Int J Clin Exp Pathol 2015;8(7):8244-8251 www.ijcep.com /ISSN:1936-2625/IJCEP0009692

Original Article Compromised natural killer cells in pulmonary embolism

Xiaoyu Zhang1*, Qiang Wang2*, Yuqin Shen2, Haoming Song2, Zhu Gong2, Lemin Wang2

1Department of Rheumatology, Tongji Hospital of Tongji University, Shanghai 200065, China; 2Department of Cardiology, Tongji Hospital of Tongji University, Shanghai 200065, China. *Equal contributors. Received April 28, 2015; Accepted June 22, 2015; Epub July 1, 2015; Published July 15, 2015

Abstract: Objective: The high morbidity, mortality and misdiagnosis rate render pulmonary embolism (PE) as a worldwide health problem. However, the etiology and pathogenesis of this disease have not been well character- ized. Increasing studies indicate infection and immunity play a crucial role in PE. Natural killer (NK) cells act as a bridge between the innate immune and acquired immune. This study aimed to investigate the possible function of NK cells in PE. Methods: Human cDNA microarray analysis was employed to detect associated with NK cells in peripheral blood mononuclear cells (PBMCs). Random variance model corrected t-test was used for statisti- cal analysis of differential expression. Flow cytometry was performed to detect the CD16+CD56+ NK cells. Results: In the present study, based on microarray analysis, we showed four inhibitory receptors (KLRB1, KLRD1, KLRF1, KLRG1) and four activating receptors (KLRC1, KLRC3, KLRK1 and NCR1) on NK cells were remarkably down-regulated and the cytological experiment demonstrated the proportion of CD16+CD56+ NK cells among PBMCs decreased in the PE group. Conclusions: We confirmed the presence of reduced expression of critical activating as well as inhibitory NK cell receptors and low proportion of CD16+CD56+ NK cells in PE. The consistence between genomic and cytological examination suggests compromised NK cells may contribute to the pathogenesis of PE.

