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Microbial Pathogenesis 61-62 (2013) 57e61

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Microbial Pathogenesis

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Short communication

ABC-cassette transporter 1 (ABCA1) expression in epithelial cellsin Chlamydia pneumoniae infection

Juha T. Korhonen a,b,c,*, Vesa M. Olkkonen d, Riitta Lahesmaa a, Mirja Puolakkainen e

a Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finlandb Institute of Biomedicine, Faculty of Medicine, University of Turku, Turku, FinlandcDrug Discovery Graduate School, FinlanddMinerva Foundation Institute for Medical Research, Helsinki, FinlandeDepartment of Virology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland

a r t i c l e i n f o

Article history:Received 11 August 2012Received in revised form10 May 2013Accepted 13 May 2013Available online 21 May 2013

Keywords:Chlamydia pneumoniaeABCA1Epithelial cells

* Corresponding author. University of Turku and ÅbCentre for Biotechnology, Tykistokatu 6, FI-20520505208482; fax: þ358 23338000.

E-mail address: juha.korhonen@utu.fi (J.T. Korhon

0882-4010/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.micpath.2013.05.006

a b s t r a c t

ATP-binding cassette transporter A1 (ABCA1) mediates reverse cholesterol transport and innate immu-nity response in different cell types. We have investigated the regulation of ABCA1 expression inresponse to intracellular Chlamydia pneumoniae infection in A549 epithelial lung carcinomacells. C. pneumoniae infection decreased ABCA1 expression in A549 cells, and the activity of the ABCA1promoter was decreased. The decreased promoter activity was dependent on its E-box and GnT-boxelements of the promoter. Chlamydial growth was decreased in ABCA1-silenced epithelial lung carci-noma cells. These data indicate an important role for ABCA1 in intracellular bacterial infection.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

ATP-binding cassette transporter A1 (ABCA1) is abundant pro-tein in macrophages [1]. It exports cellular free cholesterol andphospholipids from the Golgi apparatus to apolipoprotein A-I(apoA-I) on plasma membrane, resulting in formation of high-density lipoprotein (HDL) particles. The transcription of ABCA1 isinduced by oxysterols and 9-cis-retinoic acid (RA) through liver andretinoic X receptors (LXR and RXR). In addition, the expression ofABCA1 is responsive to many cytokines andmicrobial stimuli [1]. Inmacrophages, ABCA1 deficiency results in foam cell formation, anearly hallmark of the development of atherosclerotic lesions. Inaddition to the function on lipid transport, ABCA1 has also animportant anti-inflammatory function. ABCA1-deficient micesecrete high concentrations of pro-inflammatory cytokines,including TNF-a, interleukin-1b, IL-6, and IFN-g in lipopolysac-charide (LPS) induced sepsis [2], and the loss of ABCA1 functionin macrophages may lead to low-grade chronic inflammationin vivo [3]. The anti-inflammatory function of ABCA1 seems to be

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dependent on its interaction with apoA-I and thus to its function inlipid efflux [4].

While the function of ABCA1 in the lipid efflux is well docu-mented in macrophages, it seems to have a less wellcharacterized but equally important role in lung epithelium.McNeish and co-workers observed that alveolar type II epithelialcells of ABCA1�/� knockout mice accumulate lipids and aberrantlamellar bodies [5]. Experimental data suggest that ABCA1mediates basolateral surfactant efflux in these cells [6].

Chlamydia pneumoniae, a Gram-negative obligate intracellularbacterium, is a common cause of community-acquired pneumonia[7]. Like all Chlamydia species, it replicates only inside the hostwithin a membrane-bound intracellular vesicle, termed inclusion[7]. C. pneumoniae infection has a tendency to persist, and thepersistent infection has been suggested to contribute to chronicinflammatory states, such as atherosclerosis (for a review seeRef. [8]).

