nlrp3 inflammasome involvement in the organ damage and … · nlrp3 inflammasome involvement in the...

1521-0103/355/3/370–380$25.00 http://dx.doi.org/10.1124/jpet.115.226936THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 355:370–380, December 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

NLRP3 Inflammasome Involvement in the Organ Damage andImpaired Spermatogenesis Induced by Testicular Ischemia andReperfusion in Mice

Letteria Minutoli, Pietro Antonuccio, Natasha Irrera, Mariagrazia Rinaldi, Alessandra Bitto,Herbert Marini, Gabriele Pizzino, Carmelo Romeo, Antonina Pisani, Giuseppe Santoro,Domenico Puzzolo, Carlo Magno, Francesco Squadrito, Antonio Micali,and Domenica AltavillaDepartment of Clinical and Experimental Medicine, University of Messina, Azienda Ospedaliera Universitaria Policlinico“G. Martino”, Messina, Italy (L.M., N.I., M.R., A.B., H.M., G.P., F.S.); Department of Paediatric, Gynaecological, Microbiological,and Biomedical Sciences, University of Messina, Azienda Ospedaliera Universitaria Policlinico “G. Martino”, Messina, Italy (P.A.,C.R., D.A.); Department of Biomedical Sciences and Morphofunctional Imaging, University of Messina, Azienda OspedalieraUniversitaria Policlinico “G. Martino”, Messina, Italy (A.P., G.S., D.P., A.M.); and Department of Human Pathology, University ofMessina, Azienda Ospedaliera Universitaria Policlinico “G. Martino”, Messina, Italy (C.M.)

Received June 17, 2015; accepted September 24, 2015

ABSTRACTWe investigated the role of the nucleotide-binding oligomerizationdomain (NOD)-like receptor family pyrin domain containing 3(NLRP3) inflammasome during testis ischemia and reperfusioninjury (TI/R) in wild-type (WT) andNLRP3 knock-out (KO) mice. WTand KO mice underwent 1 hour testicular ischemia followed by4 hours and 1 and 7 days of reperfusion or a sham TI/R. Further-more, two groups of WT mice were treated at the beginning ofreperfusion and up to 7 days with two inflammasome inhibitors,BAY 11-7082 (20 mg/kg i.p.) or Brilliant Blue G (45.5mg/kg i.p.), orvehicle. Animals were killed with a pentobarbital sodium overdoseat 4 hours and 1 and 7 days, and bilateral orchidectomies wereperformed. Biochemical andmorphologic studies were carried outin all groups. TI/R in WT mice significantly increased caspase-1and interleukin (IL)-1b mRNA after 4 hours and IL-18 mRNA at 1 day

of reperfusion (P # 0.05). There was also a significant increase incaspase-3 and terminal deoxynucleotidyl transferase–mediateddigoxigenin-deoxyuridine nick-end labeling–positive cells, markedhistologic damage, and altered spermatogenesis in WT micein both testes after 1 and 7 days of reperfusion. KO TI/R mice, WTTI/R BAY 11-7082, and Brilliant Blue G treated mice showed asignificant reduced IL-1b and IL-18 mRNA expression, bluntedcaspase-1 and -3 expression, minor histologic damages, lowterminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling activity, and preserved spermato-genesis. These data suggest that the activation of NLRP3 playsa key role in TI/R, and its inhibition might represent a therapeutictarget for the management of patients with unilateral testiculartorsion.

IntroductionTestis torsion is one of themost common testicular lesions in

the pediatric population (Fan et al., 2013), with an estimatedincidence of 3.5 per 100,000 person-year (Huang et al., 2013).It causes peculiar structural damages, thus leading to in-fertility (Huang et al., 2013); therefore, it must be treatedpromptly to avoid ipsilateral testicular dysfunction (Andersonand Williamson, 1986). Events occurring during spontaneousor experimental testicular torsion and subsequent detorsionbasically depend on the extension of ischemia (Filho et al.,

2004) and are representative of the ischemia and reperfusioninjury observed in other organs (Grace, 1994). Under thesecircumstances, the testis produces several proinflammatorycytokines, including tumor necrosis factor a and interleukin(IL)-1b (Lysiak et al., 2000). The increased levels of thesecytokines can be considered indirect evidence of tissueinflammation.In addition, testicular ischemia/reperfusion (TI/R) triggers

the apoptosis cascade, leading to the degeneration of thegerminal epithelium (Tripathi et al., 2009). Apoptosis isrequired for normal spermatogenesis in mammals at differentstages of differentiation to ensure cellular homeostasis(Blanco-Rodríguez, 1998). After TI/R, increased expression ofBax and caspase-1 and -3 were observed (Lysiak, 2004),

This work was supported by departmental funding.dx.doi.org/10.1124/jpet.115.226936.

ABBREVIATIONS: ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; BBG, Brilliant Blue G; CL,contralateral; IL, interleukin; KO, knock out; MWS, Muckle-Wells syndrome; NLRP3, NOD-like receptor family pyrin domain containing 3; NO,nitric oxide; NOD, nucleotide-binding oligomerization domain; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TBS, Tris-bufferedsaline; TI/R, testicular ischemia/reperfusion; TUNEL, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling;WT, wild type.

