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hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity
J Infect Chemother xxx (2017) 1e6
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Original Article
hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity Tomoyasu Nishimura a, *, 1, Eiko Tamizu b, 1, Shunsuke Uno b, Yoshifumi Uwamino c, Hiroshi Fujiwara b, Kazumi Nishio d, Yasushi Nakano d, Hirofumi Shiono e, Ho Namkoong f, Yoshihiko Hoshino g, Satoshi Iwata h, Naoki Hasegawa b, ** a
Health Center, Keio University, Japan Center for Infectious Diseases and Infection Control, Keio University School of Medicine, Japan Department of Laboratory Medicine, Keio University School of Medicine, Japan d Department of Pulmonary Medicine, Kawasaki Municipal Ida Hospital, Japan e Medical Business Development Division, Nikon Corporation, Japan f Department of Pulmonary Medicine, Eiju General Hospital, Japan g Leprosy Research Center, National Institute of Infectious Diseases, Japan h Department of Infectious Diseases, Keio University School of Medicine, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2017 Received in revised form 20 July 2017 Accepted 31 July 2017 Available online xxx
MicroRNA (miRNA) has been recently recognized as a biomarker of various diseases; however, there are no known miRNAs associated with Mycobacterium avium complex (MAC) pulmonary disease. In addition, there are no known biomarkers to precisely reflect disease activity after the diagnosis of MAC pulmonary disease. Thus, we sought to identify a miRNA which is a candidate biomarker of MAC pulmonary disease activity. Serum hsa-miR-346 concentrations of 16 patients with M. avium pulmonary disease were significantly higher than those of 16 healthy controls (p ¼ 0.047). The secretion of hsa-miR-346 increased in a multiplicity of infection-dependent manner in M. avium-infected macrophages. Serum hsa-miR-346 levels of 5 patients with bacterial conversion at the end of follow-up were significantly lower than those at the beginning of the follow-up (p ¼ 0.043). In addition, the longitudinal change in serum hsa-miR-346 concentration correlated with bacterial load in 2 patients with M. avium pulmonary disease. Based on our results, it is supposed that MAC-infected macrophages in pulmonary lesions produce hsa-miR-346, which is then secreted into the bloodstream. The magnitude of this process could be quantitatively controlled by the bacterial load, suggesting that serum hsa-miR-346 is a potentially useful biomarker of MAC pulmonary disease activity. © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Mycobacterium avium complex MicroRNA Macrophage
1. Introduction Although the incidence of Mycobacterium avium complex (MAC) pulmonary disease continues to increase worldwide [1], the pathogenesis of MAC pulmonary disease remains unclear. Some patients with long-term MAC pulmonary disease show slight deterioration because their disease activities are stable; other
* Corresponding author. 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. ** Corresponding author. 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail addresses: [email protected] (T. Nishimura), [email protected] (N. Hasegawa). 1 These authors contributed equally to this work.
patients progress toward death faster because their disease activities are high despite intense combination chemotherapy. One of the difficulties in the management of this disease is the lack of useful biomarkers reflecting disease activity after the diagnosis of MAC pulmonary disease [2]. MAC pulmonary disease activity is typically estimated based on MAC culture results using sputum samples. However, it is difficult to precisely evaluate disease activity by microbiological examination alone because MAC culture results are relatively qualitative and dependent on the quality of the sputum sample. MicroRNAs (miRNAs) are small, non-coding RNAs that are associated with the pathophysiology of several diseases via its posttranscriptional regulation of gene expression within cells. Certain
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Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015
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miRNAs associated with carcinogenesis are used in diagnosis and therapy as biomarkers to predict cancer development [3]. Other studies reported miRNAs associated with infectious diseases [4,5]; however, there are no known miRNAs associated with MAC pulmonary disease. It has recently been determined that miRNAs produced within cells are secreted into the blood stream and subsequently circulate in the body [6,7]. Therefore, it is possible to evaluate disease activity by measuring disease-associated miRNA levels in serum. Here, we performed a preliminary study based on the hypothesis that specific serum miRNAs would be associated with the pathophysiology of MAC pulmonary disease and could be used as biomarkers to reflect disease activity. 2. Materials and methods 2.1. Study population To discover the serum miRNAs associated with MAC pulmonary disease, we conducted a study on 16 female patients with M. avium pulmonary disease who were admitted to the Keio University Hospital between July 2013 and June 2016, on the basis of the official statement for nontuberculous mycobacterial (NTM) diseases established by the American Thoracic Society (ATS) and Infectious Disease Society of America (IDSA) [2]. The patients in the study group had no history of other diseases and had never been treated for mycobacterial disease. All patients had nodular bronchiectatic M. avium pulmonary disease without apical fibrocavitary lesions. Sixteen age-matched healthy female volunteers were used as the control group. The subjects in the control group had no history of any disease, and we confirmed they had no lesions suggestive of NTM pulmonary disease by chest computed tomography. The mean age of patients in the study group was 56 years, ranging between 39 and 68 years. The mean age of healthy volunteers was 50 years, ranging between 36 and 63 years. The mean body mass index (BMI) of patients and healthy volunteers was 19.1 and 19.6 kg/m2, respectively, ranging between 16.2 and 24.7 kg/m2 and between 17.5 and 22.2 kg/m2, respectively. There were no statistically significant differences in age or BMI between patients and healthy volunteers (p ¼ 0.091 and 0.291, respectively). To determine if serum miRNA levels reflect MAC pulmonary disease activity, we conducted a study on 11 female patients with M. avium pulmonary disease who were admitted to the Keio University Hospital between July 2013 and June 2016, according to the official statement for NTM diseases established by ATS and IDSA. The patients had never been treated for mycobacterial disease and were initially administered chemotherapy for more than 6 months, which included a combination of clarithromycin (CLR), rifampicin (RIF), and ethambutol (EMB). Of the 11 patients, five whose cultures showed more than three times consecutive negative results after the chemotherapy were categorized as patients with bacterial conversion, and six patients whose cultures did not show negative after the chemotherapy were categorized as patients without bacterial conversion. Blood samples were obtained from them at the beginning and at the end of follow-up. The mean age of the patients with bacterial conversion was 61 years, ranging between 55 and 66 years. The mean age of the patients without bacterial conversion was 66 years, ranging between 51 and 74 years. The mean BMI of the patients with and without bacterial conversion was 21.3 and 19.4 kg/m2, respectively, ranging between 19.8 and 24.7 kg/m2 and between 17.0 and 22.3 kg/m2, respectively. There were no statistically significant differences in age or BMI of the two groups (p ¼ 0.240 and 0.177, respectively). In addition, we longitudinally measured the serum miRNA concentrations and sputum culture results in 2 female patients with M. avium pulmonary disease.
