RNase 7 but not psoriasin nor sPLA2-IIA associates with Mycobacterium tuberculosis during airway epithelial cell infection

RNase 7 but not psoriasin nor sPLA2-IIA associates with Mycobacterium tuberculosis during airway... Abstract Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism. RNase 7, psoriasin, sPLA2-IIA, mycobacterium, tuberculosis, antimicrobial peptides INTRODUCTION Host defense peptides (HDPs) are small peptides produced by most immune cells during infection in response to pathogen-associated molecular patterns (PAMPs) and to proinflammatory cytokines such as TNFα or IL-1β (Lehrer and Ganz 2002). These peptides have a wide antimicrobial activity against several microorganisms, including bacteria, viruses and fungi. Antimicrobial activity is not their only activity, however; several of these peptides possess remarkable immunoregulatory activity, including both anti-inflammatory and proinflammatory properties (Hancock, Haney and Gill 2016). In the last two decades, the importance of HDPs has been highlighted regarding the control of several infectious diseases (Rivas-Santiago, Serrano and Enciso-Moreno 2009), including tuberculosis, which is caused by Mycobacterium tuberculosis (Mtb). In fact this bacterial disease still has enormous impact on public health worldwide leading to 1.8 million deaths annually. Transmission occurs by the inhalation of bacilli from droplets that are expectorated by patients with active pulmonary tuberculosis. After the bacilli enter the airways, the first line of defense is the epithelial cell barrier. Epithelial cells can sense the invading microorganism and produce a wide variety of HDPs, such as β-defensins and cathelicidin LL-37 (Rivas-Santiago et al. 2005, 2008; Li, Wang and Liu 2012). Although airway epithelial cells can produce a wide variety of HDPs only a few have been associated with the innate immunity against Mtb. Phospholipases A2 are potent antimicrobial proteins that have been associated with the control of other infectious diseases. Included among these proteins is type-IIA (sPLA2-IIA) that displays the highest bactericidal activity (Touqui and Alaoui-El-Azher 2001). sPLA2-IIA exhibits potent bactericidal activity, especially against Gram-positive bacteria. This bactericidal effect is attributed to the ability of sPLA2-IIA to bind and penetrate the cell wall of Gram-positive bacteria (Wu et al. 2010). Another important HDP that has been associated with infectious disease is psoriasin (S100A7), which exhibits very strong antimicrobial activity at low concentrations (Glaser et al. 2005). Psoriasin is expressed in primary human bronchial and lung epithelial cells and human cell lines, as well as in alveolar macrophages of healthy individuals (Andresen et al. 2011); nevertheless, psoriasin has never been studied during tuberculosis infection. When healthy human skin was analyzed for the presence of endogenous HDPs, a novel 14.5 kDa peptide was identified named Ribonuclease (RNase) 7, which exhibited potent in vitro antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and yeast (Harder and Schroder 2002). RNase 7 was originally isolated from stratum corneum skin extracts; however, other tissues also express RNase 7 abundantly, such as the respiratory and urinary epithelia (Spencer et al. 2011; Becknell and Spencer 2016). Although RNase 7 is constitutively expressed, its production can be increased in the presence of PAMPs or bacterial insults. Indeed, RNase 7 concentrations are higher in patients with psoriatic skin lesions, skin infections and urinary tract infection. In the present study, we sought to determine whether these three potent antimicrobial peptides were produced in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells (bMDMs) and from the human cell line THP1 (MDMs) during Mtb infection. MATERIALS AND METHODS Mycobacterium tuberculosis growth, cell culture and infection Mtb H37Rv strain (ATCC 27294, Manassas, VA) was cultured in 25 cm2 plastic culture flasks with 10 mL of Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with 0.2% (v/v) glycerol, 10% oleic acid, albumin, dextrose and catalase (OADC enrichment media, BBL, Becton Dickinson, Franklin Lakes, NJ), and incubated at 37°C with 5% CO2 atmosphere until the bacteria reached the logarithmic phase of growth. Then the culture was divided into working aliquots of 1 × 107 cells mL−1. Suspension aliquots were centrifuged for 5 min at 6000 × g, and the resulting Mtb pellets were declumped by vortexing (5 min) with five sterile 3-mm glass beads in 1 mL of RPMI medium enriched with 10% pooled human AB serum. The remaining Mtb clumps were removed with an additional centrifugation step at 350 × g for 5 min. The Mtb suspension volumes required to obtain the desired multiplicity of infection (MOI 5) were calculated based on the colony-forming unit (CFU) numbers known to be present in the Mtb suspension supernatants. The actual CFU number used for in vitro infections was confirmed in each experiment. The human THP1 (American Type Culture Collection (ATCC) TIB-202) monocytic cell line was grown with RPMI and supplemented with 10% fetal bovine serum (FBS) and 5000 units mL−1 penicillin at 37°C with a 5% CO2 atmosphere. To produce MDMs, 2 × 105 cells were cultured in 24-well dishes or in chamber slides and incubated overnight under 5% CO2 at 37°C with 1 ng mL−1 of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO) for 72 h. Supernatants were then removed and the adherent cells were washed thoroughly with Hanks’ balanced salt solution and incubated additionally for five more days with RPMI supplemented with 10% FBS. The human type II alveolar pneumocytes (A549; ATCC reference number CCL185) and the human airway epithelial cell line (NCI-H292, ATCC reference number CRL-1848), which retain their mucoepidermoid characteristics in culture as determined by their ultrastructure and expression of multiple markers of squamous differentiation, were grown in 75 cm2 culture flasks (Costar, Ontario, Canada) with antibiotic-free RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) at 37°C with 5% CO2. Then cells were seeded onto 48-well plates at a density of 4 × 105 cells and incubated for 18 h to promote adherence. To obtain human monocytes, and after obtaining written informed consent, heparinized blood was obtained by venipuncture from three PPD-negative healthy donors, none of whom had a history of prior exposure to tuberculosis patients. Peripheral blood mononuclear cells were isolated by a Ficoll-hypaque (Nycomed Pharma AS, Oslo, Norway) density gradient. Peripheral blood mononuclear cells were cultured in RPMI medium using 24-well dishes (Costar, Corning, NY, USA) or chamber slides (Costar, Corning, NY). After 2 h, non-adherent cells were removed. The remaining adherent cells were washed at least three times with Hanks’ balanced salt solution (BioWhittaker, Walkersville, MD). Cells were incubated with homologous serum and after 7 days, bMDMs were used for infection. Cytospin preparations were prepared from adherent uninfected cells to allow evaluation of the nuclear and cellular morphology by Wright's staining. Purity and maturation of bMDMs and MDMs was assessed by flow cytometry using CD68 and HLA-DR (>90%). MDM, bMDM, A549 and NCI-H292 cells were infected using an MOI of 5. After 2 h of infection, cells were washed five times with RPMI to eliminate extracellular bacteria and then the cells were incubated for 1, 3, 6, 12, 18 and 24 h; after each time, supernatant was collected and cells were lysed with TRIzol and stored at −70°C until use. Before RNA isolation, cell culture viability was analyzed, showing 99% viability. RNA isolation, reverse transcription and real-time PCR In order to determine whether Mtb infection induced the different HDP mRNAs, reverse transcription of mRNA was performed using 5 μg of total RNA, 2 μM Oligo(dT)15 Primer (Promega, Toronto, Ontario, Canada), 10 units of ribonuclease inhibitor (10 units μL−1) (Invitrogen, Carlsbad, CA), 1 x RT Buffer, 0.5 mM of each dNTP, and 4 units Omniscript Reverse Transcriptase (Qiagen, Mexico City, Mexico). Real-time PCR was performed using a LightCycler 2.0 (Roche, Mannheim, Germany), LightCycler TaqMan Master Mix, and the specific probe for each gene that had been designed using the Universal Probe Library software (Roche) (Table 1). The relative expression of each sample was calculated using human Hypoxanthine Phosphoribosyl Transferase (HPRT) mRNA as reference gene and the 2−ΔΔCt method as described