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Inflammasome Activation Is Reactive Oxygen Species Dependent and Mediates Irinotecan-Induced Mucositis through IL-1β and IL-18 in Mice
2014; Elsevier BV; Volume: 184; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2014.03.012
ISSN1525-2191
AutoresRaquel Duque do Nascimento Arifa, Mila Fernandes Moreira Madeira, Talles Prosperi de Paula, Renata Lacerda Lima, Lívia D. Tavares, Zélia Menezes‐Garcia, Caio T. Fagundes, Milene Alvarenga Rachid, Bernhard Ryffel, Dario S. Zamboni, Mauro Martins Teixeira, Danielle G. Souza,
Tópico(s)Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
ResumoIrinotecan is a useful chemotherapeutic for the treatment of various cancers. Irinotecan treatment is associated with mucositis, which clearly limits the use of the drug. Mechanisms that account for mucositis are only partially known. This study assessed mechanisms and the role of inflammasome activation in irinotecan-induced mucositis. Mucositis in mice was induced by irinotecan injection in C57BL/6 wild-type, gp91phox−/−, il-18−/−, casp-1−/−, and asc−/− mice once a day for 4 consecutive days. In some experiments, mice received apocynin to inhibit NADPH oxidase (NOX), IL-1 receptor antagonist, or IL-18 binding protein to prevent activation of IL-1 and IL-18 receptors, respectively. Mice were euthanized 7 days after the beginning of irinotecan treatment, and small intestines were collected for analysis. Irinotecan treatment resulted in increased IL-1β and IL-18 production in ileum and NOX-2–dependent oxidative stress. gp91phox−/− and apocynin-treated mice had diminished oxidative stress and less severe mucositis. Furthermore, treatment with apocynin decreased caspase-1 activation and IL-1β and IL-18 production in the ileum. asc−/− and casp-1−/− mice also had less intestinal injury and decreased IL-1β and IL-18 production. Finally, both the absence of IL-18 and IL-1β resulted in reduced inflammatory response and attenuated intestinal injury. NOX-2–derived oxidative stress mediates inflammasome activation and inflammasome-dependent production of IL-1β and IL-18, which mediate tissue injury during irinotecan-induced mucositis in mice. Irinotecan is a useful chemotherapeutic for the treatment of various cancers. Irinotecan treatment is associated with mucositis, which clearly limits the use of the drug. Mechanisms that account for mucositis are only partially known. This study assessed mechanisms and the role of inflammasome activation in irinotecan-induced mucositis. Mucositis in mice was induced by irinotecan injection in C57BL/6 wild-type, gp91phox−/−, il-18−/−, casp-1−/−, and asc−/− mice once a day for 4 consecutive days. In some experiments, mice received apocynin to inhibit NADPH oxidase (NOX), IL-1 receptor antagonist, or IL-18 binding protein to prevent activation of IL-1 and IL-18 receptors, respectively. Mice were euthanized 7 days after the beginning of irinotecan treatment, and small intestines were collected for analysis. Irinotecan treatment resulted in increased IL-1β and IL-18 production in ileum and NOX-2–dependent oxidative stress. gp91phox−/− and apocynin-treated mice had diminished oxidative stress and less severe mucositis. Furthermore, treatment with apocynin decreased caspase-1 activation and IL-1β and IL-18 production in the ileum. asc−/− and casp-1−/− mice also had less intestinal injury and decreased IL-1β and IL-18 production. Finally, both the absence of IL-18 and IL-1β resulted in reduced inflammatory response and attenuated intestinal injury. NOX-2–derived oxidative stress mediates inflammasome activation and inflammasome-dependent production of IL-1β and IL-18, which mediate tissue injury during irinotecan-induced mucositis in mice. Irinotecan (CPT-11) is a chemotherapeutic agent used as a first- or second-line treatment for several solid tumors.1Boige V. Taieb J. Hebbar M. Malka D. Debaere T. Hannoun L. Magherini E. Mignard D. Poynard T. Ducreux M. 