C. pneumoniae infection disrupts the intracellular transport ofsurfactant lipids in type II pneumocytes [9]. Taking into accountthe central role of ABCA1 in surfactant metabolism, we investi-gated ABCA1 expression in C. pneumoniae-infected A549epithelial lung carcinoma cells. In addition, we studied theactivity of ABCA1 promoter in the infected cells. Finally, weexamined chlamydial growth in ABCA1-silenced epithelial lungcarcinoma cells.

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2. Materials and methods

2.1. Cell culture

A549 epithelial lung carcinoma cells (CCL-185) cells wereobtained from American Type Culture Collection (LGC StandardsAB, Sweden) and maintained as in Ref. [10].

2.2. Preparation of C. pneumoniae elementary bodies

C. pneumoniae elementary bodies (EBs) were prepared as pre-viously described [10]. In some instances, C. pneumoniae was heat-inactivated by incubating at 56 �C for 30min, as indicated in results.

2.3. Antibodies and reagents

The following primary antibodies were used for immunoblot-ting and microscopy: monoclonal mouse anti-ABCA1 (Abcam),rabbit polyclonal antibody against C. pneumoniae IncA [11],monoclonal anti-b-actin-peroxidase (SigmaeAldrich), and goatanti-mouse IgG HRP-conjugated (SantaCruz Biotechnology).Fluorophore-conjugated secondary antibodies (Invitrogen) wereused for confocal imaging. 9-cis-retinoic acid (RA), 22-hydroxy-R-cholesterol (22-OH), and GW3965 were purchased from Sigma.

2.4. Plasmids

A fragment of the human ABCA1 promoter (from �928 to þ101bp) linked to firefly luciferase reporter [13], and its versions withthe DR4 element, E-box, or GnT-box mutagenized have beenpreviously described [14].

2.5. ABCA1 protein expression analysis

A549 cells were plated on 24-well plates in culture media a daybefore inoculation. The cells were inoculated with viable or heat-inactivated C. pneumoniae K6 at a multiplication of infection(MOI) of 0.01. The cells were then centrifuged at 900 g at 37 �C for1 h, and incubated in culture media at 37 �C for 1 h. The cells werewashed to remove any unbound Chlamydia particles, and incubatedin culture media with or without 1 mMRA and 1 mM 22-OH or 1 mMGW3965 at 37 �C for different time periods, as indicated in results.Subsequently, the cells were harvested and immunoblotted as inRef. [10].

2.6. Quantitative RT-PCR

For ABCA1 mRNA detection total RNA was isolated withRNAeasy Kit (Qiagen) according to the manufacturer’s protocol.Universal ProbeLibrary (Roche) probe No. 11 was added to a finalconcentration of 1 mM. The primers (50-TCAGGATCAGGAAGA-TAAATAGAGG and 50-ACCTCACTTTCAGAAGAAGACAAAC) wereadded to a final concentration of 3 mM. RT-PCR amplifications wereperformed as previously described [10]. The fold change (FD) wasestimated from the threshold cycle by DDCT method [12], and theABCA1 transcript levels were normalized against those ofhypoxanthine-guanine phosphoribosyltransferase-2.

2.7. Luciferase reporter assay

A549 cells, plated on 24-well plates, were inoculatedwith viableor heat-inactivated C. pneumoniae. As a control some cells were notinoculated. Then, the cells were centrifuged at 900 g at 37 �C for 1 h,and incubated at 37 �C for 24 h, followed by co-transfection withluciferase reporter constructs and Renilla internal control plasmids

(Promega) using Lipofectamine (Invitrogen) as in Ref. [15]. Trans-fected cells were incubated with 1 mM GW3965 at 37 �C for 24 h,after which the cells were harvested. Luminescence-measurementswere carried out with Dual Luciferase Assay Reporter Systemaccording to the manufacturer’s protocol (Promega).

