Journal of Infection (2015) 70, 415e426

www.elsevierhealth.com/journals/jinf

MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG Jinli Wang a,b,e,g, Chunyu Huang a,b,f,g, Minhao Wu a,b, Qiu Zhong c, Kun Yang a,b, Miao Li a,b, Xiaoxia Zhan a,b, Jinsheng Wen d, Lin Zhou c,**, Xi Huang a,b,d,* a

Department of Immunology, Institute of Tuberculosis Control, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China b Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, China c Center for Tuberculosis Control of Guangdong Province, Guangzhou 510630, China d Department of Microbiology and Immunology, Wenzhou Medical University, Wenzhou 325035, China e Department of Laboratory Medicine, Guangzhou First Municipal People’s Hospital, Affiliated Hospital of Guangzhou Medical University, Guangzhou 510500, China f Shenzhen Key Laboratory of Reproductive Immunology for Peri-implantation, Fertility Center, Shenzhen Zhongshan Urology Hospital, Shenzhen 518045, China Accepted 15 September 2014 Available online 13 October 2014

KEYWORDS Myeloid-related protein 8/14; Mycobacterium bovis BCG; Autophagy; Bacterial killing

Summary Objective: To explore the role of myeloid-related protein 8/14 in mycobacterial infection. Methods: The mRNA and protein expression levels of MRP8 or MRP14 were measured by realtime PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry. Results: MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing

* Corresponding author. Department of Immunology, Institute of Tuberculosis Control, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China. Tel.: þ86 020 87335818; fax: þ86 020 87335818. ** Corresponding author. Tel.: þ86 020 38907752. E-mail addresses: [email protected] (L. Zhou), [email protected] (X. Huang). g The authors contribute equally to this paper. http://dx.doi.org/10.1016/j.jinf.2014.09.013 0163-4453/ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

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J. Wang et al. the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROSdependent manner. Conclusions: The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases. ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Introduction Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB).1 About one third of the world’s population is infected with Mtb and 1.4 million people die from TB annually.2 Recently, the emergence of multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) poses a serious challenge to clinical treatment.3 Nevertheless, only one tenth of the Mtb-infected population proceed to active TB, indicating that the disease outcome is largely determined by both Mtb virulence factors and the host immune response. Therefore, in addition to developing more effective antibiotics and vaccines, understanding the immune pathogenesis of TB is also urgent. Macrophage-mediated innate immune response functions as the first line of host defense against Mtb.4,5 Invading pathogens are recognized by macrophages via various evolutionarily conserved pattern-recognition receptors (PRRs), which initiate innate immune response, including the production of cytokines and chemokines,6,7 as well as recruitment and activation of phagocytes.8 The antimicrobial activity of macrophages involves two essential processes, phagocytosis and killing of pathogen. Phagocytosis is mediated by different receptors, including mannose receptors (MR), scavenger receptors (SR), Fcg receptors (FcgR) and complement receptors (CR).9 Once the pathogen is engulfed, many molecules including defensins,10 lysozymes,11 reactive oxygen species (ROS)12 and reactive nitrogen species (RNS)13 are released to fight against invading pathogens. Apart from these antimicrobial mechanisms above, it has been demonstrated that autophagy is also an important host defense mechanism in the elimination of intracellular microbes.14 Autophagy is a complicated physiological process of degradation, executing by multiple autophagy related (ATG) proteins, such as ATG5, ATG7, ATG12 and microtubuleassociated protein light chain 3 (LC3). The autophagic cascade consists of formation of an isolation membrane, engulfment of cytoplasmic material and the growth of isolation membrane to generate autophagosome. The autophagosome then fuse with lysosome to form the autolysosome, where the engulfed materials are degraded.15 Autophagy plays an important protective role in numerous infectious diseases by promoting degradation of intracellular pathogens, such as Listeria monocytogenes,14 Toxoplasma gondii16 and Mtb.14,17 Several studies have demonstrated that induction of autophagy promotes the elimination of intracellular mycobacteria in murine macrophages, such as bone-marrow derived macrophages (BMDMs) and RAW264.7 cells,14,17e20 but much less reports in human cells. Singh SB et al. demonstrated that human IRGM induces autophagy to eliminate intracellular Mycobacterium bovis BCG in human U937 cells.21 Moreover, Yuk JM et al. demonstrated that

Vitamin D3 induces autophagy to eliminate intracellular Mtb via cathelicidin in human macrophages.22 Nonetheless, the role of autophagy in Mtb control in human cells needs further investigations. Multiple factors are able to trigger autophagic responses, such as rapamycin, starvation, as well as immunological stimuli including interferon gamma (IFN-g), tumor necrosis factor (TNF).23 Recently, studies have demonstrated that myeloid-related protein (MRP) 8/14 functions as a novel autophagy inducer in tumor cells.23,24 MRP8/14 belongs to the calcium-binding S100 protein family and usually functions as a heterodimeric protein complex to execute cytokine-like activities.25 MRP8/14 is predominantly expressed in myeloid cell lineage, such as neutrophils, monocytes and macrophages, and is weakly expressed in other cell types such as endothelial and epithelial cells.26 The expression of MRP8/14 is induced by several factors, including lipopolysaccharide (LPS), double-stranded RNA, IFN-g and TNF,27,28 and is upregulated in response to bacterial challenge, such as Escherichia coli,29 Pseudomonas aeruginosa30 and Mtb.31 As a pleiotropic molecule, MRP8/14 regulates various important biological processes, including cell differentiation and motility, cell survival and death, signal transduction, as well as immune and inflammatory responses.25,26 It is reported that MRP8/14 modulates tumor cell death by inducing autophogy,24,32 but the role of MRP8/14 in autophagy during bacterial infection remains unknown. MRP8/ 14 protein has been demonstrated to inhibit the growth of E. coli, Candida albicans and L. monocytogenes, through chelating bivalent cations from microenvironment.33,34 It is reported that human MRP8/14 protein increased Mtb growth in vitro,31 but immunoregulatory functions of MRP8/14 during Mtb infection in monocytes/macrophages are largely unknown. In this study, we explored the role of MRP8/14 in the elimination of intracellular BCG. We found that the expression of MRP8/14 was up-regulated in THP1 cells and human monocyte-derived macrophages (MDMs) after challenge with mycobacteira. Importantly, our study for the first time demonstrated that MRP8/14 inhibited the survival of intracellular BCG by promoting autophagy and subsequent maturation of autophagosomes. Furthermore, MRP8/14 induced autophagy through promoting ROS production. These data provide a promising therapeutic strategy against Mtb infection.