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Bioactivation of the anticancer agent CPT-11 to SN-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions.Drug Metab Dispos. 1997; 25: 1157-1164PubMed Google Scholar This drug is a potent topoisomerase I inhibitor,5Slatter J.G. Schaaf L.J. Sams J.P. Feenstra K.L. Johnson M.G. Bombardt P.A. Cathcart K.S. Verburg M.T. Pearson L.K. Compton L.D. Miller L.L. Baker D.S. Pesheck C.V. Lord 3rd, R.S. Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following I.V. infusion of [(14)C]CPT-11 in cancer patients.Drug Metab Dispos. 2000; 28: 423-433PubMed Google Scholar, 6Li Q.Y. Zu Y.G. Shi R.Z. Yao L.P. Review camptothecin: current perspectives.Curr Med Chem. 2006; 13: 2021-2039Crossref PubMed Scopus (241) Google Scholar an effect that is essential to kill cancerous cells. However, topoisomerase I inhibition also leads to death of healthy cells with high mitotic rates, such as basal cells of the intestine.7Bowen J.M. Gibson R.J. Stringer A.M. Chan T.W. Prabowo A.S. Cummins A.G. Keefe D.M. Role of p53 in irinotecan-induced intestinal cell death and mucosal damage.Anticancer Drugs. 2007; 18: 197-210Crossref PubMed Scopus (23) Google Scholar Therefore, treatment with irinotecan is associated with several adverse effects, such as leukopenia, diarrhea, and mucositis.8Sevinc A. Kalender M.E. Altinbas M. Ozkan M. Dikilitas M. Camci C. Irinotecan as a second-line monotherapy for small cell lung cancer.Asian Pac J Cancer Prev. 2011; 12: 1055-1059PubMed Google Scholar, 9Mineur L. Sabatier R. Kirscher S. Plat F. Goubely Y. Molinari N. 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Laurence J. Keefe D.M. Irinotecan-induced mucositis is associated with changes in intestinal mucins.Cancer Chemother Pharmacol. 2009; 64: 123-132Crossref PubMed Scopus (62) Google Scholar, 12Sonis S.T. Pathobiology of mucositis.Semin Oncol Nurs. 2004; 20: 11-15Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar Irinotecan may induce mucosal damage directly; DNA damage due to topoisomerase I inhibition activates several pathways involved in apoptosis and inflammation.7Bowen J.M. Gibson R.J. Stringer A.M. Chan T.W. Prabowo A.S. Cummins A.G. Keefe D.M. Role of p53 in irinotecan-induced intestinal cell death and mucosal damage.Anticancer Drugs. 2007; 18: 197-210Crossref PubMed Scopus (23) Google Scholar However, the mechanism by which irinotecan induces mucositis is not well described, and it is likely that the inflammatory response contributes to the drug-induced mucosal damage. It has been reported that chemotherapy leads to enhanced production of reactive oxygen species (ROS) and reactive nitrogen species, which induce cell death,13Melo M.L. Brito G.A. Soares R.C. Carvalho S.B. Silva J.V. Soares P.M. Vale M.L. Souza M.H. Cunha F.Q. Ribeiro R.A. Role of cytokines (TNF-alpha, IL-1beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide.Cancer Chemother Pharmacol. 2008; 61: 775-784Crossref PubMed Scopus (83) Google Scholar, 14Lima-Junior R.C. Figueiredo A.A. Freitas H.C. Melo M.L. Wong D.V. Leite C.A. Medeiros R.P. Marques-Neto R.D. Vale M.L. Brito G.A. Oria R.B. Souza M.H. Cunha F.Q. Ribeiro R.A. Involvement of nitric oxide on the pathogenesis of irinotecan-induced intestinal mucositis: role of cytokines on inducible nitric oxide synthase activation.Cancer Chemother Pharmacol. 2012; 69: 931-942Crossref PubMed Scopus (53) Google Scholar, 15Bowen J.M. Gibson R.J. Tsykin A. Stringer A.M. Logan R.M. 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Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica.Science. 