Expression of IL-1β, HMGB1, HO-1, and LDH in malignant and non-malignant pleural effusions

Journal Pre-proof Expression of IL-1␤, HMGB1, HO-1, and LDH in malignant and non-malignant pleural effusions Kun-Ming Wu, Wen-Kuei Chang, Chih-Hao Chen, Yu Ru Kou

PII:

S1569-9048(19)30250-2

DOI:

https://doi.org/10.1016/j.resp.2019.103330

Reference:

RESPNB 103330

To appear in:

Respiratory Physiology & Neurobiology

Received Date:

16 July 2019

Revised Date:

15 October 2019

Accepted Date:

15 October 2019

Please cite this article as: Wu K-Ming, Chang W-Kuei, Chen C-Hao, Kou YR, Expression of IL-1␤, HMGB1, HO-1, and LDH in malignant and non-malignant pleural effusions, Respiratory Physiology and amp; Neurobiology (2019), doi: https://doi.org/10.1016/j.resp.2019.103330

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Expression of IL-1β, HMGB1, HO-1, and LDH in malignant and nonmalignant pleural effusions

Kun-Ming Wua,b,c,d, Wen-Kuei Changb, Chih-Hao Chenc,d,e, Yu Ru Koua,*

Institute of Physiology, School of Medicine, National Yang-Ming University, Taipei,

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a

Taiwan

Chest Division, Department of Internal Medicine, MacKay Memorial Hospital,

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b

Taipei, Taiwan

Department of Medicine, Mackay Medical College, New Taipei City, Taiwan Department of Nursing, Mackay Junior College of Medicine, Nursing and

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d

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c

Management, New Taipei City, Taiwan e

*

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Department of Thoracic Surgery, Mackay Memorial Hospital, Taipei, Taiwan

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Corresponding author: Yu Ru Kou, PhD. Department of Physiology, National Yang-

Ming University, Taipei 11221, Taiwan. E-mail: [email protected]; Phone: +886-22826-7086; Fax: +886-2-2826-4049

Highlights

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Biomarkers in pleural effusions with transudative, infectious, and malignant etiologies were studied.

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IL-1β, HMGB1, HO-1, and LDH were expressed differently in these etiologies.

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Increased IL-1β levels were associated with a decrease in cancer risk.

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Abstract IL-1β, HMGB1, HO-1, and LDH in the pleural effusions (PE) of patients with

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transudative, infectious, and malignant etiologies were determined using ELISA and enzymatic assays. IL-1β, HMGB1, HO-1, and LDH showed significant differences

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between the three etiologies. Post-hoc analysis revealed higher levels of HO-1 and

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HMGB1 in infectious versus transudative effusion. Higher levels of IL-1β were found in infectious versus transudative or malignant effusion. The comparison of LDH levels

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showed significant differences. Positive correlations were found between IL-1β,

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HMGB1, and LDH in infectious effusions. The samples were then divided into

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cancerous and non-cancerous groups, and logistic regression revealed that increasing IL-1β levels were significantly associated with a decrease in cancer risk after adjusting for HMGB1, HO-1, and LDH. Our findings suggest that IL-1β, HMGB1, HO-1, and LDH are expressed differently, with positive correlations between HMGB1, IL-1β, and LDH in infectious effusions, and low IL-1β expression in malignant effusions.

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Keywords: interleukin-1β; high mobility group box 1; heme oxygenase-1; lactate dehydrogenase; inflammation; pleural effusions

1. Introduction

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Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the immune response to infections. It is produced by neutrophils, macrophages, monocytes,

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fibroblasts, T and B lymphocytes, endothelial cells, and dendritic cells in response to stimuli, including pathogen-associated molecular patterns (PAMPs) and damage-

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associated molecular pattern (DAMP) molecules (Alexandrakis et al., 2002; Dinarello

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and Mier, 1987; Gentile and Moldawer, 2013). IL-1β acts as an immunomodulator and pro-inflammatory mediator by itself or via the induction of other cytokines and

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inflammatory mediators (Dinarello, 1986). IL-1β also has a significant role in pyogenic

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infections of the pleural space (Silva-Mejias, et al, 1995).

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High mobility group box-1 (HMGB1) was initially discovered as a nuclear DNAbinding protein. It is a key extracellular cytokine and mediates the response to infection, injury, and inflammation, and is one of the main prototypes of DAMPs molecules ( Lotze and Tracey, 2005). Upon treatment with lethal doses of LPS, HMGB1 release is delayed long after serum TNF and IL-1β have returned to basal levels in mice (Wang et al., 2001).

