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N-(3-oxododecanoyl)-l -homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells
Accepted Manuscript N-(3-oxododecanoyl)-l-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells
Shiyu Tao, Liqiong Niu, Liuping Cai, Yali Geng, Canfeng Hua, Yingdong Ni, Ruqian Zhao PII: DOI: Reference:
S0024-3205(18)30163-2 doi:10.1016/j.lfs.2018.03.049 LFS 15625
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
18 January 2018 17 March 2018 25 March 2018
Please cite this article as: Shiyu Tao, Liqiong Niu, Liuping Cai, Yali Geng, Canfeng Hua, Yingdong Ni, Ruqian Zhao , N-(3-oxododecanoyl)-l-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/j.lfs.2018.03.049
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ACCEPTED MANUSCRIPT N-(3-oxododecanoyl)-L-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells Shiyu Tao1 , Liqiong Niu1 , Liuping Cai1 , Yali Geng1 , Canfeng Hua1 , Yingdong Ni1,* ,
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Ruqian Zhao1
Key Laboratory of Animal Physiology & Biochemistry, Ministry of Agriculture,
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1
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Authors and Affiliations:
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Nanjing Agricultural University, Nanjing, Jiangsu, China.
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Running title: C12-HSL mediated mitochondrial function and proliferation in
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intestinal goblet cells
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*Corresponding author, email: [email protected]
All authors’ email address:
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Shiyu Tao: [email protected] Liqiong Niu: [email protected] Liuping Cai: [email protected] Yali Geng: [email protected] Canfeng Hua: [email protected] Yingdong Ni: [email protected] Ruqian Zhao: [email protected]
ACCEPTED MANUSCRIPT Abstract Aims: The quorum-sensing molecule N-(3-oxododecanoyl)-L- homoserine lactone (C12-HSL), produced by the Gram negative human pathogenic bacterium Pseudomonas aeruginosa, modulates mammalian cell behavior. Our previous findings suggested that C12-HSL rapidly decreases viability and induces apoptosis in LS174T
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goblet cells.
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Main methods: In this study, the effects of 100μM C12-HSL on mitochondrial
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function and cell proliferation in LS174T cells treated for 4h were evaluated by real-time PCR, enzyme- linked immunosorbent assay (ELISA) and flow cytometry.
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Key findings: The results showed that the activities of mitochondrial respiratory chain complexes IV and V were significantly increased (P<0.05) in LS174T cells
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after C12-HSL treatment, with elevated intracellular ATP generation (P<0.05). Flow cytometry analysis revealed significantly increased intracellular Ca2+ levels (P<0.05),
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as well as disrupted mitochondrial activity and cell cycle arrest upon C12-HSL
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treatment. Apoptosis and cell proliferation related genes showed markedly altered expression levels (P<0.05) in LS174T cells after C12-HSL treatment. Moreover, the paraoxonase 2 (PON2) inhibitor TQ416 (1μM) remarkably reversed the above
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C12-HSL associated effects in LS174T cells. Significance: These findings indicated that C12-HSL alters mitochondrial energy production and function, and inhibits cell proliferation in LS174T cells, with PON2 involvement. Abbreviations C12-HSL, N-(3-oxododecanoyl)-L-homoserine lactone; PON2, Paraoxonase 2; QS, quorum sensing; TQ416, Triazolo[4,3-a]quinolone. Key words: C12-HSL; mitochondria; apoptosis; cell proliferation; PON2; LS174T
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goblet cells
ACCEPTED MANUSCRIPT 1. Introduction The diverse microbial populations constituting the intestinal microbiota promote immune development and differentiation; however, differences in microbiota composition can be associated with inflammatory, metabolic, and intestinal diseases
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[1]. The gut epithelium is positioned strategically to play a key role in the host
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response by providing a physical barrier to pathogens and other environmental agents
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[2]. Intestinal epithelial cells with their tight junctions line the intestinal tube and form the main defense line between the host and the intestinal content; however, between
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the two there is a mucus layer, an often forgotten and ignored part of the defense
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system [3]. As the predominant component of the mucosal barrier, mucin2 is produced by goblet cells and forms a thick mucin layer in the colon that is coated on
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underlying epithelial cells [4]. Some pathogens and associated metabolites can
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penetrate the dense mucus layer and cause intestinal diseases [5,6]. Previous findings indicate that VvpE, an elastase encoded by Vibrio vulnificus, inhibits mucin expression by hypermethylation via lipid raft- mediated ROS signaling in intestinal
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epithelial cells [7].
Evidence reveals that some bacterial metabolites in the large intestine luminal content may have important deleterious consequences for colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism [8]. Several excessive bacterial metabolites can be considered luminal metabolic troublemakers, inducing colonocyte mitochondrial dysfunction [9]. Take hydrogen sulfide (H2 S) as an example. As a bacterial metabolite, H2 S can be produced by the intestinal microbiota
ACCEPTED MANUSCRIPT from alimentary and endogenous sulfur-containing compounds. At excessive concentration, H2 S is known to severely inhibit cytochrome c oxidase, the terminal oxidase of the mitochondrial electron transport chain, and thus mitochondrial oxygen consumption. In turn, altered mitochondrial function associated with harmful
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metabolites in the intestinal luminal content causes intestinal barrier dysfunction [10].
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Gut epithelial cell apoptosis is the main reason for intestinal barrier function
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breakdown. The mechanisms of apoptosis are very complex and mainly classified into intrinsic and extrinsic pathways [11]. The extrinsic pathway occurs in response to
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death receptors, including FAS, tumor necrosis factor (TNF) receptor 1 or
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TNF-related apoptosis- inducing ligand (TRAIL) receptor, however the intrinsic pathway is marked by a central event, i.e. mitochondrial outer membrane
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permeabilization, which results in cytochrome c release from the mitochondrial
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intermembrane space [12]. Therefore, the intrinsic pathway may be mediated by mitochondrial energy metabolism. Previous studies have demonstrated that apoptosis is induced by disrupted mitochondrial integrity or increased intracellular Ca 2+
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generation [13,14]. Moreover, the imbalanced epithelial proliferation attributed to the pathogenic development of epithelial injury induces colitis in mice [15]. Cyclin D (CCND) proteins are required for cell cycle progression and play an essential role in cell proliferation [16]. Quorum sensing (QS) is an intercellular signaling mechanism that allows bacteria to coordinate behaviors at the population level [17]. Numerous Gram- negative bacteria use small lipid-soluble and membrane-permeable molecules
ACCEPTED MANUSCRIPT as autoinducers of QS to trigger the innate immune response and induce cell apoptosis in host cells [18,19]. As a QS autoinducer, N-(3-oxododecanoyl)-homoserine lactone (C12-HSL) is produced by the Gram negative human pathogen bacterium Pseudomonas aeruginosa in the gut. Previous findings suggested that C12-HSL can
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alter epithelial integrity and the development of intestinal diseases [20]. Paraoxonase
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2 (PON2), expressed in many mammalian tissues and cell types, efficiently
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hydrolyzes C12-HSL to C12-HSL-acid [21]. Meanwhile, C12-HSL induced cell apoptosis is dependent upon PON2 activity in mouse embryonic fibroblasts [22].
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Moreover, recent evidence suggests a central role for this enzyme in modulating
previous
study
suggested
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bacterial QS and regulating the host cell response to C12-HSL [23]. Consistently, our that
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inhibition
significantly
reverse
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C12-HSL-induced cell death in LS174T cells [24].