Materials and methods Reagents Middlebrook 7H9 Broth and Middlebrook 7H10 Agar were purchased from BD Difco Laboratories (Sparks, MD).

MRP8/14 in mycobacterial infection Rapamycin, DMSO, trypan blue and b-actin antibody was purchased from SigmaeAldrich (St. Louis, MO). Texas Red and DQ-Green dyes and 20 ,70 -dichlorofluorecscin diacetate (H2DCFDA) were from Invitrogen (Carlsbad, CA). Antibodies specific for MRP8 or MRP14 were obtained from Abcam (San Francisco, CA) and Santa Cruz (Santa Cruz, CA), respectively. Antibody specific for LC3 was obtained from Novus (Littleton, CO). Bafilomycin A1 was obtained from Santa Cruz (Santa Cruz, CA). N-acetylcysteine (NAC) was obtained from Beyotime (Haimen, Jiangsu, China). pUNO1-hS100A8 (MRP8), pUNO1-hS100A9 (MRP14) plasmids and pUNO1 empty vector were purchased from Invivogen (San Diego, CA).

Cell culture THP1 cells (ATCC, TIB-202), human acute monocytic leukemia cell line, were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin (GIBCO, Grand Island, NY) and 0.05 mM 2-mercaptoethanol. THP1 cells were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 16 h to induce differentiation from monocytic to macrophage-like phenotype. A549 cells (ATCC, CCL-185), adenocarcinomic human alveolar basal epithelial cell line, were cultured in DMEM medium supplemented with 5% FBS and 1% penicillin-streptomycin. Cells were cultured in a standard tissue culture incubator at 37  C with an atmosphere of 5% CO2 and 95% air, as reported before.35

Mycobacterial culture M. bovis BCG (ATCC, 19015) and M. tuberculosis H37Rv (ATCC, 25618) were grown in Middlebrook 7H9 broth (BD-Diagnostic Systems) supplemented with 10% albumin dextrose catalase (ADC) supplement (BD-Diagnostic Systems) and 0.2% glycerol until mid logarithmic growth phase. Prior to each experiment, the homogenate of BCG or H37Rv was prepared as described by others before36 to generate a single cell suspension. The homogenate of BCG or H37Rv was used to infect cells at the indicated multiplicity of infection (MOI).

Human monocyte-derived macrophages (MDMs) isolation and culture Human peripheral blood samples from healthy donors were obtained from Guangzhou Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (TBDsciences, Tianjin) density gradient centrifugation. Human monocytes were purified from PBMCs by anti-CD14 microbeads (BD Bioscience) according to the manufacturer’s directions. Monocytes were cultured in RPMI-1640 supplemented with 10% FBS and 50 ng/ml M-CSF for a week to generate MDMs.

417 with siRNA or plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction as reported before.37

Real-time PCR Total cellular RNA was extracted from cells using TRIzol reagent following the manufacturer’s instruction, as described before.38 cDNA was synthesized from 1 mg total RNA by using RevertAid First Strand cDNA Synthesis Kit (Thermo, Glen Burnie, Maryland). Primers used for PCR amplification were: b-actin: 50 -GCTCCTCCTGAGCGCAAG-30 (forward), 50 -CATCTGCTGGAAGGTGGACA-30 (reverse); MRP8: 50 ACCTGAAGAAATTGCTAGAGACCGAGTG-30 (forward). 50 -CCACGCCCATCTTTATCACCAGAATGAG-30 (reverse); MRP14: 50 -ACCTTCCACCAATCATCTGTGAAGCTG-30 (for ward), 50 -GTCCAGGTCCTCCATGAGTGGTTCTATG-30 (reverse). Quantitative real-time PCR reactions were run by using CFX96 RT-PCR Detection System (Bio-Rad). Relative mRNA levels were calculated after normalizing to b-actin, as reported before.39

Western blot Whole cell extracts were prepared in a lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 1% (vol/ vol) protease inhibitor cocktail (Sigma, St Louis,MO), and 1 mM DTT as mentioned before.40 Proteins were separated by SDS-PAGE and transferred onto PVDF membranes or nitrocellulose membranes. Membranes were blocked in 5% BSA and incubated with primary antibodies at 4  C overnight. Then membranes were incubated at room temperature for 1 h with relevant secondary antibodies, and blots were visualized using ECL, as reported before.41

Phagocytosis assay by flow cytometry Phagocytosis assay by flow cytometry was performed as described by others before.42 BCG was labelled with FilmTracer Green Biofilm dye (FTGB, Invitrogen, Carlsbad, CA) according to the manufacturer’ instruction. THP1 cells were challenged by the FTGB-labelled BCG at an MOI of 5. After 4 h incubation, cells were washed thoroughly with PBS to remove extracellular bacteria. Cells were incubated with 0.25% trypan blue for 5 min to quench the extracellular fluorescence of FTGB, as described before.43,44 Then cells were collected and analyzed using Beckman Coulter EPICS XL/MCL flow cytometer (Beckman Coulter Inc., Fullerton, CA), as described before.45

ROS production detection siRNA preparation and transient transfection siRNA sequences were as follows: human MRP8 siRNA (siMRP8, 50 -CCUUGAACUCUAUCGACGUCUA-30 ), human MRP14 siRNA (siMRP14, 50 -CCAUCAUCAACACCUUCC ACCAAUA-30 ), human ATG7 siRNA (siATG7, 50 -GGAGUCACAGCUCUUCCUU-30 ). These siRNA were synthesized by Invitrogen, and control scrambled siRNA (siNC) was purchased from Invitrogen. Cells were transiently transfected

A549 cells stably expressing MRP8 or MRP14 were treated with NAC (10 mM), or left untreated, and then incubated with a ROS-sensitive probe 20 ,70 -dichlorofluorecscin diacetate (H2DCFDA, Invitrogen) at a final concentration of 10 mM. Cells were collected and analyzed using a Beckman Coulter EPICS XL/MCL flow cytometer, ROS levels were determined by the fluorescence of DCF, the deacetylated and oxidized product of H2DCFDA.