2.8. RNA interference assays

The following siRNA duplex with dTdT-overhangs were syn-thesized (Sigma) to target ABCA1 transcripts: (50-CUGUAUGGGUGGGUCAAUCA and 50-UGAUUGACCACCCAUACAG). A non-targetingsiRNA duplex (50-CCUACAUCCCGAUCGAUGAUG and 50-CAUCAUCGAUCGGGAUGUAGG) was used as a negative control. The cells weretransfected with siRNA duplexes as in Ref. [10]. Then, the cells wereincubated with 1 mMGW3965 for 24 h to induce ABCA1 expression.

2.9. C. pneumoniae growth assay

ABCA1-silenced cells were inoculated with C. pneumoniae K6 ata MOI of 0.01. Then the cells were centrifuged at 900 g at 37 �C for1 h, and incubated at 37 �C for 1 h. After washing, the cells wereincubated in culture media supplemented with 0.5 mg ml�1 cyclo-heximide at 37 �C for 48 h. The cells were fixed, stained, andexamined as in Ref. [10].

2.10. Confocal microscopy

To examine localization of ABCA1 in C. pneumoniae-infectedcells, the cells were fixed, stained with monoclonal ABCA1 anti-body, and examined as in Ref. [10] with LSM510 Meta ConfocalMicroscope (Zeiss).

3. Results

3.1. The expression of ABCA1 is decreased in A549 epithelial lungcarcinoma cells infected with C. pneumoniae

To study ABCA1 expression in C. pneumoniae infection, A549cells were infected with or without the RXR and LXR activators, RAand 22-OH. The ABCA1 expression was analyzed at 48 h postinfection (hpi). The basal expression of ABCA1 was low without RAand 22-OH (Fig. 1). In the infected cells, the expression of ABCA1mRNA was significantly decreased compared with non-infectedcells (Fig. 1A). In agreement with this, the expression of ABCA1protein was decreased in the infected cells (Fig. 1B).

3.2. The activity of ABCA1 promoter is suppressed uponC. pneumoniae infection, and the effect is mediated by E-box andGnT-box elements

To investigate the regulation of ABCA1 expression at a promoterlevel, A549 cells were inoculated with viable C. pneumoniae, heat-inactivated C. pneumoniae, or mock-inoculated (control). Theheat-inactivated bacteria were not able to grow inside the host(data not shown). Then, the cells were transfected with luciferasereporter plasmids linked to human wild-type ABCA1 promoter orthe ABCA1 promoter encompassing mutated DR4 element, E-box,or GnT-box. The activity of wild-type ABCA1 promoter wasdecreased to 62% and 66% in the cells that were inoculated withviable or heat-inactivated bacteria, respectively, compared with thecontrol (Fig. 2A). As expected, the activity of DR4-mutatedpromoter was low irrespective of inoculation status. The activitiesof the E-box or GnT-box mutants were comparable betweenthe cells inoculated with heat-inactivated bacteria and control(Fig. 2A), suggesting that inhibition of the promoter activity by

Fig. 1. ABCA1 expression is decreased in C. pneumoniae infection. A549 cells were infected with C. pneumoniae (Cpn) or mock-infected with and without 1 mM RA and 1 mM 22-OH(A) Quantitative RT-PCR analysis of ABCA1 mRNA expression at 48 hpi. ABCA1 mRNA expression is shown as a fold difference (FD) to basal expression before infection. Error barsdenote the standard error of the mean of 3 independent experiments. Statistically significant difference is denoted by *, which indicates p < 0.05. (B) Representative immunoblot ofnon-infected and infected A549 cells with and without RA and 22-OH. Proteins were detected with ABCA1 and beta-actin specific antibodies.

J.T. Korhonen et al. / Microbial Pathogenesis 61-62 (2013) 57e61 59

heat-inactivated C. pneumoniae requires the presence of functionalE- and GnT-boxes. In cells inoculated with viable bacteria, however,the activities of E-box or GnT-box mutants were significantlydecreased to 80% and 65%, respectively, as compared to the control(Fig. 2A). These results indicate that viable C. pneumoniae decreasesthe activity of ABCA1 promoter by an additional mechanism that isindependent of the functional E-box and GnT-box. In agreement,viable C. pneumoniae down-regulated ABCA1 protein at 48 hpi,although this could not be seen in cells infected with heat-inactivated bacteria (Fig. 2B).

