–dependent form (p17–IL-1b). Downloaded from for 1 h at room temperature and then incubated overnight at 4˚C with pri- They further showed that the aspartic protease cathepsin D is mary Abs, followed by incubation for 1 h with secondary Abs conjugated to responsible for p20–IL-1b processing. However, no information HRP. Immunoreactive bands were visualized using a Western BLoT HRP is currently available on the role of NLRP3 inflammasome and Chemiluminescent Substrate system (Takara Bio). The expression level of b IL-1b production under severe acidic conditions that simulate -actin served as an internal control for protein loading. Primary Abs were , used those against p38, phospho-p38, caspase-1 (Cell Signaling Technology, exposure to gastric fluid (pH 2.5). In the current study, we Boston, MA), IL-1b (R&D Systems, Minneapolis, MN), and anti–b-actin used mice deficient in NLRP3 and IL-1b and unexpectedly (Sigma-Aldrich, St. Louis, MO). HRP/goat anti-mouse IgG (Invitrogen, http://www.jimmunol.org/ found that the deficiency of IL-1b, but not NLRP3, resulted in Carlsbad, CA) and HRP/goat anti-rabbit IgG (Zymax Grade, Zymed Labo- fewer inflammatory responses and injury in the lung after se- ratories, South San Francisco, CA) were used as secondary Abs. The results vere acid aspiration, indicating that NLRP3 inflammasome- represent at least three independent experiments. independent IL-1b contributes to the development of acidic Immunoprecipitation stress–induced ALI. Furthermore, using mass spectrometry and processing-site mutants of pro–IL-1b,weidentifiedanovel To extract proteins with a molecular mass between 10 and 30 kDa, the b supernatants isolated from radioimmunoprecipitation assay lysate were cleavage site of pro–IL-1 in response to severe acidic stress. ultrafiltrated using Amicon Ultra (MilliporeSigma, Darmstadt, Germany). Our findings demonstrate that IL-1b plays a novel role in aspi- After the ultrafiltrated supernatants were incubated with anti–IL-1b ration pneumonitis and provide new insight into the mechanism Ab (Santa Cruz Biotechnology, Dallas, TX) for 1 h at 4˚C, Protein G by guest on September 23, 2021 underlying severe acidic stress–mediated inflammation. Sepharose (GE Healthcare, Chicago, IL) was added, and the mixture was incubated overnight at 4˚C. The proteins that bound to Protein G Sepharose were eluted by 0.1 M glycine HCl (pH 3.5). Materials and Methods b Animals and acid aspiration model IL-1 assay b All experiments in this study were approved by the Use and Care of Ex- IL-1 levels were measured using a mouse or human ELISA kit (R&D perimental Animals Committee of Jichi Medical University (permit number Systems) according to the manufacturer’s instructions. The bioactivity b 17151) and conducted in accordance with Jichi Medical University of IL-1 was analyzed using IL-1R antagonist (IL-1Ra; PeproTech, guidelines. C57BL/6J (wild-type [WT]) mice were purchased from Japan Rocky Hill, NJ). SLC (Tokyo, Japan). NLRP32/2 and IL-1b2/2 mice (C57BL/6J genetic background) were kindly provided by Dr. Vishva M. Dixit (Genentech, Flow cytometry Southern San Francisco, CA) and Dr. Yoichiro Iwakura (Tokyo University Cells collected from BALF were analyzed using flow cytometry, as de- of Science, Chiba, Japan), respectively (18, 19). Female mice aged 8214 scribed previously (22). The cells were double labeled with the following wk were used. The mice were intratracheally injected with 50 ml of 0.05 N Abs: allophycocyanin-conjugated anti-CD45 (eBioscience, San Diego, hydrochloric acid (HCl) diluted by saline or vehicle (PBS) (4, 20, 21). We CA), FITC-conjugated anti-CD45R (eBioscience), PE-conjugated anti– analyzed the lung samples at 24 h after acid aspiration. Mice were housed Ly-6G (BD Biosciences, San Jose, CA), FITC-conjugated anti-CD11c (four mice per cage; RAIR HD ventilated Micro-Isolator Animal Housing (BD Bioscience), FITC-conjugated anti-F4/80 (eBioscience), and PE- Systems, Lab Products; Seaford, DE) in an environment maintained at 23 conjugated anti-CD11b (eBioscience). The cells were examined by 6 2˚C with ad libitum access to food and water under a 12-h light and dark flow cytometry (FACS Verse; BD Biosciences); the analysis was per- cycle with lights on from 8:00 to 20:00. formed using FlowJo software (version 10; Tree Star, San Carlos, CA). Isotype control Abs were used as negative controls to exclude nonspe- Bronchoalveolar lavage fluid analysis cific staining. Dead cells were identified by the 7-AAD (BD Biosciences) Bronchoalveolar lavage fluid (BALF) was obtained by cannulating the staining. trachea with an 18-gauge catheter. After the whole lung was lavaged four times with 0.8 ml of PBS, the lavage fluid was centrifuged at 1000 rpm for Histology and immunohistochemistry 10 min at 4˚C, and cell-free supernatants were stored at 230˚C. The pellet Lungs were fixed by the intratracheal injection of 1 ml of 10% formalin was diluted in PBS, the cells were stained with trypan blue, and the and embedded in paraffin. Lung tissue sections (5-mm thick) were stained number of live cells was counted using a hemocytometer. Differential cell with H&E. ALI was scored from four points: 1) alveolar congestion, 2) analysis was performed by staining with Diff-Quik (Sysmax, Kobe, Japan) hemorrhage, 3) infiltration or aggregation of neutrophils in airspace or after a cytospin (800 rpm for 8 min at 22˚C) and by flow cytometry. the vessel wall, and 4) thickness of the alveolar wall/hyaline membrane Real-time RT-PCR analysis formation, as describing previously (23, 24). Each item was graded on a five-point scale: 0, minimal (little) damage; 1+, mild damage; 2+, RNA was extracted from the lungs perfused with PBS or cultured cells moderate damage; 3+, severe damage; and 4+, maximal damage. Lung using ISOGEN (Nippon Gene, Toyama, Japan) according to the man- tissue sections were observed excluding the peribronchial area where ufacturer’s instructions. Real-time RT-PCR analysis was performed inflammatory cells had infiltrated excessively. The Journal of Immunology 3