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To date, the effects of C12-HSL on mitochondrial energy production and cell proliferation in goblet cells remain unclear. Therefore, the objective of this study was to assess whether mitochondrial energy production and altered cell proliferation are
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involved in C12-HSL associated damage to LS174T goblet cells. We also assessed whether PON2 contributes to this process in LS174T goblet cells.
ACCEPTED MANUSCRIPT 2. Material and methods 2.1. Cell culture and treatment The well-differentiated human colonic goblet LS174T cell line has been described previously [25]. LS174T cells were cultured at 37°C in a humid environment
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containing 5% CO2 in RPMI 1640 supplemented with 10% FBS (CellMax) and antibiotics (10 U/ml penicillin G and 10 mg/ml streptomycin). LS174T cells were
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treated with C12-HSL (Sigma-Aldrich, St. Louis, MO) and PON2 inhibitor (TQ416,
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ChemDiv, San Diego, USA), respectively, as specified in the figure legends. Based on our previous study [24], C12-HSL at 100μM was used to treat LS174T cells for 4h. To
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assess the effects of the PON2 inhibitor TQ416 on C12-HSL treated LS174T cells, LS174T cells were incubated with TQ416 (1μM) and 100μM C12-HSL for 4h. The
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vehicle control DMSO was not toxic to LS174T cells.
2.2. Measure ment of intracellular Mitochondrial Respiratory Chain Complex
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activity
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The activities of intracellular mitochondrial respiratory chain complexes I-V were evaluated with Mitochondrial Respiratory Chain Complex Assay Kit (Suzhou Comin
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Biotechnology LTD, China), following the manufacturer’s instructions (catalogues nos. FHTA-2-Y, FHTB-2-Y, FHTC-2-Y, FHTD-2-Y and FHTE-2-Y, respectively). The protein concentrations in various samples were determined with a BCA protein assay kit (Pierce Thermo Scientific). 2.3. Measurement of ATP production Intracellular ATP levels were measured with the firefly luciferase ATP assay kit (S0026, Beyotime Biotechnology, China) as described previously [26]. Briefly, LS174T cells were digested by trypsin/EDTA and collected into 1.5 mL Eppendorf tubes, followed by two washes with PBS. Lysis buffer (200µL) was added to 1×10 6
ACCEPTED MANUSCRIPT cells per tube. After centrifugation (12,000×g for 5 min), 100µL of the resulting supernatant was mixed with 100µL of ATP detection solution at the working dilution in a 1.5 mL Eppendorf tube. Luminance (RLU) was measured on a GloMax 20/20 luminometer (Promega, USA), and ATP levels were determined as described by the manufacturer.
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2.4. Calcium [Ca2+] determination
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Intracellular Ca2+ levels in LS174T cells were measured using the fluorescence Ca2+
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indicator Fluo-3 A.M., as previously described [27]. After treatment, the cells were collected, washed three times with PBS and stained with 5µM of Fura-3 A.M. for 60
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min at 37°C in the dark. Then, the cells were rinsed three times with PBS and incubated for another 30 min at 37°C before fluorescence intensity detection by flow
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cytometry. The cells were resuspended in PBS and fluorescence intensity was measured for more than 10,000 cells per sample on a FACSVerse flow cytometer).
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2.5. Mitochondrial activity determination
Mitochondrial activity in LS174T cells was measured with MitoTracker Green,
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according to the manufacturer’s instructions. Briefly, after dilution to a final concentration of 200 nM with serum- free RPMI1640, MitoTracker Green was added
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to washed cells for 30 min at 37°C in the dark. Next, the cells were washed three times with PBS, and resuspended in PBS; fluorescence intensity was measured for more than 10,000 cells per sample on a FACSVerse flow cytometer. 2.6. Cell cycle analysis Cells were digested with trypsin/EDTA and collected into 1.5 mL Eppendorf tubes followed by three washes with ice-cold PBS, fixed with 75% ice-cold ethanol, and added 20µL RNase A Solution. A cell cycle assay kit (Vazyme, A411-01/02) was used for the pretreatment of cells. Briefly, after 30 min of incubation at 37°C, a 400µL
ACCEPTED MANUSCRIPT propidium iodide (PI) staining solution was added into each tube, for 1h at 4°C in the dark. Cell cycle histograms were generated automatically by the flow cytometer (FACSVerse) associated software, with at least 1×104 events recorded per sample. 2.7. RNA extraction, reverse transcription and real-time quantitative PCR Total RNA extraction and reverse transcription were performed with SuperScript III
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First-Strand Synthesis System (Invitrogen, USA), according to the manufacturer ’s
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instructions. The synthesized cDNA was used for quantitative real-time PCR,
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performed with Mx3000P (Stratagene, USA). The primers used in real-time PCR are listed in Table 1, and were synthesized by Generay Company (Shanghai, China). The
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2−ΔΔCt method was used for data analysis, with the housekeeping gene GAPDH employed as a reference control.
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2.8. Statistical analysis
All statistical procedures were computed with the SPSS statistical software (SPSS
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version 11.0 for Windows; SPSS Inc., Chicago, IL, USA). Data are mean ± standard error of the mean (SEM), and were tested for normal distribution. Group differences
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were assessed by independent samples t-test (Fig. 1 and 2) and 2-way ANOVA (Fig.
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3-6). P<0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT 3. Results 3.1. Effects of C12-HSL on the activities of mitochondrial respiratory chain complexes and ATP production in LS174T cells As shown in Figure 1 A-E, the activities of mitochondrial respiratory chain complexes
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were assessed in LS174T cells exposed 100μM C12-HSL for 4h. The results showed
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that treatment with C12-HSL significantly increased the activities of mitochondrial
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respiratory chain complexes IV (19.2942 and 94.2761 nmol/min/μg prot for the DMSO and C12-HSL groups, respectively) and V (6.9520 and 14.8952 nmol/min/μg
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prot for the DMSO and C12-HSL groups, respectively) (both P<0.05; Fig. 1 D and E).
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However, C12-HSL did not affect those of mitochondrial respiratory chain complexes I-III in LS174T cells (P>0.05; Fig. 1 A-C). Moreover, intracellular ATP levels in
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C12-HSL-treated LS174T cells were significantly higher than those of the DMSO
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control group (0.7978 and 1.2380 nmol/mg prot for the DMSO and C12-HSL groups, respectively) (P<0.05; Fig. 1 F). 3.2. Effects of C12-HSL on Ca2+ production and mitochondrial activity in
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LS174T cells
Ca2+ production as well as mitochondrial number and activity in 100μM C12-HSL-treated LS174T cells were assayed by flow cytometry. The results showed that intracellular Ca2+ levels were significantly increased (relative fluorescence intensities of 100% and 109.6144% for the DMSO and C12-HSL groups, respectively) (P<0.05; Fig. 2 A and B). Moreover, mitochondrial amounts and activity detected by MitoTracker Green fluorescence were markedly decreased in LS174T cells treated
ACCEPTED MANUSCRIPT with C12-HSL (relative fluorescence intensities of 100% and 48.8007% for the DMSO and C12-HSL groups, respectively) (P<0.05; Fig. 2 C and D). 3.3. The PON2 inhibitor TQ416 rescues the activities of mitochondrial respiratory chain complexes and ATP production in C12-HSL-treated
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LS174T cells The effects of the PON2 inhibitor TQ416 on the activities of mitochondrial
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respiratory chain complexes were evaluated. TQ416 (1μM) remarkably reversed the
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increased activities of mitochondrial respiratory chain complexes IV (21.1824,
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115.4910, 48.6712 and 29.4087 nmol/min/μg prot for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) and V (5.8721, 17.3764, 9.7317
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and 5.6992 nmol/min/μg prot for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) associated with C12-HSL in LS174T cells (P<0.05; Fig.
