Pseudomonas aeruginosa Triggers Macrophage Autophagy To EscapeIntracellular Killing by Activation of the NLRP3 Inflammasome

Qiuchan Deng,a,b Yi Wang,a,b Yuanqing Zhang,c Meiyu Li,a,b Dandan Li,a,b Xi Huang,a,b Yongjian Wu,a,b Jieying Pu,a,b Minhao Wua,b

Department of Immunology, Zhongshan School of Medicine, Institute of Human Virology, Institute of Tuberculosis Control, Sun Yat-sen University, Guangzhou, Chinaa;Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Chinab; School of Pharmaceutical Sciences, Sun Yat-senUniversity, Guangzhou, Chinac

Assembly of the inflammasome has recently been identified to be a critical event in the initiation of inflammation. However, itsrole in bacterial killing remains unclear. Our study demonstrates that Pseudomonas aeruginosa infection induces the assemblyof the NLRP3 inflammasome and the sequential secretion of caspase1 and interleukin-1� (IL-1�) in human macrophages. Moreimportantly, activation of the NLRP3 inflammasome reduces the killing of P. aeruginosa in human macrophages, without affect-ing the generation of antimicrobial peptides, reactive oxygen species, and nitric oxide. In addition, our results demonstrate thatP. aeruginosa infection increases the amount of the LC3-II protein and triggers the formation of autophagosomes in humanmacrophages. The P. aeruginosa-induced autophagy was enhanced by overexpression of NLRP3, ASC, or caspase1 but was re-duced by knockdown of these core molecules of the NLRP3 inflammasome. Treatment with IL-1� enhanced autophagy in hu-man macrophages. More importantly, IL-1� decreased the macrophage-mediated killing of P. aeruginosa, whereas knockdownof ATG7 or Beclin1 restored the IL-1�-mediated suppression of bacterial killing. Collectively, our study explores a novel mecha-nism employed by P. aeruginosa to escape from phagocyte killing and may provide a better understanding of the interactionbetween P. aeruginosa and host immune cells, including macrophages.

Pseudomonas aeruginosa is a Gram-negative bacterium whichcommonly exists in the environment and leads to diverse op-

portunistic infections. P. aeruginosa frequently infects immuno-compromised individuals with tuberculosis and cancer and inparticular infects those with cystic fibrosis (1). In recent years, theincreased emergence of drug-resistant P. aeruginosa strains hasbrought a big challenge to traditional antibiotic therapies (2);therefore, efforts to understand the immune defense of the hostagainst P. aeruginosa infection have attracted more attention.

Innate immune cells, such as macrophages and neutrophils, aswell as the cytokines/chemokines secreted by these inflammatorycells, compose the first line of host defense. Most invading patho-gens are engulfed by infiltrating phagocytes and then undergointracellular elimination via either an oxygen-dependent or -in-dependent bactericidal system, represented by reactive oxygenspecies (ROS) and reactive nitrogen species (RNS), respectively(3, 4), and via antimicrobial peptides, such as beta-defensins(BDs) and bactericidal/permeability-increasing protein (Bpi) (5).Recently, accumulating evidence demonstrates that autophagy is aprotective strategy of host defense against a variety of intracellularpathogens, including Shigella flexneri (6), Salmonella enterica se-rovar Typhimurium (7), Listeria monocytogenes (8), and Mycobac-terium tuberculosis (9, 10). However, the role of autophagy in thefight against extracellular pathogens like P. aeruginosa remainsunclear.

Autophagy is an evolutionarily conserved catabolic mecha-nism which maintains cytoplasmic homeostasis by inducing thedegradation of damaged organelles or misfolded proteins (11, 12).Initiation of autophagy is usually characterized by the formationof microtubule-associated protein light chain 3 (LC3) puncta, aswell as the conversion of LC3-I into its lipidated form, LC3-II(13). Autophagy can be activated by starvation, rapamycin, as wellas Toll-like receptor ligands and inflammatory cytokines likegamma interferon (IFN-�) and tumor necrosis factor (TNF) (13).

It is reported that P. aeruginosa can induce an autophagic responsein macrophages (14) and mast cells (15). Nonetheless, the under-lying mechanism still needs further investigation.

In addition to the induction of inflammatory cytokines andautophagy, bacterial infection also leads to the assembly of anintracellular complex called the inflammasome (16, 17). The in-flammasome is one of the most important components in theinnate immune and inflammatory response. The inflammasomeis a large complex which is composed of multiple molecules, in-cluding adapter molecules like ASC and certain types of NOD-likereceptors (NLRs) belonging to either the NLR family (e.g.,NLRP1, NLRP3, or IPAF) or the PYHIN family (e.g., AIM2) (16).Enhanced expression of some NLRs like NLRP3 has been reportedto be a critical checkpoint for inflammasome activation (17). Ac-tivation of the inflammasome finally leads to the cleavage of aninactive zymogen, procaspase1 (18). The active caspase1 then pro-motes the cleavage and secretion of interleukin-1� (IL-1�) andinterleukin-18 (IL-18). The secreted IL-1� and IL-18 not onlyfunction as proinflammatory mediators in the immune defenseagainst microbial infection but also have the potential to modulate

Received 21 July 2015 Returned for modification 25 August 2015Accepted 12 October 2015

Accepted manuscript posted online 14 October 2015

Citation Deng Q, Wang Y, Zhang Y, Li M, Li D, Huang X, Wu Y, Pu J, Wu M. 2016.Pseudomonas aeruginosa triggers macrophage autophagy to escape intracellularkilling by activation of the NLRP3 inflammasome. Infect Immun 84:56 – 66.doi:10.1128/IAI.00945-15.