Blood samples were collected after the subjects in the study group and healthy volunteers agreed to participate in the study and provided their informed consent. This study was performed in compliance with the Declaration of Helsinki and was approved by the institutional ethics review committee for human research at the Keio University School of Medicine and hospital (No. 20130134). 2.2. Microarray analysis As previously described [8], miRNA expression in human serum was measured using the 3D-Gene miRNA microarray platform (Toray Industries, Inc., Tokyo, Japan). Briefly, miRNA isolated from 300 mL of human serum was hybridized to 3D-Gene human miRNA oligo chips ver.20.0 containing 2555 miRNAs, or oligo chips ver.21 containing 2565 miRNAs. MiRNA expression was scaled by global normalization, and the resultant miRNA levels were analyzed. 2.3. RNA isolation and quantitative real-time polymerase chain reaction RNA was isolated from 300 mL of human serum using NucleoSpin miRNA Plasma (Macherey-Nagel, Düren, Germany), according to the manufacturer's protocol. RNA was eluted in 35 mL of RNasefree water. To allow for normalization of the sample-to-sample variation during RNA isolation, 1 1013 mol of synthetic Caenorhabditis (C.) elegans miRNA 39 (cel-miR-39, Bioneer, Daejeon, Republic of Korea) was added to each sample. Eluted RNA was reverse transcribed using a TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA), miRNA-specific stem-loop primers (Thermo Fisher Scientific), and Gene Amp polymerase chain reaction (PCR) System 9700 Thermal Cycler (Thermo Fisher Scientific) at 16 C for 30 min, 42 C for 30 min, and 85 C for 5 min, respectively, according to the manufacturer's protocol. Each reaction mixture for quantitative real-time PCR (qPCR) contained 10 mL of TaqMan Universal Master Mix II with UNG (Thermo Fisher Scientific) and 1 mL of a miRNA-specific primer/probe mix of TaqMan MicroRNA Assays (Thermo Fisher Scientific) in a total volume of 20 mL qPCR was completed using the Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) at 50 C for 2 min and 95 C for 10 min, followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. Data were analyzed using StepOne Software v.2.2.2 (Thermo Fisher Scientific), with the automatic Ct setting for assigning baselines and thresholds for Ct determination. A dilution series using chemically synthesized miRNAs was used to generate a standard curve that permitted the absolute quantification of miRNA. Each sample was normalized using spiked-in cel-miR-39 or hsa-miR-16-5p as controls [6,7]. 2.4. Cells and culture As previously described [9], human macrophages were obtained by culturing human peripheral blood mononuclear cells (PBMCs) from transfusion blood provided by the Japanese Red Cross Society (Tokyo, Japan) for 9 days in Iscove's modified Dulbecco's medium (Thermo Fisher Scientific) containing 10% (v/v) human AB serum (Cosmo Bio Co., Ltd., Tokyo, Japan) at a density of 6.0 106 cells/ 2 mL/well in 6-well cluster plates (Corning, NY, USA). The human bronchial epithelial cell line BEAS-2B was used as a model of airway epithelial cells. BEAS-2B cells were purchased from the European Collection of Cell Cultures (Public Health England, London, UK) and cultured in bronchial epithelial growth medium (Lonza, Basel, Switzerland). They were plated at a density of 1.0 106 cells/2 mL/well in 6-well cluster plates (Corning) and cultured to 90% confluence.
Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015
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The adherent macrophages and BEAS-2B cells were cultured with the M. avium strain 104 at a multiplicity of infection (MOI) of 10:1 or 50:1 and then washed with phosphate-buffered saline (PBS) to remove nonadherent bacilli. A fresh medium was added to the macrophage and BEAS-2B cultures after incubation for 4 h and 24 h, respectively. Supernatants were collected at 24 h after infection to measure secretory miRNA levels. The adherent macrophages were cultured with latex beads coated with FITC-labeled rabbit IgG (Cayman Chemical, MI, USA) at a final dilution of 1:500 or 1:100 and then washed with PBS to remove nonadherent beads. Fresh medium was added to the macrophage culture after incubation for 4 h. For measuring secretory miRNA levels, supernatants were collected at 24 h after addition of latex beads.
2.5. Statistical analyses The results are expressed as the mean ± SEM. The t-test or the ManneWhitney U test was used to evaluate statistical differences between two groups. The paired t-test was used to evaluate statistical differences between the groups at the beginning and the end of follow-up of the patients. Comparisons among three experimental groups were assessed with an analysis of variance followed by the Fisher's least significant difference test using IBM
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SPSS statistics 23 (IBM, NY, USA). A p value <0.05 was considered statistically different.
3. Results 3.1. hsa-miR-346 is a circulating miRNA associated with Mycobacterium avium complex pulmonary disease Two independent microarray analyses using serum samples (experiment 1 consisted of five M. avium pulmonary disease patients and five healthy controls, and experiment 2 consisted of four M. avium pulmonary disease patients and four healthy controls) repeatedly revealed that the levels of four miRNAs, including hsamiR-346, hsa-miR-532-3p, hsa-miR-4446-3p, and hsa-miR-47223p, were significantly higher (p < 0.05) in patients with MAC pulmonary disease than those found in healthy controls. To confirm the miRNAs associated with MAC pulmonary disease, we compared the serum concentrations of these miRNAs in 16 patients with M. avium pulmonary disease with those from 16 healthy controls using qPCR (Fig.1). The concentration of hsa-miR-346 was significantly higher in subjects in the MAC pulmonary disease group than in the healthy control group (mean ± SEM; 1.63 1016 ± 3.28 1017 M, and 9.42 1017 ± 1.81 1017 M, respectively, p ¼ 0.047). Unfortunately, we could not measure serum concentration of hsa-miR-
Fig. 1. Scattergrams of serum hsa-miR-346 (A), hsa-miR-532-3p (B) and hsa-miR-4446-3p (C) concentrations in patients with Mycobacterium avium complex pulmonary disease (MAC, n ¼ 16) and in subjects in control group (Control, n ¼ 16). The horizontal lines show the mean concentrations in each group. Comparisons between subjects in the MAC pulmonary disease group and in the control group were assessed using the ManneWhitney U test.