previously (Livak and Schmittgen 2001). This method was based on the expression levels of a target gene compared to a reference gene (HPRT), comparing between control group and target group. Table 1. Sequence of primers and probes used for quantitative PCR. Gene  Primers  Probe  sPLA2-IIA  F 5΄-AAATTTCTGAGCTACAAGTTTAGCAAC-3΄  GGCAGAAG    R 5΄-TTATCACACTCACACAGTTGACTTCT-3΄    RNase 7  F 5΄-GAAGACCAAGCGCAAAGC-3΄  CTCCTCCT    R 5΄-AGCAGAAGGGGGCAGAAT-3΄    Psoriasin  F 5΄-CCAAACACACACATCTCACTCA-3΄  TCCCAGCT    R 5΄-TCAGCTTGAGTGTTGCTCATC-3΄    HPRT  F 5΄-TGACCTTGATTTATTTTGCATACC-3΄  GCTGAGGA    R 5΄-CGAGCAAGACGTTCAGTCCT-3΄    Gene  Primers  Probe  sPLA2-IIA  F 5΄-AAATTTCTGAGCTACAAGTTTAGCAAC-3΄  GGCAGAAG    R 5΄-TTATCACACTCACACAGTTGACTTCT-3΄    RNase 7  F 5΄-GAAGACCAAGCGCAAAGC-3΄  CTCCTCCT    R 5΄-AGCAGAAGGGGGCAGAAT-3΄    Psoriasin  F 5΄-CCAAACACACACATCTCACTCA-3΄  TCCCAGCT    R 5΄-TCAGCTTGAGTGTTGCTCATC-3΄    HPRT  F 5΄-TGACCTTGATTTATTTTGCATACC-3΄  GCTGAGGA    R 5΄-CGAGCAAGACGTTCAGTCCT-3΄    View Large Flow cytometry To assess RNase 7 production in Mtb-infected cells, NCI-H292 cells were obtained by enzymatic disaggregation with 0.05% trypsin-EDTA in the different kinetic times mentioned above. Then, cells were treated with perm/fix kit (BD Biosciences, San Diego, CA) according to the manufacturer’s protocol. Subsequently, cells were incubated with 1% bovine serum albumin for 30 min and incubated with anti-RNase 7 antibody (1:10) (Santa Cruz Biotechnology, Dallas, TX) at 4°C for 1 h. Thereafter, cells were incubated with anti-goat IgG-PE antibody (1:10) (Santa Cruz Biotechnology) for 30 min at 4°C, fixed with 4% paraformaldehyde and analyzed with a Canto II flow cytometer using FACS-Diva software (BD Biosciences), and a standard gating strategy (Fig. S1 in the online supplementary material). Immunocytochemistry NCI-H292 cells were infected with Mtb strain H37Rv at MOI 5 in chamber slides as mentioned above. Subsequently, the slides were fixed with 4% paraformaldehyde, permeabilized with fix/perm and incubated for 1 h 30 min with 10% pig serum in HNC buffer (Hepes–NaCl2–CaCl2). Subsequently slides were incubated with anti-RNase 7 antibody, dilution 1:100 (Santa Cruz Biotechnology), overnight at 4°C, and then slides were incubated with biotinylated anti-goat antibody (Santa Cruz Biotechnology) diluted at 1:500 for 1 h 30 min at room temperature. After immunocytochemistry (ICC) slides were stained with the conventional Ziehl–Neelsen method and then analyzed by microscopy, where brown-stained cells were considered as positive for RNase 7. Confocal microscopy To assess co-localization of RNase 7 with mycobacteria, NCI-H292 cells (1 × 105) were seeded onto 4-well/chamber slides (Nunc Inc., Naperville, IL). After 18 h of adherence, cells were infected as described above with Mtb H37Rv stained with PKH67 (Sigma-Aldrich) for 1, 3 and 6 h to evaluate the co-localization of Mtb and RNase 7. Then, cells were fixed for 30 min with 4% paraformaldehyde. Subsequently, cells were permeabilized with perm/fix kit (BD Biosciences) and incubated with 3% bovine serum albumin for 30 min at room temperature to block the unspecific binding and then incubated with anti-RNase 7 antibody (1:100) (Santa Cruz Biotechnology) for 1 h at 4°C. After vigorous washing, cells were incubated with anti-goat-IgG-PE antibody (1:400) (Santa Cruz Biotechnology) and incubated for 30 min at room temperature. For nuclei staining 3 μM DAQ7 was used (Biostatus, Shepshed, UK) for 20 min at room temperature. Finally, cell preparations were mounted with Vectashield hard (Vector Laboratories, Burlingame, CA). Subcellular detection of RNase 7 by immunoelectronmicroscopy NCI-H292 cells were infected with Mtb as described above. After 6 h, infected cells were washed three times with PBS, fixed in 4% (v/v) paraformaldehyde and dissolved in 0.2 M Sörensen buffer (one vol. NaH2PO4*H2O, two vol. NaHPO4*7H2O), at pH 7.3, for 2 h at 4°C. Following a washing step of the cells with the Sörensen buffer, free aldehyde groups were blocked with 0.5 M ammonium chloride in PBS for 1 h. Fixed cells were then dehydrated in graded ethyl alcohols and embedded in LR-White hydrosoluble resin (London Resin Co., Reading, UK). Thin sections from 70 to 90 nm were placed on nickel grids. The grids were incubated overnight at 4°C with specific polyclonal goat anti-RNase 7 antibody (Santa Cruz Biotechnology) diluted to 1/200 in PBS with 1% bovine serum albumin and 0.5% Tween-20. After rinsing with PBS, the grids were incubated for 1 h at room temperature with donkey anti-goat IgG (Sigma Co., St Louis, MO) conjugated to 5 nm gold particles (Sigma) diluted 1/20 in PBS. The grids were stained with uranium salts (Electron Microscopy Sciences, Fort Washington, PA) and examined with an electron microscope FEI Technai G2 Spirit (Hillsboro, OR). As negative controls, the primary RNase 7 antibody was substituted by normal rabbit serum. Statistical analysis Statistical analyses were performed using the GraphPad Prism software for Mac (GraphPad Software version 6.01, San Diego, CA). Normal distribution was assessed using the Kolmogorov–Smirnov test for each data set, together with a non-parametric two-group comparison Mann–Whitney U or multiple comparison test of Kruskal–Wallis to identify differences among the groups. When statistical significance (P < 0.05) was found, a Dunn’s post test was performed. Two-sided P values of <0.05 were considered statistically significant. RESULTS Mycobacterium tuberculosis infection induces the expression of RNase 7 and downregulates the transcription of psoriasin in lung epithelial cells To determine if Mtb infection induced psoriasin, RNase 7 and sPLA2-IIA, mRNA expression in A549, bMDMs, MDMs and NCI-H292 was measured. Cells were infected at a MOI of 5 and after 1, 3, 6, 12, 18 and 24 h, cells were lysed and mRNA was obtained and analyzed for the expression of RNase 7, psoriasin and sPLA2-IIA. Both bMDMs from patients (n = 3, data not shown) and the MDM cell line (THP1) showed identical results: neither sPLA2-IIA, psoriasin nor RNase 7 were detected in infected cells (Fig. 1A, B and C, respectively). Similar results were obtained for A549 cells: none of the above antimicrobial peptides showed any gene transcription in all the experimental conditions analyzed (sPLA2-IIA, Fig. 1D; psoriasin, Fig. 1E; and RNase 7, Fig. 1F). Mtb-infected NCI-H292 cells did not show sPLA2-IIA gene expression (Fig. 1G), while a significant downregulation of psoriasin was detected at 1, 3, 12 and 24 h (P < 0.05), the lowest expression being at 18 h post-infection (P < 0.001, Fig. 1H). Regarding RNase 7, infection with Mtb led to an overexpression of this peptide mainly at 1, 3 and 6 h post-infection in NCI-H292 cells (P < 0.001), whereas after 12 and 18 h post-infection the induction was significantly lower (P < 0.05) when compared with non-infected cells (Fig. 1I). To assess whether other intracellular pathogens induced RNase 7 gene expression, NCI-H292 cells were infected with Salmonella typhi (clinical isolate) and analyzed for RNase 7 expression during the kinetic times. The results showed that S. typhi induces RNase 7 expression and the highest expression can be detected after 6 h of infection, which correlates with those results shown during Mtb infection (Fig. S2A in the online supplementary material). To evaluate which of both pathogens induced higher RNase 7 mRNA expression levels, results from S. typhi were compared vs. the results obtained from Mtb-infected cells under the same conditions. The results showed that infection with S. typhi induces up to 17.86 (± 6.98)-fold less RNase 7 mRNA expression levels at 6 h than the Mtb-infected cells. Similar results were observed for the rest of the kinetic points (Fig. S2B in the online supplementary material). Figure 1. View largeDownload slide RNase 7 is expressed during Mtb infection. MDMs, type II pneumocytes (A549) and airway epithelial cells (NCI-H292) were infected with Mtb and after 1, 3, 6, 12, 18 and 24 h mRNA was evaluated. Infected MDMs showed no expression for any of the HDPs studied (A–C). Similar results were obtained for type II pneumocytes (D–F). Airway epithelial cells showed no mRNA expression for sPLA2-IIA (G), whereas for psoriasin (H) and RNase 7 (I) mRNA expression change in these cells was detected. (Cp, crossing point or crossing threshold.) The positive control used for RNase 7 and psoriasin was obtained from psoriatic skin biopsies and the positive control for sPLA2-IIA was obtained