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HMGB1 release has also been found to lead to neutrophil accumulation and the upregulation of IL-1β, TNF-α, and macrophage-inflammatory protein-2 in a lung injury model (Abraham, et al., 2000).. Furthermore, DAMPs such as HMGB1 have been found to be able to bind to the cytokine IL-1β directly, forming potent hetero-complexes in vitro (Sha et al., 2008). Previous studies have found IL-1, HMGB-1, were elevated and played

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a role in inflammatory environment mostly from tissues /cell lysis and serum specimens; however, data from pleural cavity fluids has rarely been explored. This is one aim we

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want to study.

Heme oxygenase-1 (HO-1), an inducible isoform of heme oxygenase enzymes, is

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the rate-limiting enzyme of the heme catabolism ( Maines, 1988). It is induced not only

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by its substrate, heme, but also by a variety of non-heme inducers, such as endotoxin, heat shock, and inflammatory cytokines (Choi and Alam, 1996; Fujii et al., 2003). IL-1β,

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HMGB1, and HO-1 expression can be induced by LPS treatment, and HO-1 is believed

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to play a protective role by mediating the anti-inflammatory response ( Otterbein et al.,

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1995; Chen et al., 2013) The induction of HO-1 has been shown to inhibit the release of HMGB1 in LPS-activated macrophages in septic mice (Tsoyi et al., 2009), and the expression levels of HMGB1 were found to be higher in HO-1-/- mice than HO-1+/+ mice after the administration of LPS (Takamiya et al., 2009). In addition, after stimulation by transforming growth factor beta (TGF-ß), HO-1 inhibited the expression of myofibroblast

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markers of pleural mesothelial cells in a mice model (Zolak et al., 2013). This indicates that HO-1 may participate in the inflammatory process of the pleural cavity by regulating the inflammatory response. To our knowledge, the expression of HO-1 in pleural effusions (PE) has not been previously reported; its’ presence in pleural cavity and relationship with proteins aforementioned requires further elucidation.

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Light’s criteria (Light, 2013) is used to separate the transudates from the exudates in pleural effusions. The level of lactate dehydrogenase (LDH) was used by Dr. Light as an

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indication of the degree of inflammation in the pleural space (Light, 2013). Increased LDH levels in the pleural cavity has been reported in several conditions, such as pleural

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inflammation (Vergnon et al., 1984), pulmonary infarction, and malignancy ( Whitaker et

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al, 1980). Elevated LDH levels have also been used as a predictive indicator of poor prognosis in malignant pleural effusion (Bielsa et al., 2008), due to the fact that it indicates

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a high degree of necrosis in the pleural cavity. In this study, we attempted to elucidate the

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relationship between the concentration of LDH and other inflammatory proteins in

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various effusion etiologies, including those of malignant or infectious origins. Malignancy is a major concern in the evaluation of pleural effusions, in this study,

we measure these proteins and also further analysis if there is any association in cancer risk. Taking together, we proposed if these proteins (IL-1β, HMGB1, and HO-1) had high

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correlation with LHD in pleural cavity, then their expression in the effusions may indicate the severity of inflammatory like LHD does, and probable could be a surrogate for LDH. In the present study, we also aimed to determine the concentrations and relationships of IL-1β, HMGB1, HO-1, and LDH in the effusions of cancerous, infectious, and transudative etiologies and analyzed the relationship between their expression and cancer

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risk.

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2. Materials and Methods 2.1 Patients

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Forty patients (median age: 69, range: 31-94) with pleural effusions (PE) classified

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into three entities (10 transudative, 15 infectious, and 15 malignant) were included in this cross-sectional, observational study. After obtaining the approval of the institutional

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review board of MacKay Memorial Hospital (No. 11MMHIS022), this study was

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performed at the Tamsui branch of MacKay Memorial Hospital. Patients undergoing

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sonography-guided thoracentesis for PE diagnosis between February and July 2011 were recruited. Twenty to fifty milliliters of PE were extracted from each individual. Patients who were not willing to participate after providing informed consent were excluded. Our classification of transudative and exudative PE were based on Light’s criteria and clinical evaluations. The characteristics of exudates were defined as follows: pleural fluid