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4722-3p by qPCR. It was technically difficult to make the miRNAspecific primers of hsa-miR-4722-3p because it had the potential to create self-dimers due to high G þ C content. 3.2. Human macrophages infected with Mycobacterium avium complex show increased hsa-miR-346 production The primary host cells harboring MAC are macrophages while MAC can infect bronchial epithelial cells in MAC pulmonary disease [10]. Therefore, we inoculated macrophages derived from human PBMCs and BEAS-2B cells with M. avium strain 104 and then measured the level of hsa-miR-346 in their supernatants at 24 h after infection (Fig. 2). The level of hsa-miR-346 was higher in the supernatant of MAC-infected macrophages when compared with uninfected macrophages (hsa-miR-346/has-miR-16-5p ratios of infected cells at MOI of 10 and 50 compared with uninfected cells
[mean ± SEM]: 1.48 ± 0.170 [p ¼ 0.048] and 2.18 ± 0.346 [p ¼ 0.027], respectively). There was no significant difference in hsa-miR-346 levels of MAC-infected and uninfected BEAS-2B cells (hsa-miR-346/ has-miR-16-5p ratios of infected cells at MOI 10 and 50 compared with uninfected cells [mean ± SEM]: 0.962 ± 0.111 [p ¼ 0.754] and 0.727 ± 0.132 [p ¼ 0.130], respectively). To determine if hsa-miR-346 secretion in macrophages is induced by MAC infection, we treated human macrophages with latex beads coated with FITC-labeled rabbit IgG and then measured the concentration of hsa-miR-346 in their supernatants at 24 h after addition. There was no significant difference in hsa-miR-346 levels of latex beads-treated and latex beads-untreated human macrophages (hsa-miR-346/has-miR-165p ratios of treated cells at a final dilution 1:500 or 1:100 compared with untreated cells [mean ± SEM]: 1.37 ± 0.236 [p ¼ 0.174) and 1.18 ± 0.181 [p ¼ 0.374], respectively). Thus, MAC-infected macrophages are the main secretors of hsa-miR-346.
Fig. 2. hsa-miR-346 concentrations in the supernatants of human macrophages (A) and BEAS-2B cells (B) with and without Mycobacterium avium complex (MAC) infection, and human macrophages treated with latex beads coated with FITC-labeled rabbit IgG (C). Multiplicity of infection (MOI) with MAC is 10:1 or 50:1, and final concentration of latex beads is 1:500 or 1:100 dilution. Ratios between hsa-miR-346 and hsa-miR-16-5p concentrations are shown as fold changes relative to uninfected human macrophages, BEAS-2B cells, or untreated human macrophages. The data from three independent experiments are shown as the mean ± SEM. Comparisons between MAC-infected and MAC-uninfected human macrophages and BEAS-2B cells, and latex beads-treated and latex beads-untreated human macrophages were assessed using an ANOVA followed by the Fisher's least significant difference test.
Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015
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3.3. Serum hsa-miR-346 levels correlate with bacterial load in Mycobacterium avium complex pulmonary disease To determine if the concentration of hsa-miR-346 in the serum reflects MAC pulmonary disease activity, we compared serum hsamiR-346 levels at the end and the beginning of follow-up in M. avium pulmonary disease patients with and without bacterial conversion (Fig. 3). In patients with and without bacterial conversion, serum hsa-miR-346 levels were significantly lower and higher at the end of follow-up than at the beginning of follow-up, respectively (hsa-miR-346 ratios at the end of follow-up compared with those at the beginning of follow-up [mean ± SEM]: 0.604 ± 0.136 [p ¼ 0.043] and 1.40 ± 0.138 [p ¼ 0.034], respectively). In addition, we evaluated the correlation between serum hsa-miR-346 concentrations and sputum culture results in two patients. Case 1: A 55 year-old woman with a small amount of sputum visited our hospital. She had no history of other diseases and had never been treated for mycobacterial disease. We diagnosed her with a nodular bronchiectatic form of M. avium pulmonary disease, and anti-mycobacterial therapy was initiated with three antibiotics: 600 mg/day of RIF, 750 mg/day of EMB, and 800 mg/day of CLR. The serum hsa-miR-346 concentration decreased as sputum cultures results converted from positive to negative 9 months after the initiation of therapy (Fig. 4A). Case 2: A 66 year-old woman with a productive cough visited our hospital. She had no history of other diseases and had never Fig. 4. The longitudinal changes of sputum culture results and serum hsa-miR-346 concentrations in patients with Mycobacterium avium complex (MAC) pulmonary disease after initiation of therapy. RIF: rifampicin, EMB: ethambutol, CLR: clarithromycin, AMK: amikacin, (þ): positive sputum culture, (): negative sputum culture.
been treated for mycobacterial disease. We diagnosed her with a nodular bronchiectatic form of M. avium pulmonary disease, and anti-mycobacterial therapy was initiated with four antibiotics: 600 mg/day of RIF, 500 mg/day of EMB, 800 mg/day of CLR, and 600 mg/every other day of amikacin (AMK). The serum hsa-miR-346 concentration decreased as sputum cultures results converted from positive to negative 4 months after the initiation of therapy (Fig. 4B). Treatment with AMK was discontinued at this time. The serum hsa-miR-346 concentration increased as the sputum cultures reconverted from negative to positive at 30 months after the initiation of therapy. AMK was re-administered 32 months after the initiation of therapy, and sputum cultures converted from positive to negative 3 months after the re-administration of AMK. The serum hsa-miR-346 concentration decreased with the change in sputum culture results. Thus, serum hsa-miR-346 concentrations correlate with the disease activity estimated by sputum culture results. 4. Discussion
Fig. 3. The changes in serum hsa-miR-346 concentrations of 11 patients with Mycobacterium avium complex pulmonary diseases with treatment at the beginning and the end of follow-up, including five patients with bacterial conversion (A) and six patients without bacterial conversion (B). The serum hsa-miR-346 concentrations at the end of follow-up were shown as fold changes relative to that at the beginning of the followup. The data are shown as the mean ± SEM. Comparisons between the end and the beginning of follow-up were assessed using the paired t-test.
This pilot study is the first report on circulating human miRNAs associated with MAC pulmonary disease. On the basis of our results, it is supposed that MAC-infected macrophages in pulmonary lesions produce hsa-miR-346, which then reached the bloodstream. hsa-miR-346 secretion from MAC-infected macrophages was dependent on MOI, indicating that the degree of hsa-miR-346 secretion from pulmonary macrophages could be quantitatively controlled by the bacterial load in pulmonary lesions. It is reasonable to assume that the serum hsa-miR-346 concentration correlates with MAC pulmonary disease activity. In addition, we confirmed that the serum hsa-miR-346 concentrations correlated with the disease activity estimated by sputum culture results.