Immunohistochemical analyses were performed to detect the pan- 8 mg/ml polybrene (Sigma-Aldrich). Transfected FLAG/IL-1b proteins leukocyte marker CD45 and the oxidative stress marker 4-hydroxy- were purified with Flag-M2 beads from cell lysates. Humanized 2-nonenal (4-HNE). In brief, deparaffinized sections were boiled in Kusabira-Orange1 (hKO1) subcloned into CS-CA-MCS was used as a Target Retrieval Solution (Dako, Agilent Pathology Solutions, Santa negative control. Clara, CA), blocked with normal goat serum, and incubated overnight with an Ab against CD45 (BD Biosciences, Franklin Lakes, NJ). This Mass spectrometry and molecular mass analyses was followed by incubation with Histofine Simple Stain Rat MAX PO From the silver-stained gel after electrophoresis, the band at the position (Nichirei Corporation, Tokyo, Japan). The immune complexes were of the mature form of IL-1b was excised and subjected to a standard in- detected using a DAB substrate kit (Vector Laboratories, Burlingame, gel trypsin digestion protocol (28). After the enzyme digestion solu- CA). For 4-HNE immunostaining, the sections were blocked with tion was recovered, it was dried and redissolved in 0.1% formic mouse IgG blocking reagent (Mouse-On-Mouse Immunodetection acid solution. After centrifugation at 20,000 3 g for 10 min at room Kit; Vector Laboratories) and incubated overnight with an Ab against temperature, the supernatant was recovered and subjected to liquid 4-HNE (clone HNEJ-2, Japan Institute for the Control of Aging, chromatography–tandem mass spectrometry (Q Exactive Plus, Thermo Nikken SEIL, Shizuoka, Japan). This was followed by incubation with Fisher Scientific, Waltham, MA). Tandem mass spectrometry ion biotin-conjugated secondary Abs. The sections were treated with avi- search was performed using Proteome Discoverer Software (Thermo din–peroxidase (VECTASTAIN ABC Kit; Vector Laboratories). The Fisher Scientific). Extraction of the N-terminal peptide from the pep- reaction was developed using a DAB Substrate Kit (Vector Laborato- tide fragments identified by a Mascot search (Matrix Science, Boston, ries). The sections were counterstained with hematoxylin. No signals MA) suggested that the N terminus is F104 or N110. In either case, the were detected when an irrelevant IgG (Vector Laboratories) was used peptide bond on the N-terminal side of the residue cannot be cleaved instead of the primary Ab as a negative control. The images of the by trypsin; therefore, both F104 and N110 are N-terminals cleaved stained sections were obtained and analyzed using a microscope (FSX- 2 by an endogenous enzyme in the cell. Furthermore, the theoretical 100; Olympus, Tokyo, Japan). Five visual fields (0.2 mm /field) were molecular mass of cleaved IL-1b was calculated based on the predicted randomly selected to count positively stained cells. Downloaded from sequences using the “Compute pI/Mw” tool on the ExPASy server Cell culture and in vitro experiments (http://web.expasy.org/compute_pi/). Human monocytic THP-1 and human lung epithelial A549 cell lines were obtained from American Type Culture Collection (Manassas, VA). THP-1 cells were grown in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FBS. A549 cells were grown in DMEM (glucose 1000 mg/L; Wako Pure

Chemical Industries, Osaka, Japan) supplemented with 10% FBS. Murine http://www.jimmunol.org/ peritoneal macrophages were isolated using the thioglycollate elicita- tion method and cultured in RPMI 1640 supplemented with 10% FBS. For acidic stress experiments, THP-1 cells were cultured in 24-well tissue culture plates and differentiated into macrophages with PMA (200 nM; Sigma-Aldrich) for 14 h. After being primed with or without LPS (100 ng/ml; Sigma-Aldrich) for 16 h, cells were treated with control (unbuffered balanced salt solution [UBSS]; 144 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, and 0.8 mM MgCl2) or acidic solution (UBSS [pH 1.75] adjusted by HCl), as previously described (25). Subsequently, culture media were washed with 100 ml UBSS and then treated with 100 ml UBSS or UBSS (pH 1.75). After 5 min, 400 ml of 0.1% BSA/RPMI 1640 by guest on September 23, 2021 was added, and the cells were cultured for 6 h. As a positive control of NLRP3 inflammasome activation, THP-1 macrophages were treated with adenosine 39-phosphate (5 mM ATP; Sigma-Aldrich). The same protocol was used in primary murine macrophages. For inhibitor experiments, cells were pretreated for 1 h with 4- (2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche Diagnostics, Dubai, United Arab Emirates), E64 (Sigma-Aldrich), cathepsin G inhibitor I (CGI; Cayman Chemical, Ann Arbor, MI), 20 mM CA-074 Me (Wako Pure Chemical Industries), Z-Tyr-Val-Ala-Asp- fluoromethyl ketone (20 mM) (Medical and Biological Laboratories, Nagoya, Japan), and Mito-TEMPO (80 mM; Enzo Life Sciences, Farmingdale, NY), and then treated with UBSS or UBSS (pH 1.75) as described above. For experiments with mild acidic stress, THP-1 mac- rophagesweretreatedfor8hwithHEPES-bufferedsaltsolution(HBSS, 145 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,20mM HEPES, and 10 mM glucose; [pH 7.4]), HBSS (pH 6.2) (adjusted by NaOH), and lactic acid (25 mM; MP Biomedicals, Santa Ana, CA), according to the previous reports (16, 26). Cells were pretreated for 15 min with the cathepsin D inhibitor pepstatin A (50 mM; Serva Electrophoresis, Heidelberg, Germany). All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Establishment of THP-1 cells transfected with human FLAG/IL-1b mutants b Mutated IL-1bT107A, IL-1bW108A, IL-1bD109A, IL-1bN110A, IL-1bY113D, FIGURE 1. IL-1 contributes to acidic stress–induced ALI independent 2/2 IL-1bV114E, and IL-1bD116I were generated using the PrimeSTAR Muta- of NLRP3 inflammasome. Lung samples were obtained from WT, IL-1b , genesis Basal Kit (Takara Bio, Shiga, Japan) and subcloned into a and NLRP32/2 mice at 24 h after the intratracheal aspiration of acid solution CS-CA-MCS plasmid (kindly provided by Dr. H. Miyoshi, RIKEN Bio- (50 ml of 0.05-N HCl) or PBS (vehicle). (A) Lung sections were stained with 3 Resource Center, Ibaraki, Japan) (27) with a C-terminal FLAG-6 His H&E. (B) Quantitative analysis of lung injury was performed (n = 3 for each tag. To prepare the lentiviral vectors, HEK293T cells were cotransfected in vehicle, n = 4 for each in acid aspiration). (C)IL-1b levels were assessed together with CS-CA-MCS, pLP1, pLP2, and pVSVG using PEI Max 2 2 in BALF of WT and NLRP3 / mice at 24 h after the aspiration (n =4for (Polysciences, Warrington, PA). Culture media containing the lentiviral D vectors were collected and purified by ultracentrifugation. The lentiviral each in vehicle, n = 9–11 for each in acid aspiration). ( ) Il1b mRNA levels titer was measured using a Lentivirus qPCR Titer Kit (Applied Biologi- were assessed in the lung tissue of WT mice at 24 h after aspiration (n =6in cal Materials, Richmond, BC, Canada). For the production of THP-1 vehicle, n = 12 in acid aspiration). Data are expressed as the mean 6 SEM. cells expressing mutated IL-1b, cells were transduced in the presence of *p , 0.05, **p , 0.01. 4 IL-1b PROCESSING IN ACID-INDUCED INFLAMMATION