Editor: B. A. McCormick

Address correspondence to Minhao Wu, wuminhao@mail.sysu.edu.cn.

Q.D. and Y.W. contributed equally to this article.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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the autophagy response. However, the role of the inflammasomein modulating autophagy remains unclear.

In the present study, we demonstrate that the NLRP3 inflam-masome is activated during P. aeruginosa infection. More impor-tantly, silencing of the NLRP3 inflammasome reduced the level ofIL-1�/IL-18 secretion and decreased the level of autophagy, whichimpaired the elimination of P. aeruginosa in macrophages. Over-all, our results explore a novel role of the NLRP3 inflammasome inmodulating autophagy and bacterial clearance in macrophagesand may provide a better understanding of the host-pathogenrelationship during P. aeruginosa infection.

MATERIALS AND METHODSMaterials and reagents. Pseudomonas aeruginosa strain ATCC 19660 waspurchased from the American Type Culture Collection (ATCC). Nigeri-cin (an NLRP3 inflammasome inducer), glybenclamide (an NLRP3 in-flammasome inhibitor), and benzyloxycarbonyl-Val-Ala-Asp-fluorom-ethylketone (Z-VAD-FMK; a caspase1 inhibitor) were from InvivoGen(Carlsbad, CA). Rapamycin was purchased from Sigma-Aldrich (St.Louis, MO), and FilmTracer green biofilm (FTGB) was from Invitrogen(Carlsbad, CA). Recombinant human IL-1� was obtained from Cell Sig-naling Technology (Beverly, MA). Prior approval for the use of humansubjects was obtained from the Scientific Investigation Board of Sun Yat-sen University (Guangdong, China).

Cell culture. Human monocytic THP-1 cells (TIB-202; ATCC) weretreated with phorbol 12-myristate 13-acetate (PMA; 80 nM; Sigma) at37°C for 16 h to obtain PMA-differentiated THP-1 macrophages. THP-1macrophages were cultured in RPMI 1640 medium supplemented with10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen,Carlsbad, CA) and incubated at 37°C in a humidified 5% CO2 incubator.

Isolation and culture of hMDMs. Monocytes were isolated frombuffy coats from healthy human donors by density gradient centrifuga-tion as previously described (19). All the healthy donors (8 males whoranged in age from 21 to 43 years; 7 females who ranged in age from 24 to39 years) were recruited from the Guangzhou Blood Center and had nosignificant medical history. For the use of buffy coats for research pur-poses, the prior consent of the donors and approval from the InstitutionalResearch Ethics Committee, Sun Yat-sen University Zhongshan School ofMedicine, were obtained. Briefly, buffy coats from the blood of healthydonors were diluted with phosphate-buffered saline (PBS) and layered onFicoll-Paque. After centrifugation, white blood cells were collected andwashed with PBS. CD14 MicroBeads (BD Biosciences, San Jose, CA) wereused for the positive selection of human monocytes from white bloodcells. Human monocyte-derived macrophages (hMDMs) were generatedfrom monocytes by culture for 8 days in complete medium containing 100ng/ml recombinant human macrophage colony-stimulating factor.

ELISA. Cell supernatants were collected and analyzed for secreted IL-1�, caspase1, and IL-18 using specific enzyme-linked immunosorbentassay (ELISA) kits from BD Biosciences, R&D Systems, and eBioscienceSystems, respectively, and the ELISA kits were used according to the man-ufacturer’s instructions. Each sample was assayed in duplicate. The resultsshown are the means from three separate experiments. The reported sen-sitivities of these assays are �3.9 pg/ml for IL-1�, �1.24 pg/ml forcaspase1, and �9.0 pg/ml for IL-18.

Real-time PCR. Total RNA was isolated from cell pellets using theTRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufactur-er’s recommendation, 1 �g of total RNA was reverse transcribed to cDNA,and then cDNA was amplified using the SYBR green master mix (TaKaRa,Mountain View, CA) as suggested by the manufacturer. Quantitative real-time PCRs were performed using a CFX96 real-time PCR system (Bio-Rad, Hercules, CA). Relative mRNA levels were calculated after normal-ization to the level of �-actin. The primer sequences are listed in Table 1.