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indicating that both initiator and executioner caspases wereinvolved in the apoptotic cascade triggered by the experimen-tal procedure. It was suggested that the regulation of germcell apoptosis might be central in the development of exper-imental strategies to prevent their damage (Minutoli et al.,2009).

Inflammasomes are multimolecular complexes assembledin response to various activators and leading to the intracel-lular activation of the inflammatory cascade, particularly byraising the cleavage of the inactive precursor of IL-1b to its ac-tive form (Schroder and Tschopp, 2010). Several inflammasomeshave been identified (Tian et al., 2009; Duéñez-Guzmán and

Fig. 1. Representative Western blot analysis of caspase-1 at4 hours (A) and caspase-3 at 1 (B) and 7 days (C) in testes of shamWT and KO mice, TI/R + vehicle and CL + vehicle WT and KOmice, and BAY 11-7082– and BBG-treated TI/R and CL WT mice.*P , 0.05 versus sham WT mice; **P , 0.05 versus WT I/R +vehicle mice; #P , 0.05 versus sham KO mice. Bars represent themean 6 S.E. of seven experiments.

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Haig, 2014). The nucleotide-binding oligomerization domain(NOD)-like receptor family pyrin domain containing 3 (NLRP3)inflammasome is currently the most fully characterized, andit is formed by the NOD-like receptor NLRP3 by an adapterprotein called apoptosis-associated speck-like protein contain-ing a caspase recruitment domain (ASC) and procaspase-1.Furthermore, NLRP3 is of special interest because it respondsto different physical and chemical stimuli, leading to manydiseases when out of control (Chen and Sun, 2013), and can beassembled following a wide variety of stimuli, including tissuedamage (Gross et al., 2011). The actual activation of theNLRP3inflammasome is a poorly understood event, which involves theintegration of many signals indicative of tissue damage orstress (Gross et al., 2011). The inflammasome NLRP3 is animportant regulator of caspase-1 (Martinon et al., 2002; Stutzet al., 2009; Kayagaki et al., 2011), which can trigger thesequential activation of caspase-3 (Stutz et al., 2009) and reg-ulate the maturation of the proinflammatory cytokines IL-1band IL-18 or the rapid inflammatory form of cell death calledpyroptosis.Specific small molecule inhibitors, such as BAY 11-7082

(Juliana et al., 2010) and Brilliant Blue G (BBG) (Zhaoet al., 2013), are able to inhibit the proinflammatory andproapoptotic effects of the NLRP3 inflammasome. More in de-tail, BAY 11-7082, a kinase-b inhibitor, selectively inhibitedNLRP3 inflammasome activity in macrophages indepen-dently of its inhibitory effect on the priming step follow-ing nuclear factor kB activation (Juliana et al., 2010).BBG blocks the membrane-bound purinergic P2X7 receptor(Díaz-Hernández et al., 2009), which in turn suppresses theexpression of ASC and inhibits NLRP3 inflammasome activa-tion (Zhao et al., 2013).In light of this background and to better understand the role

of the NLRP3 inflammasome during testicular torsion, weinvestigated TI/R injury in wild-type (WT) and NLRP3 knock-out (KO) mice.

Materials and MethodsExperimental Protocol. All animalswere housed andmaintained

under specific pathogen-free conditions at the animal facility of theSchool of Medicine at the University of Messina, Italy. All experimentalprocedures complied with the Declaration of Helsinki, with the Guidefor the Care and Use of Laboratory Animals, as adopted and pro-mulgated by the United States National Institutes of Health and theAnimal Research: Reporting of In Vivo Experiments guidelines(Kilkenny et al., 2010) and were authorized by our local institution.WT (C57Bl6) male mice and KO (Nlrp3

tm1bhk) (25–30 g) mice werepurchased from the JacksonLaboratory (BarHarbor,ME). The animalswere provided a standard diet ad libitum, with free access to tap water,and were maintained on a 12-hour light/dark cycle. Both WT and KOmice (total number 5 126; each group consisted of seven animals)were anesthetized with an intraperitoneal injection of 80 mg/kg ofpentobarbital sodium and torsion of the left testis and spermaticcord was performed as previously described (Minutoli et al., 2009).Then, the same testis was detorted. WT and KO animals of allexperimental groups underwent testicular ischemia for 1 hour,followed by 4 hours and 1 day and 7 days reperfusion (TI/R). WTand KO sham mice were operated as previously indicated forexperimental groups, except for testicular torsion and consequentischemia. Furthermore, only WTmice were treated either with BAY11-7082 (20 mg/kg i.p.) (Bitto et al., 2014) or BBG (45.5 mg/kg i.p.)every 2 days (Díaz-Hernández et al., 2009) or vehicle (a 1:3 solutionof dimethylsulfoxide and 0.9% NaCl) for up to 7 days. Animals were

killed at the above indicated time points after reperfusion with anoverdose of pentobarbital sodium, and bilateral orchidectomies wereperformed. The testes were collected, weighted, and treated for thedifferent experimental technical procedures.

Drugs and Chemicals. The following compounds were suppliedas indicated: BAY 11-7082 by Adipogene (San Diego, CA), BBG bySigma-Aldrich (Milan, Italy), and sodium pentobarbital by Intervet(Milan, Italy).