CRISPR/Cas9-mediated genome editing in THP-1 cells mice were subjected to intratracheal aspiration of acid solution m NLRP3 and caspase-1 genes were edited by CRISPR/Cas9 system in THP-1 (50 l of 0.05-N HCl) or vehicle, and analyzed 24 h after aspiration. cells, which was performed as described previously (29). Briefly, human H&E staining showed that acid aspiration caused alveolar conges- codon–optimized Cas9 and single-guide RNA (sgRNA)–expressing vectors tion, hemorrhage, inflammatory cell infiltration, and alveolar wall (LentiCRISPRv2) were obtained from Addgene (Watertown, MA). The thickening in WT mice, compared with vehicle aspiration (Fig. 1A). sgRNA-targeting NLRP3 and caspase-1 were designed with CRISPR direct Quantitative analysis showed that all of these manifestations were (http://crispr.dbcls.jp). The target sequences of sgRNA (antisense and sense, b2/2 2/2 respectively) were as follows: 59-CACCG CAGGGTCAGCTTGCCGTAGG-39 significantly attenuated in IL-1 mice, but not in NLRP3 and 59-AAACCCTACGGCAAGCTGACCTGC-39 (GFP); 59-CACCGGATC- mice (Fig. 1B). We next assessed IL-1b production in BALF of WT GCAGCGAAGATCCACA-39 and 59-AAACTGTGGATCTTCGCTGCGAT- and NLRP32/2 mice and found that acid aspiration significantly CC-39 (NLRP3); and 59-CACCGAAGCTGTTTATCCGTTCCAT-39 and 59- increased IL-1b levels in the BALF of WT mice. However, there AAACATGGAACGGATAAACAGCTTC-39 (caspase-1). For lentiviral trans- was no significant difference in IL-1b production between WT and duction, THP-1 cells were incubated with lentiviral vectors for 16 h in the 2/2 presence of 8 mg/ml polybrene (Sigma-Aldrich). EGFP-, NLRP3-, and NLRP3 mice (Fig. 1C). Real-time RT-PCR analysis showed the caspase-1–deleted cells were further selected by incubating them with upregulation of Il1b mRNA in the lungs of WT mice after acid 2 mg/ml puromycin (Sigma-Aldrich) for at least 3 d. aspiration (Fig. 1D). These results indicate that IL-1b contributes to Statistical analysis the development of acid aspiration–induced ALI independent of NLRP3 inflammasome. Data were analyzed using IBM SPSS Statistics Version 21 Software (IBM Japan, Tokyo, Japan) and expressed as the mean 6 SEM. An unpaired t test IL-1b deficiency decreases inflammatory cell infiltration and was used to compare two groups following Levene test for homogeneity of

reactive oxygen species generation Downloaded from variance. For comparisons between multiple groups, the significance of differences between group means was determined using one-way ANOVA To investigate inflammatory cell infiltration into the lungs, we combined with Tukey test or the Games–Howell test. A p value ,0.05 was performed immunohistochemical staining for the pan-leukocyte considered to be statistically significant. marker CD45. Acid aspiration clearly increased the number of CD45+ cells in the lungs of WT mice, and this increase was signif- Results icantly prevented in IL-1b2/2 mice (Fig. 2A, 2B). We also assessed IL-1b contributes to acid aspiration–induced ALI independent the infiltration of inflammatory cells, such as alveolar macrophages http://www.jimmunol.org/ of NLRP3 inflammasome (F4/80+/CD11c+), activated macrophages (CD11b+/CD11c+), and To investigate the contributions of IL-1b and NLRP3 inflammasome neutrophils (CD45R2/Ly-6G+), in BALF. The number of total in- in gastric aspiration–induced ALI, WT, IL-1b2/2, and NLRP32/2 flammatory cells, alveolar macrophages, activated macrophages, by guest on September 23, 2021

FIGURE 2. IL-1b deficiency decreases inflammatory cell infiltration and ROS generation. Lung samples were obtained from WT, IL-1b2/2, and NLRP32/2 mice at 24 h after the intratracheal aspiration of acid solution (50 ml of 0.05-N HCl) or PBS (vehicle). (A) Lung sections were immunohistochemically stained with anti-CD45 Ab. Arrowheads indicate CD45-positive cells. (B) Quantitative analysis of CD45-positive cells was performed (n = 3 for each in vehicle, n =4 for each in acid aspiration). (C) The total cell count in BALF was determined (n = 6–8 for each in vehicle, n = 8–11 for each in acid aspiration). (D) The num- bers of alveolar macrophages (F4/80+/CD11c+), activated macrophages (CD11b+/CD11c+) and neutrophils (CD45R2/Ly-6G+) in BALF were analyzed using flow cytometry. (E) ROS generation was assessed by immunohistochemistry with anti–4-HNE Ab. Arrowheads indicate 4-HNE–positive cells. (F) Quantitative analysis of 4-HNE–positive cells was performed (n = 3 for each in vehicle, n = 4 for each in acid aspiration). Data are expressed as the mean 6 SEM. *p , 0.05, **p , 0.01. The Journal of Immunology 5 and neutrophils was significantly increased in WT mice after acid production between unprimed and LPS-primed cells; this result is aspiration (Fig. 2C, 2D). The infiltration of total inflammatory reasonable because pro–IL-1b synthesis is induced by PMA alone cells and alveolar macrophages was significantly decreased in lungs (31). To determine the contribution of NLRP3 inflammasome in of IL-1b2/2 mice. The infiltration of activated macrophages acidic stress–induced IL-1b production, the activation of caspase- and neutrophils also tended to be decreased in IL-1b2/2 mice, but 1 was determined by western blotting. Although the well-known this difference was not statistically significant. Similar changes NLRP3 inflammasome activator ATP obviously caused the were observed in the expression of inflammatory chemokines, cleavage (p20) of caspase-1, acidic stress failed to induce its Ccl2 and Cxcl1 (data not shown). Because reactive oxygen spe- cleavage (Fig. 3B), indicating that NLRP3 inflammasome is not cies (ROS) contributes to the development of gastric aspiration– involved in severe acidic stress–induced IL-1b production. Ad- induced ALI (30), we performed an immunohistochemical ditionally, although we detected weak bands of mature IL-1b in analysis for the lipid peroxidation marker 4-HNE to assess ROS severe acidic stress–treated cells, we found marked bands of generation. 4-HNE+ cells were clearly visualized in the lung of WT mature IL-1b in ATP-treated cells (Fig. 3C). To enhance the weak mice after acid aspiration (Fig. 2E, 2F), and the number of these bands of the mature form, we performed immunoprecipitation cells was markedly lower in the lungs of IL-1b2/2 mice. These using anti–IL-1b Ab and clearly showed that severe acidic stress results suggest that IL-1b plays a crucial role in the inflammatory mainly produced the 18-kDa (p18–IL-1b) mature form, which is response and ROS generation in acid aspiration–induced ALI. different from 17 kDa (p17–IL-1b) processed by caspase-1. The large majority of mature IL-1b processed by ATP and acidic stress b Acidic stress induces IL-1 processing in macrophages was p17–IL-1b and p18–IL-1b, respectively. Consistent with the