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Therefore, hsa-miR-346 is considered to be a candidate of serum biomarker for MAC pulmonary disease activity. A definitive diagnosis of MAC pulmonary disease is essentially based on multiple culture confirmations using respiratory specimens of the patients, and we have already reported the usefulness of a serum antibody test against a cell wall component for the diagnosis of MAC pulmonary disease [11]. However, there are no known sero-biomarkers to precisely reflect disease activity after diagnosis. In this regard, our current data show the potential of hsa-miR-346 as a sero-biomarker of disease activity. Therefore, sequential measurements of hsa-miR-346 levels will be useful in the management of the disease such as in taking decisions on the indication of chemotherapy or efficacy of treatment after diagnosis. Previous reports showed that hsa-miR-346 downregulated the expression of vitamin D receptor (VDR) in PBMCs, epithelial cells and smooth muscle cells [12e14]. However, it remains unclear how hsa-miR-346 plays a role in the pathophysiology of MAC pulmonary disease. Li et al. showed that hsa-miR-346 plays a role in the pathophysiology of inflammatory bowel disease [13]. Tumor necrosis factor (TNF)-a induces apoptosis of intestinal mucosal cells, which resulted in intestinal mucosal damage and ultimately inflammatory bowel disease. TNF-a-induced apoptosis of intestinal mucosal cells was blocked by the binding of 1,25-dihydroxyvitamin D (1,25(OH)2D3) to the VDR. Notably, hsa-miR-346 suppresses VDR expression, resulting in the acceleration of TNF-a-induced apoptosis. Pathological and radiological findings of MAC pulmonary disease revealed that bronchiectasis and bronchiolitis were caused by the destruction of the bronchial structure [15]. Thus, hsa-miR346 secreted from MAC-infected macrophages may accelerate the TNF-a-induced apoptosis of airway epithelial cells, leading to bronchiectasis or bronchiolitis. There are several critical limitations to this study. First, the small number of each group is a major limitation. Further clinical studies involving more cases have to be planned to confirm to our findings. Second, it remains unclear if hsa-miR-346 is specific for MAC pulmonary disease and is a candidate of diagnostic biomarker for the disease. Although currently there are no reports showing that hsamiR-346 is associated with the pathophysiology of infectious diseases, including mycobacterial diseases, or that hsa-miR-346 can act as a biomarker of disease activity, we will measure serum hsamiR-346 concentrations in patients with other diseases. Third, it remains unclear if hsa-miR-4722-3p is also a potential serum biomarker of MAC pulmonary disease activity. Although we could not measure hsa-miR-4722-3p concentration using qPCR, we will validate serum concentration of hsa-miR-4722-3p by a different way from qPCR. In conclusion, hsa-miR-346 is secreted from MAC-infected macrophages, and its serum levels could be a potentially easy and useful biomarker of MAC pulmonary disease activity. Authorship statement TN and ET designed the study and did the experiments, and collected, analyzed and interpreted data, and drafted the manuscript. SU, YU, HF, KN and YN recruited subjects, collected clinical samples, interpreted data, and revised the manuscript. HS, HN, YH and SI contributed to interpretation of data. NH conceived of and
designed the study, analyzed and interpreted the data, and revised the manuscript. All authors meet the ICMJE authorship criteria. Acknowledgments The authors thank the healthy subjects who volunteered for this study, and Dr. Takemasa Takii for the generous gift of the M. avium strain 104. The authors are grateful to Dr. Heinz G. Remold from the Brigham and Women's Hospital at Harvard Medical School (MA, USA) for his critical reading of our manuscript. This work was supported by the Japan Society for the Promotion of Science [KAKENHI 16K09942 to T.N. and 25461520 and 16K09941 to N.H.], the Daiwa Securities Health Foundation [T.N.], and the Kurozumi Medical Foundation [T.N.]. Conflicts of interests. This study was completed as part of our collaborative research with Nikon Corporation (Minato-ku, Tokyo, Japan). References [1] Prevots DR, Marras TK. Epidemiology of human pulmonary infection with nontuberculous mycobacteria: a review. Clin Chest Med 2015;36:13e34. [2] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175(4):367e416. [3] Montani F, Marzi MJ, Dezi F, Dama E, Carletti RM, Bonizzi G, et al. miR-Test: a blood test for lung cancer early detection. J Natl Cancer Inst 2015;107. djv063. [4] Murakami Y, Kawada N. MicroRNAs in hepatic pathophysiology. Hepatol Res 2017;47:60e9. [5] Xiao B, Liu Z, Li BS, Tang B, Li W, Guo G, et al. Induction of microRNA-155 during Helicobacter pylori infection and its negative regulatory role in the inflammatory response. J Infect Dis 2009;200:916e25. [6] Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, PogosovaAgadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513e8. [7] Shiotani A, Murao T, Kimura Y, Matsumoto H, Kamada T, Kusunoki H, et al. Identification of serum miRNAs as novel non-invasive biomarkers for detection of high risk for early gastric cancer. Br J Cancer 2013;109:2323e30. [8] Konishi H, Ichikawa D, Komatsu S, Shiozaki A, Tsujiura M, Takeshita H, et al. Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma. Br J Cancer 2012;106:740e7. [9] Nishimura T, Zhao X, Gan H, Koyasu S, Remold HG. The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis. FASEB J 2013;27:3827e36. [10] Honda JR, Knight V, Chan ED. Pathogenesis and risk factors for nontuberculous mycobacterial lung disease. Clin Chest Med 2015;36:1e11. [11] Nishimura T, Hasegawa N, Fujita Y, Yano I, Ishizaka A. Serodiagnostic contributions of antibody titers against mycobacterial lipid antigens in Mycobacterium avium complex pulmonary disease. Clin Infect Dis 2009;49:529e35. [12] Yi B, Huang J, Zhang W, Li AM, Yang SK, Sun J, et al. Vitamin D receptor downregulation is associated with severity of albuminuria in type 2 diabetes patients. J Clin Endocrinol Metab 2016;101:4395e404. [13] Chen Y, Du J, Zhang Z, Liu T, Shi Y, Ge X, et al. MicroRNA-346 mediates tumor necrosis factor alpha-induced downregulation of gut epithelial vitamin D receptor in inflammatory bowel diseases. Inflamm Bowel Dis 2014;20: 1910e8. [14] Martinez-Moreno JM, Herencia C, Montes de Oca A, Munoz-Castaneda JR, Rodriguez-Ortiz ME, Diaz-Tocados JM, et al. Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells. FASEB J 2016;30:1367e76. [15] Fujita J, Ohtsuki Y, Suemitsu I, Shigeto E, Yamadori I, Obayashi Y, et al. Pathological and radiological changes in resected lung specimens in Mycobacterium avium intracellulare complex disease. Eur Respir J 1999;13:535e40.