Isolation of Soluble Proteins. Isolation of soluble proteins fromtestis samples (about 30 mg) of mice was performed in 1 ml of lysisbuffer [25 mM Tris/HCl (pH 7.4), 1.0 mM EGTA, 1.0 mM EDTA,0.5 mM phenyl methylsulfonyl fluoride, 1% NP40, and aprotinin,leupeptin, and pepstatin A (10 mg/ml each)] and homogenized with aDounce homogenizer (Sigma Aldrich, Milan, Italy; Paterniti et al.,2013). The homogenate was centrifuged at 15,000g for 15minutes, andthe supernatantwas collected andused for protein determinationwiththe Bio-Rad protein assay kit (Richmond, CA).

Determination of Active Caspase-1 and -3 by Western BlotAnalysis. Total proteins (30mg) were denaturedwith reducing buffer[62 mM Tris (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate, 5%b-mercaptoethanol, and 0.003% bromophenol blue], separated byelectrophoresis, and then transferred onto a polyvinylidene difluoridemembrane using a transfer buffer [39mMglycine and 48mMTris (pH8.3)] at 200mA for 1 hour. To blockmembrane proteins, 5% nonfat drymilk in Tris-buffered saline (TBS)–0.1% Tween for 1 hour at roomtemperature was used, followed by three washes with TBS–0.1%Tween for 10 minutes each, and then incubated with a primaryantibody for caspase-1 and -3 (Abcam, Cambridge, UK) and diluted

Fig. 2. mRNA expression for IL-1b at 4 hours (A) and IL-18 at 1 day (B) inthe testes of sham WT and KO mice, TI/R + vehicle and CL + vehicle WTand KO mice, and BAY 11-7082– and BBG-treated TI/R and CL WT mice.*P, 0.05 versus shamWTmice; **P, 0.05 versus WT I/R + vehicle mice;#P , 0.05 versus sham KO mice. Bars represent the mean 6 S.E. of sevenexperiments.

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1:1000 in TBS–0.1% Tween overnight at 4°C. The membranes werewashed three times for 10 minutes each with TBS–0.1% Tween toeliminatenonspecific bindings and incubatedwith a specific peroxidase-conjugated secondary antibody (Pierce, Cambridge, UK), diluted1:2000, for 1 hour at room temperature. After three washes, themembranes were analyzed by the enhanced chemiluminescence systemaccording to the manufacturer’s protocol (Amersham, Buckingham-shire, UK) as previously described (Minutoli et al., 2005). Equal loadingof proteins was assessed on stripped blots by immunodetection ofb-actinwith a rabbitmonoclonal antibody (Cell Signaling, Beverly,MA)diluted 1:500 and peroxidase-conjugated goat antirabbit IgG (Pierce)diluted 1:20,000.

The results from each experimental group [shamWT and KOmice,TI/R1 vehicle and contralateral (CL)1 vehicle WT and KOmice, andBAY 11-7082– and BBG-treated TI/R and CLWTmice] were obtainedfrom seven samples of each experimental group. For each antibody,three different electrophoretic runs were performed at different timepoints (4 hours for caspase-1 and 1 and 7 days for caspase-3). Theresults were normalized versus b-actin and expressed as relativeintegrated intensity comparedwith those of shammicemeasuredwiththe same batch.

Real-Time PCR. For the gene expression study, the extraction oftotal mRNA was performed from testis tissue using TRIZOL (Invi-trogen, Milan, Italy) under sterile conditions following the manufac-turer’s protocol. For each sample, 5 mg of mRNA were reversetranscripted into cDNA and 3 ml of cDNA were amplified in duplicatewith the TaqMan Universal PCR Master Mix containing primer andspecific probes (TaqMan), and then using the instrument SDS 7300Real Time PCR (Applied Biosystems, Foster City, CA). The reversedtranscribed product was used to quantify the amount of IL-1b andIL-18. The result was expressed as the number of copies of the targetgene compared with the housekeeping gene (b-actin).

Histologic Evaluation. The testes were fixed in 4% paraformal-dehyde in 0.2 M phosphate-buffered saline (PBS), dehydrated ingraded ethanol, cleared in xylene, and embedded in paraffin (Para-plast; SPI Supplies,WestChester, PA). Five-micrometer sectionswerestained with hematoxylin and eosin and photographed with a ZeissPrimo Star (Carl Zeiss Inc., Oberkochen, Germany) light microscope.The images were taken with a Canon A620 Powershot camera (CanonInc., Tokyo, Japan), saved as tagged image format files, printed at thesame final magnification, and blindly assessed by two trainedobservers without knowledge of the previous treatment. The meanand S.D. of the results were recorded. Five microscopic fields, allincluding two entire seminiferous tubules from 10 not serial sectionsof each group, were considered. Morphologic evaluation was per-formed on both the tubular and extratubular compartments.