To explore the mechanisms by which acid aspiration triggers IL-1b findings in THP-1 macrophages, severe acidic stress also stimu- Downloaded from production in the lung, we examined the effects of acidic stress on lated IL-1b production by primary macrophages from WT mice. IL-1b production in macrophages in vitro because alveolar resi- This IL-1b production retained by macrophages from NLRP32/2 dent macrophages are directly exposed to acidic stress when or caspase-12/2 mice (Fig. 3D). As expected, ATP-induced IL-1b subjected to acid aspiration. Severe acidic stress, loaded by a brief production was almost completely abolished by the deficiency of exposure to acid solution (UBSS [pH 1.75]), markedly stimulated NLRP3 and caspase-1. These findings suggest that severe acidic

IL-1b production in PMA-differentiated THP-1 macrophages stress processes p18–IL-1b, which is distinct from caspase-1– http://www.jimmunol.org/ (Fig. 3A). There was no difference in acidic stress–induced IL-1b processed p17–IL-1b in macrophages. by guest on September 23, 2021

FIGURE 3. Acidic stress induces IL-1b processing in macrophages. After being primed with or without LPS (100 ng/ml) for 6 h, THP-1 macrophages were treated with UBSS (control), UBSS (pH 1.75; acidic stress), or ATP (5 mM) for 6 h. (A) IL-1b levels in the supernatants were assessed (n = 4 for each). (B) Cell lysates were prepared and analyzed by Western blotting with anti–caspase-1 Ab. p20 Indicates cleaved caspase- 1. (C) The supernatants were analyzed by Western blotting with anti–IL-1b Ab (left panel). The samples were immunoprecipitated with anti–IL-1b Ab and analyzed by Western blotting with anti–IL- 1b Ab (right panel). Arrows indicate mature IL-1b (18 and 17 kDa). (D)Afterbeingprimedwithor without LPS (100 ng/ml) for 6 h, primary murine macrophages from WT, NLRP32/2, and caspase-1 (Casp1)2/2 mice were treated with UBSS (control), UBSS (pH 1.75; acidic stress), or ATP (5 mM) for 6 h. IL-1b levels in the supernatants were assessed (n = 3–4 for each). Data are expressed as the mean 6 SEM. **p , 0.01. 6 IL-1b PROCESSING IN ACID-INDUCED INFLAMMATION

Acidic stress induces IL-1b processing independent of Serine protease(s) is responsible for acidic stress–induced NLRP3 inflammasome IL-1b processing To confirm that acidic stress produced mature IL-1b inde- Although caspase-1 is a predominant protease that processes pro– pendent of NLRP3 inflammasome, we tested the effects of in- IL-1b into its mature form, it has been reported that pro–IL-1b hibitors related to NLRP3 inflammasome: 130 mM KCl can be processed by other proteases, such as proteinase 3, (potassium efflux inhibition), Z-YVAD (caspase-1 inhibitor), neutrophil elastase, cathepsin G, chymase, and chymotrypsin CA074-Me (cathepsin B inhibitor), and Mito-TEMPO (32). To determine which protease(s) is important for acidic (mitochondria-targeted antioxidant). Although none of these stress–produced p18–IL-1b, we pretreated THP-1 macrophages inhibitors inhibited severe acidic stress–induced IL-1b pro- with serine or cysteine protease inhibitors such as AEBSF duction, all except Mito-TEMPO significantly inhibited ATP- (serine protease inhibitor) and E-64 (cysteine protease inhibi- induced IL-1b production (Fig. 4A). Consistently, Western tor). Although AEBSF inhibited severe acidic stress–produced blotting showed that these inhibitors had no effect on acidic p18–IL-1b (Fig. 5A, 5B), E-64 did not (Fig. 5C, 5D). Among stress–produced p18–IL-1b, whereas KCl, Z-YVAD, and CA074- serine proteases, cathepsin G is expressed in THP-1 cells (33) Me completely or partially inhibited ATP-produced p17–IL-1b and cleaves the N terminus of pro–IL-1b at Tyr113 (32); (Fig. 4B). To further confirm that NLRP3 inflammasomes are not therefore, we tested the CGI, but it did not affect acidic stress– involved in acidic stress–induced IL-1b production, NLRP3- and produced p18–IL-1b (Fig. 5E, 5F). caspase-1-deleted THP-1 macrophages were generated by using Because previous studies reported that mild acidic stress (pH 6.2) the CRISPER/Cas9 genome-editing system. As expected, defi- or lactic acid produces p18–IL-1b in microglia or THP-1 cells (16, ciency of NLRP3 and caspase-1 completely inhibited ATP- 17), we next investigated whether severe acidic stress (pH 1.75)– Downloaded from produced p17–IL-1b, but not severe acidic stress–produced produced p18–IL-1b would be similar to mild acidic stress p18–IL-1b (Fig. 4C). These data further support the notion that (pH 6.2)–produced p20–IL-1b. Consistent with previous reports severe acidic stress produces p18–IL-1b independent of NLRP3 (16, 17), mild acidic stress (pH 6.2)– or lactic acid–induced IL-1b inflammasome. production, which was significantly inhibited by pretreatment with http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Acidic stress induces IL-1b processing independently of NLRP3 inﬂammasomes. THP-1 macrophages were treated with UBSS (control), UBSS (pH 1.75; acidic stress), or ATP (5 mM) for 6 h in the presence or absence of 130 mM KCl (potassium efﬂux inhibition), Z-YVAD (caspase-1 inhibitor), CA074-Me (cathepsin B inhibitor), or Mito-TEMPO (mitochondria- targeted antioxidant). (A)IL-1b levels in the supernatants were assessed (n = 4 for each). (B) The supernatants were analyzed by Western blotting with anti–IL-1b Ab. Arrows indicate mature IL-1b (18 and 17 kDa). (C) NLRP3- and caspase-1–deleted THP-1 cells were generated using the CRISPR/Cas9 genome–editing system. The control (EGFP–gRNA), NLRP3-deleted (NLRP3–gRNA), and caspase-1–deleted (Casp1– gRNA) cells were treated with UBSS (control), UBSS (pH 1.75; acidic stress), or ATP (5 mM) for 6 h. The supernatants were analyzed by Western blotting with anti–IL-1b Ab. Data are expressed as the mean 6 SEM. **p , 0.01. The Journal of Immunology 7