2008; 320: 674-677Crossref PubMed Scopus (2016) Google Scholar Inflammasomes are composed of one of several NOD-like receptor (NLR) proteins, including NLRP1, NLRP3, and the non-NLR family member AIM2. NLRs function as sensors of endogenous or exogenous damage-associated molecules and lead to caspase-1 activation and subsequent cleavage of pro–IL-1β and pro–IL-18 cytokines into their mature forms.18Schroder K. Tschopp J. The inflammasomes.Cell. 2010; 140: 821-832Abstract Full Text Full Text PDF PubMed Scopus (4115) Google Scholar Importantly, IL-1β is one of the various inflammatory mediators reported to be released during mucositis.13Melo M.L. Brito G.A. Soares R.C. Carvalho S.B. Silva J.V. Soares P.M. Vale M.L. Souza M.H. Cunha F.Q. Ribeiro R.A. Role of cytokines (TNF-alpha, IL-1beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide.Cancer Chemother Pharmacol. 2008; 61: 775-784Crossref PubMed Scopus (83) Google Scholar, 19Wu Z. Han X. Qin S. Zheng Q. Wang Z. Xiang D. Zhang J. Lu H. Wu M. Zhu S. Yu Y. Wang Y. Han W. Interleukin 1 receptor antagonist reduces lethality and intestinal toxicity of 5-fluorouracil in a mouse mucositis model.Biomed Pharmacother. 2010; 65: 339-344Crossref Scopus (20) Google Scholar, 20Xiang D. Guo Y. Zhang J. Gao J. Lu H. Zhu S. Wu M. Yu Y. Han W. Interleukin-1 receptor antagonist attenuates cyclophosphamide-induced mucositis in a murine model.Cancer Chemother Pharmacol. 2011; 67: 1445-1453Crossref PubMed Scopus (17) Google Scholar, 21Wu Z.Q. Han X.D. Wang Y. Yuan K.L. Jin Z.M. Di J.Z. Yan J. Pan Y. Zhang P. Huang X.Y. Wang Z.G. Zheng Q. Interleukin-1 receptor antagonist reduced apoptosis and attenuated intestinal mucositis in a 5-fluorouracil chemotherapy model in mice.Cancer Chemother Pharmacol. 2011; 68: 87-96Crossref PubMed Scopus (47) Google Scholar However, whether the inflammasome is involved in IL-1β production and the role played by IL-18 or IL-1β in irinotecan-induced mucositis have not been reported. Thus, our aim was to assess the role of ROS for inflammasome activation in irinotecan-induced mucositis. In addition, we also investigated the role of inflammasome-derived IL-1β and IL-18 for irinotecan-induced mucositis severity. We found that ROS derived from NADPH oxidase (NOX)-2 are crucial for caspase-1 activation and consequent IL-1β and IL-18 production during mucositis. We also found that the absence of inflammasome complex components leads to diminished intestinal injury, attenuation of body weight loss, and blunted inflammatory response after mucositis induced by irinotecan. In addition, we found that IL-1β and IL-18 production plays a crucial role in mucositis. Wild-type (WT) and gene-deficient (il-18−/− and gp91phox−/−) male C57BL/6 mice (6 to 8 weeks old) were obtained from the Immunopharmacology Animal Facility of the Institute of Biological Sciences (Federal University of Minas Gerais, Belo Horizonte, Brazil). Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) knockout (asc−/−) mice were obtained from the Bioscience Unit Ribeirão Preto, Medical School, University of São Paulo (FMRP/USP), Ribeirão Preto, Brazil). Caspase-1 knockout mice (casp-1−/−) mice and their controls were bred and obtained from the Transgenose Institute of the National Center for Scientific Research (Orléans, France). All animals were housed under standard conditions in separate cages with free access to commercial chow and water. Experiments received prior approval by the animal ethics committees of the Federal University of Minas Gerais and National Center for Scientific Research. The following drugs were used: irinotecan hydrochloride (CPT-11; Camptosar, Eurofarma, Brazil; 100-mg ampule) and IL-1 receptor antagonist (IL-1Ra; Biogen, Inc., Geneva, Switzerland). Apocynin (NOX-2 inhibitor) was purchased from Sigma-Aldrich (St. Louis, MO). IL-18 binding protein (IL-18BP) was purchased from R&D Systems (Minneapolis, MN). The experimental intestinal mucositis in mice was induced as previously