Western blotting. Cells were washed three times with ice-cold PBSand then lysed in lysis buffer containing 1 mM phenylmethylsulfonyl

fluoride, 1% (vol/vol) protease inhibitor cocktail (Sigma, St. Louis, MO),and 1 mM dithiothreitol. Equal amounts (20 �g) of cell lysates (and cellsupernatants for the detection of IL-1�) were resolved by SDS-PAGE andthen transferred to polyvinylidene difluoride membranes. The mem-branes were blocked in 5% nonfat dry milk in PBS–Tween 20 and incu-bated at 4°C overnight with the respective primary antibodies (Abs), in-cluding caspase1 Ab (Santa Cruz, CA), NLRP3 Ab and ASC Ab(ProteinTech Group, IL), NLRP1 Ab (Abcam, MA), IPAF Ab and AIM2Ab (Cell Signaling Technology), caspase4 Ab (MBL International, MA),caspase5 Ab (Merck Millipore, CA), and LC3 Ab (Novus Biologicals, CO).The membranes were incubated at room temperature for 1 h with appro-priate horseradish peroxidase-conjugated secondary Abs and visualizedwith a Plus-ECL kit (KeyGEN, Nanjing, China) according to the manu-facturer’s protocol.

Transient transfection. Cells were transiently transfected with spe-cific small interfering RNAs (siRNAs), scrambled negative-control siRNA(siNC), NLRP3, ASC, and caspase1 expression vectors, or the controlpSG5 vector using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad,CA) following the manufacturer’s instruction. siRNAs were synthesizedby Invitrogen (Shanghai, China), and the sequences are as follows: forsiRNA against caspase1 (siCasp-1), CCGCAAGGTTCGATTTTCA; forsiRNA against caspase1 and caspase2 (siCasp1-2), GACTCATTGAACATATGCA; for siRNA against NLRP3 (siNLRP3), GGTGTTGGAATTAGACAAC; for siRNA against ASC (siASC), GATGCGGAAGCTCTTCAG;for siRNA against ATG7 (siATG7), GGAGTCACAGCTCTTCCTT; andfor siRNA against Beclin1 (siBeclin1), AACAGTTTGGCACAATCAATA.siNC was used as a negative control.

Immunofluorescence staining and confocal microscopy. Immuno-fluorescence staining was performed as previously described (10, 20).Cells were grown on collagen-precoated glass coverslips in 24-well plates.After transfection with the specific siRNAs and infection with P. aerugi-nosa, the cells were fixed with 4% paraformaldehyde, followed by mem-brane permeabilization using 0.2% Triton X-100. Cells were blocked with5% bovine serum albumin and sequentially incubated with primary andsecondary antibodies before mounting. The nucleus was stained withDAPI (4=,6-diamidino-2-phenylindole). Differential interference con-trast (DIC) and fluorescence images were captured on a confocal micros-copy (Zeiss Axiovert LSM710).

Bacterial killing assay. Intracellular bacterial killing was assessed bythe use of plate counts as previously described by our group (21, 22) andother groups (23). In brief, differentiated THP-1 cells were cultured in asix-well plate and then treated with rapamycin or IL-1� or transfected

TABLE 1 Nucleotide sequences of the specific primers used in PCRamplification

Gene Primer sequence (5=–3=) Orientationa

Caspase1 GGA AAC AAA AGT CGG CAG AG FACG CTG TAC CCC AGA TTT TG R

NLRP3 AGC ACT TGC TGG ACC ATC CT FGAC CTC GTT CTC CTG AAT CAG ACT R

ASC GCG AGG GTC ACA AAC GTT GA FGTG AAA CTG AAG AGC TTC CGC A R

BD2 GGG TCT TGT ATC TCC TCT TCT CGT FGAC TGG ATG ACA TAT GGC TCC ACT R

Bpi GTT CTC CAT CAG CAA CGC CAA T FCAG CCG AAA TGG ACA TGC CT R

�-Actin GCT CCT CCT GAG CGC AAG FCAT CTG CTG GAA GGT GGA CA R

a F, forward; R, reverse.

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with siRNAs against ATG7 or Beclin1, followed by P. aeruginosa challengeat a multiplicity of infection (MOI) of 25. After 1 h of infection, the cellswere treated with gentamicin (Sigma) at 300 mg/ml for 30 min andwashed with PBS three times to kill and remove the extracellular bacteria.After that, cells in one of the duplicate wells were lysed with 0.1% TritonX-100, and the number of internalized bacteria was determined by theplate count method and designated the number of CFU at 1 h [CFU(1 h)].Cells in the other of the duplicate wells were incubated for another 1 h, andthen the number of viable bacteria was determined by the plate countmethod and designated the number of CFU at 2 h [CFU(2 h)]. The killingefficiency of the internalized bacteria was calculated using the follow-ing equation: intracellular bacterial killing � {[CFU(1 h) – CFU(2 h)]/CFU(1 h)} � 100%.

ROS measurement by flow cytometry. ROS measurement was per-formed as described before (24, 25). Briefly, differentiated THP-1 cellswere transfected with specific siRNAs and siNC for 24 h. After P. aerugi-nosa infection, the cells were incubated with a ROS-sensitive probe, 2=,7=-dichlorofluorecscin diacetate (H2DCFDA; Invitrogen), at a final concen-tration of 10 �M. Then, the cells were collected and analyzed using aBeckman Coulter Epics XL/MCL apparatus (Beckman Coulter Inc.). ROSlevels were determined from the fluorescence of dichlorofluorescein(DCF), the deacetylated and oxidized product of H2DCFDA.

Griess assay. Differentiated THP-1 cells were transfected with specificsiRNAs and siNC, and the supernatant of each sample was collected at 1 h,6 h, and 24 h after P. aeruginosa infection. Nitric oxide (NO) levels weredetermined by measuring the levels of the stable end product, nitrite,using the Griess reagent (Promega, Madison, WI). The results are ex-pressed as the mean number of micromoles of nitrite per sample � stan-dard error of the mean (SEM).