As to the tubular compartment, the mean seminiferous tubulediameter was calculated by measuring the diameters of 100 separateseminiferous tubules, all showing a circular profile. A Peak ScaleLoupe 7x (GWJ Company, Hacienda Heights, CA) micrometer wasused as a scale calibration standard to calculate the diameters, whichare expressed in micrometers. Furthermore, spermatogenesis wasquantified using the Johnsen’s scoring system (Johnsen, 1970), asmodified byErdemir et al. (2012). Briefly, a score of 10 to 1was given toeach tubule according to epithelial maturation: 10, complete sper-matogenesis and perfect tubules; 9, many spermatozoa present anddisorganized spermatogenesis; 8, only a few spermatozoa present;7, no spermatozoa, but many spermatids present; 6, only a few

Fig. 3. Assessment of apoptosis with TUNEL technique at 1 and 7 daysafter the surgical procedure in TI/R WT and KO mice. (A) At 1 day, bothshamWT and KOmice testes show no TUNEL-positive cells. (B) In TI/R +vehicle WT mice at 1 day, many TUNEL-positive germ cells (arrows) arepresent in the seminiferous tubules. (C) In TI/R KO mice at 1 day, someTUNEL-positive germ cells (arrow) are gathered in small groups. (D) InBAY 11-7082–treated TI/R WT mice at 1 day, few TUNEL-positive cells(arrow) are placed in the periphery of the tubules. (E) In BBG-treated TI/RWT mice at 1 day, isolated TUNEL-positive cells (arrow) are evident. (F)At 7 days, in sham WT and KO mice testes, no TUNEL-positive cells are

observed. (G) In TI/R + vehicle WT mice at 7 days, some peripheral groupsof TUNEL-positive germ cells are evident (arrows). (H) In TI/R KOmice at7 days, occasional TUNEL-positive germ cells (arrows) are present in theouter part of the tubules. (I) In TI/R BAY 11-7082–treated WT mice at7 days, rare TUNEL-positive germ cells (arrow) are observed. (J) In TI/RBBG-treated WT mice at 7 days, few groups of TUNEL-positive germ cells(arrow) are present (scale bar: 50 mm).


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spermatids present; 5, no spermatozoa or spermatids, but manyspermatocytes present; 4, only a few spermatocytes present; 3, onlyspermatogonia present; 2, no germ cells, but only Sertoli cells present;and 1, no germ cells and no Sertoli cells present.

As to the extratubular compartment, a previously describedmethod (Minutoli et al., 2005) for the evaluation of edema,hemorrhagic extravasation, vessel dilation, and Leydig cell changeevaluation was used, and each parameter was quantified on thebasis of the following scale: 0, absent; 1, mild; 2, moderate; and 3,severe.

Terminal Deoxynucleotidyl Transferase–MediatedDigoxigenin-Deoxyuridine Nick-End Labeling Technique. Forthe terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) technique, an apoptosisdetection kit (TUNEL Universal Apoptosis Detection Kit; GenScript,Piscataway, NJ) was used on paraffin-embedded 5-mm sectionscleared with xylene and dehydrated in graded ethanol. Protein wasdigested with proteinase K (20 mg/ml PBS), and endogenous peroxi-dase activity was blocked with 3% H2O2 in methanol. Sections wereincubated with terminal deoxynucleotidyl transferase enzyme andbiotin-11-day UTP, then with the streptavidin-peroxidase substrate,and finally with the diaminobenzidine solution. Two trained

observers without knowledge of the treatment blindly assessed themicrographs, which were obtained and processed as previouslyindicated for the histologic specimens. From each group, thepercentage of tubules with apoptotic cells and the apoptotic index(mean number of TUNEL-positive cells per tubule in 100 seminifer-ous tubules) (Tsounapi et al., 2012) were calculated.

Statistical Analyses. All data are expressed as the mean 6 S.E., with the exception of the percentage of tubules with apoptoticcells, where absolute values are provided. Data were analyzed withone-way analysis of variance using Tukey’s test as the post hocprocedure or Student’s t test. A P value of #0.05 was consideredstatistically significant.

ResultsActive Caspase-1 and Caspase-3 Expression in TI/R.

No caspase-1 activity was demonstrated in the testes of eithersham WT or KO mice (Fig. 1A). After 4 hours of reperfusion,TI/R injury determined a significant increase of caspase-1 ex-pression in WT animals. This enhanced activity was higher in theipsilateral testes and lower in theCL testes (Fig. 1A). Caspase-1

TABLE 1AEffects of ischemia/reperfusion on the tubular compartment in the testes of WT and KO sham, TI/R andCL WT plus vehicle, BAY 11-7082– and BBG-treated WT, and KO mice at 1 day after the surgicalprocedureValues are expressed as the mean 6 S.E., with the exception of %TWAC, for which absolute values are provided.

Tubular Compartment

MSTD Johnsen’s Score %TWAC Apoptotic Index

mm

Sham WT 178.25 6 9.93 9.76 6 0.56 4 0.17 6 0.71Sham KO 176 6 8.37 9.51 6 0.63 5 0.22 6 0.49WT TI/R + vehicle 1 day 145 6 8.21a 5.3 6 1.07a 73a 8.3 6 1.34a

WT CL + vehicle 1 day 153 6 8.26 6.75 6 0.68 49 6.18 6 1.37WT TI/R + BAY 11-7082 1 day 166.34 6 8.52a,b 7.8 6 0.92a,b 17a,b 2.1 6 0.61a,b

WT CL + BAY 11-7082 1 day 171.2 6 6.65 8.9 6 0.59 15 1.33 6 0.22WT TI/R + BBG 1 day 165.51 6 7.75a,b 7.5 6 0.67a,b 21a,b 2.4 6 0.66a,b

WT CL + BBG 1 day 170.5 6 9.36 8.4 6 0.36 17 1.52 6 0.93KO TI/R 1 day 163.1 6 10.58c 7.9 6 0.89c 22c 1.93 6 0.99c

KO CL 1 day 166.15 6 11.17 8 6 0.78 12 1.29 6 0.81

MSTD = mean seminiferous tubule diameter; TWAC, tubules with apoptotic cells.aP , 0.05 versus sham WT mice.bP , 0.05 versus WT TI/R + vehicle mice.cP , 0.05 versus sham KO mice.