FIGURE 5. Serine protease(s) is responsible for acidic stress–induced IL-1b processing. (A–F) THP-1 macrophages were treated with UBSS (control), UBSS (pH 1.75; acidic stress), or ATP (5 mM) for 6 h in the presence or absence of AEBSF, E-64, or CGI at the indicated concentrations. (A, C, and E) IL-1b levels in the supernatants were assessed (n = 3–4 for each). (B, D, and F) The supernatants were analyzed by Western blotting with anti–IL-1b Ab. Arrows indi- Downloaded from cate mature IL-1b (18 and 17 kDa). (G and H) THP-1 macrophages were treated with control, UBSS (pH 6.2; mild acidic stress), lactic acid (25 mM), UBSS (pH 1.75; acidic stress), or ATP (5 mM) for 6 h in the presence or absence of pepstatin A (50 mM). IL- http://www.jimmunol.org/ 1b levels in the supernatants were assessed (n = 3–4 for each). Data are expressed as the mean 6 SEM. **p , 0.01. by guest on September 23, 2021

the cathepsin D inhibitor pepstatin A (Fig. 5G), indicating that previously (Y113/V114, V114/H115, and D116/A117), we mild acidic stress or lactic acid each induced IL-1b processing by performed mass spectrometry analysis to identify a cleavage cathepsin D. In contrast, neither severe acidic stress (pH 1.75)– nor site processed by acidic stress and identified two candidates: ATP-induced IL-1b production was inhibited by pepstatin A T107/W108 and D109/N110 (Fig. 7A). To identify the re- (Fig. 5H). These findings suggest that severe acidic stress–induced sponsible cleavage site, we constructed FLAG/pro–IL-1b mu- production of p18–IL-1b is mediated by serine protease(s), but not tants IL-1bT107A,IL-1bW108A,IL-1bD109A, and IL-1bN110A. by cathepsin G or cathepsin D in macrophages. After confirming the equal expression levels of each FLAG/ pro–IL-1b mutant in THP-1 macrophages (Fig. 7B), we tested Identification of pro–IL-1b cleavage sites by acidic stress the effect of ATP- or severe acidic stress–induced pro–IL-1b To explore the cleavage site(s) of IL-1b by acidic stress, we processing of each of these mutants. ATP processed all of these constructed several FLAG-tagged human IL-1b mutants. Previous mutants (Fig. 7C, 7D). However, acidic stress processed IL- b studies identified that pro–IL-1 is recognized by proteases at 1bT107A, IL-1bW108A, and IL-1bN110A (Fig. 7E, 7F), but failed to amino acids E111, Y113, V114, D116, and R120 (32). Of these, process IL-1bD109A, indicating that D109 is the cleavage site of E111 is cleaved by the metalloprotease meprin b and the mo- pro–IL-1b by acidic stress. Furthermore, through use of the lecular mass of IL-1b cleaved at R120 would be less than that “Compute pI/Mw” tool, the theoretical molecular mass of acidic cleaved between D116 and A117; thus, three human IL-1b mu- stress–induced mature IL-1b was calculated to be 18.2 kDa. tants were constructed: IL-1b , IL-1b , and IL-1b . Y113D V114E D116I b The expression levels of each FLAG/pro–IL-1b mutant in THP-1 Acidic stress–processed IL-1 exerts bioactivity in alveolar macrophages were confirmed to be equal (Fig. 6A). Caspase-1 has epithelial cells been shown to cleave pro–IL-1b between D116 and A117. Indeed, Finally, to confirm whether acidic stress–produced p18–IL-1b ATP treatment processed IL-1bWT, IL-1bY113D, and IL-1bV114E, could exert bioactivity, we examined the effects of the super- but not IL-1bD116I, into p17–IL-1b (Fig. 6B). In contrast, severe natants prepared from acidic stress–treated THP-1 macrophages acidic stress processed all of these IL-1b mutants into p18–IL-1b on p38 phosphorylation in alveolar epithelial A549 cells. The (Fig. 6C). supernatants from the acidic stress condition clearly promoted Because our data suggested that cleavage site(s) of pro–IL-1b p38 phosphorylation, which is consistent with previous reports by severe acidic stress are different from the sites described that IL-1b stimulates p38 phosphorylation in A549 cells 8 IL-1b PROCESSING IN ACID-INDUCED INFLAMMATION

FIGURE 6. IL-1b is processed by acidic stress at a previously unreported cleavage site. THP-1 macrophages transfected with human FLAG/IL-1bWT, FLAG/IL-1bY113D,FLAG/IL-1bV114E,andFLAG/ IL-1bD116I were prepared. Transfection of hKO1 was used as a negative control. (A)ExpressionofFLAG/ IL-1b mutants was assessed by Western blotting in each transfected THP-1 macrophage. (B and C) Downloaded from Each transfected THP-1 macrophage was treated with UBSS (control), ATP (5 mM), or UBSS (pH 1.75; acidic stress) for 6 h. The supernatants were analyzed by Western blotting with anti–IL-1b Ab. http://www.jimmunol.org/ by guest on September 23, 2021