Statistical analysis. The differences between two groups were com-pared by using an unpaired two-tailed Student’s t test, while differencesbetween three or more groups were compared by using analysis of vari-ance (ANOVA) with Bonferroni’s posttest. Data were considered statisti-cally significant when the P value was �0.05.

RESULTSThe NLRP3 inflammasome is activated after P. aeruginosa in-fection. We first examined caspase1 activation and IL-1� secre-tion in P. aeruginosa-infected THP-1 macrophages. ELISA datashowed that the protein levels of caspase1 and IL-1� in the culturesupernatants of THP-1 macrophages were obviously upregulatedin a time- and dose-dependent manner (Fig. 1A to D). Similarly,in primary human monocyte-derived macrophages (hMDMs),we also detected the upregulation of secreted caspase1 and IL-1�after P. aeruginosa infection at various time points postinfection(p.i.) (Fig. 1E and F). Moreover, silencing of caspase1 significantlydecreased the secretion of IL-1� at 1 h, 6 h, and 24 h p.i. in bothTHP-1 macrophages (Fig. 1G) and hMDMs (Fig. 1I), and theknockdown efficacy of siRNA against caspase1 (siCasp1) was con-firmed by Western blotting (Fig. 1H and J). These data togetherimplicate that P. aeruginosa infection activates an inflammasomein macrophages.

To further determine which type of inflammasome is activatedby P. aeruginosa, we tested the expression of the central moleculesof different canonical and noncanonical inflammasomes inTHP-1 macrophages before and at 6 h, 12 h, 24 h, and 48 h after P.aeruginosa infection. The Western blotting data showed that atprotein levels, these molecules were all constitutively expressed inTHP-1 macrophages, but only NLRP3 and caspase1 expressionwas increased after P. aeruginosa infection, while IPAF expressionwas decreased, and the levels of expression of the other proteins(including NLRP1, AIM2, ASC, caspase4, and caspase5) were un-changed (Fig. 2A). We further tested NLRP3 expression at earlier

time points (1 h, 3 h, and 6 h) postinfection by real-time PCR andWestern blotting and found that NLRP3 expression was enhancedin THP-1 macrophages after P. aeruginosa infection in a time-dependent manner (Fig. 2B and C).

Since NLRP3 upregulation usually indicates the activation ofthe NLRP3 inflammasome, we next examined whether P. aerugi-nosa infection activates the NLRP3 inflammasome in distinct celltypes. First, THP-1 macrophages were transiently transfected withsiNLRP3, siASC, or siNC, infected with P. aeruginosa for the timesindicated below, and then analyzed for the knockdown efficiencyas well as the secretion of IL-1� and caspase1. The knockdownefficiency of siNLRP3 and siASC was confirmed by Western blot-ting and real-time PCR at different times postinfection (Fig. 3A toD). The ELISA data indicated that the expression of IL-1� (Fig.3E), caspase1 (Fig. 3F), and IL-18 (Fig. 3G) was significantlydownregulated at the various time points compared to their levelsof expression after the control treatment, suggesting that NLRP3and ASC are required for the P. aeruginosa-mediated activation ofthe inflammasome. Consistent with the observations with THP-1macrophages, silencing of NLRP3, ASC, or caspase1 also resultedin a significant inhibition of IL-1� secretion in hMDMs after P.aeruginosa infection (Fig. 3H). We further confirmed the P.aeruginosa-induced activation of the NLRP3 inflammasome byimmunofluorescence staining. Confocal microscopy resultsshowed the apparent colocalization of NLRP3 and caspase1 inTHP-1 differentiated macrophages after P. aeruginosa infection,as well as in cells treated with nigericin (a pore-forming toxinwhich activates the NLRP3 inflammasome used as a positive con-trol) (Fig. 4A and B). Furthermore, P. aeruginosa-induced IL-1�secretion was abrogated by treatment with the NLRP3 inflam-masome inhibitor glybenclamide (a proton pump inhibitor) orthe caspase1-specific inhibitor Z-VAD-FMK at the concentra-tions indicated in Fig. 4C and D. Taken together, these data indi-cate that the NLRP3 inflammasome is activated in macrophagesafter P. aeruginosa infection.

The NLRP3 inflammasome affects bacterial killing in P.aeruginosa-infected macrophages. We further assessed the effectof the NLRP3 inflammasome on bacterial clearance by overex-pressing or silencing core molecules of the NLRP3 inflammasome.For the overexpression assay, THP-1 macrophages were trans-fected with an NLRP3, ASC, or caspase1 expression plasmid or thecontrol pSG5 vector, followed by P. aeruginosa infection at anMOI of 25. The efficiency of overexpression was confirmed byWestern blotting (Fig. 5A). Bacterial clearance was assessed usinga bacterial killing assay based on plate counts, and the resultsshowed that overexpression of NLRP3, ASC, or caspase1 signifi-cantly decreased the macrophage-mediated killing of P. aerugi-nosa (Fig. 5B). Data from the knockdown assay consistentlyshowed that silencing of NLRP3, ASC, or caspase1 significantly en-hanced the macrophage-mediated killing of internalized P. aerugi-nosa (Fig. 5C). To further explore the underlying microbicidalmechanisms, we first examined the generation of oxygen-depen-dent and -independent bactericidal components in siNLRP3-,siASC-, siCasp1-, or siNC-transfected THP-1 macrophages afterP. aeruginosa infection. PCR data showed that levels of mRNA forantimicrobial peptides such as BDs and Bpi were comparable in allthe tested groups (Fig. 5D). We further examined the levels of NOand ROS generation by the Griess assay and flow cytometry, re-spectively. No differences in NO levels (Fig. 5E; as determinedfrom the amount of the stable end product nitrite) or the levels of