Keywords: Pulmonary embolism, gene,

Introduction in the pathogenesis of PE. In 2006, Smeeth et al [4] reported infection was associated with an Venous thromboembolism (VTE) represents a increased VTE risk. The association was stron- spectrum of conditions that include pulmonary gest within the first 2 weeks after infection embolism (PE), chronic thromboembolic pulmo- onset. In 2012, Schmidt et al retrospectively nary hypertension (CTEPH) and deep venous investigated 15009 cases of VTE and showed thrombosis (DVT). The high morbidity, mortality that risk for VTE was increased two-fold in and misdiagnosis rate render VTE as a world- patients with infection, especially within two wide health problem [1]. The American College weeks of infection, gradually declining thereaf- of Chest Physicians has recommended nine ter [5]. Since 2004, we have been engaged in editions of the guideline for the diagnosis and basic and clinical investigation of VTE. We prevention of VTE since 1995 and also pro- observed virus-like microorganism [6], rod-sha- posed the concept of risk stratification for pro- ped bacteria like microorganisms [7] and SARS phylaxis of VTE [2]. However, the incidence of coronavirus infection [8] in patients with VTE. symptomatic VTE is increasing gradually ins- All these findings indicate that onset of VTE is tead of reducing, partly because the etiology closely related with microorganisms infections. and pathogenesis of this disease have not In addition, we discovered the impaired cyto- been well characterized [3]. toxic activity of T cells [9] and compromised complement system in VTE patients [10], It has been demonstrated that genetic and strongly supporting an involvement of the environmental factors are involved in the devel- immune system in the pathogenesis of VTE. opment of VTE and increasing studies indicate Natural killer (NK) cells are a type of cytotoxic that infection and immunity play a crucial role lymphocyte critical to the innate immune sys- Natural killer cell-mediated immune compromise in pulmonary embolism tem. What’s more, numerous experiments have was 50-108 mmHg in CTEPH patients. In the worked to demonstrate their ability to readily control group, 20 patients (11 males and 9 adjust to the immediate environment and for- females) with a mean age of 72±14 years mulate antigen-specific immunological memo- (44~91 years) were enrolled during the same ry, fundamental for responding to secondary period. No significant difference in age was infections with the same antigen. On the basis found between PE patients and controls (P> of previous findings, this study was undertaken 0.05). to detect the mRNA expression levels of NK cells in PBMCs isolated from PE patients and Cellular immunology controls by the whole microar- ray and analysis. In addition, A total of 41 clinically proven VTE patients (31 flow cytometry was performed to investigate with APE, 9 with DVT and 10 with CTEPH) aged the changes in NK cells in PE patients, which 67±12 years (31~88 years) were recruited from aimed to validate the results from genome Tongji Hospital, including 18 males and 23 analysis. females. The proportion of CD16+CD56+ NK cells in VTE patients were all compared with the Materials and methods detection intervals of normal population (8.6- 21.1%). Subjects Extraction of total RNA All subjects were from Tongji Hospital of Tongji University. The diagnostic criteria for pulmo- A total of 5 ml of venous EDTA anti-coagulated nary embolism were any two of the following: blood was obtained from patients of both (1) selective pulmonary angiography showing groups and mononuclear cells were isolated by pulmonary artery occlusion or filling defect; (2) density gradient centrifugation. Red blood cell pulmonary ventilation-perfusion scan showing lysis buffer (Qiagen, Hilden, Germany) was used single or multiple perfusion defect, normal or to isolate mononuclear cells and total RNA was abnormal ventilation, and ventilation-perfusion extracted from mononuclear cells with TRIzol mismatch; (3) clinical diagnosis: risk factors for (Invitrogen, Carlsbad, USA) followed by purifica- PE are present and other cardiovascular dis- tion with RNeasy column (QIAGEN). Treatment eases may be excluded by clinical performance, with DNase was performed to avoid the influ- electrocardiogram and chest film; arterial blood ence by genomic DNA. Quantification of extract- gas analysis, D-dimer detection, echocardiog- ed RNA was performed with Nanodrop ND- raphy and/or chest computed tomography sup- 1000 spectrophotometer (Nanodrop Techno- port PE diagnosis. The diagnosis of DVT was logy, Cambridge, UK). based on the criteria previously reported [11] and the criteria for CTEPH was used in accor- Gene expression profiling dance with the diagnosis of CTEPH [12]. Mali- gnancies, use of immunosuppresants or auto- Agilent G4112A Whole Human Genome Oligo immune diseases were excluded in all subjects. Microarrays were purchased from Agilent (USA). Healthy controls underwent physical examina- A microarray is composed of 44,290 spots tion before they were recruited from the outpa- including 41675 genes or transcripts, 314 neg- tient clinic. The present study was approved by ative control spots, 1924 positive control spots the Ethics Committee of Tongji Hospital and and 359 blank spots. The functions of more informed consent was obtained before study. than 70% of genes in the microarray have been known. All patients of both groups were sub- Genomic study jected to microarray analysis.

Twenty PE inpatients and 20 controls were Sample marking and hybridization randomly selected in Cardiology Department, Tongji Hospital of Tongji University. In the PE Indirect approach was applied to mark sam- group, there were 11 males and 9 females, ples. About 1 μg of total RNA was reversely with a mean age of 70±14 years (44~89 years). transcribed into double strand cDNA. After puri- There were 13 patients with acute PE and 7 fication, in vitro amplification was performed with CTEPH. The pulmonary artery pressure with Agilent Low RNA Input Linear Amplification

8245 Int J Clin Exp Pathol 2015;8(7):8244-8251 Natural killer cell-mediated immune compromise in pulmonary embolism

Figure 1. Microarray results of NK cell surface receptors. A. KLR receptors were down-regulated totally in PE group. The difference of KLRC1, KLRC3 and KLRK1 showed statistical significance (*P<0.05), and KLRB1, KLRD1, KLRF1 and KLRG1 were remarkably down-regulated (**P<0.01). B. The mRNA expression of KIR receptors was lower than that of controls consistently, but the difference had no statistical significance. C. NCR receptors were down- regulated and NCR1 was remarkably down-regulated (**P<0.01).