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3 A and B). Moreover, TQ416 (1μM) significantly reversed C12-HSL associated increased intracellular ATP levels in LS174T cells (0.7978, 1.2380, 1.0006 and 0.9182
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nmol/mg prot for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05; Fig. 3 C).
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3.4. The PON2 inhibitor TQ416 rescues Ca2+ production and mitochondrial activity in C12-HSL-treated LS174T cells The effects of the PON2 inhibitor TQ416 on intracellular Ca2+ production and mitochondrial activity were assessed. The results showed that TQ416 (1μM) significantly inhibited C12-HSL-induced intracellular Ca2+ production (relative fluorescence intensities of 100%, 128.0254%, 110.1271% and 104.4379% for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05; Fig. 4 A and B) and restored C12-HSL-associated decreased mitochondrial activity (relative fluorescence intensities of 100%, 49.0322%, 102.3002% and 106.3039% for
ACCEPTED MANUSCRIPT the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05; Fig. 4 C and D) in LS174T cells. 3.5. Effects of C12-HSL on mRNA expression levels of apoptosis-related genes in LS174T cells
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Compared with the DMSO control group, C12-HSL-treated LS174T cells showed
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significantly increased caspase-6, 8, 9 and 10 mRNA expression levels (all P<0.05;
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Fig. 5). In addition, TQ416 (1μM) markedly inhibited C12-HSL associated increased caspase-6 (relative abundance rates of 1, 1.6745, 0.9539 and 1.0154 for the DMSO,
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C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively), caspase-8 (relative
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abundance rates of 1, 2.0977, 1.0949 and 0.8911 for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively), caspase-9 (relative abundance
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rates of 1, 3.3133, 1.3402 and 1.2528 for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) and caspase-10 (relative abundance rates of 1,
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2.9879, 1.3909 and 1.2030 for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) mRNA expression levels in LS174T cells (P<0.05; Fig. 5).
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3.6. Effects of C12-HSL on cell proliferation in LS174T cells Flow cytometry revealed cell cycle arrest at the G0/G1 phase in the C12-HSL group, indicated by significantly increased proportion of cells in the G0/G1 phase (relative fluorescence intensities of 66.8400%, 78.1133%, 67.5267% and 67.5267% for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P< 0.05) and markedly decreased proportion of cells in the S+G2/M phase (relative fluorescence intensities of 33.1600%, 21.8867%, 32.4733% and 34.1233% for the DMSO,
ACCEPTED MANUSCRIPT C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05), compared with the C12-HSL group (Fig. 6 A and B). Moreover, C12-HSL-treated LS174T cells showed significantly decreased CCND1 and CCND2 mRNA expression levels (P<0.05; Fig. 6 C) compared with the DMSO control group. Meanwhile, TQ416
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(1μM) markedly repaired C12-HSL- induced cell cycle alteration as well as CCND1
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(relative abundance rates of 1, 0.4262, 0.7975 and 0.8651 for the DMSO, C12-HSL,
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C12-HSL+TQ416 and TQ416 groups, respectively) and 2 (relative abundance rates of 1, 0.4706, 1.0444 and 0.8766 for the DMSO, C12-HSL, C12-HSL+TQ416 and
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TQ416 groups, respectively) mRNA expression changes in LS174T cells (P<0.05; Fig.
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6).
ACCEPTED MANUSCRIPT 4. Discussion Pseudomonas aeruginosa is a human pathogen with high environmental adaptability, and traditionally considered a common cause of lung infections. However, these bacteria are found even in the intestinal tract, and often replace the
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normal resident flora in critically ill and immuno-compromised patients [28,29].
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Using a human colonic epithelial cell model it has been demonstrated that clinical
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isolates of P. aeruginosa can adhere to, penetrate and disrupt the barrier function of intestinal epithelial cells, in addition to forming biofilms [30,31]. Intestinal P.
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aeruginosa creates a porous leaky barrier to different toxins, eventually resulting in
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lethal sepsis or other intestinal diseases [32]. The interactions between P. aeruginosa and host cells are controlled by the QS machinery [33,34]. Evidence suggests that
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quorum sensing molecules accumulate at very high levels in biofilms grown in vitro,
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yielding up to 300–600μM C12-HSL concentrations [35,36]. Previous studies have shown that C12-HSL affects various host cell types, including fibroblasts and epithelial cells [37,38]. However, little is known about the effects of P. aeruginosa
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derived C12-HSL on mitochondrial function in human intestinal epithelial cells. This study assessed whether the activities of mitochondrial respiratory chain complexes were affected by P. aeruginosa C12-HSL. The mitochondrial respiratory chain complex IV, also termed cytochrome c oxidase, is a large transmembrane protein complex found in eukaryotic mitochondria. It is the final enzyme in the respiratory electron transport chain of the mitochondria, helping establish a transmembrane electrochemical potential, which is then used by the mitochondrial
ACCEPTED MANUSCRIPT complex V (ATP synthase) for ATP synthesis [39]. Previous evidence suggests that increased mitochondrial complex IV and V activities, and enhanced ATP production contribute to apoptosis in HeLa and HT-22 cells [26]. The above data clearly showed that C12-HSL-treatment resulted in significantly increased activities of mitochondrial
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respiratory chain complexes IV and V in LS174T cells. Moreover, intracellular ATP
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production levels were markedly increased in C12-HSL-treated LS174T cells.
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Consistently, a previous study indicated that release of cytochrome c, acting as an electron donor to cytochrome c oxidase, can generate an electrochemical proton
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gradient and induce the synthesis of ATP, which is required for apoptosis occurrence
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[40]. The activities of mitochondrial respiratory chain complexes I-III showed no changes in LS174T cells treated with C12-HSL. Recently, Neely et al reported that
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C12-HSL increases mitochondrial permeability, subsequently activating apoptosis in
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fibroblasts [41]. The current data further demonstrated that C12-HSL might act upstream the mitochondrial respiratory chain to alter mitochondrial energy production.
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Cellular responses to external stimuli often involve Ca 2+-dependent signaling cascades. As a second messenger, Ca2+ is essential for many physiological processes, including the epithelial response to bacteria. Moreover, it was reported that alteration of cellular calcium homeostasis affects gastrointestinal epithelial cell integrity [42]. Therefore, we further assessed the effect of P. aeruginosa C12-HSL on intracellular Ca2+ levels in LS174T cells. A previous study suggested that P. aeruginosa C12-HSL-treatment increases intracellular free Ca2+ amounts in intestinal epithelial
ACCEPTED MANUSCRIPT cells; in addition, depletion of Ca2+ from the culture medium reduces the response of intestinal epithelial cells to the auto- inducer [2]. Consistently, the present study demonstrated that C12-HSL-treatment resulted in significantly elevated intracellular Ca2+ levels in LS174T cells.