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Confocal microscopy For immunofluorescence experiments, A549 cells stably expressing MRP8 or MRP14 were cultured on collagenprecoated glass coverslips in 24-well plates. THP1-derived macrophages were transiently transfected with siNC or siMRP8/14 for 24 h using Lipofectamine 2000, and then infected with BCG (or Texas Red-labelled BCG) (MOI 10) for 1 h. Cells were fixed with 4% paraformaldehyde followed by membrane permeabilization using 0.2% Triton X-100. Cells were blocked with 5% BSA and incubated with primary and then secondary antibodies before mounting, as described before.46 In fluorescence experiments, THP1derived macrophages were transiently transfected with siNC or siMRP8/14 for 24 h, and then infected with Redlabelled BCG (MOI 10) for 1 h. DQ-Green staining was performed by adding DQ-Green (50 mg/ml) to cells and incubating at 37  C for 30 min. The cells were fixed in 4% paraformaldehyde for 10 min and viewed by confocal microscopy (Zeiss Axiovert, LSM710). The observer was blinded to the treatment of each sample. The fields were randomly selected and photographed. LC3 puncta (1 mm) were quantified, and cell with more than 10 puncta was defined as LC3 puncta positive. The percentage of LC3 punctae positive cell was calculated by the following equation: LC3 punctae positive (%) Z (LC3 puncta positive cells)/(total cells in the field).

J. Wang et al. then analyzed for MRP8 and MRP14 expression by real-time PCR. The mRNA levels of MRP8 and MRP14 were significantly up-regulated after BCG infection in a dose- (Figs. 1A, B) and time-dependent manner (Figs. 1C, D). Moreover, the protein levels of MRP8 (Fig. 1E) and MRP14 (Fig. 1F) were detected by flow cytometry, and the results showed that the protein expressions of MRP8 and MRP14 were also induced in response to BCG infection.

MRP8 and MRP14 were induced in human monocyte-derived macrophages after mycobacterial infection To determine the expression levels of MRP8 and MRP14 in primary macrophages after BCG infection, human monocyte-derived macrophages (MDMs) were challenged with BCG, and then analyzed for MRP8 and MRP14 expression by real-time PCR. The mRNA levels of MRP8 and MRP14 were significantly up-regulated after BCG infection in a

Colony forming units (CFU) Intracellular killing assay by bacterial plate count were performed as described by others before.47,48 THP1 cells or MDMs were transfected with siMRP8, siMRP14, siMRP8/14 or siNC for 24 h, and then challenged with BCG at an MOI of 5. After 4 h incubation, cells were washed thoroughly with cold PBS and centrifuged to remove extracellular bacteria. Then infected cells were incubated for the indicated time. Cells were lysed with sterile water containing 0.01% Trinton X-100, and serial 10-fold dilutions of each sample were plated on Middlebrook 7H10 agar plates. Colonies were counted after three-week incubation. The percentage of BCG survival was calculated according to intracellular bacillary load after initial 4-h uptake.

Statistical analysis Unpaired Student’s t test or one-way analysis of variation was used to determine the significance of the results from real-time RT-PCR experiments and CFU assays, the quantification of GFP-LC3 puncta and the colocalization of BCG with lysosomes or autophagosomes. Data were considered statistically significant at p < 0.05.

Results MRP8 and MRP14 were induced in THP1 cells after BCG infection To explore the expression patterns of MRP8 and MRP14 after BCG infection, THP1 cells were challenged with BCG, and

Figure 1 MRP8 and MRP14 expression in THP1 cells after BCG infection. (AeD) THP1 cells were infected with BCG at the indicated MOIs for 24 h or at an MOI of 2 for the indicated time points. mRNA expression levels of MRP8 (A, C) and MRP14 (B, D) were measured by real-time PCR. Data are shown as the mean  SEM of at least three independent experiments. (E, F) THP1 cells were infected with BCG at an MOI of 2 for 48 h. MRP8 (E) and MRP14 (F) protein levels were measured by flow cytometry. Mean fluorescent intensity (MFI) of MRP8 (E) and MRP14 (F) are shown above histograms.

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Figure 2 MRP8 and MRP14 expression in human MDMs after mycobacterial infection. (AeD) MDMs were infected with BCG at the indicated MOIs for 24 h (A, B) or at an MOI of 2 for the indicated time points (C, D). (E, F) MDMs cells were infected with H37Rv at the indicated MOIs for 24 h mRNA expression levels of MRP8 (A, C and E) and MRP14 (B, D and F) were measured by real-time PCR. Data are shown as the mean  SEM of at least three independent experiments.

dose- (Figs. 2A, B) and time-dependent manner (Figs. 2C, D). Moreover, we challenged MDMs with M. tuberculosis H37Rv, and found that both MRP8 and MRP14 were induced in a dose-dependent manner after H37Rv infection (Figs. 2E, F).