(Fig. 8A) (34). In addition, p38 phosphorylation was inhibited by alveolar epithelial cells. These results clearly indicate that acidic pretreatment with IL-1Ra (Fig. 8B). Similarly, the supernatants stress processes pro–IL-1b at a site different from that previously from the acidic condition significantly induced the expression of reported and promotes IL-1b–driven inflammation independent of chemokines, CCL2 and CXCL1, and this increased expression NLRP3 inflammasome, and contributes to the development of acid was significantly inhibited by IL-1Ra (Fig. 8C). These results aspiration–induced ALI. Our study also shows a novel mechanism indicate that acidic stress–produced p18–IL-1b exerts bioactivity of acid-induced IL-1b production and inflammation, and provides as its mature form. new insight into the therapeutic strategies for gastric aspiration pneumonitis and ALI. Discussion Increasing evidence indicates that sterile inflammation con- The major findings of this study are as follows: 1) acid aspiration tributes to the development of aspiration pneumonitis and ALI induced inflammatory responses, ROS generation, and ALI in WT (1, 2). Indeed, several inflammatory cytokines and chemokines mice, and these effects were significantly attenuated in IL-1b2/2 including TNF-a and MIP-2 have been shown to be involved in its mice; 2) acid aspiration–induced ALI and IL-1b production were pathogenesis (35, 36). Although IL-1b plays a pivotal role in the not inhibited in NLRP32/2 mice; 3) in vitro experiments revealed process of sterile inflammation, its role in gastric aspiration–in- that severe acidic stress (pH 1.75; similar to gastric fluid) produced pneumonitis and ALI has not yet been demonstrated. In the cessed pro–IL-1b into p18–IL-1b, which was different from p17– current study, we clearly showed that IL-1b levels were ele- IL-1b processed by caspase-1, in human THP-1 macrophages vated after acid aspiration and that IL-1b deficiency significantly and primary murine macrophages; 4) deficiency of NLRP3 and improved acid aspiration–induced inflammatory responses and caspase-1 had no effect on acidic stress–produced p18–IL-1b in ALI, indicating that IL-1b is required for its pathogenesis. Re- macrophages; 5) acidic stress–induced production of p18–IL-1b garding IL-1b production, NLRP3 inflammasome has received was prevented by inhibitors for serine proteases (AEBSF), but not much attention because it contributes to the development of sterile cysteine proteases (E-64), cathepsin G, cathepsin D, or NLRP3 inflammatory diseases (5, 6). Therefore, we hypothesized that inflammasome; 6) acidic stress processed pro–IL-1b with known NLRP3 inflammasome could mediate acid aspiration–induced IL- cleavage site mutations (IL-1bY113D, IL-1bV114E, and IL-1bD116I); 1b production in the lung. Contrary to our expectation, deficiency 7) using mass spectrometry and processing-site mutants of pro– of NLRP3 failed to prevent acid aspiration–induced inflammatory IL-1b, we identified D109/110 as a novel cleavage site of pro–IL- responses and ALI. Furthermore, acid aspiration–induced IL-1b 1b in response to severe acidic stress; and 8) the bioactivity production was not decreased by NLRP3 deficiency. Previous of acidic stress–produced p18–IL-1b was confirmed by its ability studies have suggested that NLRP3 inflammasome plays a key to induce p38 phosphorylation and chemokine upregulation in role in several ALI models, such as LPS alone and combined with The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. Identification of pro–IL-1b cleavage site by acidic stress. (A) Acidic stress–induced cleavage site candidates in pro–IL-1b.(B–E) THP-1 macrophages transfected with human FLAG/IL-1bWT,IL-1bT107A,IL-1bW108A,IL-1bD109A,andIL-1bN110A were prepared. Transfection of hKO1 was used as a negative control. (B) Expression of FLAG/IL-1b mutants was assessed. (C and D) Each of the transfected THP-1 macrophages was treated with ATP (5 mM) for 6 h. The supernatants were analyzed by Western blotting with an anti–IL-1b Ab. (E and F) Each of the transfected THP-1 macrophages were treated with UBSS (control) or UBSS (pH 1.75; acidic stress) for 6 h. The supernatants were analyzed by Western blotting with an anti–IL-1b Ab. mechanical ventilation (37, 38). Grailer et al. (39) also reported To our knowledge, this study is the first to demonstrate that that extracellular histones released from neutrophils activates NLRP3 inflammasome–independent IL-1b production contributes NLRP3 inflammasome and induces inflammatory responses and to the development of aspiration pneumonitis and subsequent ALI. lung injury in C5a- and IgG immune complex–induced ALI. In Homeostasis of cellular pH is essential for the proper cell contrast, we have recently reported that hyperoxia induces ALI function and stability. Previous studies have shown that changes in dependent of NLRP3, but independent of IL-1b (22). Further- the cellular pH environment influence inflammatory responses (15, more, Cheng et al. (40) recently highlighted the role of caspase-11 17, 25, 26). With respect to IL-1b, two studies have reported that noncanonical inflammasome in a murine model of LPS-induced cathepsin D–dependent production of p20–IL-1b occurs under ALI. Therefore, we postulate that the contribution of NLRP3 mild acidic conditions, and suggested that this p20–IL-1b is dis- inflammasome or IL-1b may depend on the ALI model employed. tinct from p17–IL-1b processed by caspase-1 (16, 17). In these 10 IL-1b PROCESSING IN ACID-INDUCED INFLAMMATION

IL-1bV114E, and IL-1bD116I; however, severe acidic stress processed all of these IL-1b mutants into p18–IL-1b, indicating that acidic stress–produced p18–IL-1b is cleaved at sites other than Y113, V114, and D116. The results prompted us to perform mass spectrometry, and subsequent analysis using cleavage mutations of pro–IL-1b revealed that D109 is a novel cleavage site of pro–IL- 1b in response to acidic stress. We further calculated that the theoretical molecular mass of acidic stress–induced mature IL-1b was 18.2 kDa. These ﬁndings suggest that this cleavage site is a novel potential therapeutic target for aspiration pneumonitis. Several limitations of this study should be noted. First, we showed that macrophages are the predominant effector cells that produce IL-1b under severe acidic conditions because alveolar macrophages are directly exposed to acidic stress in the setting of gastric aspiration; however, the role of other cell types (e.g., ﬁbroblasts, epithelial cells, and endothelial cells) in the acidic stress–induced production of IL-1b remains to be determined. Second, we showed that severe acidic stress–induced p18–IL-

1b clearly induces p38 phosphorylation and chemokine ex- Downloaded from pression, indicating its bioactivity. Edye et al. (26) recently reported that mild acidic stress–induced p20–IL-1b was mini- mally active in the transduction of IL-1 signaling and suggested that cathepsin D–mediated p20–IL-1b may act as a negative regulator of p17–IL-1b that limits the amount of pro–IL-1b

available for caspase-1 processing. Third, although several http://www.jimmunol.org/ interventions including platelet depletion and IL-17Ra deletion have been shown to prevent the development of acid aspiration–induced ALI (41, 42), there does not seem to be speciﬁc FIGURE 8. Acidic stress–processed IL-1b exerts bioactivity in alveolar clinical intervention that is accepted for the treatment of acid epithelial cells. (A and B) Alveolar epithelial A549 cells were treated with aspiration–induced ALI. In this regard, gastric ﬂuid commonly the supernatants (Sup) from acidic stress–treated THP-1 macrophages for contains particulate food matter in the clinical setting, and 20 min in the presence or absence of IL-1Ra at the indicated concentra- several experimental studies have reported that aspiration of tions. Cell lysates were prepared and analyzed by Western blotting with acidic stress with gastric particles synergistically exacerbated