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protein/serum protein ratio of >0.5; pleural fluid lactate dehydrogenase (LDH)/serum LDH ratio of >0.6; or pleural fluid LDH level over two-thirds of the upper limit of the normal serum value. 2.2 Diagnosis and definitions The definitive clinical diagnosis was obtained from the patient medical records and

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clinical manifestation. Effusions occurring secondary to lung cancer (diagnosed by the presence of malignant cells in cytological examination) and other malignancies (effusions

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that were clearly secondary to other malignancies due to the presence of malignant cells) were considered as malignant pleural effusion (MPE). Parapneumonic effusion was

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diagnosed when there was acute febrile illness with purulent sputum, pulmonary

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infiltrates, responsiveness to antibiotic treatment, or identification of the organism in the PE by culture. Empyema was defined by the presence of purulent effusions in the pleural

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cavity and the presence of bacteria, detected by Gram stain or fluid culture. Tuberculosis

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(TB) pleurisy was diagnosed using a positive acid-fast stain in the PE or by the presence

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of caseous granulomas in pleural biopsies, and the culture of above specimens should be positive for Mycobacterium tuberculosis. PE due to congestive heart failure (CHF) was characterized by an enlarged cardiac shadow, pulmonary congestion in the chest X-ray exam, and peripheral edema in response to CHF treatment, compatible with the presence of at least two major criteria or one major criterion in conjunction with two minor criteria

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as defined by the Framingham criteria (McKee et al., 1971), and the absence of any other cause of PE. Patients with co-existing CHF, infectious, and malignant diseases (any two or all of them) were excluded from this study. 2.3 Measurement of biomarkers The levels of IL-1β, HMGB1, and HO-1 in the pleural fluid were determined using

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commercial enzyme-linked immunosorbent assay kits (Human IL-1R&D Systems, MN, USA; HMGB1: Shino-Test Corporation, Kanagawa, Japan; HO-1: Enzo Life

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Sciences, Inc., PA, USA) according to the manufacturer’s instructions. The optical density was measured at a wavelength of 450 nm using a plate reader (TECAN Infinite 200; Tecan

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Group Ltd., Männedorf, Switzerland), with the reference wavelength set to 540 nm. All

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assays were performed using recommended buffers, diluents, and substrates. The levels of IL-1β, HMGB1, and HO-1 were estimated by comparison with the standard

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concentration curves. The LDH level was measured using an enzymatic method

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(Beckman UniCel® DxC 800) using the pleural fluid samples.

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2.4 Statistical analysis

The results are expressed as the median with an interquartile range (25%, 75%).

Kruskal-Wallis one-way analysis was used to predict significance among the groups, and a post-hoc Dunn’s method was used for pair-wise comparisons. Spearman’s rank correlation was used for correlation analysis. Regression lines were plotted using simple

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linear regression. Binary logistic regression was used to predict malignancy. Differences were considered significant when the p-value was less than 0.05. All data were analyzed using IBM SPSS Statistics 24.0 (IBM Corporation, Armonk, NY, USA) and SigmaPlot 12 software for Windows (Systat Software, Inc. San Jose, CA, USA). All relevant data

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are presented in either the manuscript or the Supporting Information.

3. Results

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3.1 Patient characteristics

During the study period, 40 patients (20 male and 20 female) were enrolled in the

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study and divided into three groups based on the criteria described in the Materials and

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methods. Within the patient cohort, 10 patients had transudative PE caused by congestive heart failure and 30 patients had exudative PE (15 infectious and 15 malignancy) (Table

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1). Of the 15 patients with infectious effusions, eight had parapneumonic effusions, four

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tuberculosis, and three empyema. Of the 15 patients with malignant pleural effusion, 11

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were caused by lung cancer (adenocarcinoma) and four by metastatic malignancy. 3.2 Cellular profiles and laboratory studies The white blood cell (WBC) counts of the effusions varied significantly between the

different groups (P = 0.003) (Table 2). The number of WBCs in the subgroup of infectious (parapneumonic and empyema) was greater than the rest groups. Likewise, the number

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of neutrophils in this group was the highest among the four categories (P = 0.007). The red blood cell (RBC) counts were higher in the malignant effusion group (P = 0.034). Although the number of peripheral WBCs was highest in the infectious group (10100.0 × 109/L), there was no statistical difference among the different groups (P = 0.668). However, the protein and glucose levels differed significantly between the transudative,

3.3 IL-1β, HMGB1, HO-1, and LDH in pleural effusions

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infectious, and malignant effusions (Table 2).