MRP8/14 enhanced intracellular killing but not phagocytosis of BCG in THP1 cells To examine whether MRP8 and MRP14 play a role in phagocytosis and killing of BCG, the expressions of MRP8 and MRP14 were knocked down with specific siRNAs. Firstly, THP1 cells were transfected with siMRP8, siMRP14, or scrambled control siRNA (siNC), and the silencing efficiency of MRP8 and MRP14 was examined by real-time PCR. Results showed that the mRNA levels of MRP8 and MRP14 were decreased to 30% and 10%, respectively (Figs. 3A, B). Next, the effects of MRP8 and MRP14 on the phagocytosis and killing of BCG were examined by flow cytometry and colony

Figure 3 Silencing of MRP8/14 inhibited the killing of intracellular BCG in THP1 cells. (A, B) THP1 cells were transfected with siMRP8, siMRP14 or siNC for 24 h. MRP8 (A) and MRP14 (B) mRNA expression levels were measured by realtime PCR. Data are shown as the mean  SEM of at least three independent experiments. (C, D) THP1 cells were transfected with siMRP8, siMRP14, siMRP8/14 or siNC for 24 h, then infected with BCG at an MOI of 5. Phagocytosis of BCG was detected by flow cytometry at 4 h postinfection (p.i.) (C) and intracellular bacterial survival was detected by CFU at 8 h p.i. (D). Data represent three independent experiments. *p < 0.05, ***p < 0.001.

forming units (CFU) assay, respectively. Results showed that silencing of MRP8, MRP14, or both had no influence on the phagocytosis of BCG (Fig. 3C). However, the survival of intracellular BCG was markedly increased in cells transfected with siRNA against MRP8, MRP14 or both (Fig. 3D, all p < 0.05), indicating that MRP8 and MRP14 promote the elimination of intracellular BCG.

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MRP8/14 enhanced intracellular killing of BCG in MDMs To further examine whether MRP8/14 play a role in mycobacterial killing in primary macrophages, intracellular BCG survival was tested in MDMs in which MRP8 and MRP14 were knocked down with specific siRNAs. Firstly, MDMs were transfected with siMRP8, siMRP14, or siNC respectively, and the silencing efficiency of MRP8 and MRP14 was examined. Results showed that the mRNA levels of MRP8 and MRP14 were decreased to 10% and 1%, respectively (Figs. 4A, B). Next, the effects of MRP8 and MRP14 on the killing of BCG were examined by CFU assay. Results showed that the bacterial clearance was decreased in the cells transfected with siMRP8 and siMRP14 at 8 and 24 h post-infection compared with siNC-treated cells (Fig. 4C, all p < 0.05). At the later time points, intracellular BCG increased markedly in MRP8/14-knocked down cells, while bacillary growth was restricted in cells transfected with siNC (Fig. 4C, all p < 0.01). These results indicated that MRP8/14 played an important role in both clearance of BCG and restriction of bacillary growth.

Silencing of MRP8/14 suppressed autophagy To explore the mechanism involved in the increased survival of BCG after silencing MRP8/14, we examined the effect of MRP8/14 on autophagy, a well-defined defense mechanism in inhibiting intracellular mycobacterial survival. The activation of autophagy is indicated by the amount of microtubule-associated protein light chain 3-II (LC3-II) and the formation of LC3 puncta. THP1 cells were transfected with siMRP8/14 or siNC, and the amount of LC3 was detected by using Western blot. Data showed that silencing of MRP8/14 decreased the protein levels of LC3-II in THP1 cells (Fig. 5A, lane 1 and 2). Since autophagy is a dynamic process, and LC3-II accumulation during the

J. Wang et al. process of autophagy depends on both the conversion rate of from LC3-I to LC3-II, and the degradation rate of LC3-II by autolysosomes. To rule out the possibility that MRP8/14 affects LC3-II degradation, we employed bafilomycin A1 (Baf.A1), an antagonist of vacuolar Hþ ATPase, to block the degradation of LC3-II by lysosomes. Our results showed that silencing of MRP8/14 still decreased the amount of LC3-II in the presence of Baf.A1 (Fig. 5A, lane 3 and 4), indicating that silencing of MRP8/14 suppresses the induction of autophagy. Moreover, the formation of LC3 puncta in THP1-derived macrophages after MRP8/14 silencing was detected by immunofluorescence microscopy. Results showed that BCG infection increased the formation of LC3 puncta (Figs. 5C, D). However, LC3 puncta positive cells reduced when MRP8/14 were knocked down in THP1-derived macrophages (Figs. 5C, D).

Overexpression of MRP8/14 increased autophagy in A549 cells To further explore the induction of autophagy by MRP8/14, we constructed MRP8 and MRP14 stably expressing A549 cell line, as the expression of MRP8 and MRP14 was intrinsically low in A549 cells. Our results showed that overexpression of either MRP8 or MRP14 in A549 cells markedly increased autophagy, as indicated by the elevated amount of LC3-II protein (Figs. 6A, B), with or without BCG infection. In addition, the distribution of LC3 from diffuse pattern to LC3 puncta was greatly enhanced in the A549 cells stably expressing MRP8 (Figs. 6C, D) or MRP14 (Figs. 6E, F). These results indicated that MRP8 and MRP14 induced autophagy in A549 cells.

MRP8/14 promoted the elimination of intracellular BCG by inducing autophagy Autophagy has been reported to promote the maturation of mycobacterial phagosome and the elimination of

Figure 4 Silencing of MRP8/14 inhibited the killing of intracellular BCG in MDMs. (A, B) MDMs were transfected with siMRP8, siMRP14 or siNC for 24 h. MRP8 (A) and MRP14 (B) expression levels were measured by real-time PCR. (C) MDMs were transfected with siMRP8/14 or siNC for 24 h, then infected with BCG at an MOI of 5. Intracellular bacterial survival was detected by CFU at the indicated time points (C). Data are shown as the mean  SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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Figure 5 Silencing of MRP8/14 suppressed autophagy. (A) THP1cells were transfected with siMRP8/14 or siNC for 24 h, and then treated with DMSO or bafilomycin A1 (Baf.A1, 100 nM) for 2 h. The amount of LC3 was detected by Western blot. The ratios of LC3II/b-actin were calculated as shown below the representative blot. (B, C) THP1-derived macrophages were transiently transfected with siNC or siMRP8/14 for 24 h, followed by BCG challenge. The endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated second antibody (Green), and LC3 puncta (>1 mm) were detected by confocal microscopy (B) and quantified (C). Data are shown as the mean  SEM of three independent experiments. Scale bar, 10 mm **p < 0.01.