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Original Article
hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity Tomoyasu Nishimura a, *, 1, Eiko Tamizu b, 1, Shunsuke Uno b, Yoshifumi Uwamino c, Hiroshi Fujiwara b, Kazumi Nishio d, Yasushi Nakano d, Hirofumi Shiono e, Ho Namkoong f, Yoshihiko Hoshino g, Satoshi Iwata h, Naoki Hasegawa b, ** a
Health Center, Keio University, Japan Center for Infectious Diseases and Infection Control, Keio University School of Medicine, Japan Department of Laboratory Medicine, Keio University School of Medicine, Japan d Department of Pulmonary Medicine, Kawasaki Municipal Ida Hospital, Japan e Medical Business Development Division, Nikon Corporation, Japan f Department of Pulmonary Medicine, Eiju General Hospital, Japan g Leprosy Research Center, National Institute of Infectious Diseases, Japan h Department of Infectious Diseases, Keio University School of Medicine, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2017 Received in revised form 20 July 2017 Accepted 31 July 2017 Available online xxx
MicroRNA (miRNA) has been recently recognized as a biomarker of various diseases; however, there are no known miRNAs associated with Mycobacterium avium complex (MAC) pulmonary disease. In addition, there are no known biomarkers to precisely reflect disease activity after the diagnosis of MAC pulmonary disease. Thus, we sought to identify a miRNA which is a candidate biomarker of MAC pulmonary disease activity. Serum hsa-miR-346 concentrations of 16 patients with M. avium pulmonary disease were significantly higher than those of 16 healthy controls (p ¼ 0.047). The secretion of hsa-miR-346 increased in a multiplicity of infection-dependent manner in M. avium-infected macrophages. Serum hsa-miR-346 levels of 5 patients with bacterial conversion at the end of follow-up were significantly lower than those at the beginning of the follow-up (p ¼ 0.043). In addition, the longitudinal change in serum hsa-miR-346 concentration correlated with bacterial load in 2 patients with M. avium pulmonary disease. Based on our results, it is supposed that MAC-infected macrophages in pulmonary lesions produce hsa-miR-346, which is then secreted into the bloodstream. The magnitude of this process could be quantitatively controlled by the bacterial load, suggesting that serum hsa-miR-346 is a potentially useful biomarker of MAC pulmonary disease activity. © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Mycobacterium avium complex MicroRNA Macrophage
1. Introduction Although the incidence of Mycobacterium avium complex (MAC) pulmonary disease continues to increase worldwide [1], the pathogenesis of MAC pulmonary disease remains unclear. Some patients with long-term MAC pulmonary disease show slight deterioration because their disease activities are stable; other
* Corresponding author. 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. ** Corresponding author. 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail addresses: [email protected] (T. Nishimura), [email protected] (N. Hasegawa). 1 These authors contributed equally to this work.
patients progress toward death faster because their disease activities are high despite intense combination chemotherapy. One of the difficulties in the management of this disease is the lack of useful biomarkers reflecting disease activity after the diagnosis of MAC pulmonary disease [2]. MAC pulmonary disease activity is typically estimated based on MAC culture results using sputum samples. However, it is difficult to precisely evaluate disease activity by microbiological examination alone because MAC culture results are relatively qualitative and dependent on the quality of the sputum sample. MicroRNAs (miRNAs) are small, non-coding RNAs that are associated with the pathophysiology of several diseases via its posttranscriptional regulation of gene expression within cells. Certain
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Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015
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miRNAs associated with carcinogenesis are used in diagnosis and therapy as biomarkers to predict cancer development [3]. Other studies reported miRNAs associated with infectious diseases [4,5]; however, there are no known miRNAs associated with MAC pulmonary disease. It has recently been determined that miRNAs produced within cells are secreted into the blood stream and subsequently circulate in the body [6,7]. Therefore, it is possible to evaluate disease activity by measuring disease-associated miRNA levels in serum. Here, we performed a preliminary study based on the hypothesis that specific serum miRNAs would be associated with the pathophysiology of MAC pulmonary disease and could be used as biomarkers to reflect disease activity. 2. Materials and methods 2.1. Study population To discover the serum miRNAs associated with MAC pulmonary disease, we conducted a study on 16 female patients with M. avium pulmonary disease who were admitted to the Keio University Hospital between July 2013 and June 2016, on the basis of the official statement for nontuberculous mycobacterial (NTM) diseases established by the American Thoracic Society (ATS) and Infectious Disease Society of America (IDSA) [2]. The patients in the study group had no history of other diseases and had never been treated for mycobacterial disease. All patients had nodular bronchiectatic M. avium pulmonary disease without apical fibrocavitary lesions. Sixteen age-matched healthy female volunteers were used as the control group. The subjects in the control group had no history of any disease, and we confirmed they had no lesions suggestive of NTM pulmonary disease by chest computed tomography. The mean age of patients in the study group was 56 years, ranging between 39 and 68 years. The mean age of healthy volunteers was 50 years, ranging between 36 and 63 years. The mean body mass index (BMI) of patients and healthy volunteers was 19.1 and 19.6 kg/m2, respectively, ranging between 16.2 and 24.7 kg/m2 and between 17.5 and 22.2 kg/m2, respectively. There were no statistically significant differences in age or BMI between patients and healthy volunteers (p ¼ 0.091 and 0.291, respectively). To determine if serum miRNA levels reflect MAC pulmonary disease activity, we conducted a study on 11 female patients with M. avium pulmonary disease who were admitted to the Keio University Hospital between July 2013 and June 2016, according to the official statement for NTM diseases established by ATS and IDSA. The patients had never been treated for mycobacterial disease and were initially administered chemotherapy for more than 6 months, which included a combination of clarithromycin (CLR), rifampicin (RIF), and ethambutol (EMB). Of the 11 patients, five whose cultures showed more than three times consecutive negative results after the chemotherapy were categorized as patients with bacterial conversion, and six patients whose cultures did not show negative after the chemotherapy were categorized as patients without bacterial conversion. Blood samples were obtained from them at the beginning and at the end of follow-up. The mean age of the patients with bacterial conversion was 61 years, ranging between 55 and 66 years. The mean age of the patients without bacterial conversion was 66 years, ranging between 51 and 74 years. The mean BMI of the patients with and without bacterial conversion was 21.3 and 19.4 kg/m2, respectively, ranging between 19.8 and 24.7 kg/m2 and between 17.0 and 22.3 kg/m2, respectively. There were no statistically significant differences in age or BMI of the two groups (p ¼ 0.240 and 0.177, respectively). In addition, we longitudinally measured the serum miRNA concentrations and sputum culture results in 2 female patients with M. avium pulmonary disease.