from umbilical cord sections. *P < 0.05, ***P < 0.001. Data are presented as mean ± standard deviation. n = 6. Figure 1. View largeDownload slide RNase 7 is expressed during Mtb infection. MDMs, type II pneumocytes (A549) and airway epithelial cells (NCI-H292) were infected with Mtb and after 1, 3, 6, 12, 18 and 24 h mRNA was evaluated. Infected MDMs showed no expression for any of the HDPs studied (A–C). Similar results were obtained for type II pneumocytes (D–F). Airway epithelial cells showed no mRNA expression for sPLA2-IIA (G), whereas for psoriasin (H) and RNase 7 (I) mRNA expression change in these cells was detected. (Cp, crossing point or crossing threshold.) The positive control used for RNase 7 and psoriasin was obtained from psoriatic skin biopsies and the positive control for sPLA2-IIA was obtained from umbilical cord sections. *P < 0.05, ***P < 0.001. Data are presented as mean ± standard deviation. n = 6. Intracellular RNase 7 detection by flow cytometry Once it was determined that RNase 7 was upregulated during Mtb infection in airway epithelial cells NCI-H292, we determined the RNase 7 mean fluorescence intensity and percentage of positive cells by flow cytometry along the kinetics used in the mRNA expression experiments. The results indicated that after 6 h of infection the mean fluorescence intensity was higher than for non-infected cells (P < 0.01); similar results were observed after 3 and 12 h of infection (P < 0.05) (Fig. 2A). Intracellular RNase 7 was detected in all kinetic times, but only after 6 h with 39 ± 6% (P < 0.01) and after 3 h with 28 ± 8% (P < 0.05) was there statistical significance when compared with non-infected cells (Fig. 2B). Figure 2. View largeDownload slide Intracellular RNase 7 detection by flow cytometry. Mean fluorescence intensity for RNase 7 was evaluated in airway epithelial cells NCI-H292 (A) as well as percentage of RNase 7-positive cells (B). Data are presented as percentage of RNase 7-positive cells and as geometric mean of fluorescence intensity. *P < 0.05, **P < 0.01. Data are presented as mean ± standard deviation. n = 6. Figure 2. View largeDownload slide Intracellular RNase 7 detection by flow cytometry. Mean fluorescence intensity for RNase 7 was evaluated in airway epithelial cells NCI-H292 (A) as well as percentage of RNase 7-positive cells (B). Data are presented as percentage of RNase 7-positive cells and as geometric mean of fluorescence intensity. *P < 0.05, **P < 0.01. Data are presented as mean ± standard deviation. n = 6. RNase 7 associates intracellularly with Mycobacterium tuberculosis in infected airway epithelial cells Although we showed that Mtb induces RNase 7 upregulation, mainly after 6 h of infection, it was also important to determine whether RNase 7 was associated with intracellular Mtb. First, Mtb-infected cells were submitted for ICC analysis; subsequently slides were stained with Ziehl-Neelsen. Results showed that infected cells were positive for RNase 7 immunostaining (Fig. 3A); the Ziehl–Neelsen staining after ICC showed that apparently RNase 7 was associated with Mtb. It is of note that all infected cells produced RNase 7 (Fig. 3B). To assess the intracellular association between RNase 7 and Mtb, confocal microscopy was used. Results indicated that RNase 7 is produced constitutively by non-infected cells, and after 6 h of infection RNase 7 increased substantially. Fluorescence-labeled Mtb was detected intracellularly and was associated with RNase 7 at the three time-points studied. The main difference observed was the fluorescence intensity among times (Fig. 4). To evaluate the RNase 7 expression, we used a quantitative analysis by confocal microscopy, showing similar results to those obtained in the flow cytometry analysis (Fig. S3 in the online supplementary material). Co-localization was confirmed by ultrastructural immunolabeling at 6 h after infection; the results showed that RNase 7 was dispersed throughout the cytoplasm of bronchial epithelial cells NCI-H292, being more concentrated in vacuoles, and particularly high amounts associated with the Mtb cell wall (Fig. 5A and B); approximately 90 ± 5% of the total immunogold particles were associated with Mtb (data not shown). Some lysed mycobacteria associated with RNase 7 were found inside vacuoles (Fig. 5C). Figure 3. View largeDownload slide Immunocytochemistry in airway epithelial cells infected with Mtb for 6 h. Airway epithelial cells NCI-H292 were seeded onto chamber slides and infected for 6 h with Mtb and then fixed and submitted for ICC. Infected cells showed positive staining for RNase 7 (A, ×200), secondary antibody showed no unspecific binding (inset). After the ICC, cells were stained with Ziehl–Neelsen (B, ×1000): mycobacteria are traditionally stained in fuchsia (arrows) while immunostained cells remain brown. Microphotographs are representative of at least three independent experiments. Figure 3. View largeDownload slide Immunocytochemistry in airway epithelial cells infected with Mtb for 6 h. Airway epithelial cells NCI-H292 were seeded onto chamber slides and infected for 6 h with Mtb and then fixed and submitted for ICC. Infected cells showed positive staining for RNase 7 (A, ×200), secondary antibody showed no unspecific binding (inset). After the ICC, cells were stained with Ziehl–Neelsen (B, ×1000): mycobacteria are traditionally stained in fuchsia (arrows) while immunostained cells remain brown. Microphotographs are representative of at least three independent experiments. Figure 4. View largeDownload slide Immunofluorescence of RNase 7 and Mtb in airway epithelial cells NCI-H292. Mtb was stained with PKH67 and used to infect airway epithelial cells. At 1, 3 and 6 h post-infection, cells were fixed and incubated with anti-RNase 7 antibody for 1 h. Then cell preparations were incubated with anti-goat-IgG-PE antibody for 30 min. For nuclei staining, DAQ7 was used. On the merge lane, asterisks show immunostaining for RNase 7 and arrows show Mtb co-localization with RNase 7. Microphotographs are representative of at least three independent experiments. (×400). Figure 4. View largeDownload slide Immunofluorescence of RNase 7 and Mtb in airway epithelial cells NCI-H292. Mtb was stained with PKH67 and used to infect airway epithelial cells. At 1, 3 and 6 h post-infection, cells were fixed and incubated with anti-RNase 7 antibody for 1 h. Then cell preparations were incubated with anti-goat-IgG-PE antibody for 30 min. For nuclei staining, DAQ7 was used. On the merge lane, asterisks show immunostaining for RNase 7 and arrows show Mtb co-localization with RNase 7. Microphotographs are representative of at least three independent experiments. (×400). Figure 5. View largeDownload slide Subcellular association of RNase 7 and Mtb in NCI-H92 cells. Representative immunoelectronmicroscopy micrographs showing the subcellular localization of RNase 7 labeled with 5 nm gold particles (black dots). (A) Image shows cytoplasmic mild diffuse distribution of RNase 7, more concentration of this peptide in cytoplasmic vacuoles (V) and particularly strong labelling in the mycobacterium cell wall (arrow). Magnification, ×20,000. (B) Longitudinal section of intracellular Mycobacterium tuberculosis (M) showing co-localization with RNase 7 labeled with gold particles. Magnification, ×50,000. (C) Mycobacterium tuberculosis (M) inside cytoplasmic vacuole (V); a lysed mycobacterium is shown inside the same vacuole (L). Magnification ×30,000. Figure 5. View largeDownload slide Subcellular association of RNase 7 and Mtb in NCI-H92 cells. Representative immunoelectronmicroscopy micrographs showing the subcellular localization of RNase 7 labeled with 5 nm gold particles (black dots). (A) Image shows cytoplasmic mild diffuse distribution of RNase 7, more concentration of this peptide in cytoplasmic vacuoles (V) and particularly strong labelling in the mycobacterium cell wall (arrow). Magnification, ×20,000. (B) Longitudinal section of intracellular Mycobacterium tuberculosis (M) showing co-localization with RNase 7 labeled with gold particles. Magnification, ×50,000. (C) Mycobacterium tuberculosis (M) inside cytoplasmic vacuole (V); a lysed mycobacterium is shown inside the same vacuole (L). Magnification ×30,000. Supernatants from Mycobacterium tuberculosis-infected MDMs do not induce RNase 7 in airway epithelial cells To determine whether any molecule secreted by infected macrophages might induce RNase 7 in airway epithelial cells as a mechanism