intracellular mycobacteria in murine macrophages.14 To explore whether MRP8/14 plays a role in modulating the process of mycobacterial phagosome maturation, the colocalization of BCG with autolysosome was detected with confocal microscopy. THP1-derived macrophages were transfected with siMRP8/14 or siNC, followed by Texas red-labelled BCG infection. Our results showed that silencing of MRP8/14 significantly reduced the colocalization of BCG with LC3 positive autophagosome (Figs. 7A, B), the specific molecular marker of autophagosome. In addition, the colocalization of BCG with lysosome (staining with DQ Green BSA, a fluorogenic substrate for proteases) was also decreased in MRP8/14 silenced THP1 cells (Figs. 7C, D). Moreover, silencing of MRP8/14 enhanced the survival of intracellular BCG (Fig. 7E), while induction of autophagy by rapamycin significantly abrogated the increase of BCG survival in the cells transfected with MRP8/14 siRNA (Fig. 7E). To further confirm that MRP8/14 enhances the elimination of intracellular BCG via autophagy, we blocked autophagy by silencing autophagy-related (ATG) genes, ATG7. Our results showed that overexpression of MRP8/14 decreased intracellular BCG survival, while silencing ATG7 increased intracellular BCG survival and attenuated MRP8/14-mediated mycobactericidal activity (Fig. 7F). Together, these data suggested that MRP8/14 induced autophagy and promoted the maturation of BCG phagosome, thus facilitating the elimination of the intracellular BCG.

MRP8/14 induced autophagy though promoting ROS production To elucidate the mechanism underlying MRP8/14-induced autophagy, we first tested whether MRP8/14 modulates the production of ROS, which had been demonstrated to be an autophagy inducer. Our results showed that overexpression of either MRP8 or MRP14 in A549 cells markedly increased ROS production (Figs. 8A, B). Whereas treatment with antioxidant N-acetylcysteine (NAC) dramatically decreased the ROS production in control A549 cells and cells stably

expressing MRP8 or MRP14 (Figs. 8A, B). Moreover, the amount of LC3 was detected with Western blot in the presence or absence of NAC. Overexpression of MRP8 or MRP14 in A549 cells greatly induced autophagy, as indicating by the increase of the amount of LC3-II (Figs. 8C, D). But treatment with NAC, apparently decreased autophagy in both control cells and cells stably expressing MRP8 or MRP14 (Figs. 8C, D). These results indicated that MRP8 and MRP14 induced autophagy through promoting ROS production.

Discussion MRP8/14 has broad-spectrum antimicrobial activities against various microorganisms, such as E. coli, Staphylococcus aureus, Staphylococcus epidermidis, and C. albicans,26 while its role in antimycobacterial defense remains unknown. In the present study, we examined the role of MRP8/14 in killing of intracellular Mtb, and explored the underlying mechanism involved in this process. Studies have demonstrated that MRP8/14 expression is induced in monocytes or macrophages by various inflammatory stimulators, such as LPS, dsRNA polyinosinic:polycytidylic acid and TNF,27,28 and is inhibited by combined treatment with anti-inflammatory cytokines IL-10 and IL4.49 Our previous study demonstrated that MRP8/14 was dramatically enhanced in both human and murine ocular surface after P. aeruginosa infection, and functions as an inflammatory amplifier.50 In the present study, both mRNA and protein levels of MRP8/14 were dramatically upregulated in THP1 cells and MDMs after mycobacterial challenge, indicating the involvement of MRP8/14 in the host immune response to mycobacterial infection. MRP8/14 has been identified as an important endogenous damage-associated molecular pattern (DAMP), and functions as TLR4 agonist to amplify the inflammatory response in infectious and autoimmune diseases, as well as tumor growth and metastasis.51 It is also demonstrated that MRP8/14 inhibits the binding and uptake of

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Figure 6 Overexpression of MRP8/14 induced autophagy in A549 cells. (A, B) A549 cells stably expressing MRP8 (A) or MRP14 (B) were left uninfected or infected with BCG, and the amount of LC3 was detected by Western blot. The ratios of LC3-II/b-actin were calculated as shown below the representative blot. (CeF) A549 cells stably expressing MRP8 (C, D) or MRP14 (E, F) were left uninfected or infected with BCG. The endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated second antibody (Green), and LC3 puncta (>1 mm) were detected by confocal microscopy (C, E) and quantified (D, F). Data are shown as the mean  SEM of three independent experiments. Scale bar, 10 mm *p < 0.05, **p < 0.01, ***p < 0.001.

L. monocytogenes and Salmonella enterica serovar Typhimurium in mucosal epithelial cells.52 While our data demonstrated that MRP8/14 has no influence on the phagocytosis of BCG in THP1 cells, which is consistent with

previous study showing that MRP8/14 did not affect the phagocytosis of P. aeruginosa.50 Substantial evidence has shown that phagocytosis is largely determined by the interaction between host cells and invading pathogens, and

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Figure 7 MRP8/14 enhanced intracellular BCG elimination by inducing autophagy. (AeD) THP1-derived macrophages were transiently transfected with siNC or siMRP8/14 for 24 h, and then infected with Texas red-labeled BCG. The endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated second antibody (Green) (A). Lysosomes were labeled with a fluorogenic substrate for proteases, DQ-Green BSA (Green) (C). Arrows indicate the colocalization of BCG with autophagosome (A) or lysosomes (C). Scale bar, 10 mm. The percentage of colocalization of BCG with LC3 positive autophagosome (B) or DQGreen labeled lysosome (D) was quantified. Quantification of data was shown as the mean  SEM of three independent experiments. (EeF) THP1 cells were transfected with siMRP8/14 or siNC for 24 h, treated with rapamycin (rap), and then infected with BCG at an MOI of 5 for 4 h (E). THP1 cells were co-transfected with empty vector or plasmids expressing MRP8 and MRP14 together with siNC or siATG7 for 24 h, and then infected with BCG at an MOI of 5 for 4 h (F). Intracellular bacterial survival was detected by CFU at 3 d p.i. Data are shown as the mean  SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

different receptors are involved in this process. For example, E-cadherin serves as a receptor to facilitate uptake of L. monocytogenes,53 while the phagocytosis of Mtb is mediated by complement receptor 3 (CR3).54 In this regard, the distinct effects of MRP8/14 on bacterial phagocytosis may be closely related to the bacterial and cellular specificity.