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Mitochondria play crucial roles in cell proliferation and death [43]. Apoptosis,
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also referred to as programmed cell death, comprises a series of well-coordinated and receptors or
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strictly controlled processes in which ligand binding to specific
cytotoxic insults result in the activation of several proteases and other hydrolytic
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enzymes, leading to cell shrinkage and membrane blebbing, as well as DNA
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fragmentation and chromatin condensation [44]. Apoptosis occurs via extrinsic or intrinsic pathways; extrinsic signals bind to their receptors and trigger intracellular
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signaling leading cell programmed death, while intrinsic pro-apoptotic signals
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translocate to mitochondrial and cause mitochondrial membrane permeabilization [45]. To confirm the involvement of the mitochondrial apoptotic pathway in C12-HSL treated LS174T cells, we assessed mitochondrial activity using MitoTracker Green (a
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stain which enters only actively respiring mitochondria in live cells); flow cytometry showed significantly decreased fluorescence intensity in LS174T cells after C12-HSL treatment. Moreover, mitochondrial apoptotic pathway related pro-apoptotic caspases, i.e. caspases 6, 8, 9 and 10, showed markedly increased expression levels in C12-HSL treated LS174T cells. Previous findings suggested that CCND 1 and 2 regulate cell proliferation in mammalian cells [46,47]. In the present study, C12-HSL induced cell cycle arrest and reduced CCND 1 and 2 gene expression levels in LS174T cells.
ACCEPTED MANUSCRIPT These results suggest that the mitochondrial apoptotic pathway and cell proliferation alteration are involved in C12-HSL associated damage in LS174T goblet cells. Paraoxonase 2 (PON2), expressed intracellularly, is found widely in many tissues and cell types of mammalians, independent of its hydrolytic activity; PON2 rapidly
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hydrolyzes C12-HSL to C12-HSL-acid, and mediates a series of biological effects
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such as oxidative stress, immune response and apoptosis [21,22,48]. Additionally, a
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previous study demonstrated a novel PON2-dependent mechanism by which C12-HSL elicits biological effects in mammalian cells [23]. Therefore, in the present
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study, we also assessed whether PON2 contributes to C12-HSL induced mitochondrial
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dysfunction in LS174T goblet cells. Previous findings showed that the PON2 inhibitor TQ416 may prevent the negative effects of C12-HSL by inhibiting PON2 activity at
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1μM [23]. Moreover, our previous report demonstrated that the rescuing effect of TQ416 significantly decreases with co-administration of 5μM TQ416 and 100μM
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C12-HSL for 4h, and completely lost after administration of 10μM TQ416 [24]. As shown above, the PON2 inhibitor TQ416 (1μM) efficiently restored the altered
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activities of the mitochondrial respiratory chain complexes IV and V, as well as intracellular ATP and Ca2+ levels, and the gene expression levels of mitochondrial apoptotic pathway related pro-apoptotic caspases, which were associated with 100μM C12-HSL treatment in LS174T cells. Consistently, TQ416 also restored C12-HSL induced deficiency of mitochondrial activity and cell proliferation in LS174T cells. These results suggest that PON2 mediates C12-HSL induced mitochondrial apoptotic pathway and cell proliferation alteration in LS174T cells.
ACCEPTED MANUSCRIPT 5. Conclusions A major finding of this study is that P. aeruginosa C12-HSL (100μM) activates various events associated with the mitochondrial apoptotic pathway and cell proliferation, including activity alteration of mitochondrial respiratory chain
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complexes IV and V, increased intracellular ATP and Ca2+ levels, enhanced cell
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apoptosis and expression levels of proliferation related genes, and disrupted
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mitochondrial activity. Meanwhile, the PON2 inhibitor TQ416 (1μM) could rescue these effects on mitochondrial function in goblet LS174T cells.
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Conflict of Interest statement
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The authors declare that there are no conflicts of interest. Authors’ contributions
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S.T. Performed experiments and drafted the manuscript. L.N., L.C., Y.G. and C.H.
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performed experiments and analyzed the data. R.Z. contributed to experimental design and manuscript revision. Y.N. conceived the study, designed the experiments, and finalized the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
This work was supported by the National Nature Science Foundation of China (project no. 31572433), the National Basic Research Program of China (project no. 2011CB100802), the Program for New Century Excellent Talents in University (NCET-13-0862), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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ACCEPTED MANUSCRIPT Figure legends Figure 1: Effects of C12-HSL on the activities of mitochondrial respiratory chain complexes and ATP production in LS174T cells The activities of mitochondrial respiratory chain complexes I-V and intracellular ATP
independent experiments. *p<0.05 versus DMSO group.
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production were assayed as described in Methods. Data are mean±SEM of three
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Figure 2: Effects of C12-HSL on Ca2+ production and mitochondrial integrity in
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LS174T cells
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(A, B) Flow cytometric analysis of intracellular Ca2+ production in LS174T cells treated with 100μM C12-HSL for 4h. (C, D) Flow cytometric analysis of
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mitochondrial integrity in LS174T cells treated with 100μM C12-HSL for 4h. Values
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are mean±SEM (n= 3) in (B) Fluo-3 A.M. fluorescence and (D) MitoTracker Green fluorescence intensities. *p<0.05 versus DMSO group.
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Figure 3: Effects of the PON2 inhibitor TQ416 on the activities of mitochondrial respiratory chain complexes and ATP production in C12-HSL-treated LS174T cells
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The activities of mitochondrial respiratory chain complexes IV and V as well as intracellular ATP levels were assessed as described in Methods. Data are mean±SEM of three independent experiments. *p<0.05 versus DMSO group, &p<0.05 versus C12-HSL treatment group. Figure 4: Effects of the PON2 inhibitor TQ416 on Ca2+ production and mitochondrial integrity in C12-HSL-treated LS174T cells (A, B) Flow cytometric analysis of intracellular Ca2+ production in LS174T cells. (C, D) Flow cytometric analysis of mitochondrial integrity in LS174T cells. Values are
ACCEPTED MANUSCRIPT mean±SEM (n=3) for (B) Fluo-3 A.M. and (D) MitoTracker Green fluorescence intensities. *p<0.05 versus DMSO group, &p<0.05 versus C12-HSL treatment group. Figure 5: Effects of C12-HSL on expression levels of apoptosis-related genes in LS174T cells
&
p<0.05 versus C12-HSL
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are mean±SEM (n=3). *p<0.05 versus DMSO group,
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Relative mRNA expression levels of caspases 6, 8, 9 and 10 in LS174T cells. Values
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treatment group.
Figure 6: Effects of C12-HSL on cell proliferation in LS174T cells
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(A, B) Flow cytometric analysis of the cell cycle in LS174T cells. (C) Relative mRNA
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expression levels of CCND1 and CCND2 in LS174T cells. Values are mean±SEM
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(n=3). *p<0.05 versus DMSO group, &p<0.05 versus C12-HSL treatment group.
ACCEPTED MANUSCRIPT Table 1 PCR primers used in this study
Caspase-6
NM_001226.3
Caspase-8
NM_001080125.1
Caspase-9
NM_001278054.1
Caspase-10
NM_032974.4
CCND1
NM_053056.2
CCND2
M90813.1
Primer sequences F: 5'-TGCACCACCAACTGCTTAGC-3' R: 5'-GGCATGGACTGTGGTCATGAG-3' F: 5'-ATTCTCACCGGGAAACTGTG-3' R: 5'-AATTGCACTTGGGTCTTTGC-3' F: 5'-AAGCAAACCTCGGGGATACT-3' R: 5'-GGGGCTTGATCTCAAAATGA-3' F: 5'-GTTGAGACCCTGGACGACAT-3' R: 5'-GCATTAGCGACCCTAAGCAG-3' F: 5'-AGTGCCCTAGACTGGCTGAA-3' R: 5'-ATCTGCTTCGATGGATACGG-3' F: 5'-CAGATCATCCGCAAACACGC-3' R: 5'-GACAGGAAGTTGTTGGGGCT-3' F: 5'-TACCTGGACCGTTTCTTGGC-3' R: 5'-AGGCTTGATGGAGTTGTCGG-3'
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GenBank accession M17851.1
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Target gene GAPDH
Graphics Abstract
Figure 1
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Figure 3
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Shiyu Tao, Liqiong Niu, Liuping Cai, Yali Geng, Canfeng Hua, Yingdong Ni, Ruqian Zhao PII: DOI: Reference:
S0024-3205(18)30163-2 doi:10.1016/j.lfs.2018.03.049 LFS 15625
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
18 January 2018 17 March 2018 25 March 2018
Please cite this article as: Shiyu Tao, Liqiong Niu, Liuping Cai, Yali Geng, Canfeng Hua, Yingdong Ni, Ruqian Zhao , N-(3-oxododecanoyl)-l-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/j.lfs.2018.03.049
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ACCEPTED MANUSCRIPT N-(3-oxododecanoyl)-L-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells Shiyu Tao1 , Liqiong Niu1 , Liuping Cai1 , Yali Geng1 , Canfeng Hua1 , Yingdong Ni1,* ,
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Ruqian Zhao1
Key Laboratory of Animal Physiology & Biochemistry, Ministry of Agriculture,
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1
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Authors and Affiliations:
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Nanjing Agricultural University, Nanjing, Jiangsu, China.