TABLE 2AEffects of ischemia/reperfusion on the tubular compartment in the testes of WT and KO sham, TI/R andCL of WT plus vehicle, BAY 11-7082– and BBG-treated WT, and KO mice at 7 days after the surgicalprocedureValues are expressed as the mean 6 S.E., with the exception of %TWAC, for which absolute values are provided.

Tubular Compartment

MSTD Johnsen’s Score %TWAC Apoptotic Index

mm

Sham WT 179.4 6 8.87 9.61 6 0.33 3 0.23 6 0.34Sham KO 177.3 6 9.12 9.49 6 0.47 5 0.29 6 0.55WT TI/R + vehicle 7 days 136.9 6 8.81a 4.95 6 0.82a 63a 6.96 6 1.73a

WT CL + vehicle 7 days 150.6 6 8.89 6.98 6 0.64 42 4.4 6 1.22WT TI/R + BAY 11-70882 7 days 161 6 7.73a,b 7.48 6 0.83a,b 12a,b 1.24 6 0.52a,b

WT CL + BAY 11-70882 7 days 168.5 6 6.38 8.11 6 0.97 6 0.67 6 1.12WT TI/R + BBG 7 days 160.2 6 8.12a,b 7.44 6 0.52a,b 11b 1.16 6 0.44a,b

WT CL + BBG 7 days 168.3 6 9.15 7.81 6 0.67 9 0.82 6 0.71KO TI/R 7 days 159 6 9.23c 7.35 6 0.6c 15c 1.21 6 0.95c

KO CL 7 days 168.15 6 11.87 8.25 6 0.72 10 0.92 6 0.68

MSTD = mean seminiferous tubule diameter; TWAC, tubules with apoptotic cells.aP , 0.05 versus sham WT mice.bP , 0.05 versus WT TI/R + vehicle mice.cP , 0.05 versus sham KO mice.

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expression was significantly lower in the ipsilateral and CLtestes ofWTmice treated with both BAY 11-7082 and BBG andin KO mice (Fig. 1A).No caspase-3 expression was observed in the testes of both

shamWTandKOmice at 1 and 7 days (Fig. 1, B andC). On thecontrary, TI/R injury induced an increased expression of

caspase-3 in the ipsilateral testis of WT animals after 1 and7 days of reperfusion (Fig. 1, B and C). At 7 days, the levels ofcaspase-3 further increased when compared with those at1 day (Fig. 1, B and C). Caspase-3 expression was significantlylower in the ipsilateral testes of WT mice treated with bothBAY 11-7082 and BBG and in KO mice (Fig. 1, B and C).

Fig. 4. Histologic findings at 1 day after the surgical procedure inseminiferous tubules stained with hematoxylin and eosin. (A and B)Sham WT and KO mice show a normal morphology. (C) In TI/R +vehicle WT mice, degenerative changes of the germinal cells(arrowhead) and edema of the extratubular compartment (*) areevident. (D) CL testes of WT mice show disorganized germinalepithelium (arrows) and hemorrhagic extravasation (h). (E, G, and I)BAY 11-7082– and BBG-treated WT TI/R and KO TI/R testes. Onlymild changes of the germinal epithelium and a normal extratubularcompartment are present. (F, H, and J) BAY 11-7082– and BBG-treatedWTCL and KOCL testes. No evident morphologic changes arerecognized in both the tubular and extratubular compartments (scalebar: 50 mm).


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Similar results were observed in the CL testes of WT and KOmice even if the absolute values were lower.IL-1b mRNA and IL-18 mRNA Expression in TI/R.

Testes from sham animals of both strains had negligible basallevels of IL-1b mRNA expression (Fig. 2A). TI/R injuryinduced an increased expression of IL-1b mRNA in theipsilateral testes of WT animals following 4 hours of reperfu-sion (Fig. 2A). IL-1bmRNA expression was significantly lowerin the ipsilateral testes of WTmice treated with both BAY 11-7082 and BBG and in KO mice (Fig. 2A). Similar results wereobserved in the CL testes of WT and KO mice even if theabsolute values were lower.Testes from sham animals of both strains had very low

basal levels of IL-18 mRNA expression (Fig. 2B). After 1 dayof reperfusion, the expression of IL-18 mRNA was signifi-cantly increased in WT animals (Fig. 2B). IL-18 mRNAexpression was significantly lower in the ipsilateral testesof WT mice treated with both BAY 11-7082 and BBG and inKO mice (Fig. 2B). Similar results were observed in the CLtestes of WT and KO mice even if the absolute values werelower.Morphologic Evaluation of Apoptosis. No TUNEL-

positive germ cells were observed in the seminiferous tubulesfrom the sham groups of both the WT and KO mice at 1 and 7days of reperfusion (Fig. 3, A and F; Tables 1A and 2A).After 1 day of reperfusion, in TI/R WT mice, many TUNEL-

positive germ cells were observed in the wall of the seminiferoustubules (Fig. 3B; Table 1A). In TI/R KO mice, few TUNEL-positive germ cells were present in the periphery of theseminiferous tubules (Fig. 3C; Table 1A). Similarly, in BAY 11-7082– (Fig. 3D; Table 1A) and BBG-treated TI/R WT mice (Fig.3D; Table 1A), testes showed a low number of TUNEL-positivecells only in the peripheral layer of the tubules.After 7 days of reperfusion, in TI/R WT mice, many

peripheral TUNEL-positive germ cells were evident (Fig.3G; Table 2A). On the contrary, in TI/R KO mice (Fig. 3H;Table 2A) and BAY 11-7082– (Fig. 3I; Table 2A) and BBG-treated TI/R WT mice (Fig. 3J; Table 2A), few isolatedTUNEL-positive germ cells were observed in the peripheryof the seminiferous tubules.