C by guest on September 23, 2021 Abs against p38 and phospho-p38. ( ) Alveolar epithelial A549 cells were acid aspiration–induced ALI (2, 35). Thus, further investiga- treated with the supernatants for 6 h in the presence or absence of IL-1Ra tions are necessary to elucidate the mechanism and role of (6 ng/ml). CCL2 and CXCL1 mRNA expression was assessed by real-time b RT-PCR analysis. Data are expressed as the mean 6 SEM. **p , 0.01. IL-1 in the pathophysiology of aspiration pneumonitis. In conclusion, we demonstrated that NLRP3 inflammasome– independent IL-1b production contributes to the development of studies, however, cells were subjected to mild acidic conditions gastric aspiration pneumonitis. We also showed that severe acidic (pH 6–7) because tissue ischemia has been shown to drop at ap- stress induces the serine protease–dependent production of mature proximately pH 6–6.5 (13, 14). Therefore, IL-1b production under IL-1b in macrophages and identified a novel cleavage site of severe acidic conditions similar to the gastric environment has IL-1b in response to severe acidic stress. The findings of the not yet been clarified. In the current study, we showed that acidic current study demonstrate that IL-1b plays a pivotal role in acid stress (pH 1.75) induced the production of p18–IL-1b, indepen- aspiration–induced inflammation and ALI and identify the novel dent of NLRP3 inflammasome, in macrophages. Interestingly, the mechanism underlying acidic stress–mediated IL-1b processing. well-known NLRP3 inflammasome activator ATP produced not Furthermore, this study provides new insight into therapeutic only p17–IL-1b, but also p18–IL-1b; the amount of p18–IL-1b strategies for aspiration pneumonitis. was much less than that of p17–IL-1b. In contrast, although acidic stress produced both p17–IL-1b and p18–IL-1b, the large ma- Acknowledgments jority was p18–IL-1b. These results are partly consistent with We thank Dr. Tsukasa Ohmori (Jichi Medical University) for invaluable previous reports that danger signals, such as ATP, monosodium suggestions, and Dr. Dirk Bohmann (University of Rochester, Rochester, urate, and calcium pyrophosphate dehydrate, produce p20–IL-1b NY) for providing the plasmids. under mild acidic conditions in microglial cells (16, 17). In addition, we showed that severe acidic stress produced p18–IL-1b in Disclosures a serine protease–dependent, but cathepsin D–independent, man- The authors have no financial conflicts of interest. ner, whereas mild acidic stress produced p20–IL-1b in a cathepsin D–dependent manner. Therefore, we assumed that although se- References vere and mild acidic stress produce the mature form of IL-1b 1. Marik, P. E. 2001. Aspiration pneumonitis and aspiration pneumonia. N. Engl. J. with similar molecular mass, the responsible protease(s) differs Med. 344: 665–671. depending on the pH level. Because V113, Y114, and D116 are 2. Raghavendran, K., J. Nemzek, L. M. Napolitano, and P. R. Knight. 2011. Aspiration-induced lung injury. Crit. Care Med. 39: 818–826. known to be cleaved by proteinases (neutrophil elastase, cathepsin 3. Knight, P. R., G. Druskovich, A. R. Tait, and K. J. Johnson. 1992. The role of G, chymase, and chymotrypsin proteinase 3 for V113; proteinase neutrophils, oxidants, and proteases in the pathogenesis of acid pulmonary in- b jury. Anesthesiology 77: 772–778. 3 for Y114; and meprin A/ for D116) (32), we tested whether 4. Matute-Bello, G., C. W. Frevert, and T. R. Martin. 2008. Animal models of acute acidic stress–induced processing could be inhibited in IL-1bY113D, lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 295: L379–L399. The Journal of Immunology 11

5. Takahashi, M. 2014. NLRP3 inflammasome as a novel player in myocardial 23. Nishina, K., K. Mikawa, Y. Takao, M. Shiga, N. Maekawa, and H. Obara. 1998. infarction. Int. Heart J. 55: 101–105. Intravenous lidocaine attenuates acute lung injury induced by hydrochloric acid 6. Latz, E., T. S. Xiao, and A. Stutz. 2013. Activation and regulation of the aspiration in rabbits. Anesthesiology 88: 1300–1309. inflammasomes. Nat. Rev. Immunol. 13: 397–411. 24. Belperio, J. A., M. P. Keane, M. D. Burdick, V. Londhe, Y. Y. Xue, K. Li, 7. Kuida, K., J. A. Lippke, G. Ku, M. W. Harding, D. J. Livingston, M. S. Su, R. J. Phillips, and R. M. Strieter. 2002. Critical role for CXCR2 and CXCR2 and R. A. Flavell. 1995. Altered cytokine export and apoptosis in mice ligands during the pathogenesis of ventilator-induced lung injury. J. Clin. Invest. deficient in interleukin-1 beta converting enzyme. Science 267: 2000– 110: 1703–1716. 2003. 25. Fernandez, S. F., C. Fung, J. D. Helinski, R. Alluri, B. A. Davidson, and 8. Yajima, N., M. Takahashi, H. Morimoto, Y. Shiba, Y. Takahashi, J. Masumoto, P. R. Knight, III. 2013. Low pH environmental stress inhibits LPS and LTA- H. Ise, J. Sagara, J. Nakayama, S. Taniguchi, and U. Ikeda. 2008. Critical role of stimulated proinflammatory cytokine production in rat alveolar macrophages. bone marrow apoptosis-associated speck-like protein, an inflammasome adaptor BioMed Res. Int. 2013: 742184. molecule, in neointimal formation after vascular injury in mice. Circulation 117: 26. Edye, M. E., D. Brough, and S. M. Allan. 2015. Acid-dependent interleukin-1 3079–3087. (IL-1) cleavage limits available pro-IL-1b for caspase-1 cleavage. J. Biol. Chem. 9. Kawaguchi, M., M. Takahashi, T. Hata, Y. Kashima, F. Usui, H. Morimoto, 290: 25374–25381. A. Izawa, Y. Takahashi, J. Masumoto, J. Koyama, et al. 2011. Inflammasome 27. Miyoshi, H., U. Blo¨mer, M. Takahashi, F. H. Gage, and I. M. Verma. 1998. activation of cardiac fibroblasts is essential for myocardial ischemia/reperfusion Development of a self-inactivating lentivirus vector. J. Virol. 72: 8150–8157. injury. Circulation 123: 594–604. 28. Shevchenko, A., H. Tomas, J. Havlis, J. V. Olsen, and M. Mann. 2006. In-gel 10. Usui, F., K. Shirasuna, H. Kimura, K. Tatsumi, A. Kawashima, T. Karasawa, digestion for mass spectrometric characterization of proteins and proteomes. K. Yoshimura, H. Aoki, H. Tsutsui, T. Noda, et al. 2015. Inflammasome acti- Nat. Protoc. 1: 2856–2860. vation by mitochondrial oxidative stress in macrophages leads to the develop- 29. Shirasuna, K., T. Karasawa, F. Usui, M. Kobayashi, T. Komada, H. Kimura, ment of angiotensin II-induced aortic aneurysm. Arterioscler. Thromb. Vasc. A. Kawashima, A. Ohkuchi, S. Taniguchi, and M. Takahashi. 2015. NLRP3 Biol. 35: 127–136. deficiency improves angiotensin II-induced hypertension but not fetal growth 11. Komada, T., F. Usui, A. Kawashima, H. Kimura, T. Karasawa, Y. Inoue, restriction during pregnancy. Endocrinology 156: 4281–4292. M. Kobayashi, Y. Mizushina, T. Kasahara, S. Taniguchi, et al. 2015. Role of 30. Segal, B. H., B. A. Davidson, A. D. Hutson, T. A. Russo, B. A. Holm, B. Mullan, NLRP3 inflammasomes for rhabdomyolysis-induced acute kidney injury. Sci. M. Habitzruther, S. M. Holland, and P. R. Knight, III. 2007. Acid aspiration- Rep. 5: 10901. induced lung inflammation and injury are exacerbated in NADPH oxidase- Downloaded from 12. Usui, F., K. Shirasuna, H. Kimura, K. Tatsumi, A. Kawashima, T. Karasawa, deficient mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 292: L760–L768. S. Hida, J. Sagara, S. Taniguchi, and M. Takahashi. 2012. Critical role of 31. Martinon, F., K. Burns, and J. Tschopp. 2002. The inflammasome: a molecular caspase-1 in vascular inflammation and development of atherosclerosis in platform triggering activation of inflammatory caspases and processing of proIL- Western diet-fed apolipoprotein E-deficient mice. Biochem. Biophys. Res. beta. Mol. Cell 10: 417–426. Commun. 425: 162–168. 32. Netea, M. G., F. L. van de Veerdonk, J. W. van der Meer, C. A. Dinarello, and 13. Nemoto, E. M., and S. Frinak. 1981. Brain tissue pH after global brain ischemia L. A. Joosten. 2015. Inflammasome-independent regulation of IL-1-family cy- and barbiturate loading in rats. Stroke 12: 77–82. tokines. Annu. Rev. Immunol. 33: 49–77.