Studies have demonstrated that MRP8/14 executes both direct and indirect antimicrobial activities against various extracellular and intracellular microorganisms. For example, MRP8/14 directly inhibits the growth of C. albicans, through restriction of mycelial growth and inhibition of glucose incorporation.33 It is also reported that MRP8/14 decreased the number of bacteria via

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Figure 8 MRP8/14 induced autophagy by promoting ROS production. A549 cells stably expressing MRP8 (A, C) or MRP14 (B, D) were left untreated or treated with NAC (10 mM). Cells were collected and incubated with ROS-sensitive probe H2DCFDA. ROS production was determined by flow cytometry, and mean fluorescent intensity (MFI) was shown above representative histograms (A, B). The amount of LC3 was measured by Western blot, and the ratios of LC3-II/b-actin were calculated as shown below the representative blot (C, D).

bacteriostasis rather than a direct lethal effect.55 Our previous study showed that MRP8/14 enhanced macrophagemediated killing of P. aeruginosa by triggering the production of ROS, but not nitric oxide.50 However, little is known regarding the role of MRP8/14 in controlling the mycobacterial survival in macrophages. As the major phagocytes and antigen presenting cells in the antimicrobial immune system, macrophages play a critical role in the host defense against mycobacteria.56,57 The present study showed that MRP8/14 suppressed the survival of intracellular BCG in THP1 cells and MDMs, without affecting the phagocytosis of BCG, suggesting a protective role of MRP8/14 in the host defense against intracellular BCG. Pechkovsky et al. reported that addition of MRP8/14 into the liquid medium accelerated the growth rate of Mtb,31 indicating that MRP8/14 may play distinct roles in intracellular vs extracellular microenvironment. Moreover, unlike BCG, virulent Mtb strain employs multiple strategies to evade host immune response, such as limitation of the acidification and maturation of mycobacterial phagosomes to escape degradation by lysosomal hydrolases.58 Although it is reported that induction of autophagy promotes the elimination of intracellular Mtb as well as BCG in murine macrophages,14 evidence about equivalent control of Mtb and BCG in human cells is absent. Therefore, the role of MRP8/14 in human macrophages-mediated Mtb killing needs to be investigated in future. We further explored the mechanisms by which MRP8/14 inhibited the mycobacterial survival in THP1 cells and MDMs. As an intracellular pathogen, Mtb can persist within macrophages for a long time by interfering with phagolysosome biogenesis, whereas stimulation of autophagic pathways in macrophages promotes the maturation of

phagolysosomes and subsequential degradation of mycobacteria.14 Therefore, autophagy is an important host defense mechanism inhibiting mycobacterial survival in infected macrophages. Several effectors have been demonstrated to enhance the elimination of intracellular Mtb by inducing autophagy, including rapamycin,14 IFN-g14 and microRNA-155.17 Our data showed that silencing of MRP8/14 suppressed autophagy in THP1 cells, while overexpression of MRP8 and MRP14 greatly induced autophagy in A549 cells. These findings are consistent with previous study showing that MRP8/14 induced autophagy in various cells of different origins, such as human breast cancer cell line MCF-7,24 acute promyelocytic leukemia HL-60, chronic myelogenous leukemia K562 cells,32 and murine fibroblasts L929 cell line.24 However, little is known about the role of MRP8/14-mediated autophagy in bacterial infection. Our results showed that blocking autophagy by silencing ATG7 attenuated MRP8/14-mediated intracellular BCG killing, while induced autophagy by rapamycin promoted the elimination of intracellular BCG. Our results indicate that MRP8/14 play an important role in the elimination of intracellular BCG by inducing autophagy. Previous study has demonstrated that ROS positively regulates autophagy.59 In the present study, we found that the MRP8/14-mediated autophagy was greatly decreased in the presence of antioxidant N-acetylcysteine (NAC), suggesting that MRP8/14 induced autophagy via a ROS-dependent manner, which is consistent with other’s observation.24 In addition to ROS generation, other molecular mechanisms may also be involved in the MRP8/14triggered autophagy. For example, Ghavami et al. demonstrated that MRP8/14 increased the expression of Beclin1, an important autophagy related gene, and enhanced the

MRP8/14 in mycobacterial infection formation of, Atg12-Atg5 complex, which is one of the key stages during the execution of autophagy.24 While Yang et al. demonstrated that MRP8 can directly interact with Beclin1, thereby promoting the disassociation of Beclin1 from Beclin1-Bcl-2 complex and facilitating the activation of autophagic pathways.32 We didn’t observe any significant difference in Beclin1 expression after overexpressing or silencing MRP8/14 in THP1 cells (data not shown), however, other possible mechanisms still need further investigation. In summary, our results demonstrated a novel function of MRP8/14 in the host antimycobaterial defense. MRP8/14 was induced in THP1 cells and MDMs in response to BCG challenge and in turn enhanced the elimination of intracellular BCG through inducing autophagy. Furthermore, our study also demonstrated that MRP8/14 enhanced ROS generation and promoted autophagy in a ROS-dependent manner. Collectively, our observations described herein have broadened the function of MRP8/14 in antimycobacterial response, which may provide a potential therapeutic target for TB.