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The data distribution of IL-1β, HMGB1, HO-1, and LDH in the transudative, infectious, and malignant effusions is shown in Fig. 1. The three cases of empyema

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classified as empyema (Table 1) are indicated by arrows. There were significant

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differences in the levels of IL-1β, HMGB1, HO-1, and LDH between the three groups (Table 3). Post-hoc analysis using Dunn’s method showed higher levels of HO-1 and

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HMGB1 in the infectious effusions compared to the transudates (P < 0.05). The

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concentration of IL-1β was also greater in infectious PE compared to transudative or

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malignant effusions (P < 0.05). The LDH levels in all three groups were significantly differences (P < 0.05) (Fig. 1). 3.4 Correlations between HMGB1, IL-1β, and LDH in infectious effusions We used SPLOM (scatter plot matrix) to determine compare the proteins levels in the effusions of the three patient groups (transudative, infectious, and malignant effusions)

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and found correlations between protein expression in the infectious effusion group (Fig. 2). However, there was no correlation between HO-1 and the other three proteins in the infectious effusion, as shown in Fig. 2. For the infectious effusions, the pair-wise and 3D correlations between HMGB1, IL-1β, and LDH were plotted (Fig. 3) and a high positive correlation was found between IL-1β and HMGB1 (Spearman’s rank correlation

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coefficient ()= 0.779, P < 0.001); IL-1β and LDH (= 0.793, P <0.001); HMGB1 and LDH (= 0.836, P < 0.001). However, during the analysis of the association between two

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continuous variables, extreme data can affect the standard sample correlation coefficient

(Mukaka, 2012). Our study included three empyema cases with extremely high values

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compared to the other infectious cases (arrows in Fig. 3). As such, we removed these three

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values to perform further analysis (Table S1) but found that, after removing this data, correlations in both statistics (Spearman’s and Pearson’s) still existed. The relationships

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between these three proteins are also depicted by a parallel plot (Fig. S1).

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3.5 Binary logistic regression for cancerous effusion analysis

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Furthermore, we divided the exudative samples into cancerous and non-cancerous groups and used binary logistic regression to demonstrate that increasing levels of IL-1β were significantly associated with a decrease in cancer risk after adjusting for HMGB1, HO-1, and LDH (odds ratio = 0.71, 95% CI: 0.522-0.966, P = 0.029) (Table 4).

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4. Discussion IL-1β, a pro-inflammatory cytokine with pleiotropic effects, plays a key role in infections. It was not until 2002 that Tschopp (Martinon et al., 2002) introduced the concept of the inflammasome complex and IL-1β processing machinery, which is the molecular basis of a spectrum of inflammasome-associated disorders (Dagenais et al.,

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2012). Previous studies have demonstrated that IL-1β is comparatively higher in the empyema group than other causes (Silva-Mejias, et al., 1995). Alexandrakis et al. found

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that the expression of IL-1β in pleural effusions were higher in the exudate group than in

the transudate group (Alexandrakis et al., 2002). In our study, we found that the

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concentration of IL-1β is significantly higher in infectious effusions than the other types

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of effusions. However, there was no difference in IL-1β expression between the malignant and transudative effusions, which is consistent with previous studies, which have shown

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lower levels of IL-1β in malignant effusions compared to in infectious ones (Silva-Mejias

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et al., 1995; Yanagawa et al., 1996; Hua et al., 1999). However, these studies didn’t use

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regression model to analysis the risk between cancerous effusions and concentration of IL-1β or other proteins. High mobility group box-1 (HMGB1) was initially discovered as a nuclear DNA-

binding protein with rapid electrophoretic migration. It was recently discovered that activated macrophages (Wang et al., 1999), mature dendritic cells, and natural killer cells

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can release HMGB1 in response to these stimuli.

DAMPs and cytokines participate in

the immune dysfunction in response to burns, sepsis, and trauma, and their interaction is critical for the regulation of innate immune function (Coleman et al., 2018). Coleman et al. found HMGB1/cleaved-IL-1β complexes in vivo and in human brain tissue (Coleman et al., 2018). Previous studies have indicated that compared to transudates, the average

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level of HMGB1 is significantly higher in exudative effusions/fluids (Winter et al., 2009). In our study, the concentration of HMGB1, similar to that of IL-1β, was significantly

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higher in infectious effusions. We found there is a highly positive correlation between IL-

1β and HMGB1 in infectious effusions, indicating that both play an important role in the

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towards inflammatory onslaughts.