3.3. Silencing of ABCA-1 inhibits C. pneumoniae growth in epitheliallung carcinoma cells

To investigate whether silencing of ABCA1 had an effect onC. pneumoniae infection, A549 cells were transfected with siRNA

Fig. 2. ABCA1 promoter is suppressed in C. pneumonie infection. (A) A549 cells were inoculcontrol, cells were treated identically without inoculation (control). After 24 h incubationpromoter (WT) or ABCA1 promoter encompassing mutated DR4 element (DR4 m), E-box (E-incubated with 1 mM GW3965 for 24 h, after which cells were harvested and luminescence wtreated cells, luminescence of WT reporter was marked as one. Error bars denote standard esignificant difference is denoted by *, which indicates p < 0.05. (B) ABCA1 expression was ininactivated bacteria. As a control cells were mock-inoculated. ABCA1 protein expression wa

targeted against ABCA1 (siABCA1), or control siRNA. The expressionof ABCA1 was then induced with 1 mM GW3965 for 24 h. Afterwhich the cells were infected, and bacterial growth was measuredas formation of IFUs at 48 hpi. Silencing of ABCA1 reduced bacterialgrowth to 58% compared to the control (Fig. 3A). Representativeimages of siRNA-transfected and C. pneumoniae-infected cells areshown (Fig. 3C). C. pneumoniae grew equally in A549 cells that wereincubated with or without GW3965, indicating that up-regulationof ABCA1 had no effect on the number of C. pneumoniae in-clusions (data not shown).

4. Discussion

We observed that the expression of ABCA1 was decreased inepithelial lung carcinoma cells in response to C. pneumoniaeinfection. This finding differs with a previous study where an

ated with viable C. pneumoniae (Cpn) or heat-inactivated C. pneumoniae (HI-Cpn). As a, cells were transfected with luciferase-reporter plasmids linked to wild-type ABCA1box m), or GnT-box (GnT-box m) with an internal vector control. Transfected cells wereas measured. Data from each sample was normalized to the internal control. In control-rror of the mean of 3 independent experiments measured in quadruplicate. Statisticallyduced with 1 mM GW3965 in A549 cells, and cells were inoculated with viable or heat-s analyzed at 48 hpi.

Fig. 3. Silencing of ABCA1 inhibits C. pneumoniae growth in epithelial lung carcinoma cells. (A) A549 cells were transfected with siRNA directed against ABCA1 (siABCA1), or withnon-targeting oligonucleotide (control) for 2 days, and inoculated with C. pneumoniae in the presence of 1 mM GW3965. Inclusions were stained and counted at 48 hpi. Inclusioncounts were marked as one in control-treated cells. Error bars denote the standard error of the mean of 3 independent experiments measured in triplicate. Statistically significantdifference is denoted by **, which indicates p < 0.01. (B) Representative immunoblot of siABCA1-transfected and control-transfected cells with ABCA1 and b-actin specific anti-bodies. (C) Representative confocal images of the siRNA-transfected and C. pneumoniae infected cells. Inclusions were detected with an antibody specific for IncA and anti-rabbitAlexa Fluor 647-conjugated secondary antibody and are shown in red. Bacterial and host DNA were stained with DAPI and are shown in blue (bar 20 mm).

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increase of ABCA1 expression is seen in Pseudomonas aeruginosa-infected rodent lung epithelial cells [16], but is in keeping withstudies showing ABCA1 down-regulation in macrophages infectedwith Escherichia coli, Influenza A, or C. pneumoniae [17,18].