Acknowledgements The authors wish to thank Dr. Linda Hazlett from Wayne State University School of Medicine for her useful comments and language editing. We thank Ping Zhang and Siyu Wu (Zhongshan School of Medicine, Sun Yat-sen University) for kindly providing the A549 cells stably expressing MRP8 and MRP14. This work was supported by National Science and Technology Key Projects for Major Infectious Diseases (2013ZX10003001, 2014ZX10003002, 2012ZX10004903), the 111 Project (No. B13037), National Natural Science Foundation of China (31470877, 81261160323, U0832006, 30972763), Guangdong Innovative Research Team Program (NO. 2009010058), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (NO. 2009), the Natural Science Foundation of Zhejiang (LY13H160035).

References 1. Russell DG, Barry 3rd CE, Flynn JL. Tuberculosis: what we don’t know can, and does, hurt us. Science 2010;328:852e6. 2. Korbel DS, Schneider BE, Schaible UE. Innate immunity in tuberculosis: myths and truth. Microbes Infect 2008;10: 995e1004. 3. Lynch JB. Multidrug-resistant tuberculosis. Med Clin North Am 2013;97:553e79. ix-x. 4. Liu PT, Modlin RL. Human macrophage host defense against Mycobacterium tuberculosis. Curr Opin Immunol 2008;20: 371e6. 5. Persson YA, Blomgran-Julinder R, Rahman S, Zheng L, Stendahl O. Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria. Microbes Infect 2008;10:233e40. 6. Kawai T, Akira S. TLR signaling. Semin Immunol 2007;19: 24e32. 7. Wu MH, Zhang P, Huang X. Toll-like receptors in innate immunity and infectious diseases. Front Med China 2010;4:385e93. 8. Liu Y, Yang B, Ma J, Wang H, Huang F, Zhang J, et al. Interleukin-21 maintains the expression of CD16 on monocytes via the production of IL-10 by human naive CD4þ T cells. Cell Immunol 2011;267:102e8.

425 9. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 1999;17:593e623. 10. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710e20. 11. Niyonsaba F, Ogawa H. Protective roles of the skin against infection: implication of naturally occurring human antimicrobial agents beta-defensins, cathelicidin LL-37 and lysozyme. J Dermatol Sci 2005;40:157e68. 12. Miller RA, Britigan BE. Role of oxidants in microbial pathophysiology. Clin Microbiol Rev 1997;10:1e18. 13. Chan J, Xing Y, Magliozzo RS, Bloom BR. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med 1992;175:1111e22. 14. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004;119:753e66. 15. Ravikumar B, Sarkar S, Davies JE, Futter M, GarciaArencibia M, Green-Thompson ZW, et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 2010;90:1383e435. 16. Ling YM, Shaw MH, Ayala C, Coppens I, Taylor GA, Ferguson DJ, et al. Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages. J Exp Med 2006;203:2063e71. 17. Wang J, Yang K, Zhou L, Minhaowu, Wu Y, Zhu M, et al. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. PLoS Pathog 2013;9:e1003697. 18. Harris J, De Haro SA, Master SS, Keane J, Roberts EA, Delgado M, et al. T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis. Immunity 2007;27:505e17. 19. Delgado MA, Elmaoued RA, Davis AS, Kyei G, Deretic V. Tolllike receptors control autophagy. EMBO J 2008;27:1110e21. 20. Kim JJ, Lee HM, Shin DM, Kim W, Yuk JM, Jin HS, et al. Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action. Cell Host Microbe 2012;11:457e68. 21. Singh SB, Davis AS, Taylor GA, Deretic V. Human IRGM induces autophagy to eliminate intracellular mycobacteria. Science 2006;313:1438e41. 22. Yuk JM, Shin DM, Lee HM, Yang CS, Jin HS, Kim KK, et al. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host Microbe 2009;6:231e43. 23. Delgado M, Singh S, De Haro S, Master S, Ponpuak M, Dinkins C, et al. Autophagy and pattern recognition receptors in innate immunity. Immunol Rev 2009;227:189e202. 24. Ghavami S, Eshragi M, Ande SR, Chazin WJ, Klonisch T, Halayko AJ, et al. S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3. Cell Res 2010;20:314e31. 25. Hsu K, Champaiboon C, Guenther BD, Sorenson BS, Khammanivong A, Ross KF, et al. Anti-infective protective properties of S100 Calgranulins. Antiinflamm Antiallergy Agents Med Chem 2009;8:290e305. 26. Striz I, Trebichavsky I. Calprotectin - a pleiotropic molecule in acute and chronic inflammation. Physiol Res 2004;53:245e53. 27. Endoh Y, Chung YM, Clark IA, Geczy CL, Hsu K. IL-10dependent S100A8 gene induction in monocytes/macrophages by double-stranded RNA. J Immunol 2009;182:2258e68. 28. Xu K, Geczy CL. IFN-gamma and TNF regulate macrophage expression of the chemotactic S100 protein S100A8. J Immunol 2000;164:4916e23. 29. Abtin A, Eckhart L, Glaser R, Gmeiner R, Mildner M, Tschachler E. The antimicrobial heterodimer S100A8/S100A9 (calprotectin) is upregulated by bacterial flagellin in human epidermal keratinocytes. J Invest Dermatol 2010;130:2423e30.