to promote innate immunity, MDMs were infected as described above. After 18 h of infection supernatants were collected and used immediately to stimulate epithelial cells (NCI-H292). Airway epithelial cells were grown in 24-well plates and left for 18 h to allow adhesion. After this time, the supernatant was discarded and substituted with supernatants from Mtb-infected MDMs; after 6 h of stimulation (given that RNase 7 expression was mostly detected at this time in previous experiments), cells were lysed with TRIzol and submitted for mRNA expression analyses. The results showed that there were no statistical differences between non-infected cells and cells stimulated with MDM supernatant (Fig. 6), suggesting that the induction of RNase 7 is dependent on the interaction between airway epithelial cells and Mtb. Figure 6. View largeDownload slide Airway epithelial cells treated with filtrated supernatants from Mtb-infected cells. MDMs were infected for 18 h with Mtb, then supernatant was collected, filtrated and subsequently transferred to NCI-H292 cells (EpCs + SN). For the ‘None’ condition, instead of using supernatants, NCI-H292 cells were stimulated with PBS and after 6 h cells were lysed and submitted for mRNA analysis for RNase 7 expression. Data are presented as mean ± standard deviation. n = 6. Figure 6. View largeDownload slide Airway epithelial cells treated with filtrated supernatants from Mtb-infected cells. MDMs were infected for 18 h with Mtb, then supernatant was collected, filtrated and subsequently transferred to NCI-H292 cells (EpCs + SN). For the ‘None’ condition, instead of using supernatants, NCI-H292 cells were stimulated with PBS and after 6 h cells were lysed and submitted for mRNA analysis for RNase 7 expression. Data are presented as mean ± standard deviation. n = 6. DISCUSSION Although there are approximately 122 HDPs (most of them identified by bioinformatic approaches), fewer than 20 have been associated with infectious diseases (Wang 2014). In the last decade, HDPs such as defensins and cathelicidin have been associated with the elimination of Mtb (Rivas-Santiago et al. 2005, 2008a,b; Liu et al. 2007). Nevertheless, there are other HDPs with clinical importance such as psoriasin, RNase 7 and sPLA2-IIA (Jia et al. 2014; Becknell and Spencer 2016; Tan and Goh 2017) that have not been studied in tuberculosis pathogenesis. To study the relationship of these peptides with innate immunity against tuberculosis, we first determined whether infection with Mtb increased the mRNA expression of RNase 7 and sPLA2-IIA in an in vitro model. Our results showed that neither MDMs from THP1 nor bMDMs (data not shown) showed mRNA expression for RNase 7 and sPLA2-IIA. Similar results were observed in A549 and NCI-H292 cells in the case of sPLA2-IIA. Although these antimicrobial proteins have been described in several infectious pathologies, during Mtb infection we could not detect RNase 7 or sPLA2-IIA upregulation. Indeed, we do not know why the mycobacteria did not induce sPLA2. It is plausible that the type of cells used for this study do not express sPLA2. In the case of psoriasin neither MDMs, bMDMs nor A549 cells expressed this peptide during infection with Mtb; however, NCI-H292 cells constitutively express psoriasin and mycobacterial infection downregulated the expression of this HDP showing statistical significance almost in all the studied times. This suggests that mycobacteria could use this mechanism to avoid innate immunity; however, further studies need to be done to unveil whether downregulation of psoriasin is important for Mtb pathogenesis and whether this phenomenon is related to mycobacterial virulence. Regarding RNase 7, we only found mRNA expression in NCI-H292 cells, which was importantly upregulated during the first 6 h after infection and then went back to basal expression. It has been reported that RNase 7 is an antimicrobial peptide produced mainly by epithelial cells (Becknell et al. 2015); the absence of RNase 7 in human monocytes has been showed previously (Becknell and Spencer 2016), and its expression in macrophages has not been documented. Based on our results, we can suggest that MDMs do not express this peptide. Our results showed that RNase 7 gene expression is induced at early time points and after 24 h its expression is similar to that in non-infected cells. Similar studies have reported that RNase 7 was induced by IL-1β in keratinocytes, showing that higher expression levels can be detected after 24 h of stimulation (Becknell and Spencer 2016), whereas our results showed that the highest expression of RNase 7 can be observed at 6 h post-infection. This could be explained because Mtb activates different signaling pathways such as those induced by (Toll-like receptor) TLR-2, TLR-4 and TLR-9 mostly a few hours after infection (Kleinnijenhuis et al. 2011), which is the main pathway to induce RNase 7 expression. To assess whether any other molecule secreted by Mtb-infected macrophages such as cytokines or other AMPs induced RNase 7 mRNA expression, epithelial cells were treated with the filtered supernatant from infected MDMs. Our results suggest that the Mtb by itself induced RNase 7 production and that other molecules produced during infection are not involved. To determine whether mRNA expression was translated into protein, we detected intracellular RNase 7 by cytofluorometry and immunocytochemistry. Results from both studies were consistent, showing a peak in RNase 7 production after 6 h of infection in NCI-H292 cells. Then, we evaluated whether the produced RNase 7 was associated with intracellular Mtb, which could suggest a direct antimicrobial effect on Mtb. A mild association was seen in ICC-stained slides subsequently stained with Ziehl–Neelsen, which was confirmed by confocal microscopy and immunoelectron microscopy. Previous studies have described the structure of RNase 7 and its electrostatic properties, and also the antimicrobial mechanism of RNase 7 generated by the high positive charge (Huang et al. 2007). Antimicrobial activity is generated by electrostatic interaction produced by the high cationicity of RNase 7 with the mycobacterial negative phospholipids, mainly by peptidoglycan, which is one of the main components of the Mtb cell wall (Zhang, Dyer and Rosenberg 2003; Huang et al. 2007). However, even though RNase 7 evidently associates with Mtb during infection and we found lysed mycobacteria associated with RNase 7 by ultrastructural analysis, it cannot be certain that this peptide is involved in mycobacteria elimination. It remains unknown whether deficient RNase 7 production increases infection risk or if increased RNase 7 production shields the host from microbial challenge. Further studies need to be done to elucidate this. In conclusion, our studies suggest for the first time that Mtb infection induces the production of RNase 7 in NCI-H292 cells (airway epithelial cells) and that the produced HDP binds to the Mtb cell wall. These results propose another mechanism used by the host to counterattack Mtb infection and new insights to understand the innate immunity in tuberculosis. The relevance of RNase 7 in the resistance to tuberculosis needs to be further investigated. It is important to explore whether RNase 7 could be used for the development of new therapies or as a biomarker for diagnostics. SUPPLEMENTARY DATA Supplementary data are available at FEMSPD online. Acknowledgements We acknowledge Julio Castañeda-Delgado for confocal microscopy technical support and Alejandra Montoya-Rosales for her invaluable assistance in BSL3 facilities. FUNDING This work was supported by Mexican Institute of Social Security (IMSS) FIS/IMSS/PROT/G14/1318. BRS has a scholarship from Fundación IMSS. Conflict of interest. None declared. REFERENCES Andresen E, Lange C, Strodthoff D et al.   S100A7/psoriasin expression in the human lung: unchanged in patients with COPD, but upregulated upon positive S. aureus detection. BMC Pulm Med  2011; 11: 10. Google Scholar CrossRef Search ADS PubMed  Becknell B, Eichler TE, Beceiro S et al.   Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract. Kidney Int  2015; 87: 151– 61. 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Google Scholar CrossRef Search ADS PubMed  Wu Y, Raymond B, Goossens PL et al.   Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host. Biochimie  2010; 92: 583– 7. Google Scholar CrossRef Search ADS PubMed  Zhang J, Dyer KD, Rosenberg HF. Human RNase 7: a new cationic ribonuclease of the RNase A superfamily. Nucleic Acids Res  2003; 31: 602– 7. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pathogens and Disease Oxford University Press