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pro-inflammatory environment and in the subcellular mechanism involved in the response

Heme oxygenase-1 (HO-1) plays an important role in the regulation and function of

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the heme metabolism; it breaks down the pro-oxidant heme to generate CO and

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biliverdin/bilirubin and is a rate limiting enzyme ( Maines, 1988; Choi and Alam, 1996).

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It has been reported that HO-1 is induced by LPS administration in various organs, including the lungs (Camhi et al., 1995; Suzuki et al., 2000), liver, kidneys ( Suzuki et al., 2000), and intestine ( Fujii et al., 2003; Otani et al., 2000) in animal models. Upregulated IL-1β, HMGB1, and HO-1 were observed post-LPS treatment (Chen et al., 2013), and HO-1 is thought to mediate the anti-inflammatory response and play a protective role

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against LPS-induced endotoxic injury (Otterbein et al., 1995), as well as immunologic reactions in acute lung injury, smoking, and COPD ( Raval and Lee, 2010). The induction of HO-1 has been shown to inhibit the release of HMGB1 in LPS-activated macrophages in septic mice ( Tsoyi et al., 2009) and in the rat myocardial ischemia/reperfusion injury model ( Wang et al., 2014). These findings indicate that HO-1 could be part of the

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inflammatory process of the pleural cavity via inflammatory regulation. In our study, we showed that the level of HO-1 is higher in infectious effusions than in other groups,

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indicating that in the pleural effusion caused by infectious stimulus, HO-1 is upregulated,

consistent with the results of previous studies performed in regions other than the pleural

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space. However, HO-1 is dispersedly distributed and showed no correlations with IL-1β,

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HMGB1, or HO-1 in the pleural space. This could be partly due to the limited sample size of our study, the complex microenvironment of the pleural space, such that further

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stimuli.

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research is required to clarify the role of HO-1 in response to inflammatory or malignant

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In the evaluation of pleural effusions, Light’s criteria is still used in the separation of transudates from exudates and as an indication of the degree of inflammation in the pleural space (Light, 2013). In inflammatory processes, activated, damaged mesothelial cells and inflammatory cells that have migrated into the pleural space are all sources of pleural fluid LDH (Paavonen et al., 1991; Whitaker et al., 1982). Thus, increasing

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concentrations of LDH in the pleural fluid is an indicator of the underlying exudative process. Serum LDH measurement is clinically significant in cancer as it is a consequence of tissue destruction caused by the neoplastic growth (Miao et al., 2013), and an increase in the concentration of LDH in the pleural cavities has been found in several medical conditions. Lassos. et al. proposed using pleural fluid LDH isoenzyme pattern for the

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differential diagnosis of PE (Lossos et al., 1997), and Xie et al. demonstrated that LDHA (LDH-5) inhibits tumorigenesis and progression in mouse models of lung cancer (Xie

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et al., 2014). In this study, we found that the expression level of LDH is higher in

infectious effusions than malignant and transudative effusions, and has a highly positive

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correlation with IL-1β and HMGB1 in infectious fluids, suggesting that these two proteins,

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especially HMGB1 (= 0.836, P < 0.001), might be used to predict inflammatory severity in the pleural space like LDH. As such, there may be crosstalk between these proteins in

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the molecular pathways involved in the response of inflammatory damage in the pleural

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cavity.

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In the analysis of malignant effusions, among the four proteins measured (IL-1β, HMGB1, HO-1, and LDH), we found only in LDH study that there are statistical significance in all three pairwise comparisons (infectious vs. malignant; infectious vs. transudative; transudative vs. malignant). Elevated LDH levels have been used as a predictive factor of poor prognosis in malignant pleural effusion (Bielsa et al., 2008), so

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further research is required to see if LDH could be used to discriminate malignant form infectious effusions. In the present study, we used binary logistic regression to analyze cancerous and noncancerous exudative effusions, and only found increasing levels of IL-1β were significantly associated with a decreased in cancer risk, indicates the concentration of IL-

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1β is low in cancerous effusion. However, large scale samples studies are required for more advanced results.