No significant variations were observed in the CL testes ofboth strains of mice (morphologic data not shown).Histologic Evaluation. After 1 day of reperfusion, the

sham groups of both the WT and KO mice showed seminifer-ous tubules and extratubular compartments with normalmorphology (Fig. 4, A and B; Tables 1A and 1B).In the WT mice, the TI/R group showed massive tubular

disorganization, with degenerative changes of the germinalcells, interstitial edema, hemorrhagic extravasation fromslightly dilated vessels, and mild Leydig cell changes (Fig.4C; Tables 1A and 1B). CL testes of the same mice showedsmall tubules with disorganized germinal epithelium andvascular dilation with hemorrhagic extravasation (Fig. 4D;Tables 1A and 1B).In BAY 11-7082– (Fig. 4E; Tables 1A and 1B) and BBG-

treated WT mice (Fig. 4G; Tables 1A and 1B) and KO mice(Fig. 4I; Tables 1A and 1B), TI/R testes showed only mildchanges of the germinal epithelium, but a normal extra-tubular compartment. In the same groups (Fig. 4, F, H, and J;Tables 1A and 1B), CL testes showed well preserved spermcells and a normal extratubular compartment.After 7 days of reperfusion, the sham groups showed normal

morphology of both the seminiferous tubules and extratubularcompartment (Fig. 5, A and B; Tables 2A and 2B).In the WT mice, the TI/R group presented small tubules

with a highly disorganized germinal epithelium and somespermatogonia along their periphery. The extratubular com-partment was damaged, and Leydig cells showed mild-to-moderate structural changes consisting of pale cytoplasm anddarker and condensed nuclei (Fig. 5C; Tables 2A and 2B). CLtestes of WT mice at the same time had larger, less damagedtubules, while the extratubular compartment showed wellevident edema (Fig. 5D; Tables 2A and 2B).In BAY 11-7082– (Fig. 5E; Tables 2A and 2B) and BBG-

treated WT mice (Fig. 5G; Tables 2A and 2B) and KO mice(Fig. 5I; Tables 2A and 2B), TI/R testes showed a germinalepithelium, with many immature spermatids, reduced sper-matozoa, and some intercellular clefts. The extratubularcompartment exhibited mild edema and few dilated vessels.In the same groups (Fig. 5, F, H, and J; Tables 2A and 2B), CL

TABLE 1BEffects of ischemia/reperfusion on the extratubular compartment in the testes of WT and KO sham, TI/R and CL of WT plusvehicle, BAY 11-7082– and BBG-treated WT, and KO mice at 1 day after the surgical procedureValues are expressed as the mean 6 S.E. Histologic grading of the extratubular compartment changes was based on the following scale: 0, absent;1, mild; 2, moderate; and 3, severe.

Extratubular Compartment

Edema Hemorrhagic Extravasation Vascular Dilation Leydig Cell Changes

Sham WT 0 0 0 0Sham KO 0 0 0 0WT TI/R + vehicle 1 day 2.1 6 0.64a 1.55 6 0.6a 1.15 6 0.55a 1.25 6 0.68a

WT CL + vehicle 1 day 0.95 6 0.51 1.44 6 0.59 1.05 6 0.36 0.8 6 0.55WT TI/R + BAY 11-70821 day

0.41 6 0.34a,b 0.41 6 0.11a,b 0.38 6 0.19a,b 0.19 6 0.22a,b

WT CL + BAY 11-70821 day

0.3 6 0.13 0.2 6 0.14 0.33 6 0.21 0.22 6 0.19

WT TI/R + BBG 1 day 0.48 6 0.33a,b 0.66 6 0.37a,b 0.55 6 0.53a,b 0.23 6 0.22a,b

WT CL + BBG 1 day 0.29 6 0.11 0.31 6 0.14 0.32 6 0.16 0.14 6 0.24KO TI/R 1 day 0.45 6 0.51c 0.55 6 0.44c 0.4 6 0.41c 0.25 6 0.36KO CL 1 day 0.34 6 0.47 0.25 6 0.48 0.31 6 0.5 0.25 6 0.51

aP , 0.05 versus sham WT mice.bP , 0.05 versus WT TI/R + vehicle mice.cP , 0.05 versus sham KO mice.

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testes showed better preserved sperm cells and a normalextratubular compartment.