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Running title: C12-HSL mediated mitochondrial function and proliferation in
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intestinal goblet cells
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*Corresponding author, email: [email protected]
All authors’ email address:
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Shiyu Tao: [email protected] Liqiong Niu: [email protected] Liuping Cai: [email protected] Yali Geng: [email protected] Canfeng Hua: [email protected] Yingdong Ni: [email protected] Ruqian Zhao: [email protected]
ACCEPTED MANUSCRIPT Abstract Aims: The quorum-sensing molecule N-(3-oxododecanoyl)-L- homoserine lactone (C12-HSL), produced by the Gram negative human pathogenic bacterium Pseudomonas aeruginosa, modulates mammalian cell behavior. Our previous findings suggested that C12-HSL rapidly decreases viability and induces apoptosis in LS174T
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goblet cells.
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Main methods: In this study, the effects of 100μM C12-HSL on mitochondrial
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function and cell proliferation in LS174T cells treated for 4h were evaluated by real-time PCR, enzyme- linked immunosorbent assay (ELISA) and flow cytometry.
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Key findings: The results showed that the activities of mitochondrial respiratory chain complexes IV and V were significantly increased (P<0.05) in LS174T cells
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after C12-HSL treatment, with elevated intracellular ATP generation (P<0.05). Flow cytometry analysis revealed significantly increased intracellular Ca2+ levels (P<0.05),
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as well as disrupted mitochondrial activity and cell cycle arrest upon C12-HSL
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treatment. Apoptosis and cell proliferation related genes showed markedly altered expression levels (P<0.05) in LS174T cells after C12-HSL treatment. Moreover, the paraoxonase 2 (PON2) inhibitor TQ416 (1μM) remarkably reversed the above
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C12-HSL associated effects in LS174T cells. Significance: These findings indicated that C12-HSL alters mitochondrial energy production and function, and inhibits cell proliferation in LS174T cells, with PON2 involvement. Abbreviations C12-HSL, N-(3-oxododecanoyl)-L-homoserine lactone; PON2, Paraoxonase 2; QS, quorum sensing; TQ416, Triazolo[4,3-a]quinolone. Key words: C12-HSL; mitochondria; apoptosis; cell proliferation; PON2; LS174T
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goblet cells
ACCEPTED MANUSCRIPT 1. Introduction The diverse microbial populations constituting the intestinal microbiota promote immune development and differentiation; however, differences in microbiota composition can be associated with inflammatory, metabolic, and intestinal diseases
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[1]. The gut epithelium is positioned strategically to play a key role in the host
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response by providing a physical barrier to pathogens and other environmental agents
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[2]. Intestinal epithelial cells with their tight junctions line the intestinal tube and form the main defense line between the host and the intestinal content; however, between
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the two there is a mucus layer, an often forgotten and ignored part of the defense
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system [3]. As the predominant component of the mucosal barrier, mucin2 is produced by goblet cells and forms a thick mucin layer in the colon that is coated on
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underlying epithelial cells [4]. Some pathogens and associated metabolites can
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penetrate the dense mucus layer and cause intestinal diseases [5,6]. Previous findings indicate that VvpE, an elastase encoded by Vibrio vulnificus, inhibits mucin expression by hypermethylation via lipid raft- mediated ROS signaling in intestinal
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epithelial cells [7].
Evidence reveals that some bacterial metabolites in the large intestine luminal content may have important deleterious consequences for colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism [8]. Several excessive bacterial metabolites can be considered luminal metabolic troublemakers, inducing colonocyte mitochondrial dysfunction [9]. Take hydrogen sulfide (H2 S) as an example. As a bacterial metabolite, H2 S can be produced by the intestinal microbiota
ACCEPTED MANUSCRIPT from alimentary and endogenous sulfur-containing compounds. At excessive concentration, H2 S is known to severely inhibit cytochrome c oxidase, the terminal oxidase of the mitochondrial electron transport chain, and thus mitochondrial oxygen consumption. In turn, altered mitochondrial function associated with harmful
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metabolites in the intestinal luminal content causes intestinal barrier dysfunction [10].
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Gut epithelial cell apoptosis is the main reason for intestinal barrier function
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breakdown. The mechanisms of apoptosis are very complex and mainly classified into intrinsic and extrinsic pathways [11]. The extrinsic pathway occurs in response to
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death receptors, including FAS, tumor necrosis factor (TNF) receptor 1 or
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TNF-related apoptosis- inducing ligand (TRAIL) receptor, however the intrinsic pathway is marked by a central event, i.e. mitochondrial outer membrane
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permeabilization, which results in cytochrome c release from the mitochondrial
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intermembrane space [12]. Therefore, the intrinsic pathway may be mediated by mitochondrial energy metabolism. Previous studies have demonstrated that apoptosis is induced by disrupted mitochondrial integrity or increased intracellular Ca 2+
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generation [13,14]. Moreover, the imbalanced epithelial proliferation attributed to the pathogenic development of epithelial injury induces colitis in mice [15]. Cyclin D (CCND) proteins are required for cell cycle progression and play an essential role in cell proliferation [16]. Quorum sensing (QS) is an intercellular signaling mechanism that allows bacteria to coordinate behaviors at the population level [17]. Numerous Gram- negative bacteria use small lipid-soluble and membrane-permeable molecules
ACCEPTED MANUSCRIPT as autoinducers of QS to trigger the innate immune response and induce cell apoptosis in host cells [18,19]. As a QS autoinducer, N-(3-oxododecanoyl)-homoserine lactone (C12-HSL) is produced by the Gram negative human pathogen bacterium Pseudomonas aeruginosa in the gut. Previous findings suggested that C12-HSL can
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alter epithelial integrity and the development of intestinal diseases [20]. Paraoxonase
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2 (PON2), expressed in many mammalian tissues and cell types, efficiently
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hydrolyzes C12-HSL to C12-HSL-acid [21]. Meanwhile, C12-HSL induced cell apoptosis is dependent upon PON2 activity in mouse embryonic fibroblasts [22].
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Moreover, recent evidence suggests a central role for this enzyme in modulating
previous
study
suggested
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bacterial QS and regulating the host cell response to C12-HSL [23]. Consistently, our that
PON2
inhibition
significantly
reverse
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C12-HSL-induced cell death in LS174T cells [24].
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To date, the effects of C12-HSL on mitochondrial energy production and cell proliferation in goblet cells remain unclear. Therefore, the objective of this study was to assess whether mitochondrial energy production and altered cell proliferation are
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involved in C12-HSL associated damage to LS174T goblet cells. We also assessed whether PON2 contributes to this process in LS174T goblet cells.