Blood samples were collected after the subjects in the study group and healthy volunteers agreed to participate in the study and provided their informed consent. This study was performed in compliance with the Declaration of Helsinki and was approved by the institutional ethics review committee for human research at the Keio University School of Medicine and hospital (No. 20130134). 2.2. Microarray analysis As previously described [8], miRNA expression in human serum was measured using the 3D-Gene miRNA microarray platform (Toray Industries, Inc., Tokyo, Japan). Briefly, miRNA isolated from 300 mL of human serum was hybridized to 3D-Gene human miRNA oligo chips ver.20.0 containing 2555 miRNAs, or oligo chips ver.21 containing 2565 miRNAs. MiRNA expression was scaled by global normalization, and the resultant miRNA levels were analyzed. 2.3. RNA isolation and quantitative real-time polymerase chain reaction RNA was isolated from 300 mL of human serum using NucleoSpin miRNA Plasma (Macherey-Nagel, Düren, Germany), according to the manufacturer's protocol. RNA was eluted in 35 mL of RNasefree water. To allow for normalization of the sample-to-sample variation during RNA isolation, 1 1013 mol of synthetic Caenorhabditis (C.) elegans miRNA 39 (cel-miR-39, Bioneer, Daejeon, Republic of Korea) was added to each sample. Eluted RNA was reverse transcribed using a TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA), miRNA-specific stem-loop primers (Thermo Fisher Scientific), and Gene Amp polymerase chain reaction (PCR) System 9700 Thermal Cycler (Thermo Fisher Scientific) at 16 C for 30 min, 42 C for 30 min, and 85 C for 5 min, respectively, according to the manufacturer's protocol. Each reaction mixture for quantitative real-time PCR (qPCR) contained 10 mL of TaqMan Universal Master Mix II with UNG (Thermo Fisher Scientific) and 1 mL of a miRNA-specific primer/probe mix of TaqMan MicroRNA Assays (Thermo Fisher Scientific) in a total volume of 20 mL qPCR was completed using the Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) at 50 C for 2 min and 95 C for 10 min, followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. Data were analyzed using StepOne Software v.2.2.2 (Thermo Fisher Scientific), with the automatic Ct setting for assigning baselines and thresholds for Ct determination. A dilution series using chemically synthesized miRNAs was used to generate a standard curve that permitted the absolute quantification of miRNA. Each sample was normalized using spiked-in cel-miR-39 or hsa-miR-16-5p as controls [6,7]. 2.4. Cells and culture As previously described [9], human macrophages were obtained by culturing human peripheral blood mononuclear cells (PBMCs) from transfusion blood provided by the Japanese Red Cross Society (Tokyo, Japan) for 9 days in Iscove's modified Dulbecco's medium (Thermo Fisher Scientific) containing 10% (v/v) human AB serum (Cosmo Bio Co., Ltd., Tokyo, Japan) at a density of 6.0 106 cells/ 2 mL/well in 6-well cluster plates (Corning, NY, USA). The human bronchial epithelial cell line BEAS-2B was used as a model of airway epithelial cells. BEAS-2B cells were purchased from the European Collection of Cell Cultures (Public Health England, London, UK) and cultured in bronchial epithelial growth medium (Lonza, Basel, Switzerland). They were plated at a density of 1.0 106 cells/2 mL/well in 6-well cluster plates (Corning) and cultured to 90% confluence.
Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015
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The adherent macrophages and BEAS-2B cells were cultured with the M. avium strain 104 at a multiplicity of infection (MOI) of 10:1 or 50:1 and then washed with phosphate-buffered saline (PBS) to remove nonadherent bacilli. A fresh medium was added to the macrophage and BEAS-2B cultures after incubation for 4 h and 24 h, respectively. Supernatants were collected at 24 h after infection to measure secretory miRNA levels. The adherent macrophages were cultured with latex beads coated with FITC-labeled rabbit IgG (Cayman Chemical, MI, USA) at a final dilution of 1:500 or 1:100 and then washed with PBS to remove nonadherent beads. Fresh medium was added to the macrophage culture after incubation for 4 h. For measuring secretory miRNA levels, supernatants were collected at 24 h after addition of latex beads.
2.5. Statistical analyses The results are expressed as the mean ± SEM. The t-test or the ManneWhitney U test was used to evaluate statistical differences between two groups. The paired t-test was used to evaluate statistical differences between the groups at the beginning and the end of follow-up of the patients. Comparisons among three experimental groups were assessed with an analysis of variance followed by the Fisher's least significant difference test using IBM
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SPSS statistics 23 (IBM, NY, USA). A p value <0.05 was considered statistically different.
3. Results 3.1. hsa-miR-346 is a circulating miRNA associated with Mycobacterium avium complex pulmonary disease Two independent microarray analyses using serum samples (experiment 1 consisted of five M. avium pulmonary disease patients and five healthy controls, and experiment 2 consisted of four M. avium pulmonary disease patients and four healthy controls) repeatedly revealed that the levels of four miRNAs, including hsamiR-346, hsa-miR-532-3p, hsa-miR-4446-3p, and hsa-miR-47223p, were significantly higher (p < 0.05) in patients with MAC pulmonary disease than those found in healthy controls. To confirm the miRNAs associated with MAC pulmonary disease, we compared the serum concentrations of these miRNAs in 16 patients with M. avium pulmonary disease with those from 16 healthy controls using qPCR (Fig.1). The concentration of hsa-miR-346 was significantly higher in subjects in the MAC pulmonary disease group than in the healthy control group (mean ± SEM; 1.63 1016 ± 3.28 1017 M, and 9.42 1017 ± 1.81 1017 M, respectively, p ¼ 0.047). Unfortunately, we could not measure serum concentration of hsa-miR-
Fig. 1. Scattergrams of serum hsa-miR-346 (A), hsa-miR-532-3p (B) and hsa-miR-4446-3p (C) concentrations in patients with Mycobacterium avium complex pulmonary disease (MAC, n ¼ 16) and in subjects in control group (Control, n ¼ 16). The horizontal lines show the mean concentrations in each group. Comparisons between subjects in the MAC pulmonary disease group and in the control group were assessed using the ManneWhitney U test.