By promoter reporter assay we could confirm that ABCA1expression is downregulated at the transcriptional level byC. pneumoniae. When the cells were inoculated with viable or heat-inactivated C. pneumoniae, the activity of ABCA1 promoter reporterwas decreased. The down-regulative effect of heat-inactivatedC. pneumoniae was abolished, however, when the E-box or GnT-box was mutated. A current model suggests that both E-box andGnT-box co-operate in ABCA1 gene regulation [1] and therefore, itwasnot surprise that bothof these regulatoryelements had an effecton ABCA1 expression upon bacterial stimuli. Lipopolysaccharidedown-regulates ABCA1 transcription [1], and it could have beenresponsible for this effect. In contrast, viable C. pneumoniae still hadan inhibitory effect on the ABCA1 promoter reporter with mutatedE-box or GnT-box. This latter observation suggests that the viablebacteria attenuated ABCA1 expression by an additional mechanism,which is independent of E-box and GnT-box. The suppression ofABCA1 expression with heat-inactivated C. pneumoniae was tran-sient because ABCA1 was not down-regulated in protein level at48 hpi. Therefore, it seems that bacterial invasion and intracellularinfection result in a more persistent down-regulation of ABCA1.

The precise molecular mechanism, by which ABCA1 promoter isregulated upon C. pneumoniae infection, remains to be elucidated.

Hypoxia-inducible factor 1 alpha (HIF-1a), an established trans-activator of ABCA1 [19,20], is destabilized upon C. pneumoniaeinfection [21] and thus may play a role in the modulation of ABCA1expression. In this study, wewere unable to address this hypothesisbecause HIF-1a protein was undetectable in A549 cells by immu-noblotting (data not shown).

We observed that the growth of C. pneumoniaewas decreased inABCA1-silenced cells. Like most of the other intracellular patho-gens, Chlamydia is dependent on the cellular lipids of the host [22].Therefore, it is conceivable that C. pneumoniae intersects ABCA1-mediated lipid transport from Golgi to plasma membrane, andthe down-regulation of this pathway results in decreased chla-mydial growth. Of particular interest, intracellular lipid accumula-tion, a known consequence of ABCA1 deficiency [1], limitsC. pneumoniae infection of macrophages [23]. Emerging evidenceshows a direct link between innate immune system and the ABCA1-mediated cholesterol efflux [3,4]. Our data point to a possibility thatdown-regulation of ABCA1 and foam cell formation may haveevolved to limit infections with C. pneumoniae and other intracel-lular bacteria.

It is important to note that ABCA1 interacts directly with amembrane-associated host protein, flotillin-1 [24], which contrib-utes to C. pneumoniae growth and targets the chlamydial inclusionmembrane as we have previously shown [10]. We did not, however,find ABCA1 in the inclusion membranes by confocal microscopy(data not shown).

J.T. Korhonen et al. / Microbial Pathogenesis 61-62 (2013) 57e61 61

ABCA1 has an important role in surfactant transport andsecretion [6]. In addition, it has been reported that C. pneumoniaeinterferes with surfactant secretion in lung epithelial cells [9].These data and our findings suggest that dysfunctional surfactanttrafficking may result from C. pneumoniae-induced ABCA1 down-regulation. This possibility needs to be addressed in future studies.

In conclusion, our results indicate that ABCA1 is down-regulatedin C. pneumoniae infected epithelial lung carcinoma cells at thetranscriptional level. In addition, ABCA1 expression seems to beimportant in the early phase of chlamydial infection. Our findingsalso suggest a possible role for ABCA1 in the pathogenesis ofC. pneumoniae infection.

Acknowledgments

This study was supported by Academy of Finland (Microbes andMan Research Program, MICMAN project number 202491, andproject numbers 118391, 217554/EBIBUG, and 130043/CHLAMY-TRANS) and Turku University Hospital Fund. J.K. was supported byDrug Discovery Graduate School, Finland, as well as Siiri Suominenfund. Andrew Brown (University of New South Wales, Sydney,Australia) is thanked for kindly providing the ABCA1 promoter-luciferase constructs and Robert Moulder for valuable commentson the manuscript.

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