RNase 7 but not psoriasin nor sPLA2-IIA associates with Mycobacterium tuberculosis during airway epithelial cell infection

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Abstract

Abstract Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism. RNase 7, psoriasin, sPLA2-IIA, mycobacterium, tuberculosis, antimicrobial peptides INTRODUCTION Host defense peptides (HDPs) are small peptides produced by most immune cells during infection in response to pathogen-associated molecular patterns (PAMPs) and to proinflammatory cytokines such as TNFα or IL-1β (Lehrer and Ganz 2002). These peptides have a wide antimicrobial activity against several microorganisms, including bacteria, viruses and fungi. Antimicrobial activity is not their only activity, however; several of these peptides possess remarkable immunoregulatory activity, including both anti-inflammatory and proinflammatory properties (Hancock, Haney and Gill 2016). In the last two decades, the importance of HDPs has been highlighted regarding the control of several infectious diseases (Rivas-Santiago, Serrano and Enciso-Moreno 2009), including tuberculosis, which is caused by Mycobacterium tuberculosis (Mtb). In fact this bacterial disease still has enormous impact on public health worldwide leading to 1.8 million deaths annually. Transmission occurs by the inhalation of bacilli from droplets that are expectorated by patients with active pulmonary tuberculosis. After the bacilli enter the airways, the first line of defense is the epithelial cell barrier. Epithelial cells can sense the invading microorganism and produce a wide variety of HDPs, such as β-defensins and cathelicidin LL-37 (Rivas-Santiago et al. 2005, 2008; Li, Wang and Liu 2012). Although airway epithelial cells can produce a wide variety of HDPs only a few have been associated with the innate immunity against Mtb. Phospholipases A2 are potent antimicrobial proteins that have been associated with the control of other infectious diseases. Included among these proteins is type-IIA (sPLA2-IIA) that displays the highest bactericidal activity (Touqui and Alaoui-El-Azher 2001). sPLA2-IIA exhibits potent bactericidal activity, especially against Gram-positive bacteria. This bactericidal effect is attributed to the ability of sPLA2-IIA to bind and penetrate the cell wall of Gram-positive bacteria (Wu et al. 2010). Another important HDP that has been associated with infectious disease is psoriasin (S100A7), which exhibits very strong antimicrobial activity at low concentrations (Glaser et al. 2005). Psoriasin is expressed in primary human bronchial and lung epithelial cells and human cell lines, as well as in alveolar macrophages of healthy individuals (Andresen et al. 2011); nevertheless, psoriasin has never been studied during tuberculosis infection. When healthy human skin was analyzed for the presence of endogenous HDPs, a novel 14.5 kDa peptide was identified named Ribonuclease (RNase) 7, which exhibited potent in vitro antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and yeast (Harder and Schroder 2002). RNase 7 was originally isolated from stratum corneum skin extracts; however, other tissues also express RNase 7 abundantly, such as the respiratory and urinary epithelia (Spencer et al. 2011; Becknell and Spencer 2016). Although RNase 7 is constitutively expressed, its production can be increased in the presence of PAMPs or bacterial insults. Indeed, RNase 7 concentrations are higher in patients with psoriatic skin lesions, skin infections and urinary tract infection. In the present study, we sought to determine whether these three potent antimicrobial peptides were produced in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells (bMDMs) and from the human cell line THP1 (MDMs) during Mtb infection. MATERIALS AND METHODS Mycobacterium tuberculosis growth, cell culture and infection Mtb H37Rv strain (ATCC 27294, Manassas, VA) was cultured in 25 cm2 plastic culture flasks with 10 mL of Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with 0.2% (v/v) glycerol, 10% oleic acid, albumin, dextrose and catalase (OADC enrichment media, BBL, Becton Dickinson, Franklin Lakes, NJ), and incubated at 37°C with 5% CO2 atmosphere until the bacteria reached the logarithmic phase of growth. Then the culture was divided into working aliquots of 1 × 107 cells mL−1. Suspension aliquots were centrifuged for 5 min at 6000 × g, and the resulting Mtb pellets were declumped by vortexing (5 min) with five sterile 3-mm glass beads in 1 mL of RPMI medium enriched with 10% pooled human AB serum. The remaining Mtb clumps were removed with an additional centrifugation step at 350 × g for 5 min. The Mtb suspension volumes required to obtain the desired multiplicity of infection (MOI 5) were calculated based on the colony-forming unit (CFU) numbers known to be present in the Mtb suspension supernatants. The actual CFU number used for in vitro infections was confirmed in each experiment. The human THP1 (American Type Culture Collection (ATCC) TIB-202) monocytic cell line was grown with RPMI and supplemented with 10% fetal bovine serum (FBS) and 5000 units mL−1 penicillin at 37°C with a 5% CO2 atmosphere. To produce MDMs, 2 × 105 cells were cultured in 24-well dishes or in chamber slides and incubated overnight under 5% CO2 at 37°C with 1 ng mL−1 of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO) for 72 h. Supernatants were then removed and the adherent cells were washed thoroughly with Hanks’ balanced salt solution and incubated additionally for five more days with RPMI supplemented with 10% FBS. The human type II alveolar pneumocytes (A549; ATCC reference number CCL185) and the human airway epithelial cell line (NCI-H292, ATCC reference number CRL-1848), which retain their mucoepidermoid characteristics in culture as determined by their ultrastructure and expression of multiple markers of squamous differentiation, were grown in 75 cm2 culture flasks (Costar, Ontario, Canada) with antibiotic-free RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) at 37°C with 5% CO2. Then cells were seeded onto 48-well plates at a density of 4 × 105 cells and incubated for 18 h to promote adherence. To obtain human monocytes, and after obtaining written informed consent, heparinized blood was obtained by venipuncture from three PPD-negative healthy donors, none of whom had a history of prior exposure to tuberculosis patients. Peripheral blood mononuclear cells were isolated by a Ficoll-hypaque (Nycomed Pharma AS, Oslo, Norway) density gradient. Peripheral blood mononuclear cells were cultured in RPMI medium using 24-well dishes (Costar, Corning, NY, USA) or chamber slides (Costar, Corning, NY). After 2 h, non-adherent cells were removed. The remaining adherent cells were washed at least three times with Hanks’ balanced salt solution (BioWhittaker, Walkersville, MD). Cells were incubated with homologous serum and after 7 days, bMDMs were used for infection. Cytospin preparations were prepared from adherent uninfected cells to allow evaluation of the nuclear and cellular morphology by Wright's staining. Purity and maturation of bMDMs and MDMs was assessed by flow cytometry using CD68 and HLA-DR (>90%). MDM, bMDM, A549 and NCI-H292 cells were infected using an MOI of 5. After 2 h of infection, cells were washed five times with RPMI to eliminate extracellular bacteria and then the cells were incubated for 1, 3, 6, 12, 18 and 24 h; after each time, supernatant was collected and cells were lysed with TRIzol and stored at −70°C until use. Before RNA isolation, cell culture viability was analyzed, showing 99% viability. RNA isolation, reverse transcription and real-time PCR In order to determine whether Mtb infection induced the different HDP mRNAs, reverse transcription of mRNA was performed using 5 μg of total RNA, 2 μM Oligo(dT)15 Primer (Promega, Toronto, Ontario, Canada), 10 units of ribonuclease inhibitor (10 units μL−1) (Invitrogen, Carlsbad, CA), 1 x RT Buffer, 0.5 mM of each dNTP, and 4 units Omniscript Reverse Transcriptase (Qiagen, Mexico City, Mexico). Real-time PCR was performed using a LightCycler 2.0 (Roche, Mannheim, Germany), LightCycler TaqMan Master Mix, and the specific probe for each gene that had been designed using the Universal Probe Library software (Roche) (Table 1). The relative expression