described22Ikuno N. Soda H. Watanabe M. Oka M. Irinotecan (CPT-11) and characteristic mucosal changes in the mouse ileum and cecum.J Natl Cancer Inst. 1995; 87: 1876-1883Crossref PubMed Scopus (160) Google Scholar and modified for our experimental conditions. Saline or irinotecan (75 mg/kg) was administered i.p. once per day for 4 consecutive days. Mice were weighed daily. Seven days after beginning irinotecan treatment, mice were euthanized by cervical dislocation. The small intestine was removed to measure the length, and ileum was used to further analysis. To evaluate the role of the IL-1 receptor, WT or il-18−/− mice were treated with 4 mg/kg of IL-1Ra or saline s.c. 8 hours before the first dose of irinotecan. Subsequently, mice were treated every 8 hours during the following 7 days. Another WT mouse group received one i.p. injection of 20 mg/kg of apocynin 24 hours before the first dose of irinotecan and a daily i.p. injection of apocynin throughout the experimental period. Another group received 250 μg/kg of IL-18BP daily 1 hour before irinotecan treatment during 4 consecutive days. To analyze the role of IL-1β and IL-18 in the later phase of mucositis, mice were kept alive for 21 days after the beginning of irinotecan treatment. In these experiments, mice received irinotecan and were treated with 4 mg/kg IL-1Ra (8 hours before the first dose of irinotecan and subsequently every 8 hours for 21 days) or vehicle (saline, as for the IL-1Ra treatment). In parallel experiments, il-18−/− mice were given irinotecan and evaluated for 21 days. Control WT or il-18−/− mice received saline (the vehicle for irinotecan). Because there was no difference between WT and the il-18−/− mice not exposed to irinotecan (given vehicle only), these groups were pooled and are represented together for ease of visualization. The levels of the cytokines IL-1β and IL-18 in ileum samples were measured as previously described.13Melo M.L. Brito G.A. Soares R.C. Carvalho S.B. Silva J.V. Soares P.M. Vale M.L. Souza M.H. Cunha F.Q. Ribeiro R.A. Role of cytokines (TNF-alpha, IL-1beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide.Cancer Chemother Pharmacol. 2008; 61: 775-784Crossref PubMed Scopus (83) Google Scholar The cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits from R&D Systems and MBL International Corporation (Woburn, MA), respectively. Experiments were reproduced three times, and one representative experiment is presented in the Results section. The extent of the neutrophil accumulation in the ileum tissue was measured by the quantification of the myeloperoxidase (MPO) activity, a neutrophil enzyme marker, as previously described.23Souza D.G. Soares A.C. Pinho V. Torloni H. Reis L.F. Teixeira M.M. Dias A.A. Increased mortality and inflammation in tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia and reperfusion injury.Am J Pathol. 2002; 160: 1755-1765Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar Briefly, a portion of the animals' ileums was removed and snap frozen in liquid nitrogen. On thawing and processing, the tissue was assayed for MPO activity by measuring the change in optical density at 450 nm using tetramethylbenzidine (Sigma-Aldrich). Results were expressed as relative units that denote activity of MPO compared with casein-elicited murine peritoneal neutrophils processed in the same way. The cleaved form of caspase-1 was detected in supernatants of ileum homogenates using a Western blotting assay with rat anti–caspase-1 p20 monoclonal antibody clone 4B4 (Genentech, South San Francisco, CA), as previously described.24Sousa L.P. Lopes F. Silva D.M. Tavares L.P. Vieira A.T. Rezende B.M. Carmo A.F. Russo R.C. Garcia C.C. Bonjardim C.A. Alessandri A.L. Rossi A.G. Pinho V. Teixeira M.M. PDE4 inhibition drives resolution of neutrophilic inflammation by inducing apoptosis in a