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ROS production (Fig. 5F and G; as calculated from the percentageof DCF-positive cells) were detected among the tested groups.These data suggest that the NLRP3 inflammasome reduces themacrophage-mediated intracellular killing of P. aeruginosa inde-pendently of antimicrobial peptides, NO, and ROS.

The NLRP3 inflammasome promotes autophagy during P.aeruginosa infection. Since autophagy has been identified to bean efficient means for host cells to eradicate intracellular patho-gens, we next tested the role of the NLRP3 inflammasome onmodulating the autophagic response in P. aeruginosa-infectedTHP-1 macrophages. Western blotting and fluorescence micros-copy were used to test the amount of the LC3-II protein and thenumber of LC3 puncta, respectively. Western blotting resultsshowed that overexpression of NLRP3, ASC, or caspase1 in THP-1cells enhanced autophagy in P. aeruginosa-infected cells, as indi-cated by the increased amount of the LC3-II protein (Fig. 6A),while silencing of NLRP3, ASC, or caspase1 in THP-1 macro-phages decreased LC3-II levels (Fig. 6B). Immunofluorescentstaining results consistently showed that silencing of NLRP3, ASC,or caspase1 dramatically decreased the levels of formation of LC3puncta (Fig. 6C and D), as well as the level of formation of au-tophagosomes containing P. aeruginosa, as indicated by the colo-calization of LC3 and FTGB-labeled P. aeruginosa (Fig. 6E and F).These data together demonstrate that the NLRP3 inflammasomeelevates autophagy in THP-1 macrophages.

IL-1�-mediated autophagy impaired bacterial eradicationin P. aeruginosa-infected macrophages. Next, THP-1-derivedmacrophages were transfected with specific siRNAs against ATG7and Beclin1 or treated with rapamycin (an autophagy inducer)

and then analyzed by a bacterial killing assay to determine theinfluence of autophagy on P. aeruginosa elimination. The knock-down efficiency of siATG7 and siBeclin1 was confirmed by West-ern blotting (Fig. 7A). Data on the numbers of CFU showed thatsilencing of ATG7 or Beclin1 significantly enhanced the efficiencyof macrophage-mediated bacterial killing compared to the controltreatment (Fig. 7B), while treatment with rapamycin attenuated

FIG 1 Caspase1 activation and IL-1� secretion are induced after P. aeruginosa infection. (A to D) THP-1 macrophages were infected with P. aeruginosa (PA) atan MOI of 1 for 6 h, 12 h, and 24 h (A, B) or at MOIs of 1, 5, and 10 for 24 h (C, D). Caspase1 (A, C) and IL-1� (B, D) protein levels in the indicated cellsupernatants were examined by ELISA. (E, F) hMDMs were infected with P. aeruginosa at an MOI of 1 for the indicated times, and caspase1 (E) and IL-1� (F)protein levels in the indicated cell supernatants were examined by ELISA. (G to J) THP-1 macrophages (G) and hMDMs (I) were transfected with siCasp1 for 24h, followed by P. aeruginosa infection at an MOI of 1 for 1 h, 6 h, and 24 h, and then the protein levels of IL-1� in the cell supernatants were tested by ELISA. Theknockdown efficiency of caspase1 in THP-1 cells (H) and hMDMs (J) was confirmed by Western blotting. PA inf., time of P. aeruginosa infection. Data are shownas the mean � SEM and represent the results from three individual experiments. *, P � 0.05; **, P � 0.01.

FIG 2 Expression of inflammasome molecules in P. aeruginosa-infected hu-man macrophages. (A) THP-1 macrophages were infected with P. aeruginosaat an MOI of 1 for 6 h, 12 h, 24 h, and 48 h, and then the levels of NLRP1,NLRP3, IPAF, AIM2, ASC, caspase1, caspase4, and caspase5 protein expres-sion were tested by Western blotting. Furthermore, NLRP3 mRNA (B) andprotein (C) levels in P. aeruginosa-infected THP-1 macrophages at 1 h, 3 h, and6 h postinfection were examined using real-time PCR and Western blotting,respectively. Data are shown as the mean � SEM and represent the resultsfrom three individual experiments.