of each sample was calculated using human Hypoxanthine Phosphoribosyl Transferase (HPRT) mRNA as reference gene and the 2−ΔΔCt method as described previously (Livak and Schmittgen 2001). This method was based on the expression levels of a target gene compared to a reference gene (HPRT), comparing between control group and target group. Table 1. Sequence of primers and probes used for quantitative PCR. Gene  Primers  Probe  sPLA2-IIA  F 5΄-AAATTTCTGAGCTACAAGTTTAGCAAC-3΄  GGCAGAAG    R 5΄-TTATCACACTCACACAGTTGACTTCT-3΄    RNase 7  F 5΄-GAAGACCAAGCGCAAAGC-3΄  CTCCTCCT    R 5΄-AGCAGAAGGGGGCAGAAT-3΄    Psoriasin  F 5΄-CCAAACACACACATCTCACTCA-3΄  TCCCAGCT    R 5΄-TCAGCTTGAGTGTTGCTCATC-3΄    HPRT  F 5΄-TGACCTTGATTTATTTTGCATACC-3΄  GCTGAGGA    R 5΄-CGAGCAAGACGTTCAGTCCT-3΄    Gene  Primers  Probe  sPLA2-IIA  F 5΄-AAATTTCTGAGCTACAAGTTTAGCAAC-3΄  GGCAGAAG    R 5΄-TTATCACACTCACACAGTTGACTTCT-3΄    RNase 7  F 5΄-GAAGACCAAGCGCAAAGC-3΄  CTCCTCCT    R 5΄-AGCAGAAGGGGGCAGAAT-3΄    Psoriasin  F 5΄-CCAAACACACACATCTCACTCA-3΄  TCCCAGCT    R 5΄-TCAGCTTGAGTGTTGCTCATC-3΄    HPRT  F 5΄-TGACCTTGATTTATTTTGCATACC-3΄  GCTGAGGA    R 5΄-CGAGCAAGACGTTCAGTCCT-3΄    View Large Flow cytometry To assess RNase 7 production in Mtb-infected cells, NCI-H292 cells were obtained by enzymatic disaggregation with 0.05% trypsin-EDTA in the different kinetic times mentioned above. Then, cells were treated with perm/fix kit (BD Biosciences, San Diego, CA) according to the manufacturer’s protocol. Subsequently, cells were incubated with 1% bovine serum albumin for 30 min and incubated with anti-RNase 7 antibody (1:10) (Santa Cruz Biotechnology, Dallas, TX) at 4°C for 1 h. Thereafter, cells were incubated with anti-goat IgG-PE antibody (1:10) (Santa Cruz Biotechnology) for 30 min at 4°C, fixed with 4% paraformaldehyde and analyzed with a Canto II flow cytometer using FACS-Diva software (BD Biosciences), and a standard gating strategy (Fig. S1 in the online supplementary material). Immunocytochemistry NCI-H292 cells were infected with Mtb strain H37Rv at MOI 5 in chamber slides as mentioned above. Subsequently, the slides were fixed with 4% paraformaldehyde, permeabilized with fix/perm and incubated for 1 h 30 min with 10% pig serum in HNC buffer (Hepes–NaCl2–CaCl2). Subsequently slides were incubated with anti-RNase 7 antibody, dilution 1:100 (Santa Cruz Biotechnology), overnight at 4°C, and then slides were incubated with biotinylated anti-goat antibody (Santa Cruz Biotechnology) diluted at 1:500 for 1 h 30 min at room temperature. After immunocytochemistry (ICC) slides were stained with the conventional Ziehl–Neelsen method and then analyzed by microscopy, where brown-stained cells were considered as positive for RNase 7. Confocal microscopy To assess co-localization of RNase 7 with mycobacteria, NCI-H292 cells (1 × 105) were seeded onto 4-well/chamber slides (Nunc Inc., Naperville, IL). After 18 h of adherence, cells were infected as described above with Mtb H37Rv stained with PKH67 (Sigma-Aldrich) for 1, 3 and 6 h to evaluate the co-localization of Mtb and RNase 7. Then, cells were fixed for 30 min with 4% paraformaldehyde. Subsequently, cells were permeabilized with perm/fix kit (BD Biosciences) and incubated with 3% bovine serum albumin for 30 min at room temperature to block the unspecific binding and then incubated with anti-RNase 7 antibody (1:100) (Santa Cruz Biotechnology) for 1 h at 4°C. After vigorous washing, cells were incubated with anti-goat-IgG-PE antibody (1:400) (Santa Cruz Biotechnology) and incubated for 30 min at room temperature. For nuclei staining 3 μM DAQ7 was used (Biostatus, Shepshed, UK) for 20 min at room temperature. Finally, cell preparations were mounted with Vectashield hard (Vector Laboratories, Burlingame, CA). Subcellular detection of RNase 7 by immunoelectronmicroscopy NCI-H292 cells were infected with Mtb as described above. After 6 h, infected cells were washed three times with PBS, fixed in 4% (v/v) paraformaldehyde and dissolved in 0.2 M Sörensen buffer (one vol. NaH2PO4*H2O, two vol. NaHPO4*7H2O), at pH 7.3, for 2 h at 4°C. Following a washing step of the cells with the Sörensen buffer, free aldehyde groups were blocked with 0.5 M ammonium chloride in PBS for 1 h. Fixed cells were then dehydrated in graded ethyl alcohols and embedded in LR-White hydrosoluble resin (London Resin Co., Reading, UK). Thin sections from 70 to 90 nm were placed on nickel grids. The grids were incubated overnight at 4°C with specific polyclonal goat anti-RNase 7 antibody (Santa Cruz Biotechnology) diluted to 1/200 in PBS with 1% bovine serum albumin and 0.5% Tween-20. After rinsing with PBS, the grids were incubated for 1 h at room temperature with donkey anti-goat IgG (Sigma Co., St Louis, MO) conjugated to 5 nm gold particles (Sigma) diluted 1/20 in PBS. The grids were stained with uranium salts (Electron Microscopy Sciences, Fort Washington, PA) and examined with an electron microscope FEI Technai G2 Spirit (Hillsboro, OR). As negative controls, the primary RNase 7 antibody was substituted by normal rabbit serum. Statistical analysis Statistical analyses were performed using the GraphPad Prism software for Mac (GraphPad Software version 6.01, San Diego, CA). Normal distribution was assessed using the Kolmogorov–Smirnov test for each data set, together with a non-parametric two-group comparison Mann–Whitney U or multiple comparison test of Kruskal–Wallis to identify differences among the groups. When statistical significance (P < 0.05) was found, a Dunn’s post test was performed. Two-sided P values of <0.05 were considered statistically significant. RESULTS Mycobacterium tuberculosis infection induces the expression of RNase 7 and downregulates the transcription of psoriasin in lung epithelial cells To determine if Mtb infection induced psoriasin, RNase 7 and sPLA2-IIA, mRNA expression in A549, bMDMs, MDMs and NCI-H292 was measured. Cells were infected at a MOI of 5 and after 1, 3, 6, 12, 18 and 24 h, cells were lysed and mRNA was obtained and analyzed for the expression of RNase 7, psoriasin and sPLA2-IIA. Both bMDMs from patients (n = 3, data not shown) and the MDM cell line (THP1) showed identical results: neither sPLA2-IIA, psoriasin nor RNase 7 were detected in infected cells (Fig. 1A, B and C, respectively). Similar results were obtained for A549 cells: none of the above antimicrobial peptides showed any gene transcription in all the experimental conditions analyzed (sPLA2-IIA, Fig. 1D; psoriasin, Fig. 1E; and RNase 7, Fig. 1F). Mtb-infected NCI-H292 cells did not show sPLA2-IIA gene expression (Fig. 1G), while a significant downregulation of psoriasin was detected at 1, 3, 12 and 24 h (P < 0.05), the lowest expression being at 18 h post-infection (P < 0.001, Fig. 1H). Regarding RNase 7, infection with Mtb led to an overexpression of this peptide mainly at 1, 3 and 6 h post-infection in NCI-H292 cells (P < 0.001), whereas after 12 and 18 h post-infection the induction was significantly lower (P < 0.05) when compared with non-infected cells (Fig. 1I). To assess whether other intracellular pathogens induced RNase 7 gene expression, NCI-H292 cells were infected with Salmonella typhi (clinical isolate) and analyzed for RNase 7 expression during the kinetic times. The results showed that S. typhi induces RNase 7 expression and the highest expression can be detected after 6 h of infection, which correlates with those results shown during Mtb infection (Fig. S2A in the online supplementary material). To evaluate which of both pathogens induced higher RNase 7 mRNA expression levels, results from S. typhi were compared vs. the results obtained from Mtb-infected cells under the same conditions. The results showed that infection with S. typhi induces up to 17.86 (± 6.98)-fold less RNase 7 mRNA expression levels at 6 h than the Mtb-infected cells. Similar results were observed for the rest of the kinetic points (Fig. S2B in the online supplementary material). Figure 1. View largeDownload slide RNase 7 is expressed during Mtb infection. MDMs, type II pneumocytes (A549) and airway epithelial cells (NCI-H292) were infected with Mtb and after 1, 3, 6, 12, 18 and 24 h mRNA was evaluated. Infected MDMs showed no expression for any of the HDPs studied (A–C). Similar results were obtained for type II pneumocytes (D–F). Airway epithelial cells showed no mRNA expression for sPLA2-IIA (G), whereas for psoriasin (H) and RNase 7 (I) mRNA expression change in these cells was detected. (Cp, crossing point or crossing threshold.) The