426 30. Bastonero S, Le Priol Y, Armand M, Bernard CS, ReynaudGaubert M, Olive D, et al. New microbicidal functions of tracheal glands: defective anti-infectious response to Pseudomonas aeruginosa in cystic fibrosis. PLoS One 2009;4:e5357. 31. Pechkovsky DV, Zalutskaya OM, Ivanov GI, Misuno NI. Calprotectin (MRP8/14 protein complex) release during mycobacterial infection in vitro and in vivo. FEMS Immunol Med Microbiol 2000;29:27e33. 32. Yang L, Yang M, Zhang H, Wang Z, Yu Y, Xie M, et al. S100A8targeting siRNA enhances arsenic trioxide-induced myeloid leukemia cell death by down-regulating autophagy. Int J Mol Med 2012;29:65e72. 33. Murthy AR, Lehrer RI, Harwig SS, Miyasaki KT. In vitro candidastatic properties of the human neutrophil calprotectin complex. J Immunol 1993;151:6291e301. 34. Zaia AA, Sappington KJ, Nisapakultorn K, Chazin WJ, Dietrich EA, Ross KF, et al. Subversion of antimicrobial calprotectin (S100A8/S100A9 complex) in the cytoplasm of TR146 epithelial cells after invasion by Listeria monocytogenes. Mucosal Immunol 2009;2:43e53. 35. Wu S, He L, Li Y, Wang T, Feng L, Jiang L, et al. miR-146a facilitates replication of dengue virus by dampening interferon induction by targeting TRAF6. J Infect 2013;67:329e41. 36. Engele M, Stossel E, Castiglione K, Schwerdtner N, Wagner M, Bolcskei P, et al. Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis. J Immunol 2002;168:1328e37. 37. Ng MH, Ho TH, Kok KH, Siu KL, Li J, Jin DY. MIP-T3 is a negative regulator of innate type I IFN response. J Immunol 2011;187: 6473e82. 38. Chi W, Yang P, Li B, Wu C, Jin H, Zhu X, et al. IL-23 promotes CD4þ T cells to produce IL-17 in Vogt-Koyanagi-Harada disease. J Allergy Clin Immunol 2007;119:1218e24. 39. Zhao Q, Kuang DM, Wu Y, Xiao X, Li XF, Li TJ, et al. Activated CD69þ T cells foster immune privilege by regulating IDO expression in tumor-associated macrophages. J Immunol 2012;188:1117e24. 40. Chen K, Yin L, Nie X, Deng Q, Wu Y, Zhu M, et al. Beta-Catenin promotes host resistance against Pseudomonas aeruginosa keratitis. J Infect 2013;67:584e94. 41. Wu M, Peng A, Sun M, Deng Q, Hazlett LD, Yuan J, et al. TREM1 amplifies corneal inflammation after Pseudomonas aeruginosa infection by modulating Toll-like receptor signaling and Th1/Th2-type immune responses. Infect Immun 2011;79: 2709e16. 42. Mariencheck WI, Savov J, Dong Q, Tino MJ, Wright JR. Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain of P. aeruginosa. Am J Physiol 1999;277: L777e86. 43. Caulfield JP, Chiang CP, Yacono PW, Smith LA, Golan DE. Low density lipoproteins bound to Schistosoma mansoni do not alter the rapid lateral diffusion or shedding of lipids in the outer surface membrane. J Cell Sci 1991;99(Pt 1):167e73. 44. Vranic S, Boggetto N, Contremoulins V, Mornet S, Reinhardt N, Marano F, et al. Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry. Part Fibre Toxicol 2013;10:2.

J. Wang et al. 45. Yang K, Wu M, Li M, Li D, Peng A, Nie X, et al. miR-155 suppresses bacterial clearance in Pseudomonas aeruginosa keratitis by targeting Rheb. J Infect Dis 2014;210:89e98. 46. Yang K, Wang J, Xiang AP, Zhan X, Wang Y, Wu M, et al. Functional RIG-I-like receptors control the survival of mesenchymal stem cells. Cell Death Dis 2013;4:e967. 47. Thurlow LR, Thomas VC, Fleming SD, Hancock LE. Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion. Infect Immun 2009;77:5551e7. 48. Feterl M, Govan BL, Ketheesan N. The effect of different Burkholderia pseudomallei isolates of varying levels of virulence on toll-like-receptor expression. Trans R Soc Trop Med Hyg 2008;102(Suppl. 1):S82e8. 49. Lugering N, Kucharzik T, Lugering A, Winde G, Sorg C, Domschke W, et al. Importance of combined treatment with IL-10 and IL-4, but not IL-13, for inhibition of monocyte release of the Ca(2þ)-binding protein MRP8/14. Immunology 1997;91:130e4. 50. Deng Q, Sun M, Yang K, Zhu M, Chen K, Yuan J, et al. MRP8/14 enhances corneal susceptibility to Pseudomonas aeruginosa infection by amplifying inflammatory responses. Invest Ophthalmol Vis Sci 2013;54:1227e34. 51. Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J. The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 2009;86:557e66. 52. Nisapakultorn K, Ross KF, Herzberg MC. Calprotectin expression inhibits bacterial binding to mucosal epithelial cells. Infect Immun 2001;69:3692e6. 53. Mengaud J, Ohayon H, Gounon P, Mege RM, Cossart P. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell 1996; 84:923e32. 54. Hetland G, Wiker HG. Antigen 85C on Mycobacterium bovis, BCG and M. tuberculosis promotes monocyte-CR3-mediated uptake of microbeads coated with mycobacterial products. Immunology 1994;82:445e9. 55. Lusitani D, Malawista SE, Montgomery RR. Calprotectin, an abundant cytosolic protein from human polymorphonuclear leukocytes, inhibits the growth of Borrelia burgdorferi. Infect Immun 2003;71:4711e6. 56. Li Q, Li L, Liu Y, Fu X, Wang H, Lao S, et al. Biological functions of Mycobacterium tuberculosis-specific CD4þT cells were impaired by tuberculosis pleural fluid. Immunol Lett 2011;138:113e21. 57. Qiao D, Yang BY, Li L, Ma JJ, Zhang XL, Lao SH, et al. ESAT-6and CFP-10-specific Th1, Th22 and Th17 cells in tuberculous pleurisy may contribute to the local immune response against Mycobacterium tuberculosis infection. Scand J Immunol 2011; 73:330e7. 58. Bhatt K, Salgame P. Host innate immune response to Mycobacterium tuberculosis. J Clin Immunol 2007;27:347e62. 59. Scherz-Shouval R, Shvets E, Elazar Z. Oxidation as a posttranslational modification that regulates autophagy. Autophagy 2007;3:371e3.