DiscussionExperimental studies have suggested that innate pattern

recognition receptors may prime tissue and organ damage

during ischemic states (Shigeoka et al., 2007, 2010;Haneklauset al., 2013).Testicular torsion is characterized by tissue damage and

represents a urological emergency (Ringdahl and Teague,2006), which, when misdiagnosed and inappropriately treat-ed, can lead to male infertility (Antonuccio et al., 2006).Several mechanisms have been implicated in the development

Fig. 5. Histologic findings at 7 days after the surgical procedure inseminiferous tubules stained with hematoxylin and eosin. (A and B)ShamWT and KOmice show a normal morphology. (C) TI/R + vehicleWTmice. Tubules with disorganized germinal epithelium (arrow) anda widened extratubular compartment (*) are evident. (D) CL testes ofWT mice. A well evident edema (*) is present in the extratubularcompartment. (E, G, and I) BAY 11-7082–and BBG-treated WT TI/Rand KO TI/R testes. Many immature spermatids (s), some sperma-tozoa (z), occasional mitoses (m), and intercellular clefts (arrow) areevident. The extratubular compartment exhibits mild edema and fewdilated vessels. (F, H, and J) BAY 11-7082– and BBG-treated WT CLand KO CL testes. Better preserved sperm cells and a normalextratubular compartment are present (scale bar: 50 mm).


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of testicular damage following the torsion and detorsionprocesses (Lysiak et al., 2000; Nadiri et al., 2006; Srinivasanet al., 2007; Minutoli et al., 2009, 2012), which have beenconsidered similar to ischemia-reperfusion injuries observedin different organs (Eltzschig and Eckle, 2011).Ischemia and reperfusion is a pathologic event character-

ized, at first, by a reduction of blood supply followed by thereestablishment of perfusion and related reoxygenation(Eltzschig and Eckle, 2011). It induces early tissue injuries,such as reactive oxygen species (ROS) generation (Lei et al.,2015), interstitial edema (Widgerow, 2014), and a damagedbarrier function of endothelial cells, due to reduced adenylatecyclase activity and intracellular cAMP levels. In particular,during ischemia and reperfusion, nucleotides in the form ofATP promote tissue inflammation and activate the NLRP3inflammasome (McDonald et al., 2010).Ischemia and reperfusion activates various programs of cell

death, among which necrosis and apoptosis play a significantrole. Necrosis is characterized by a swelling of cells andorganelles, with rupture of their membranes and release oftheir contents. These cells trigger the inflammatory processand cytokine production. In contrast, apoptosis is character-ized by a cellular and nuclear shrinkage and plasma mem-brane integrity (Hotchkiss et al., 2009).In particular, TI/R results in exaggerated production of

ROS, activates the mitogen-activated protein kinases family,and triggers the inflammatory cascade and apoptosis machin-ery. This pathologic cascade is responsible for the testicularatrophy and impaired spermatogenesis observed at a laterstage (Filho et al., 2004; Antonuccio et al., 2006; Nadiri et al.,2006; Srinivasan et al., 2007;Minutoli et al., 2009, 2012). Evenif the importance of early events is well recognized, the latephase of TI/R is crucial from a clinical point of view as thetestis morphology and function could be severely impaired(Altavilla et al., 2012; Cvetkovic et al., 2015).Increased levels of tumor necrosis factor a and IL-1b have

been measured during testicular ischemia and reperfusion(Lysiak et al., 2000), thus confirming a role of inflammation intestis twisting. Inflammation works in concert with apoptosisto induce late organ damage and may be responsible forinfertility.

Apoptosis, further than a physiologic process that entailsthe programmed cell death, can also induce testicular injury.In fact, this process is essential during normal spermatogen-esis in both humans and animals; however, TI/R injury causesapoptosis of germ cells (Tripathi et al., 2009). All thesefindings, taken together, clearly suggest that inflammation andapoptosis are important components of the organ damageand impaired spermatogenesis induced by testicular twistingand untwisting.Muckle-Wells syndrome (MWS) is an inflammatory disease

consisting of recurrent symptoms of ocular and joint inflam-mation, fever, and skin rash, which is due to NLRP3 genemutations causing an impairment in the activity of the geneproduct cryopyrin (Agostini et al., 2004). Cryopyrin inducescaspase-1 and, in turn, the cleavage of IL-1b and IL-18 thatcauses the typical inflammatory disease of MWS (Agostiniet al., 2004; Hoffman et al., 2001).Recently, a study suggested that the NLRP3/ASC/caspase-1

axis regulated the proinflammatory cytokines IL-1b and IL-18(Xie et al., 2014).Interestingly, patients suffering from MWS have oligo-

zoospermia and/or azoospermia and show subfertility or in-fertility (Tran et al., 2012). On the basis of this observationand of other experimental data (Lech et al., 2010), as NLRP3is involved in the molecular cascade of inflammation andapoptosis in response to cellular ischemia, we thought it wasworthy of interest to investigate its role in the alteredspermatogenesis induced by TI/R injury.IL-1b and IL-18 were markedly reduced in the TI/R

inflammasome–deficient mice compared with the TI/R WTanimals. These data suggest that the inflammatory cascadeinduced by TI/R injury is blunted in the absence of thismolecular platform.Also, contralateral testes showed evident structural dam-

ages, even if they were milder than those of the I/R testes.Several mechanisms have been proposed to understand thecontralateral testicular damage, such as a decrease in testic-ular blood flow after an afferent stimulus (Prillaman andTurner, 1997; Andiran et al., 2000; Minutoli et al., 2005),ROS generation after detorsion and overproduction of nitricoxide (NO), or autoimmunization against the spermatogonia

TABLE 2BEffects of ischemia/reperfusion on the extratubular compartment in the testes of WT and KO sham, TI/R and CL of WT plusvehicle, BAY 11-7082– and BBG-treated WT, and KO mice at 7 days after the surgical procedureValues are expressed as the mean 6 S.E. Histologic grading of the extratubular compartment changes was based on the following scale: 0, absent;1, mild; 2, moderate; and 3, severe.