PKA-PI3K/Akt-dependent and NF-kappaB-independent manner.J Leukoc Biol. 2010; 87: 895-904Crossref PubMed Scopus (99) Google Scholar Protein levels of caspase-1 were quantified by using densitometric analysis LabImage 1D software (LabImage, Leipzig, Germany). The protein levels were normalized by levels of β-actin in the same sample, and results are expressed as caspase-1/β-actin ratio in arbitrary units. Ileum samples from adult euthanized mice were obtained at day 7 after mice were exposed to irinotecan. Afterward, they were immediately fixed in 10% buffered formalin for 24 hours and embedded in paraffin. Tissue sections (5 μm thick) were stained with H&E and evaluated under a microscope (Olympus, Tokyo, Japan) adapted with a digital camera (Moticon, Munich, Germany). The height of the mucosal villous or crypt depth of each sample was measured using digitalized images obtained with a 10× objective of a light microscope (Olympus) adapted with a digital camera (Moticon) and the villus/crypt ratio calculated. The histologic score was obtained based on the intensity of mononuclear and polymorphonuclear infiltrates in the lamina propria, changes in the architecture of the mucosa, decreased villus height, and ulceration. For each parameter, the changes were graded according to the following scale: 0, absent; 1, mild; 2, moderate; and 3, intense. The inflammation score was represented by numbers from 0 (normal) to 12 (highly altered). The histologic analysis was performed by a single examiner (M.A.R.) masked to the experimental groups status. None of control mice had any alteration in histopathologic analysis, and their scores were zero. The results were expressed as the means ± SEM of the inflammation score for each experimental group.25Usselmann B. Newbold M. Morris A.G. Nwokolo C.U. Deficiency of colonic telomerase in ulcerative colitis.Am J Gastroenterol. 2001; 96: 1106-1112Crossref PubMed Google Scholar The total thiobarbituric acid–reactive substance (TBARS) levels in the ileum, an index of malonyldialdehyde production, were determined as previously described.26Ohkawa H. Ohishi N. Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23164) Google Scholar The reduced glutathione (GSH) levels were measured as previously described.27Sedlak J. Lindsay R.H. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent.Anal Biochem. 1968; 25: 192-205Crossref PubMed Scopus (6553) Google Scholar Briefly, ileum was homogenized, mixed with trichloroacetic acid, and kept on ice for 30 minutes to allow protein precipitation and then centrifuged. Then 30 μL of the clear supernatant were mixed with 270 μL of phosphate buffer 0.1 mol/L (pH 8.5) and 5 μL of 5,5-dithiobis-(2-nitrobenzoicacid) in methanol. The absorbance of the reaction solution was measured at 415 nm. The results were analyzed using GraphPad Prism 4 software (GraphPad Inc., San Diego, CA). Data are presented as means ± SEM and compared using one-way analysis of variance with posttest Newman-Keuls multiple comparisons. Student's t-test was used to assess whether the means of two groups were statistically different from each other. P < 0.05 was considered statistically significant. Our results indicate that treatment with irinotecan for 4 days is associated with body weight loss from day 2 of treatment (Figure 1A). On day 7, mice were euthanized, and analysis of the small intestine revealed shortening of the intestinal length (Figure 1B) and increase in neutrophil influx to the ileum, as assessed by MPO assay (Figure 1C). Consistent with the findings from the MPO assay, histopathologic analysis revealed neutrophil infiltration in the ileum and further marked changes in tissue architecture and ulceration (Supplemental Figure 1A), leading to an important increase in the histopathologic score (Figure 1D). Interestingly, the inflammatory response in the ileum