ACCEPTED MANUSCRIPT 2. Material and methods 2.1. Cell culture and treatment The well-differentiated human colonic goblet LS174T cell line has been described previously [25]. LS174T cells were cultured at 37°C in a humid environment
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containing 5% CO2 in RPMI 1640 supplemented with 10% FBS (CellMax) and antibiotics (10 U/ml penicillin G and 10 mg/ml streptomycin). LS174T cells were
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treated with C12-HSL (Sigma-Aldrich, St. Louis, MO) and PON2 inhibitor (TQ416,
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ChemDiv, San Diego, USA), respectively, as specified in the figure legends. Based on our previous study [24], C12-HSL at 100μM was used to treat LS174T cells for 4h. To
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assess the effects of the PON2 inhibitor TQ416 on C12-HSL treated LS174T cells, LS174T cells were incubated with TQ416 (1μM) and 100μM C12-HSL for 4h. The
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vehicle control DMSO was not toxic to LS174T cells.
2.2. Measure ment of intracellular Mitochondrial Respiratory Chain Complex
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activity
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The activities of intracellular mitochondrial respiratory chain complexes I-V were evaluated with Mitochondrial Respiratory Chain Complex Assay Kit (Suzhou Comin
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Biotechnology LTD, China), following the manufacturer’s instructions (catalogues nos. FHTA-2-Y, FHTB-2-Y, FHTC-2-Y, FHTD-2-Y and FHTE-2-Y, respectively). The protein concentrations in various samples were determined with a BCA protein assay kit (Pierce Thermo Scientific). 2.3. Measurement of ATP production Intracellular ATP levels were measured with the firefly luciferase ATP assay kit (S0026, Beyotime Biotechnology, China) as described previously [26]. Briefly, LS174T cells were digested by trypsin/EDTA and collected into 1.5 mL Eppendorf tubes, followed by two washes with PBS. Lysis buffer (200µL) was added to 1×10 6
ACCEPTED MANUSCRIPT cells per tube. After centrifugation (12,000×g for 5 min), 100µL of the resulting supernatant was mixed with 100µL of ATP detection solution at the working dilution in a 1.5 mL Eppendorf tube. Luminance (RLU) was measured on a GloMax 20/20 luminometer (Promega, USA), and ATP levels were determined as described by the manufacturer.
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2.4. Calcium [Ca2+] determination
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Intracellular Ca2+ levels in LS174T cells were measured using the fluorescence Ca2+
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indicator Fluo-3 A.M., as previously described [27]. After treatment, the cells were collected, washed three times with PBS and stained with 5µM of Fura-3 A.M. for 60
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min at 37°C in the dark. Then, the cells were rinsed three times with PBS and incubated for another 30 min at 37°C before fluorescence intensity detection by flow
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cytometry. The cells were resuspended in PBS and fluorescence intensity was measured for more than 10,000 cells per sample on a FACSVerse flow cytometer).
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2.5. Mitochondrial activity determination
Mitochondrial activity in LS174T cells was measured with MitoTracker Green,
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according to the manufacturer’s instructions. Briefly, after dilution to a final concentration of 200 nM with serum- free RPMI1640, MitoTracker Green was added
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to washed cells for 30 min at 37°C in the dark. Next, the cells were washed three times with PBS, and resuspended in PBS; fluorescence intensity was measured for more than 10,000 cells per sample on a FACSVerse flow cytometer. 2.6. Cell cycle analysis Cells were digested with trypsin/EDTA and collected into 1.5 mL Eppendorf tubes followed by three washes with ice-cold PBS, fixed with 75% ice-cold ethanol, and added 20µL RNase A Solution. A cell cycle assay kit (Vazyme, A411-01/02) was used for the pretreatment of cells. Briefly, after 30 min of incubation at 37°C, a 400µL
ACCEPTED MANUSCRIPT propidium iodide (PI) staining solution was added into each tube, for 1h at 4°C in the dark. Cell cycle histograms were generated automatically by the flow cytometer (FACSVerse) associated software, with at least 1×104 events recorded per sample. 2.7. RNA extraction, reverse transcription and real-time quantitative PCR Total RNA extraction and reverse transcription were performed with SuperScript III
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First-Strand Synthesis System (Invitrogen, USA), according to the manufacturer ’s
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instructions. The synthesized cDNA was used for quantitative real-time PCR,
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performed with Mx3000P (Stratagene, USA). The primers used in real-time PCR are listed in Table 1, and were synthesized by Generay Company (Shanghai, China). The
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2−ΔΔCt method was used for data analysis, with the housekeeping gene GAPDH employed as a reference control.
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2.8. Statistical analysis
All statistical procedures were computed with the SPSS statistical software (SPSS
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version 11.0 for Windows; SPSS Inc., Chicago, IL, USA). Data are mean ± standard error of the mean (SEM), and were tested for normal distribution. Group differences
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were assessed by independent samples t-test (Fig. 1 and 2) and 2-way ANOVA (Fig.
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3-6). P<0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT 3. Results 3.1. Effects of C12-HSL on the activities of mitochondrial respiratory chain complexes and ATP production in LS174T cells As shown in Figure 1 A-E, the activities of mitochondrial respiratory chain complexes
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were assessed in LS174T cells exposed 100μM C12-HSL for 4h. The results showed
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that treatment with C12-HSL significantly increased the activities of mitochondrial
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respiratory chain complexes IV (19.2942 and 94.2761 nmol/min/μg prot for the DMSO and C12-HSL groups, respectively) and V (6.9520 and 14.8952 nmol/min/μg
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prot for the DMSO and C12-HSL groups, respectively) (both P<0.05; Fig. 1 D and E).
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However, C12-HSL did not affect those of mitochondrial respiratory chain complexes I-III in LS174T cells (P>0.05; Fig. 1 A-C). Moreover, intracellular ATP levels in
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C12-HSL-treated LS174T cells were significantly higher than those of the DMSO
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control group (0.7978 and 1.2380 nmol/mg prot for the DMSO and C12-HSL groups, respectively) (P<0.05; Fig. 1 F). 3.2. Effects of C12-HSL on Ca2+ production and mitochondrial activity in
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LS174T cells
Ca2+ production as well as mitochondrial number and activity in 100μM C12-HSL-treated LS174T cells were assayed by flow cytometry. The results showed that intracellular Ca2+ levels were significantly increased (relative fluorescence intensities of 100% and 109.6144% for the DMSO and C12-HSL groups, respectively) (P<0.05; Fig. 2 A and B). Moreover, mitochondrial amounts and activity detected by MitoTracker Green fluorescence were markedly decreased in LS174T cells treated
ACCEPTED MANUSCRIPT with C12-HSL (relative fluorescence intensities of 100% and 48.8007% for the DMSO and C12-HSL groups, respectively) (P<0.05; Fig. 2 C and D). 3.3. The PON2 inhibitor TQ416 rescues the activities of mitochondrial respiratory chain complexes and ATP production in C12-HSL-treated
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LS174T cells The effects of the PON2 inhibitor TQ416 on the activities of mitochondrial
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respiratory chain complexes were evaluated. TQ416 (1μM) remarkably reversed the
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increased activities of mitochondrial respiratory chain complexes IV (21.1824,
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115.4910, 48.6712 and 29.4087 nmol/min/μg prot for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) and V (5.8721, 17.3764, 9.7317
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and 5.6992 nmol/min/μg prot for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) associated with C12-HSL in LS174T cells (P<0.05; Fig.