FIG 3 The NLRP3 inflammasome is activated in human macrophages after P.aeruginosa infection. (A to D) THP-1 macrophages were transfected withsiNLRP3 or siASC for 24 h, followed by P. aeruginosa infection for the indi-cated times, and the knockdown efficiency was confirmed by Western blotting(A and B) and real-time PCR (C and D). (E to G) IL-1� (E), caspase1 (F), andIL-18 (G) levels in cell supernatants were tested by ELISA. (H) hMDMs weretransfected with siNLRP3, siASC, siCasp1, or siNC for 24 h, followed by P.aeruginosa infection, and IL-1� levels were tested by ELISA. Data are shown asthe mean � SEM and represent the results from three individual experiments.*, P � 0.05; **, P � 0.01; ***, P � 0.001.

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the efficiency of macrophage-mediated bacterial killing comparedto the control treatment (Fig. 7C). These results suggest that in-duction of autophagy reduces bacterial clearance during P. aerugi-nosa infection.

To further confirm the relationship between the inflam-masome and autophagy, THP-1 macrophages were treated or nottreated with IL-1� (the final product of inflammasome activation)and analyzed for the amount of the LC3-II protein and bacterialkilling efficiency, respectively. Treatment with recombinant IL-1�enhanced the amount of the LC3-II protein in THP-1 cells (Fig.7D). In addition, treatment with IL-1� decreased the macro-phage-mediated killing of P. aeruginosa (Fig. 7E), while knock-down of ATG7 or Beclin1 restored the IL-1�-mediated suppres-

sion of bacterial killing (Fig. 7F). Collectively, these results suggestthat IL-1� impairs the clearance of P. aeruginosa by promotingautophagy in human macrophages.

DISCUSSION

As an important component of the innate immune system, theinflammasome is mainly expressed in myeloid cells and is respon-sible for the initiation of inflammatory processes. Different typesof inflammasomes can be activated in innate immunocytes duringbacterial infection (26). Studies have demonstrated that P. aerugi-nosa infection mainly activates the IPAF inflammasome in murinemacrophages (27–29). It has recently been reported that P. aerugi-nosa also triggers activation of the NLRP3 inflammasome in hu-

FIG 4 The NLRP3 inflammasome is detected by immunofluorescence. (A) THP-1 macrophages were infected with P. aeruginosa (MOI, 25) or nigericin (Nig.;10 �M) for 1 h, and then the localization of NLRP3 and caspase1 was visualized by immunofluorescent staining. The colocalization of NLRP3 (green) andcaspase1 (red) was detected by confocal microscopy. Arrows, colocalization of NLRP3 and caspase1. Bars � 10 �m. Cells were also visualized by differentialinterference contrast (DIC) microscopy. (B) The percentage of cells with colocalization of NLRP3 and caspase1 was determined. ***, P � 0.001. (C, D) Cells werepretreated with glybenclamide (Glyben) (C) or Z-VAD-FMK (Z-VAD) (D) at different concentrations or the vehicle control (DMSO) for 30 min, followed byP. aeruginosa infection (MOI, 1) for 6 h, and the level of IL-1� secretion was tested by ELISA. Data are shown as the mean � SEM and represent the results fromthree independent experiments. Ctrl., control.

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man bronchial epithelial cells derived from patients with cysticfibrosis (30). Nonetheless, the machinery in human macrophagesthat is activated by P. aeruginosa infection remains unknown. Ourstudy demonstrates that P. aeruginosa triggers the assembly of the

NLRP3 inflammasome in human macrophages and explored therole of the P. aeruginosa-triggered NLRP3 inflammasome in mod-ulating autophagy and bacterial eradication, providing evidenceof a novel mechanism of P. aeruginosa-induced immune evasion.

FIG 5 The NLRP3 inflammasome suppresses bacterial killing of macrophages independently of antimicrobial peptides, NO, and ROS. (A, B) THP-1 macro-phages were transiently transfected with an NLRP3, ASC, or caspase1 expression plasmid or the pSG5 control vector for 24 h, followed by P. aeruginosa infection,and the efficiency of overexpression was determined by Western blotting (A). (B) The bacterial killing efficiency in the indicated groups was assessed by a killingassay by the plate count method. (C to F) THP-1 macrophages were transfected with the indicated siRNAs for 24 h, followed by P. aeruginosa infection. (C) Thekilling efficiency of P. aeruginosa was determined by a killing assay by the plate count method. (D) The levels of expression of mRNA for the antimicrobialpeptides BD and Bpi were tested using real-time PCR. hBD, human BD. (E) NO production (as indicated by the nitrite concentration) was tested using the Griessassay at the indicated time points. (F, G) ROS levels were analyzed by flow cytometry at 1 h and 6 h p.i. Data are shown as the mean � SEM and represent theresults from three independent experiments. ***, P � 0.001.