Kit (Agilent, Pal alto, USA) and modified UTP rescent products were fragmented at 60°C and [aaUTP, 5-(3-aminoally1)-UTP] was used to hybridization was conducted in Human Whole- replace UTP. The integrated aaUTP can interact Genome 60-mer oligo-chips (G4112F, Agilent with Cy3 NHS ester forming fluorescent prod- Technologies) at 60°C for 17 h at 10 rpm. After ucts which are then used for hybridization. The hybridization, the chips were washed with integration rate of fluorescence can be deter- Agilent Gene Expression Wash Buffer accord- mined with a NanodropND-1000 spectropho- ing to manufacturer’s instructions. Original sig- tometer. Then, hybridization mixture was pre- nals were obtained Agilent scanner and Feature pared with Agilent oligonucleotide microarray in Extraction software. The standardization of situ hybridization plus . About 750 ng of fluo- original signals was carried out with RMA stan-

8246 Int J Clin Exp Pathol 2015;8(7):8244-8251 Natural killer cell-mediated immune compromise in pulmonary embolism

8247 Int J Clin Exp Pathol 2015;8(7):8244-8251 Natural killer cell-mediated immune compromise in pulmonary embolism

Figure 2. Microarray results of remarkably down-regulated receptors of NK cells. Crucial receptors including KLRB1 (A), KLRC1 (B), KLRC3(C), KLRD1 (D), KLRF1 (E), KLRG1 (F), KLRK1 (G) and NCR1 (H) were significantly down- regulated.

model. Differentially express- ed genes were identified from whole genomes. Independent- Samples T Test was used to compare mRNA levels in sam- ples from PE patients and con- trols. Statistical tests were performed using SPSS 17.0, and P values <0.05 were con- sidered significant. Before t test, test for equality of vari- ances was performed, if vari- ances were not equal, t test result would be corrected.

Detection of differentiation antigens on NK cells

Figure 3. Analysis of peripheral CD16+CD56+ NK cells proportion by flow cy- Sample collection: the fasting tometry. The CD16+CD56+ NK cells were decreased in 16 out of 41 patients venous blood (2 ml) was col- compared with the detection intervals of normal population (8.6-21.1%), in- lected in the morning and dicating that the proportion of activated NK cells declined. added to the ET tube. Flow cytometry was performed to dardized method and standardized signal val- detect the differentiation antigens on NK cells ues were used for screening of differentially with BECKMANCOULTER EPICS XL-II flow cytom- expressed genes. eter. The NK cell marker CD16+CD56+ was detected in 41PE patients. RT-PCR Results The spots in the microarray were randomly selected and their expressions were confirmed mRNA expressions of NK cells related genes by RT-PCR. Among genes with differential expressions, 3 genes were randomly selected NK cells related surface receptors, including and these genes and house-keeping gene killer cell lectin-like receptor (KLR), killer cell (GAPDH) were subjected to RT-PCR. The rela- immunoglobulin-like receptor (KIR) and natural tive expressions were expressed as the expres- cytotoxicity receptor (NCR) were uniformly sions of target genes normalized by that of down-regulated. In the PE group, four inhibitory GAPDH (2-ΔΔCt). Melting curve and 2-ΔΔCt method receptors (KLRB1, KLRD1, KLRF1, KLRG1) and were used to compare the difference in the four activating receptors (KLRC1, KLRC3, NCR1 expressions between control group and PE and KLRK1) which positively correlate with NK group. Results from RT-PCR were consistent cytotoxicity, were remarkably down-regulated with microarray analysis. compared with control group (P<0.05), while the difference of KIR receptors, KLRC2, KLRC4, Significant differential gene expression analy- NCR2 and NCR3 between two groups had no sis statistical significance Figures( 1, 2).