positive control used for RNase 7 and psoriasin was obtained from psoriatic skin biopsies and the positive control for sPLA2-IIA was obtained from umbilical cord sections. *P < 0.05, ***P < 0.001. Data are presented as mean ± standard deviation. n = 6. Figure 1. View largeDownload slide RNase 7 is expressed during Mtb infection. MDMs, type II pneumocytes (A549) and airway epithelial cells (NCI-H292) were infected with Mtb and after 1, 3, 6, 12, 18 and 24 h mRNA was evaluated. Infected MDMs showed no expression for any of the HDPs studied (A–C). Similar results were obtained for type II pneumocytes (D–F). Airway epithelial cells showed no mRNA expression for sPLA2-IIA (G), whereas for psoriasin (H) and RNase 7 (I) mRNA expression change in these cells was detected. (Cp, crossing point or crossing threshold.) The positive control used for RNase 7 and psoriasin was obtained from psoriatic skin biopsies and the positive control for sPLA2-IIA was obtained from umbilical cord sections. *P < 0.05, ***P < 0.001. Data are presented as mean ± standard deviation. n = 6. Intracellular RNase 7 detection by flow cytometry Once it was determined that RNase 7 was upregulated during Mtb infection in airway epithelial cells NCI-H292, we determined the RNase 7 mean fluorescence intensity and percentage of positive cells by flow cytometry along the kinetics used in the mRNA expression experiments. The results indicated that after 6 h of infection the mean fluorescence intensity was higher than for non-infected cells (P < 0.01); similar results were observed after 3 and 12 h of infection (P < 0.05) (Fig. 2A). Intracellular RNase 7 was detected in all kinetic times, but only after 6 h with 39 ± 6% (P < 0.01) and after 3 h with 28 ± 8% (P < 0.05) was there statistical significance when compared with non-infected cells (Fig. 2B). Figure 2. View largeDownload slide Intracellular RNase 7 detection by flow cytometry. Mean fluorescence intensity for RNase 7 was evaluated in airway epithelial cells NCI-H292 (A) as well as percentage of RNase 7-positive cells (B). Data are presented as percentage of RNase 7-positive cells and as geometric mean of fluorescence intensity. *P < 0.05, **P < 0.01. Data are presented as mean ± standard deviation. n = 6. Figure 2. View largeDownload slide Intracellular RNase 7 detection by flow cytometry. Mean fluorescence intensity for RNase 7 was evaluated in airway epithelial cells NCI-H292 (A) as well as percentage of RNase 7-positive cells (B). Data are presented as percentage of RNase 7-positive cells and as geometric mean of fluorescence intensity. *P < 0.05, **P < 0.01. Data are presented as mean ± standard deviation. n = 6. RNase 7 associates intracellularly with Mycobacterium tuberculosis in infected airway epithelial cells Although we showed that Mtb induces RNase 7 upregulation, mainly after 6 h of infection, it was also important to determine whether RNase 7 was associated with intracellular Mtb. First, Mtb-infected cells were submitted for ICC analysis; subsequently slides were stained with Ziehl-Neelsen. Results showed that infected cells were positive for RNase 7 immunostaining (Fig. 3A); the Ziehl–Neelsen staining after ICC showed that apparently RNase 7 was associated with Mtb. It is of note that all infected cells produced RNase 7 (Fig. 3B). To assess the intracellular association between RNase 7 and Mtb, confocal microscopy was used. Results indicated that RNase 7 is produced constitutively by non-infected cells, and after 6 h of infection RNase 7 increased substantially. Fluorescence-labeled Mtb was detected intracellularly and was associated with RNase 7 at the three time-points studied. The main difference observed was the fluorescence intensity among times (Fig. 4). To evaluate the RNase 7 expression, we used a quantitative analysis by confocal microscopy, showing similar results to those obtained in the flow cytometry analysis (Fig. S3 in the online supplementary material). Co-localization was confirmed by ultrastructural immunolabeling at 6 h after infection; the results showed that RNase 7 was dispersed throughout the cytoplasm of bronchial epithelial cells NCI-H292, being more concentrated in vacuoles, and particularly high amounts associated with the Mtb cell wall (Fig. 5A and B); approximately 90 ± 5% of the total immunogold particles were associated with Mtb (data not shown). Some lysed mycobacteria associated with RNase 7 were found inside vacuoles (Fig. 5C). Figure 3. View largeDownload slide Immunocytochemistry in airway epithelial cells infected with Mtb for 6 h. Airway epithelial cells NCI-H292 were seeded onto chamber slides and infected for 6 h with Mtb and then fixed and submitted for ICC. Infected cells showed positive staining for RNase 7 (A, ×200), secondary antibody showed no unspecific binding (inset). After the ICC, cells were stained with Ziehl–Neelsen (B, ×1000): mycobacteria are traditionally stained in fuchsia (arrows) while immunostained cells remain brown. Microphotographs are representative of at least three independent experiments. Figure 3. View largeDownload slide Immunocytochemistry in airway epithelial cells infected with Mtb for 6 h. Airway epithelial cells NCI-H292 were seeded onto chamber slides and infected for 6 h with Mtb and then fixed and submitted for ICC. Infected cells showed positive staining for RNase 7 (A, ×200), secondary antibody showed no unspecific binding (inset). After the ICC, cells were stained with Ziehl–Neelsen (B, ×1000): mycobacteria are traditionally stained in fuchsia (arrows) while immunostained cells remain brown. Microphotographs are representative of at least three independent experiments. Figure 4. View largeDownload slide Immunofluorescence of RNase 7 and Mtb in airway epithelial cells NCI-H292. Mtb was stained with PKH67 and used to infect airway epithelial cells. At 1, 3 and 6 h post-infection, cells were fixed and incubated with anti-RNase 7 antibody for 1 h. Then cell preparations were incubated with anti-goat-IgG-PE antibody for 30 min. For nuclei staining, DAQ7 was used. On the merge lane, asterisks show immunostaining for RNase 7 and arrows show Mtb co-localization with RNase 7. Microphotographs are representative of at least three independent experiments. (×400). Figure 4. View largeDownload slide Immunofluorescence of RNase 7 and Mtb in airway epithelial cells NCI-H292. Mtb was stained with PKH67 and used to infect airway epithelial cells. At 1, 3 and 6 h post-infection, cells were fixed and incubated with anti-RNase 7 antibody for 1 h. Then cell preparations were incubated with anti-goat-IgG-PE antibody for 30 min. For nuclei staining, DAQ7 was used. On the merge lane, asterisks show immunostaining for RNase 7 and arrows show Mtb co-localization with RNase 7. Microphotographs are representative of at least three independent experiments. (×400). Figure 5. View largeDownload slide Subcellular association of RNase 7 and Mtb in NCI-H92 cells. Representative immunoelectronmicroscopy micrographs showing the subcellular localization of RNase 7 labeled with 5 nm gold particles (black dots). (A) Image shows cytoplasmic mild diffuse distribution of RNase 7, more concentration of this peptide in cytoplasmic vacuoles (V) and particularly strong labelling in the mycobacterium cell wall (arrow). Magnification, ×20,000. (B) Longitudinal section of intracellular Mycobacterium tuberculosis (M) showing co-localization with RNase 7 labeled with gold particles. Magnification, ×50,000. (C) Mycobacterium tuberculosis (M) inside cytoplasmic vacuole (V); a lysed mycobacterium is shown inside the same vacuole (L). Magnification ×30,000. Figure 5. View largeDownload slide Subcellular association of RNase 7 and Mtb in NCI-H92 cells. Representative immunoelectronmicroscopy micrographs showing the subcellular localization of RNase 7 labeled with 5 nm gold particles (black dots). (A) Image shows cytoplasmic mild diffuse distribution of RNase 7, more concentration of this peptide in cytoplasmic vacuoles (V) and particularly strong labelling in the mycobacterium cell wall (arrow). Magnification, ×20,000. (B) Longitudinal section of intracellular Mycobacterium tuberculosis (M) showing co-localization with RNase 7 labeled with gold particles. Magnification, ×50,000. (C) Mycobacterium tuberculosis (M) inside cytoplasmic vacuole (V); a lysed mycobacterium is shown inside the same vacuole (L). Magnification ×30,000. Supernatants from Mycobacterium tuberculosis-infected MDMs do not induce RNase 7 in airway