14. Garlick, P. B., G. K. Radda, and P. J. Seeley. 1979. Studies of acidosis in the 33. Rivera-Marrero, C. A., J. Stewart, W. M. Shafer, and J. Roman. 2004. The down- http://www.jimmunol.org/ ischaemic heart by phosphorus nuclear magnetic resonance. Biochem. J. 184: regulation of cathepsin G in THP-1 monocytes after infection with Mycobac- 547–554. terium tuberculosis is associated with increased intracellular survival of bacilli. 15. Rajama¨ki, K., T. Nordstro¨m, K. Nurmi, K. E. Akerman,˚ P. T. Kovanen, K. O¨ o¨rni, Infect. Immun. 72: 5712–5721. and K. K. Eklund. 2013. Extracellular acidosis is a novel danger signal alerting 34. Lin, C. C., C. T. Kuo, C. Y. Cheng, C. Y. Wu, C. W. Lee, H. L. Hsieh, I. T. Lee, innate immunity via the NLRP3 inﬂammasome. J. Biol. Chem. 288: 13410– and C. M. Yang. 2009. IL-1 beta promotes A549 cell migration via MAPKs/AP- 13419. 1- and NF-kappaB-dependent matrix metalloproteinase-9 expression. Cell. Sig- 16. Takenouchi, T., Y. Iwamaru, S. Sugama, M. Tsukimoto, M. Fujita, A. Sekigawa, nal. 21: 1652–1662. K. Sekiyama, M. Sato, S. Kojima, B. Conti, et al. 2011. The activation of P2X7 35. Davidson, B. A., P. R. Knight, J. D. Helinski, N. D. Nader, T. P. Shanley, and receptor induces cathepsin D-dependent production of a 20-kDa form of IL-1b K. J. Johnson. 1999. The role of tumor necrosis factor-alpha in the pathogenesis under acidic extracellular pH in LPS-primed microglial cells. J. Neurochem. 117: of aspiration pneumonitis in rats. Anesthesiology 91: 486–499. 712–723. 36. Shanley, T. P., B. A. Davidson, N. D. Nader, N. Bless, N. Vasi, P. A. Ward,

17. Edye, M. E., G. Lopez-Castejon, S. M. Allan, and D. Brough. 2013. Acidosis K. J. Johnson, and P. R. Knight. 2000. Role of macrophage inflammatory by guest on September 23, 2021 drives damage-associated molecular pattern (DAMP)-induced interleukin-1 se- protein-2 in aspiration-induced lung injury. Crit. Care Med. 28: 2437–2444. cretion via a caspase-1-independent pathway. J. Biol. Chem. 288: 30485–30494. 37. Wang, S., J. Zhao, H. Wang, Y. Liang, N. Yang, and Y. Huang. 2015. Blockage of 18. Horai, R., M. Asano, K. Sudo, H. Kanuka, M. Suzuki, M. Nishihara, P2X7 attenuates acute lung injury in mice by inhibiting NLRP3 inflammasome. M. Takahashi, and Y. Iwakura. 1998. Production of mice deficient in genes for Int. Immunopharmacol. 27: 38–45. interleukin (IL)-1alpha, IL-1beta, IL-1alpha/beta, and IL-1 receptor antagonist 38. Jones, H. D., T. R. Crother, R. A. Gonzalez-Villalobos, M. Jupelli, S. Chen, shows that IL-1beta is crucial in turpentine-induced fever development and J. Dagvadorj, M. Arditi, and K. Shimada. 2014. The NLRP3 inflammasome is glucocorticoid secretion. J. Exp. Med. 187: 1463–1475. required for the development of hypoxemia in LPS/mechanical ventilation acute 19. Lamkanfi, M., J. L. Mueller, A. C. Vitari, S. Misaghi, A. Fedorova, K. Deshayes, lung injury. Am. J. Respir. Cell Mol. Biol. 50: 270–280. W. P. Lee, H. M. Hoffman, and V. M. Dixit. 2009. Glyburide inhibits the 39. Grailer, J. J., B. A. Canning, M. Kalbitz, M. D. Haggadone, R. M. Dhond, Cryopyrin/Nalp3 inflammasome. J. Cell Biol. 187: 61–70. A. V. Andjelkovic, F. S. Zetoune, and P. A. Ward. 2014. Critical role for the 20. Davidson, B. A., and R. Alluri. 2013. Gastric aspiration models. Bio Protoc. 3: NLRP3 inflammasome during acute lung injury. J. Immunol. 192: 5974–5983. e968. 40. Cheng, K. T., S. Xiong, Z. Ye, Z. Hong, A. Di, K. M. Tsang, X. Gao, S. An, 21. Vergadi, E., K. Vaporidi, E. E. Theodorakis, C. Doxaki, E. Lagoudaki, M. Mittal, S. M. Vogel, et al. 2017. Caspase-11-mediated endothelial pyroptosis E. Ieronymaki, V. I. Alexaki, M. Helms, E. Kondili, B. Soennichsen, et al. 2014. underlies endotoxemia-induced lung injury. J. Clin. Invest. 127: 4124–4135. Akt2 deficiency protects from acute lung injury via alternative macrophage ac- 41. Zarbock, A., K. Singbartl, and K. Ley. 2006. Complete reversal of acid-induced tivation and miR-146a induction in mice. J. Immunol. 192: 394–406. acute lung injury by blocking of platelet-neutrophil aggregation. J. Clin. Invest. 22. Mizushina, Y., K. Shirasuna, F. Usui, T. Karasawa, A. Kawashima, H. Kimura, 116: 3211–3219. M. Kobayashi, T. Komada, Y. Inoue, N. Mato, et al. 2015. NLRP3 protein de- 42. Crowe, C. R., K. Chen, D. A. Pociask, J. F. Alcorn, C. Krivich, R. I. Enelow, ficiency exacerbates hyperoxia-induced lethality through Stat3 protein signaling T. M. Ross, J. L. Witztum, and J. K. Kolls. 2009. Critical role of IL-17RA in independent of interleukin-1b. J. Biol. Chem. 290: 5065–5077. immunopathology of influenza infection. J. Immunol. 183: 5301–5310.