Agilent Feature extraction software was used Flow cytometry analysis of CD16+CD56+ NK to collect original data from microarray followed cells by analysis with robust multichip average (RMA). Gene intensity data between PE group Flow cytometry was performed to detect periph- and control group were compared with t test eral blood NK cell surface marker CD16+CD56+. after calibration with a stochastic variance The CD16+CD56+ NK cells were decreased in

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16 out of 41 patients (39.02%), suggesting that we have been engaged in basic and clinical the proportion of activated NK cells decreased investigation of VTE. In 2010, we reported (Figure 3). virus-like microorganisms observed in cyto- plasm and intercellular substance of lympho- Discussion cytes from peripheral venous blood in VTE patients with pulmonary hypertension and T Based on gene expression microarray analysis, cell immune dysfunction/disorder [6]. We also we have shown in the present study four inhibi- observed rod-shaped bacteria like microorgan- tory receptors (KLRB1, KLRD1, KLRF1, KLRG1) isms in apoptotic phagocytes from peripheral and four activating receptors (KLRC1, KLRC3, venous blood in patients with repeated PE/DVT KLRK1 and NCR1) on NK cells were remarkably and T cell immune dysfunction/disorder [7]. down-regulated and the cytological experiment Besides, we also reported VTE in multiple or- demonstrated the proportion of CD16+CD56+ gans of a patient who died of SARS, suggesting NK cells among PBMCs reduced in the PE viral infection is closely related with VTE [8]. All group. The activating and inhibitory receptors these findings indicate that viral infections of NK cells are key mediators in controlling the associated with decreased NK cell activity con- immune reaction to viral infection [13]; thus, it tribute to the onset of VTE. Yet most of the is likely that both the low gene expression of NK molecular mechanisms whereby compromised cell receptors and the reduced proportion of NK cells interact with virus to promote the activated NK cells may lead to impairment of occurrence of VTE remain largely undefined. It NK cell function in this disease. On the other was reported previously that certain viruses hand, our results showed no significant differ- have developed strategies that interfere with ences in the expression of KLRA1, KLRC2, NK cell cytotoxicity. For example, through mod- KLRC4, NCR2, NCR3 and KIR receptors in this ulation of NK cell receptor ligands on the sur- study. Considered together, while it was face of infected cells or down-regulation of cer- unknown if they were correlated with each tain activating NK cell receptors to reduce in other, these findings indicated that the reduced their cytolytic effectiveness with consequent receptors might play a role at least in part, immune evasion [13]. On basis of our previous among the molecules related to NK cell dys- findings, we suppose that the impaired NK cells function in the development of PE. immune function may be the internal cause of susceptibility to acquired VTE, and infection It is well known that individuals with defective acts as a triggering factor. When the pathogens NK cells are susceptible to virus infection and invade the subjects with NK cell dysfunction, cancer formation. In this study, malignant the pathogens cannot be completely removed tumors or use of immunosuppressive drugs by the immune system. Thus, patients with were all excluded, so we speculated compro- compromised NK cell function are susceptible mised NK cells in PE patients might be associ- to infection, especially viral infection, which is a ated with virus infection. Successive scholars high risk factor for VTE. The current study pro- demonstrate that individuals who are infected vided a new insight into a novel role for NK cells special virus, such as influenza virus [14], her- in the pathogenesis of venous thrombosis from pes simplex virus [15], Epstein-Barr virus [16], an immune perspective. However, the method- cytomegalovirus [17], human immunodeficien- ology of this study is relatively simple and cy virus [18], have high tendency to VTE this understanding the cellular mechanisms where- decade. Zhu et al [19] conducted a case-con- by NK cells dysfunction contribute to VTE is still trol study involving 1,454 adults enrolled in 11 an important challenge of biomedical research. French centers to investigate whether influenza Furthermore, Long term follow-up is needed as vaccination reduced the risk of VTE. They con- regards the restoration of pulmonary perfusion, cluded that influenza vaccination was associ- the rate of survival, and the occurrence of new ated with a reduced risk of VTE. Viral infections diseases such as cancer in future. associated with decreased NK cell activity in other human diseases have also been report- Acknowledgements ed. Nakata S et al pointed that the presence of reduced NK activating receptor gene expres- This study was supported by “12th five year” sion and low proportion of NK cells was involved National Science and Technology Supporting in fulminant type 1 diabetes [20]. Since 2004, Program (2011BAI11B16).

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