epithelial cells To determine whether any molecule secreted by infected macrophages might induce RNase 7 in airway epithelial cells as a mechanism to promote innate immunity, MDMs were infected as described above. After 18 h of infection supernatants were collected and used immediately to stimulate epithelial cells (NCI-H292). Airway epithelial cells were grown in 24-well plates and left for 18 h to allow adhesion. After this time, the supernatant was discarded and substituted with supernatants from Mtb-infected MDMs; after 6 h of stimulation (given that RNase 7 expression was mostly detected at this time in previous experiments), cells were lysed with TRIzol and submitted for mRNA expression analyses. The results showed that there were no statistical differences between non-infected cells and cells stimulated with MDM supernatant (Fig. 6), suggesting that the induction of RNase 7 is dependent on the interaction between airway epithelial cells and Mtb. Figure 6. View largeDownload slide Airway epithelial cells treated with filtrated supernatants from Mtb-infected cells. MDMs were infected for 18 h with Mtb, then supernatant was collected, filtrated and subsequently transferred to NCI-H292 cells (EpCs + SN). For the ‘None’ condition, instead of using supernatants, NCI-H292 cells were stimulated with PBS and after 6 h cells were lysed and submitted for mRNA analysis for RNase 7 expression. Data are presented as mean ± standard deviation. n = 6. Figure 6. View largeDownload slide Airway epithelial cells treated with filtrated supernatants from Mtb-infected cells. MDMs were infected for 18 h with Mtb, then supernatant was collected, filtrated and subsequently transferred to NCI-H292 cells (EpCs + SN). For the ‘None’ condition, instead of using supernatants, NCI-H292 cells were stimulated with PBS and after 6 h cells were lysed and submitted for mRNA analysis for RNase 7 expression. Data are presented as mean ± standard deviation. n = 6. DISCUSSION Although there are approximately 122 HDPs (most of them identified by bioinformatic approaches), fewer than 20 have been associated with infectious diseases (Wang 2014). In the last decade, HDPs such as defensins and cathelicidin have been associated with the elimination of Mtb (Rivas-Santiago et al. 2005, 2008a,b; Liu et al. 2007). Nevertheless, there are other HDPs with clinical importance such as psoriasin, RNase 7 and sPLA2-IIA (Jia et al. 2014; Becknell and Spencer 2016; Tan and Goh 2017) that have not been studied in tuberculosis pathogenesis. To study the relationship of these peptides with innate immunity against tuberculosis, we first determined whether infection with Mtb increased the mRNA expression of RNase 7 and sPLA2-IIA in an in vitro model. Our results showed that neither MDMs from THP1 nor bMDMs (data not shown) showed mRNA expression for RNase 7 and sPLA2-IIA. Similar results were observed in A549 and NCI-H292 cells in the case of sPLA2-IIA. Although these antimicrobial proteins have been described in several infectious pathologies, during Mtb infection we could not detect RNase 7 or sPLA2-IIA upregulation. Indeed, we do not know why the mycobacteria did not induce sPLA2. It is plausible that the type of cells used for this study do not express sPLA2. In the case of psoriasin neither MDMs, bMDMs nor A549 cells expressed this peptide during infection with Mtb; however, NCI-H292 cells constitutively express psoriasin and mycobacterial infection downregulated the expression of this HDP showing statistical significance almost in all the studied times. This suggests that mycobacteria could use this mechanism to avoid innate immunity; however, further studies need to be done to unveil whether downregulation of psoriasin is important for Mtb pathogenesis and whether this phenomenon is related to mycobacterial virulence. Regarding RNase 7, we only found mRNA expression in NCI-H292 cells, which was importantly upregulated during the first 6 h after infection and then went back to basal expression. It has been reported that RNase 7 is an antimicrobial peptide produced mainly by epithelial cells (Becknell et al. 2015); the absence of RNase 7 in human monocytes has been showed previously (Becknell and Spencer 2016), and its expression in macrophages has not been documented. Based on our results, we can suggest that MDMs do not express this peptide. Our results showed that RNase 7 gene expression is induced at early time points and after 24 h its expression is similar to that in non-infected cells. Similar studies have reported that RNase 7 was induced by IL-1β in keratinocytes, showing that higher expression levels can be detected after 24 h of stimulation (Becknell and Spencer 2016), whereas our results showed that the highest expression of RNase 7 can be observed at 6 h post-infection. This could be explained because Mtb activates different signaling pathways such as those induced by (Toll-like receptor) TLR-2, TLR-4 and TLR-9 mostly a few hours after infection (Kleinnijenhuis et al. 2011), which is the main pathway to induce RNase 7 expression. To assess whether any other molecule secreted by Mtb-infected macrophages such as cytokines or other AMPs induced RNase 7 mRNA expression, epithelial cells were treated with the filtered supernatant from infected MDMs. Our results suggest that the Mtb by itself induced RNase 7 production and that other molecules produced during infection are not involved. To determine whether mRNA expression was translated into protein, we detected intracellular RNase 7 by cytofluorometry and immunocytochemistry. Results from both studies were consistent, showing a peak in RNase 7 production after 6 h of infection in NCI-H292 cells. Then, we evaluated whether the produced RNase 7 was associated with intracellular Mtb, which could suggest a direct antimicrobial effect on Mtb. A mild association was seen in ICC-stained slides subsequently stained with Ziehl–Neelsen, which was confirmed by confocal microscopy and immunoelectron microscopy. Previous studies have described the structure of RNase 7 and its electrostatic properties, and also the antimicrobial mechanism of RNase 7 generated by the high positive charge (Huang et al. 2007). Antimicrobial activity is generated by electrostatic interaction produced by the high cationicity of RNase 7 with the mycobacterial negative phospholipids, mainly by peptidoglycan, which is one of the main components of the Mtb cell wall (Zhang, Dyer and Rosenberg 2003; Huang et al. 2007). However, even though RNase 7 evidently associates with Mtb during infection and we found lysed mycobacteria associated with RNase 7 by ultrastructural analysis, it cannot be certain that this peptide is involved in mycobacteria elimination. It remains unknown whether deficient RNase 7 production increases infection risk or if increased RNase 7 production shields the host from microbial challenge. Further studies need to be done to elucidate this. In conclusion, our studies suggest for the first time that Mtb infection induces the production of RNase 7 in NCI-H292 cells (airway epithelial cells) and that the produced HDP binds to the Mtb cell wall. These results propose another mechanism used by the host to counterattack Mtb infection and new insights to understand the innate immunity in tuberculosis. The relevance of RNase 7 in the resistance to tuberculosis needs to be further investigated. It is important to explore whether RNase 7 could be used for the development of new therapies or as a biomarker for diagnostics. SUPPLEMENTARY DATA Supplementary data are available at FEMSPD online. Acknowledgements We acknowledge Julio Castañeda-Delgado for confocal microscopy technical support and Alejandra Montoya-Rosales for her invaluable assistance in BSL3 facilities. FUNDING This work was supported by Mexican Institute of Social Security (IMSS) FIS/IMSS/PROT/G14/1318. BRS has a scholarship from Fundación IMSS. Conflict of interest. None declared. REFERENCES Andresen E, Lange C, Strodthoff D et al.   S100A7/psoriasin expression in the human lung: unchanged in patients with COPD, but upregulated upon positive S. aureus detection. BMC Pulm Med  2011; 11: 10. Google Scholar CrossRef Search ADS PubMed  Becknell B, Eichler TE, Beceiro S et al.   Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract. Kidney Int  2015; 87: 151– 61. 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Pathogens and DiseaseOxford University Press

Published: Mar 1, 2018

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