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Our results show that infection with P. aeruginosa ATCC 19660(a cytotoxic P. aeruginosa strain) results in activation of theNLRP3 inflammasome in THP-1 macrophages and hMDMs. Pre-treatment with glybenclamide (a potent NLRP3 inflammasomeinhibitor) dramatically impaired IL-1� secretion in P. aeruginosa-infected human macrophages, further supporting this conclusion.Infection with another invasive P. aeruginosa stain, PAO1, alsoconsistently led to the activation of the NLRP3 inflammasome(data not shown). To the best of our knowledge, this is the firstreport of the P. aeruginosa-induced activation of the NLRP3 in-flammasome in bone marrow-derived cells. Studies have demon-strated that P. aeruginosa can activate IPAF but not the NLRP3

inflammasome in murine macrophages (27–29). In the presentstudy, silencing of IPAF reduced the level of IL-1� secretion inhMDMs within 6 h p.i., but this effect was not detected in P.aeruginosa-infected THP-1 cells (data not shown). Studies havedemonstrated that the NLRP3 inflammasome is activated by var-ious particles, crystals, toxins, and pathogens (27–29), as well asmitochondrial Ca2 (30), while the IPAF inflammasome is re-sponsive to a narrower spectrum of activators, such as DNAreleased from mitochondrial damage (31) and cytoplasmically de-livered bacterial flagellin (32). Therefore, the type of inflam-masomes activated in host cells largely depends on the cell’s abilityto uptake, generate, or recognize cytoplasmic inducers. This abil-

FIG 6 The NLRP3 inflammasome promotes autophagy during P. aeruginosa infection. (A, B) THP-1 macrophages were transiently transfected with an NLRP3,ASC, or caspase1 expression plasmid or the pSG5 control vector (A) or were transfected with siNLRP3, siASC, siCasp1, or siNC (B), followed by P. aeruginosainfection, and then the levels of the LC3 protein were detected by Western blotting. (C to F) THP-1 macrophages were transfected with the indicated siRNAs andthen infected with unlabeled (C, D) or FTGB-labeled (E, F) P. aeruginosa. Endogenous LC3 was stained with anti-LC3 antibody followed by Alexa Fluor488-conjugated goat anti-rabbit IgG (green), and the LC3 puncta were detected by confocal microscopy (C) and quantified (D). Endogenous LC3 was stainedwith anti-LC3 antibody followed by Cy3-conjugated goat anti-rabbit lgG, and the colocalization of FTGB-labeled P. aeruginosa (green) with an LC3-positive(LC3) autophagosome (red) was detected by confocal microscopy (E) and quantified (F). Data are shown as the mean � SEM and represent the results fromthree independent experiments. ***, P � 0.001. Bars � 10 �m.

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ity is predisposed to the species specificity, which may explain thedifference in P. aeruginosa-induced inflammasome activation be-tween human and murine macrophages.

It is worth mentioning that, in the present study, silencing ofcaspase1 only partially, albeit significantly, decreased the level ofsecretion of IL-1�, which is consistent with the findings of otherinvestigators showing that nonclassical IL-1� activation duringmammary gland infection is pathogen dependent but caspase-1independent (33). Recently, it has been reported that cytosoliclipopolysaccharide induces noncanonical activation of the NLRP3inflammasome and IL-1� secretion in myeloid cells in a manner

that is independent of caspase1 but dependent on caspase4 andcaspase11 in human and mouse cells, respectively (34, 35). OurPCR data showed that caspase4 and caspase5 expression was un-changed after P. aeruginosa infection (Fig. 2A), indicating that thenoncanonical inflammasome was not activated or at least is notthe major inflammasome type during P. aeruginosa infection. Arecent study reported that after injection of anti-COL7 IgG, theextent of skin lesions, which is mediated by IL-1� release, is sim-ilar in caspase1/11-deficient and wild-type mice (36). In this re-gard, P. aeruginosa may promote IL-1� secretion through bothinflammasome-dependent and -independent pathways, thereforeleading to the partial role of siCasp1 (as well as siASC) in IL-1�secretion.

The role of the inflammasome in host defense, especially inbacterial eradication, remains unclear. It is reported that IPAFdeficiency leads to higher bacterial burdens in P. aeruginosa-in-fected mice without affecting bacterial survival (28). However, invivo studies using a murine model of P. aeruginosa pneumoniaprovide substantive evidence that IL-1� and IL-18 impair thehost’s ability to eradicate P. aeruginosa from infected lungs (37,38). Moreover, Hazlett’s group (39, 40) previously reported thatknockout of IL-1�-converting enzyme (ICE), which is actuallycaspase1/ caspase 11/ (41), or treatment with an ICE inhib-itor dramatically decreases the ocular inflammatory response andbacterial load after P. aeruginosa corneal infection, whereas Pearl-man’s group found that IL-1� processing in the P. aeruginosa-infected corneas was dependent on neutrophil serine proteasesbut independent of caspase1 and NLRC4 (42). Their in vitro stud-ies also demonstrated that P. aeruginosa-induced IL-1� secretionwas ablated in caspase-1/ and NLRC4/ mouse macrophagescompared with that in wild-type C57BL/6 mouse macrophages,whereas in neutrophils, IL-1� secretion was partially dependenton caspase-1 and NLRC4 (42). Polymorphonuclear leukocytes(PMNs) are the predominant inflammatory cells early in the re-sponse to P. aeruginosa infection, while macrophages are presentand participate at later time points after infection (43). Therefore,the differences in the results between these in vivo studies may bedue to the activation of different types of inflammasomes in neu-trophils versus macrophages. Our in vitro study demonstrates thatsilencing of NLRP3/ASC/caspase1 enhances macrophage-medi-ated P. aeruginosa clearance, while treatment with recombinantIL-1� attenuates the killing efficiency. This finding indicates thatactivation of the NLRP3 inflammasome may be an immune eva-sion strategy employed by P. aeruginosa, but the underlying mech-anism remains unknown.