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3 A and B). Moreover, TQ416 (1μM) significantly reversed C12-HSL associated increased intracellular ATP levels in LS174T cells (0.7978, 1.2380, 1.0006 and 0.9182
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nmol/mg prot for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05; Fig. 3 C).
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3.4. The PON2 inhibitor TQ416 rescues Ca2+ production and mitochondrial activity in C12-HSL-treated LS174T cells The effects of the PON2 inhibitor TQ416 on intracellular Ca2+ production and mitochondrial activity were assessed. The results showed that TQ416 (1μM) significantly inhibited C12-HSL-induced intracellular Ca2+ production (relative fluorescence intensities of 100%, 128.0254%, 110.1271% and 104.4379% for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05; Fig. 4 A and B) and restored C12-HSL-associated decreased mitochondrial activity (relative fluorescence intensities of 100%, 49.0322%, 102.3002% and 106.3039% for
ACCEPTED MANUSCRIPT the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05; Fig. 4 C and D) in LS174T cells. 3.5. Effects of C12-HSL on mRNA expression levels of apoptosis-related genes in LS174T cells
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Compared with the DMSO control group, C12-HSL-treated LS174T cells showed
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significantly increased caspase-6, 8, 9 and 10 mRNA expression levels (all P<0.05;
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Fig. 5). In addition, TQ416 (1μM) markedly inhibited C12-HSL associated increased caspase-6 (relative abundance rates of 1, 1.6745, 0.9539 and 1.0154 for the DMSO,
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C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively), caspase-8 (relative
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abundance rates of 1, 2.0977, 1.0949 and 0.8911 for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively), caspase-9 (relative abundance
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rates of 1, 3.3133, 1.3402 and 1.2528 for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) and caspase-10 (relative abundance rates of 1,
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2.9879, 1.3909 and 1.2030 for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) mRNA expression levels in LS174T cells (P<0.05; Fig. 5).
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3.6. Effects of C12-HSL on cell proliferation in LS174T cells Flow cytometry revealed cell cycle arrest at the G0/G1 phase in the C12-HSL group, indicated by significantly increased proportion of cells in the G0/G1 phase (relative fluorescence intensities of 66.8400%, 78.1133%, 67.5267% and 67.5267% for the DMSO, C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P< 0.05) and markedly decreased proportion of cells in the S+G2/M phase (relative fluorescence intensities of 33.1600%, 21.8867%, 32.4733% and 34.1233% for the DMSO,
ACCEPTED MANUSCRIPT C12-HSL, C12-HSL+TQ416 and TQ416 groups, respectively) (P<0.05), compared with the C12-HSL group (Fig. 6 A and B). Moreover, C12-HSL-treated LS174T cells showed significantly decreased CCND1 and CCND2 mRNA expression levels (P<0.05; Fig. 6 C) compared with the DMSO control group. Meanwhile, TQ416
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(1μM) markedly repaired C12-HSL- induced cell cycle alteration as well as CCND1
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(relative abundance rates of 1, 0.4262, 0.7975 and 0.8651 for the DMSO, C12-HSL,
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C12-HSL+TQ416 and TQ416 groups, respectively) and 2 (relative abundance rates of 1, 0.4706, 1.0444 and 0.8766 for the DMSO, C12-HSL, C12-HSL+TQ416 and
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TQ416 groups, respectively) mRNA expression changes in LS174T cells (P<0.05; Fig.
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6).
ACCEPTED MANUSCRIPT 4. Discussion Pseudomonas aeruginosa is a human pathogen with high environmental adaptability, and traditionally considered a common cause of lung infections. However, these bacteria are found even in the intestinal tract, and often replace the
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normal resident flora in critically ill and immuno-compromised patients [28,29].
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Using a human colonic epithelial cell model it has been demonstrated that clinical
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isolates of P. aeruginosa can adhere to, penetrate and disrupt the barrier function of intestinal epithelial cells, in addition to forming biofilms [30,31]. Intestinal P.
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aeruginosa creates a porous leaky barrier to different toxins, eventually resulting in
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lethal sepsis or other intestinal diseases [32]. The interactions between P. aeruginosa and host cells are controlled by the QS machinery [33,34]. Evidence suggests that
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quorum sensing molecules accumulate at very high levels in biofilms grown in vitro,
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yielding up to 300–600μM C12-HSL concentrations [35,36]. Previous studies have shown that C12-HSL affects various host cell types, including fibroblasts and epithelial cells [37,38]. However, little is known about the effects of P. aeruginosa
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derived C12-HSL on mitochondrial function in human intestinal epithelial cells. This study assessed whether the activities of mitochondrial respiratory chain complexes were affected by P. aeruginosa C12-HSL. The mitochondrial respiratory chain complex IV, also termed cytochrome c oxidase, is a large transmembrane protein complex found in eukaryotic mitochondria. It is the final enzyme in the respiratory electron transport chain of the mitochondria, helping establish a transmembrane electrochemical potential, which is then used by the mitochondrial
ACCEPTED MANUSCRIPT complex V (ATP synthase) for ATP synthesis [39]. Previous evidence suggests that increased mitochondrial complex IV and V activities, and enhanced ATP production contribute to apoptosis in HeLa and HT-22 cells [26]. The above data clearly showed that C12-HSL-treatment resulted in significantly increased activities of mitochondrial
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respiratory chain complexes IV and V in LS174T cells. Moreover, intracellular ATP
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production levels were markedly increased in C12-HSL-treated LS174T cells.
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Consistently, a previous study indicated that release of cytochrome c, acting as an electron donor to cytochrome c oxidase, can generate an electrochemical proton
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gradient and induce the synthesis of ATP, which is required for apoptosis occurrence
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[40]. The activities of mitochondrial respiratory chain complexes I-III showed no changes in LS174T cells treated with C12-HSL. Recently, Neely et al reported that
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C12-HSL increases mitochondrial permeability, subsequently activating apoptosis in
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fibroblasts [41]. The current data further demonstrated that C12-HSL might act upstream the mitochondrial respiratory chain to alter mitochondrial energy production.
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Cellular responses to external stimuli often involve Ca 2+-dependent signaling cascades. As a second messenger, Ca2+ is essential for many physiological processes, including the epithelial response to bacteria. Moreover, it was reported that alteration of cellular calcium homeostasis affects gastrointestinal epithelial cell integrity [42]. Therefore, we further assessed the effect of P. aeruginosa C12-HSL on intracellular Ca2+ levels in LS174T cells. A previous study suggested that P. aeruginosa C12-HSL-treatment increases intracellular free Ca2+ amounts in intestinal epithelial
ACCEPTED MANUSCRIPT cells; in addition, depletion of Ca2+ from the culture medium reduces the response of intestinal epithelial cells to the auto- inducer [2]. Consistently, the present study demonstrated that C12-HSL-treatment resulted in significantly elevated intracellular Ca2+ levels in LS174T cells.
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Mitochondria play crucial roles in cell proliferation and death [43]. Apoptosis,
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also referred to as programmed cell death, comprises a series of well-coordinated and receptors or
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strictly controlled processes in which ligand binding to specific
cytotoxic insults result in the activation of several proteases and other hydrolytic
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enzymes, leading to cell shrinkage and membrane blebbing, as well as DNA
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fragmentation and chromatin condensation [44]. Apoptosis occurs via extrinsic or intrinsic pathways; extrinsic signals bind to their receptors and trigger intracellular
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signaling leading cell programmed death, while intrinsic pro-apoptotic signals
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translocate to mitochondrial and cause mitochondrial membrane permeabilization [45]. To confirm the involvement of the mitochondrial apoptotic pathway in C12-HSL treated LS174T cells, we assessed mitochondrial activity using MitoTracker Green (a
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stain which enters only actively respiring mitochondria in live cells); flow cytometry showed significantly decreased fluorescence intensity in LS174T cells after C12-HSL treatment. Moreover, mitochondrial apoptotic pathway related pro-apoptotic caspases, i.e. caspases 6, 8, 9 and 10, showed markedly increased expression levels in C12-HSL treated LS174T cells. Previous findings suggested that CCND 1 and 2 regulate cell proliferation in mammalian cells [46,47]. In the present study, C12-HSL induced cell cycle arrest and reduced CCND 1 and 2 gene expression levels in LS174T cells.