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4722-3p by qPCR. It was technically difficult to make the miRNAspecific primers of hsa-miR-4722-3p because it had the potential to create self-dimers due to high G þ C content. 3.2. Human macrophages infected with Mycobacterium avium complex show increased hsa-miR-346 production The primary host cells harboring MAC are macrophages while MAC can infect bronchial epithelial cells in MAC pulmonary disease [10]. Therefore, we inoculated macrophages derived from human PBMCs and BEAS-2B cells with M. avium strain 104 and then measured the level of hsa-miR-346 in their supernatants at 24 h after infection (Fig. 2). The level of hsa-miR-346 was higher in the supernatant of MAC-infected macrophages when compared with uninfected macrophages (hsa-miR-346/has-miR-16-5p ratios of infected cells at MOI of 10 and 50 compared with uninfected cells
[mean ± SEM]: 1.48 ± 0.170 [p ¼ 0.048] and 2.18 ± 0.346 [p ¼ 0.027], respectively). There was no significant difference in hsa-miR-346 levels of MAC-infected and uninfected BEAS-2B cells (hsa-miR-346/ has-miR-16-5p ratios of infected cells at MOI 10 and 50 compared with uninfected cells [mean ± SEM]: 0.962 ± 0.111 [p ¼ 0.754] and 0.727 ± 0.132 [p ¼ 0.130], respectively). To determine if hsa-miR-346 secretion in macrophages is induced by MAC infection, we treated human macrophages with latex beads coated with FITC-labeled rabbit IgG and then measured the concentration of hsa-miR-346 in their supernatants at 24 h after addition. There was no significant difference in hsa-miR-346 levels of latex beads-treated and latex beads-untreated human macrophages (hsa-miR-346/has-miR-165p ratios of treated cells at a final dilution 1:500 or 1:100 compared with untreated cells [mean ± SEM]: 1.37 ± 0.236 [p ¼ 0.174) and 1.18 ± 0.181 [p ¼ 0.374], respectively). Thus, MAC-infected macrophages are the main secretors of hsa-miR-346.
Fig. 2. hsa-miR-346 concentrations in the supernatants of human macrophages (A) and BEAS-2B cells (B) with and without Mycobacterium avium complex (MAC) infection, and human macrophages treated with latex beads coated with FITC-labeled rabbit IgG (C). Multiplicity of infection (MOI) with MAC is 10:1 or 50:1, and final concentration of latex beads is 1:500 or 1:100 dilution. Ratios between hsa-miR-346 and hsa-miR-16-5p concentrations are shown as fold changes relative to uninfected human macrophages, BEAS-2B cells, or untreated human macrophages. The data from three independent experiments are shown as the mean ± SEM. Comparisons between MAC-infected and MAC-uninfected human macrophages and BEAS-2B cells, and latex beads-treated and latex beads-untreated human macrophages were assessed using an ANOVA followed by the Fisher's least significant difference test.
Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015
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3.3. Serum hsa-miR-346 levels correlate with bacterial load in Mycobacterium avium complex pulmonary disease To determine if the concentration of hsa-miR-346 in the serum reflects MAC pulmonary disease activity, we compared serum hsamiR-346 levels at the end and the beginning of follow-up in M. avium pulmonary disease patients with and without bacterial conversion (Fig. 3). In patients with and without bacterial conversion, serum hsa-miR-346 levels were significantly lower and higher at the end of follow-up than at the beginning of follow-up, respectively (hsa-miR-346 ratios at the end of follow-up compared with those at the beginning of follow-up [mean ± SEM]: 0.604 ± 0.136 [p ¼ 0.043] and 1.40 ± 0.138 [p ¼ 0.034], respectively). In addition, we evaluated the correlation between serum hsa-miR-346 concentrations and sputum culture results in two patients. Case 1: A 55 year-old woman with a small amount of sputum visited our hospital. She had no history of other diseases and had never been treated for mycobacterial disease. We diagnosed her with a nodular bronchiectatic form of M. avium pulmonary disease, and anti-mycobacterial therapy was initiated with three antibiotics: 600 mg/day of RIF, 750 mg/day of EMB, and 800 mg/day of CLR. The serum hsa-miR-346 concentration decreased as sputum cultures results converted from positive to negative 9 months after the initiation of therapy (Fig. 4A). Case 2: A 66 year-old woman with a productive cough visited our hospital. She had no history of other diseases and had never Fig. 4. The longitudinal changes of sputum culture results and serum hsa-miR-346 concentrations in patients with Mycobacterium avium complex (MAC) pulmonary disease after initiation of therapy. RIF: rifampicin, EMB: ethambutol, CLR: clarithromycin, AMK: amikacin, (þ): positive sputum culture, (): negative sputum culture.
been treated for mycobacterial disease. We diagnosed her with a nodular bronchiectatic form of M. avium pulmonary disease, and anti-mycobacterial therapy was initiated with four antibiotics: 600 mg/day of RIF, 500 mg/day of EMB, 800 mg/day of CLR, and 600 mg/every other day of amikacin (AMK). The serum hsa-miR-346 concentration decreased as sputum cultures results converted from positive to negative 4 months after the initiation of therapy (Fig. 4B). Treatment with AMK was discontinued at this time. The serum hsa-miR-346 concentration increased as the sputum cultures reconverted from negative to positive at 30 months after the initiation of therapy. AMK was re-administered 32 months after the initiation of therapy, and sputum cultures converted from positive to negative 3 months after the re-administration of AMK. The serum hsa-miR-346 concentration decreased with the change in sputum culture results. Thus, serum hsa-miR-346 concentrations correlate with the disease activity estimated by sputum culture results. 4. Discussion
Fig. 3. The changes in serum hsa-miR-346 concentrations of 11 patients with Mycobacterium avium complex pulmonary diseases with treatment at the beginning and the end of follow-up, including five patients with bacterial conversion (A) and six patients without bacterial conversion (B). The serum hsa-miR-346 concentrations at the end of follow-up were shown as fold changes relative to that at the beginning of the followup. The data are shown as the mean ± SEM. Comparisons between the end and the beginning of follow-up were assessed using the paired t-test.
This pilot study is the first report on circulating human miRNAs associated with MAC pulmonary disease. On the basis of our results, it is supposed that MAC-infected macrophages in pulmonary lesions produce hsa-miR-346, which then reached the bloodstream. hsa-miR-346 secretion from MAC-infected macrophages was dependent on MOI, indicating that the degree of hsa-miR-346 secretion from pulmonary macrophages could be quantitatively controlled by the bacterial load in pulmonary lesions. It is reasonable to assume that the serum hsa-miR-346 concentration correlates with MAC pulmonary disease activity. In addition, we confirmed that the serum hsa-miR-346 concentrations correlated with the disease activity estimated by sputum culture results.