We next tested the role of the NLRP3 inflammasome on mi-crobicidal mechanisms. Silencing of NLRP3/ASC/caspase1 hadno influence on the production of ROS, NO, and antimicrobialpeptides but dramatically reduced the autophagy response inmacrophages after P. aeruginosa infection. Overexpression of theNLRP3 inflammasome core molecules or treatment with IL-1�promoted autophagy in P. aeruginosa-infected macrophages,which is consistent with the findings of others showing thatcaspase1 increases the autophagic influx by upregulating Beclin1expression (43). Jabir et al. demonstrated that the DNA releasedfrom damaged mitochondria contributes to P. aeruginosa-medi-ated activation of the IPAF inflammasome and is downregulatedby autophagy (31). These findings indicate the cross talk betweeninflammasome and autophagy.

Autophagy not only protects host cells from nutrient depriva-

FIG 7 Autophagy and IL-1� impaired bacterial eradication in P. aeruginosa-infected macrophages. (A, B) THP-1 macrophages were transfected with si-ATG7 or siBeclin1 for 24 h, followed by P. aeruginosa infection at an MOI of25, and then knockdown efficiency (A) and bacterial killing (B) were examinedby Western blotting and an assay counting the numbers of CFU, respectively.(C) THP-1 macrophages were pretreated with rapamycin (8 �M) for 6 h,followed by P. aeruginosa infection at an MOI of 25, and then the killingefficiency was determined by an assay counting the numbers of CFU. (D, E)THP-1 macrophages were incubated with recombinant IL-1� (rIL-1�; 100ng/ml) for 1 h and 3 h, and LC3 protein levels were detected by Westernblotting (D). (E) Bacterial killing was tested by an assay counting the numbersof CFU after incubation with IL-1� for 3 h. (F) THP-1 macrophages weretransfected with siNC, siATG7, or siBeclin1 for 24 h, followed by treatmentwith recombinant IL-1� (100 ng/ml) for 3 h, and then the killing efficiency ofeach group was determined by an assay counting the numbers of CFU. Dataare shown as the mean � SEM and represent the results from three indepen-dent experiments. ns., not significant; **, P � 0.01; ***, P � 0.001.

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tion and other cellular stresses but also accelerates the degradationof intracellular pathogens by promoting the maturation of au-tophagolysosomes (44). Our study demonstrates that P. aerugi-nosa induces autophagy in human macrophages, thereby sup-pressing the bacterial killing activity of macrophages. This isconsistent with the findings of other studies showing that au-tophagy deficiency in macrophages increases the phagocytosis ofvarious bacterial species through enhancement of the expressionof scavenger receptors (45). Yuan et al. reported that P. aeruginosainduces autophagy in murine macrophages, which in turn de-creases the number of intracellular P. aeruginosa bacteria (14).They roughly conclude that the decrease in the levels of intracel-lular P. aeruginosa bacteria is an indicator of enhanced bacterialkilling, ignoring the possibility that the suppression of phagocy-tosis also leads to a reduced number of bacteria being taken up bymacrophages. Studies using a murine model of P. aeruginosa ker-atitis have demonstrated that treatment with rapamycin (an au-tophagy inducer) diminished the bactericidal activity of PMNsand increased the bacterial load in P. aeruginosa-infected corneas(22). These in vitro and in vivo studies together indicate that acti-vation of autophagy inhibits bacterial killing in phagocytes. Notethat our previous studies found that overexpression of Rheb,which inhibits autophagy by activating mTOR, enhanced the kill-ing of P. aeruginosa bacteria (46) yet reduced the killing of Myco-bacterium bovis BCG bacteria in murine macrophages (10). Thedifferent roles of autophagy in modulating the macrophage-me-diated killing of P. aeruginosa and BCG bacteria may be explainedby the distinct receptors involved in the process of phagocytosis ofextracellular versus intracellular bacteria.

Collectively, our study demonstrates for the first time the acti-vation of the NLRP3 inflammasome in response to P. aeruginosainfection and unravels a novel role of the NLRP3 inflammasomein modulating the autophagic response and bacterial eliminationin macrophages. These data may provide useful information forthe development of potential therapeutic interventions against P.aeruginosa and other bacterial infections.

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural ScienceFoundation of China (31470877, 31200662, 31370868, 81172811,81261160323), the 111 Project (no. B13037), the Open Project Grantat the State Key Laboratory of Ophthalmology (Zhongshan Ophthal-mic Center), the Guangdong Innovative Research Team Program(2009010058, 2011Y035), the Specialized Research Fund for the Doc-toral Program of Higher Education of China (20120171120064), theGuangdong Natural Science Foundation (10251008901000013,S2012040006680), Guangdong Province Universities and Colleges PearlRiver Scholar Funded Scheme (no. 2009), and the National Science and Tech-nology Key Projects for Major Infectious Diseases (2013ZX10003001).

FUNDING INFORMATIONNational Natural Science Foundation of China (NSFC) provided fundingto Meiyu Li, Xi Huang, and Minhao Wu under grant numbers 31470877,31200662, 31370868, 81172811, and 81261160363.

The funders had no role in study design, data collection and interpreta-tion, or the decision to submit the work for publication.

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