due to irinotecan treatment was associated with an increase in IL-1β (Figure 1E) and IL-18 (Figure 1F) production. Next, we evaluated the oxidative stress in ileum of irinotecan-treated mice by assessing levels of TBARS and levels of reduced GSH in tissues. Irinotecan-induced mucositis led to a marked increase in TBARS levels (Figure 2A) and reduced GSH levels (Figure 2B) in the ileum. Experiments in gp91phox−/− mice or apocynin-treated mice revealed no increase in TBARS production after irinotecan in these mice (Figure 2A). Furthermore, gp91phox−/− mice, but not apocynin-treated animals, presented a partial reversion of the decrease in GSH levels (Figure 2B). These results suggest that NOX-2–derived ROS are associated to oxidative stress during irinotecan-induced mucositis. We then evaluated the role of NOX-2–derived ROS for disease progression and tissue damage induced by irinotecan. gp91phox−/− and apocynin-treated mice had diminished weight loss on day 7 after administration of irinotecan (Figure 2C) and no intestinal shortening (Figure 2D) compared with WT mice given irinotecan. Furthermore, damage in the mucosa architecture, ulceration, and consequently the histopathologic score were decreased in the absence of NOX-2 (Figure 2E). Our results clearly indicate the important role played by ROS for irinotecan-induced experimental mucositis in mice. ROS has been suggested to be involved in inflammasome activation in several pathologic conditions.28Morishige T. Yoshioka Y. Inakura H. Tanabe A. Yao X. Narimatsu S. Monobe Y. Imazawa T. Tsunoda S. Tsutsumi Y. Mukai Y. Okada N. Nakagawa S. The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL-1beta production, ROS production and endosomal rupture.Biomaterials. 2010; 31: 6833-6842Crossref PubMed Scopus (122) Google Scholar, 29Said-Sadier N. Padilla E. Langsley G. Ojcius D.M. Aspergillus fumigatus stimulates the NLRP3 inflammasome through a pathway requiring ROS production and the Syk tyrosine kinase.PLoS One. 2010; 5: e10008Crossref PubMed Scopus (234) Google Scholar, 30Martinon F. Signaling by ROS drives inflammasome activation.Eur J Immunol. 2010; 40: 616-619Crossref PubMed Scopus (456) Google Scholar, 31Tschopp J. Schroder K. NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production?.Nat Rev Immunol. 2010; 10: 210-215Crossref PubMed Scopus (1261) Google Scholar Next we evaluated whether ROS was involved in inflammasome activation during irinotecan-induced mucositis in mice. Inhibition of NOX by treatment with apocynin or use of gp91phox−/− mice was associated with a decrease in caspase-1 activation (Figure 3A). More importantly, inhibition of NOX activation with apocynin caused inhibition of IL-1β (Figure 3B) and IL-18 (Figure 3C) production in the ileum of mice exposed to irinotecan. These results suggest that NOX-2–derived ROS are involved in both inflammasome activation and IL-1β/IL-18 production during irinotecan-induced mucositis. Interestingly, in the absence of inflammasome components there is a decrease in TBARS levels (data not shown), suggesting that inflammasome activation feeds back to the system and enhances ROS production. Both IL-1β and IL-18 are important proinflammatory mediators that are generated at sites of injury by coordinated pathways: proinflammatory stimuli induce expression of inactive cytokine proforms, whereas inflammasome complexes control cytokine maturation and release.32Sims J.E. Smith D.E. The IL-1 family: regulators of immunity.Nat Rev Immunol. 2010; 10: 89-102Crossref PubMed Scopus (995) Google Scholar, 33Henao-Mejia J. Elinav E. Strowig T. Flavell R.A. Inflammasomes: far beyond inflammation.Nat Immunol. 2012; 13: 321-324Crossref PubMed Scopus (144) Google Scholar, 34Martinon F. Mayor A. Tschopp J. The inflammasomes: guardians of the body.Annu Rev Immunol. 