ACCEPTED MANUSCRIPT These results suggest that the mitochondrial apoptotic pathway and cell proliferation alteration are involved in C12-HSL associated damage in LS174T goblet cells. Paraoxonase 2 (PON2), expressed intracellularly, is found widely in many tissues and cell types of mammalians, independent of its hydrolytic activity; PON2 rapidly
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hydrolyzes C12-HSL to C12-HSL-acid, and mediates a series of biological effects
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such as oxidative stress, immune response and apoptosis [21,22,48]. Additionally, a
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previous study demonstrated a novel PON2-dependent mechanism by which C12-HSL elicits biological effects in mammalian cells [23]. Therefore, in the present
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study, we also assessed whether PON2 contributes to C12-HSL induced mitochondrial
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dysfunction in LS174T goblet cells. Previous findings showed that the PON2 inhibitor TQ416 may prevent the negative effects of C12-HSL by inhibiting PON2 activity at
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1μM [23]. Moreover, our previous report demonstrated that the rescuing effect of TQ416 significantly decreases with co-administration of 5μM TQ416 and 100μM
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C12-HSL for 4h, and completely lost after administration of 10μM TQ416 [24]. As shown above, the PON2 inhibitor TQ416 (1μM) efficiently restored the altered
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activities of the mitochondrial respiratory chain complexes IV and V, as well as intracellular ATP and Ca2+ levels, and the gene expression levels of mitochondrial apoptotic pathway related pro-apoptotic caspases, which were associated with 100μM C12-HSL treatment in LS174T cells. Consistently, TQ416 also restored C12-HSL induced deficiency of mitochondrial activity and cell proliferation in LS174T cells. These results suggest that PON2 mediates C12-HSL induced mitochondrial apoptotic pathway and cell proliferation alteration in LS174T cells.
ACCEPTED MANUSCRIPT 5. Conclusions A major finding of this study is that P. aeruginosa C12-HSL (100μM) activates various events associated with the mitochondrial apoptotic pathway and cell proliferation, including activity alteration of mitochondrial respiratory chain
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complexes IV and V, increased intracellular ATP and Ca2+ levels, enhanced cell
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apoptosis and expression levels of proliferation related genes, and disrupted
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mitochondrial activity. Meanwhile, the PON2 inhibitor TQ416 (1μM) could rescue these effects on mitochondrial function in goblet LS174T cells.
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Conflict of Interest statement
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The authors declare that there are no conflicts of interest. Authors’ contributions
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S.T. Performed experiments and drafted the manuscript. L.N., L.C., Y.G. and C.H.
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performed experiments and analyzed the data. R.Z. contributed to experimental design and manuscript revision. Y.N. conceived the study, designed the experiments, and finalized the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
This work was supported by the National Nature Science Foundation of China (project no. 31572433), the National Basic Research Program of China (project no. 2011CB100802), the Program for New Century Excellent Talents in University (NCET-13-0862), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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ACCEPTED MANUSCRIPT Figure legends Figure 1: Effects of C12-HSL on the activities of mitochondrial respiratory chain complexes and ATP production in LS174T cells The activities of mitochondrial respiratory chain complexes I-V and intracellular ATP
independent experiments. *p<0.05 versus DMSO group.
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production were assayed as described in Methods. Data are mean±SEM of three
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Figure 2: Effects of C12-HSL on Ca2+ production and mitochondrial integrity in
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LS174T cells
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(A, B) Flow cytometric analysis of intracellular Ca2+ production in LS174T cells treated with 100μM C12-HSL for 4h. (C, D) Flow cytometric analysis of
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mitochondrial integrity in LS174T cells treated with 100μM C12-HSL for 4h. Values
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are mean±SEM (n= 3) in (B) Fluo-3 A.M. fluorescence and (D) MitoTracker Green fluorescence intensities. *p<0.05 versus DMSO group.
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Figure 3: Effects of the PON2 inhibitor TQ416 on the activities of mitochondrial respiratory chain complexes and ATP production in C12-HSL-treated LS174T cells
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The activities of mitochondrial respiratory chain complexes IV and V as well as intracellular ATP levels were assessed as described in Methods. Data are mean±SEM of three independent experiments. *p<0.05 versus DMSO group, &p<0.05 versus C12-HSL treatment group. Figure 4: Effects of the PON2 inhibitor TQ416 on Ca2+ production and mitochondrial integrity in C12-HSL-treated LS174T cells (A, B) Flow cytometric analysis of intracellular Ca2+ production in LS174T cells. (C, D) Flow cytometric analysis of mitochondrial integrity in LS174T cells. Values are
ACCEPTED MANUSCRIPT mean±SEM (n=3) for (B) Fluo-3 A.M. and (D) MitoTracker Green fluorescence intensities. *p<0.05 versus DMSO group, &p<0.05 versus C12-HSL treatment group. Figure 5: Effects of C12-HSL on expression levels of apoptosis-related genes in LS174T cells
&
p<0.05 versus C12-HSL
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are mean±SEM (n=3). *p<0.05 versus DMSO group,
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Relative mRNA expression levels of caspases 6, 8, 9 and 10 in LS174T cells. Values
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treatment group.
Figure 6: Effects of C12-HSL on cell proliferation in LS174T cells
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(A, B) Flow cytometric analysis of the cell cycle in LS174T cells. (C) Relative mRNA
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expression levels of CCND1 and CCND2 in LS174T cells. Values are mean±SEM
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(n=3). *p<0.05 versus DMSO group, &p<0.05 versus C12-HSL treatment group.
ACCEPTED MANUSCRIPT Table 1 PCR primers used in this study
Caspase-6
NM_001226.3
Caspase-8
NM_001080125.1
Caspase-9
NM_001278054.1
Caspase-10
NM_032974.4
CCND1
NM_053056.2
CCND2
M90813.1
Primer sequences F: 5'-TGCACCACCAACTGCTTAGC-3' R: 5'-GGCATGGACTGTGGTCATGAG-3' F: 5'-ATTCTCACCGGGAAACTGTG-3' R: 5'-AATTGCACTTGGGTCTTTGC-3' F: 5'-AAGCAAACCTCGGGGATACT-3' R: 5'-GGGGCTTGATCTCAAAATGA-3' F: 5'-GTTGAGACCCTGGACGACAT-3' R: 5'-GCATTAGCGACCCTAAGCAG-3' F: 5'-AGTGCCCTAGACTGGCTGAA-3' R: 5'-ATCTGCTTCGATGGATACGG-3' F: 5'-CAGATCATCCGCAAACACGC-3' R: 5'-GACAGGAAGTTGTTGGGGCT-3' F: 5'-TACCTGGACCGTTTCTTGGC-3' R: 5'-AGGCTTGATGGAGTTGTCGG-3'
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GenBank accession M17851.1
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Target gene GAPDH
Graphics Abstract
Figure 1
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Figure 3
Figure 4
Figure 5
Figure 6