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Therefore, hsa-miR-346 is considered to be a candidate of serum biomarker for MAC pulmonary disease activity. A definitive diagnosis of MAC pulmonary disease is essentially based on multiple culture confirmations using respiratory specimens of the patients, and we have already reported the usefulness of a serum antibody test against a cell wall component for the diagnosis of MAC pulmonary disease [11]. However, there are no known sero-biomarkers to precisely reflect disease activity after diagnosis. In this regard, our current data show the potential of hsa-miR-346 as a sero-biomarker of disease activity. Therefore, sequential measurements of hsa-miR-346 levels will be useful in the management of the disease such as in taking decisions on the indication of chemotherapy or efficacy of treatment after diagnosis. Previous reports showed that hsa-miR-346 downregulated the expression of vitamin D receptor (VDR) in PBMCs, epithelial cells and smooth muscle cells [12e14]. However, it remains unclear how hsa-miR-346 plays a role in the pathophysiology of MAC pulmonary disease. Li et al. showed that hsa-miR-346 plays a role in the pathophysiology of inflammatory bowel disease [13]. Tumor necrosis factor (TNF)-a induces apoptosis of intestinal mucosal cells, which resulted in intestinal mucosal damage and ultimately inflammatory bowel disease. TNF-a-induced apoptosis of intestinal mucosal cells was blocked by the binding of 1,25-dihydroxyvitamin D (1,25(OH)2D3) to the VDR. Notably, hsa-miR-346 suppresses VDR expression, resulting in the acceleration of TNF-a-induced apoptosis. Pathological and radiological findings of MAC pulmonary disease revealed that bronchiectasis and bronchiolitis were caused by the destruction of the bronchial structure [15]. Thus, hsa-miR346 secreted from MAC-infected macrophages may accelerate the TNF-a-induced apoptosis of airway epithelial cells, leading to bronchiectasis or bronchiolitis. There are several critical limitations to this study. First, the small number of each group is a major limitation. Further clinical studies involving more cases have to be planned to confirm to our findings. Second, it remains unclear if hsa-miR-346 is specific for MAC pulmonary disease and is a candidate of diagnostic biomarker for the disease. Although currently there are no reports showing that hsamiR-346 is associated with the pathophysiology of infectious diseases, including mycobacterial diseases, or that hsa-miR-346 can act as a biomarker of disease activity, we will measure serum hsamiR-346 concentrations in patients with other diseases. Third, it remains unclear if hsa-miR-4722-3p is also a potential serum biomarker of MAC pulmonary disease activity. Although we could not measure hsa-miR-4722-3p concentration using qPCR, we will validate serum concentration of hsa-miR-4722-3p by a different way from qPCR. In conclusion, hsa-miR-346 is secreted from MAC-infected macrophages, and its serum levels could be a potentially easy and useful biomarker of MAC pulmonary disease activity. Authorship statement TN and ET designed the study and did the experiments, and collected, analyzed and interpreted data, and drafted the manuscript. SU, YU, HF, KN and YN recruited subjects, collected clinical samples, interpreted data, and revised the manuscript. HS, HN, YH and SI contributed to interpretation of data. NH conceived of and
designed the study, analyzed and interpreted the data, and revised the manuscript. All authors meet the ICMJE authorship criteria. Acknowledgments The authors thank the healthy subjects who volunteered for this study, and Dr. Takemasa Takii for the generous gift of the M. avium strain 104. The authors are grateful to Dr. Heinz G. Remold from the Brigham and Women's Hospital at Harvard Medical School (MA, USA) for his critical reading of our manuscript. This work was supported by the Japan Society for the Promotion of Science [KAKENHI 16K09942 to T.N. and 25461520 and 16K09941 to N.H.], the Daiwa Securities Health Foundation [T.N.], and the Kurozumi Medical Foundation [T.N.]. Conflicts of interests. This study was completed as part of our collaborative research with Nikon Corporation (Minato-ku, Tokyo, Japan). References [1] Prevots DR, Marras TK. Epidemiology of human pulmonary infection with nontuberculous mycobacteria: a review. Clin Chest Med 2015;36:13e34. [2] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175(4):367e416. [3] Montani F, Marzi MJ, Dezi F, Dama E, Carletti RM, Bonizzi G, et al. miR-Test: a blood test for lung cancer early detection. J Natl Cancer Inst 2015;107. djv063. [4] Murakami Y, Kawada N. MicroRNAs in hepatic pathophysiology. Hepatol Res 2017;47:60e9. [5] Xiao B, Liu Z, Li BS, Tang B, Li W, Guo G, et al. Induction of microRNA-155 during Helicobacter pylori infection and its negative regulatory role in the inflammatory response. J Infect Dis 2009;200:916e25. [6] Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, PogosovaAgadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513e8. [7] Shiotani A, Murao T, Kimura Y, Matsumoto H, Kamada T, Kusunoki H, et al. Identification of serum miRNAs as novel non-invasive biomarkers for detection of high risk for early gastric cancer. Br J Cancer 2013;109:2323e30. [8] Konishi H, Ichikawa D, Komatsu S, Shiozaki A, Tsujiura M, Takeshita H, et al. Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma. Br J Cancer 2012;106:740e7. [9] Nishimura T, Zhao X, Gan H, Koyasu S, Remold HG. The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis. FASEB J 2013;27:3827e36. [10] Honda JR, Knight V, Chan ED. Pathogenesis and risk factors for nontuberculous mycobacterial lung disease. Clin Chest Med 2015;36:1e11. [11] Nishimura T, Hasegawa N, Fujita Y, Yano I, Ishizaka A. Serodiagnostic contributions of antibody titers against mycobacterial lipid antigens in Mycobacterium avium complex pulmonary disease. Clin Infect Dis 2009;49:529e35. [12] Yi B, Huang J, Zhang W, Li AM, Yang SK, Sun J, et al. Vitamin D receptor downregulation is associated with severity of albuminuria in type 2 diabetes patients. J Clin Endocrinol Metab 2016;101:4395e404. [13] Chen Y, Du J, Zhang Z, Liu T, Shi Y, Ge X, et al. MicroRNA-346 mediates tumor necrosis factor alpha-induced downregulation of gut epithelial vitamin D receptor in inflammatory bowel diseases. Inflamm Bowel Dis 2014;20: 1910e8. [14] Martinez-Moreno JM, Herencia C, Montes de Oca A, Munoz-Castaneda JR, Rodriguez-Ortiz ME, Diaz-Tocados JM, et al. Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells. FASEB J 2016;30:1367e76. [15] Fujita J, Ohtsuki Y, Suemitsu I, Shigeto E, Yamadori I, Obayashi Y, et al. Pathological and radiological changes in resected lung specimens in Mycobacterium avium intracellulare complex disease. Eur Respir J 1999;13:535e40.
Please cite this article in press as: Nishimura T, et al., hsa-miR-346 is a potential serum biomarker of Mycobacterium avium complex pulmonary disease activity, J Infect Chemother (2017), http://dx.doi.org/10.1016/j.jiac.2017.07.015