Extratubular Compartment

Edema Hemorrhagic Extravasation Vascular Dilation Leydig Cell Changes

Sham WT 0 0 0 0Sham KO 0 0 0 0WT TI/R + vehicle 7 days 2.25 6 0.44a 0.92 6 0.61a 1.07 6 0.59a 0.55 6 0.51a

WT CL + vehicle 7 days 1.85 6 0.71 0.72 6 0.3 0.65 6 0.49 0.7 6 0.65WT TI/R + BAY 11-70827 days

0.66 6 0.25a,b 0.62 6 0.27a 0.53 6 0.14a 0.53 6 0.4a

WT CL + BAY 11-70827 days

0.33 6 0.19 0.49 6 0.18 0.39 6 0.22 0.41 6 0.27

WT TI/R + BBG 7 days 0.55 6 0.49a,b 0.71 6 0.35a 0.54 6 0.44a,b 0.44 6 0.41a

WT CL + BBG 7 days 0.46 6 0.33 0.42 6 0.26 0.33 6 0.31 0.26 6 0.5KO TI/R 7 days 0.62 6 0.51c 0.65 6 0.6c 0.65 6 0.51c 0.35 6 0.6KO CL 7 days 0.47 6 0.5 0.4 6 0.41 0.35 6 0.48 0.4 6 0.41

aP , 0.05 versus sham WT mice.bP , 0.05 versus WT TI/R + vehicle mice.cP , 0.05 versus sham KO mice.

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(Harrison et al., 1981; Sarica et al., 1997; Shiraishi et al.,2001). In this context, the role of NO is of primary importanceas the endothelial nitric oxide synthase and neuronal nitricoxide synthase are: 1) constitutively expressed in the testis, 2)activated transiently in response to the increase in intracel-lular Ca21 mobilization, and 3) contribute to the physiologicregulation of vascular tone in both the testes and other targetorgans (Shiraishi et al., 2001; Esposito and Cuzzocrea, 2009).Moreover, the delicate balance between NO and peroxynitritecatalysts appears as an interesting therapeutic target inischemia/reperfusion injury (Esposito and Cuzzocrea, 2009).In the present study, we also studied caspase-1 and -3

expression after reperfusion in both WT and KO animals. Ourresults demonstrated that KO mice exhibited a reducedexpression of both caspases when compared with WT animalsduring TI/R. Accordingly, a large number of TUNEL-positivegerm cells were observed in the seminiferous tubules fromTI/RWTmice either at 1 or 7 days, mostly in the outer layer ofthe seminiferous tubules, suggesting that apoptosis is acutelyincreased in the tubular tissue of the twisted testes. NLRP3KO mice showed a reduced number of TUNEL-positive cells,thus suggesting that the absence of this inflammasomereduces the activation of the apoptotic machinery. Therefore,an important finding of our paper is that NLRP3-deficientmice respond to the ischemia and reperfusion insult, with alower activation of the inflammatory and apoptosis cascadethan in WT animals.The most susceptible function to testicular twisting is

spermatogenesis. In fact, WT animals subjected to TI/R hada very poor Johnsen’s score, which is a method to evaluatespermatogenesis (Johnsen, 1970; Erdemir et al., 2012). In-terestingly, the lack of NLRP3 produced an improvement inspermatogenesis following the surgical insult of TI/R. Thisfinding is of particular interest. In fact, it is possible tospeculate that this molecular platform represents one of themost prominent mechanisms for the development of the latecomplications associated with testicular torsion.To confirm the crucial role of the NLRP3 inflammasome in

TI/R, we used another way of activation blocking by means ofspecific inhibitors, such as BAY 11-7082 (Juliana et al., 2010)and BBG (Zhao et al., 2013), which were administered in TI/RWT mice. Our data showed biochemical and morphologicpatterns similar to those of KO mice, thus indicating thatthe absence of changes can be related to the blockade of eitherthe priming step (Juliana et al., 2010) or themembrane-boundpurinergic P2X7 receptor (Díaz-Hernández et al., 2009).Furthermore, our results indicate that NLRP3 is an interest-ing target for innovative drugs aimed to treat altered sper-matogenesis and male fertility and subfertility.In conclusion, our study is the first to our knowledge to

suggest that the NLRP3 inflammasome signals inflammatoryand apoptotic responses in the testis during low-flow states.

Acknowledgments

The authors thank Mr. Sebastiano Brunetto from the Departmentof Biomedical Sciences and Morphofunctional Images of the Univer-sity of Messina for technical assistance.

Authorship Contributions

Participated in research design: Squadrito, Minutoli, Altavilla,Micali.

Conducted experiments: Puzzolo, Irrera, Rinaldi, Bitto, Santoro,Pisani, Pizzino, Antonuccio.

Performed data analysis: Marini, Magno, Bitto.Wrote or contributed to the writing of the manuscript: Squadrito,

Altavilla, Minutoli, Romeo, Marini, Micali.

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