2009; 27: 229-265Crossref PubMed Scopus (1863) Google Scholar Thus, because irinotecan induced inflammasome activation and an increase in IL-1β and IL-18 levels, we hypothesized that the inflammasome complex may play a role in irinotecan-induced damage. Using asc or casp-1–deficient mice, we found that mucositis induced by irinotecan treatment was dependent on inflammasome activation, as seen by lack of body weight loss after irinotecan treatment (Figures 4A and 5A ), abrogation of intestine shortening (Figures 4B and 5B), and decrease in neutrophil influx (Figures 4C and 5C) compared with irinotecan-treated WT mice. The histopathologic analysis revealed that asc−/− and casp-1−/− mice had reduced inflammatory cell infiltrate, less damage in the mucosa architecture, and, therefore, less ulceration and consequently a decrease in histologic score (Figures 4D and 5D). Interestingly, lack of ASC or caspase-1 was associated with abrogation of IL-1β (Figures 4E and 5E) and IL-18 (Figures 4F and 5F) production. Together, these results indicate that activation of ASC/caspase-1 plays an important role in the inflammatory response and IL-1β and IL-18 production in irinotecan-induced mucositis.Figure 5Caspase-1 contributes to IL-1β and IL-18 production and is associated with ileum damage induced by irinotecan. A: Casp-1 mice have attenuation of body weight loss on days 5, 6, and 7 after irinotecan treatment compared with WT mice. B and C: On day 7, there was less intestinal shortening (B) and decreased neutrophil influx (C). D: Absence of caspase-1 resulted in reductions in inflammatory cell influx, mucosal damage, and ulceration; these alterations resulted in a decrease in histologic score when compared with irinotecan-injected WT mice. Control mice do not have any histopathologic alteration and had a score of 0. The line above control indicates a score of 0. E and F: casp-1−/− mice had a decrease in IL-1β (E) and IL-18 (F) production. Results are the means ± SEM of 3 to 10 animals in each group. ∗P < 0.05 compared with the control group; †P < 0.05 compared with the WT irinotecan-treated group. Scale bar = 100 μm (D). Irin, irinotecan.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next we assessed the role of IL-1β and IL-18 in this mucositis model. Experiments in mice that lack IL-18 or in mice treated with the soluble IL-1Ra suggest that both cytokines are important for the damage induced by irinotecan (Figure 6). In the absence of IL-18 or IL-1 receptor activation, mice had reductions in body weight loss after day 5 of treatment (Figure 6A), intestinal shortening (Figure 6B), and neutrophil influx into the ileum (Figure 6C) compared with irinotecan-treated WT mice. In addition, histologic analysis of the ileum revealed that both the absence of IL-18 or blockade of IL-1 receptor resulted in reductions in inflammatory cell infiltration, damage in the architecture of the mucosa, and ulceration. Overall, these changes resulted in reduced histopathologic score (Figure 6D). Both strategies were able to decrease oxidative stress induced by irinotecan treatment, as assessed by decrease of TBARS and increase of GSH (Supplemental Figure 2, A and B, respectively). To confirm the results obtained in mice genetically deficient for Il-18, we treated WT mice with IL-18BP. IL-18BP treatment was also able to decrease the damage induced by irinotecan, as assessed by improvement in body weight loss (Supplemental Figure 3A) and in intestinal shortening (Supplemental Figure 3B). In addition, there was reductions in neutrophil influx (Supplemental Figure 3C), histologic damage (Supplemental Figure 3D), and IL-1β production (Supplemental Figure 3E) when compared with vehicle-treated mice. Previous studies have found that IL-18 deficiency may impair epithelial reconstitution.35Saleh M. Trinchieri G. 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