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In our study, there were three empyema cases with extremely high values. Assuming that these correlations may be mainly attributed to these extreme data, we removed them

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and performed another analysis as outliers can distort the output, leading to wrong

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conclusions. After removing these data, although correlations still exist, these changed from a high positive (0.70 to 0.90) to a moderate positive (0.50 to 0.70) (Mukaka, 2012),

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suggesting that the correlations between these proteins in infectious effusions are not

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caused solely by the high values of empyema samples (Table 1).

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In clinical observation, TB attacks hosts and elicits immune responses in a distinct way, different chemical agents are thus given and clinical course is much longer in the treatment of such patients compared with pneumonia caused by other common pathogens. Thus we should enroll larger case numbers in the future study to analyze separately if there were any differences between TB and other common infection in this settings.

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However, there are limitations in our study. This is a preliminary study with a relatively small sample size, which is associated with low statistical power and a reduced chance of detecting a true effect. Another concern is sample selection bias, which could affect the interpretation of results from sampling errors. Nonetheless, as the patients’ data were collected from daily practices at a teaching hospital, all the cases represent

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real-world clinical scenarios and the results were consistent with those of similar previous studies regarding the concentrations of IL-1β, HMGB1, and LDH in pleural

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effusions (Silva-Mejias et al., 1995; Winter et al., 2009; Light, 2013). As such, the bias

should be limited. Furthermore, significant results were obtained even after removing

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the extreme data from the correlation analysis (Table S1).

5. Conclusions

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In this study, we measured the concentrations of IL-1β, HMGB1, HO-1, and LDH

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in pleural effusions obtained from patients of various etiologies and found a correlation

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between the levels of HMGB1, IL-1β, and LDH in infectious effusions. These findings suggest that these proteins, especially HMGB1, might be used to as a surrogate in determining the severity of inflammation in the pleural cavity like LDH. Moreover, our data indicate that these proteins may share common molecular pathways in the inflammatory response in closed pleural cavities. We also found that malignant effusions

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had low levels of IL-1β expression. However, the efficacy and accuracy should be evaluated in a larger study with more patients involved, and further research is required to elucidate the underlying mechanisms of our results.

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Funding

This study was supported by the Ministry of Science and Technology, Taiwan [grant

numbers MOST 104-2320-B-010-014-MY3 and MOST 107-2320-B-010-027-MY3].

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The funders had no role in study design, data collection and analysis, decision to

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publish, or preparation of the manuscript.

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Disclosures

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No conflicts of interest, financial or otherwise, are declared by the authors.

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Author Contributions

KMW and YRK conceived and designed the experiments. KMW, WKC, and CHC performed the experiments. KMW, WKC, and CHC analyzed the data. KMW, WKC,

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CHC, and YRK drafted the manuscript. The final version of the manuscript was approved by all the authors.

Acknowledgements

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We would like to thank Editage (www.editage.com) for English language editing.

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27

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ro of

Figures

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Fig. 1. Levels of IL-1β, HMGB1, HO-1, and LDH in transudative, infectious, and malignant

lP

effusions. The expression of these four proteins were measured in each study sample from the three patient groups (transudative (n=10), infectious (n=15), and malignant (n=15)).

na

(a) IL-1β, (b) HMGB1, (c) HO-1, and (d) LDH. Significant differences of each protein in the three

ur

groups (transudative, infectious, and malignant) are indicated by *P < 0.05, ***P < 0.001,

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Kruskal-Wallis one-way analysis. Significant differences in post-hoc pair-wise comparisons are indicated by †P < 0.05 vs. infectious effusion, ††P < 0.05 vs. infectious and transudative effusion, Dunn’s method. Numbers in the plots represent the median values. Arrows indicate the three empyema cases (see Table 1).

28

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Fig. 2. Scatterplot matrix of infectious effusions. SPLOM (scatter plot matrix) analysis was used to determine the correlation of HMGB1, IL-1β, and LDH in transudative, infectious, and

na

malignant effusions groups respectively. Correlation was found in infectious effusions. Strong correlations between HMGB1, IL-1β, and LDH were found. Poor correlations were

Jo

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observed between HO-1 and these three proteins.

29

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Fig. 3. Correlations between HMGB1, IL-1β, and LDH in infectious effusions. In infectious pleural effusion, the concentration of HMGB1, IL-1β, and LDH was plotted pairwise, and their

na

coefficient were analyzed. 3 D graphic showed each sample’s value of these three proteins. (a) IL-1β vs. HMGB1: Spearman’s rank correlation coefficient  (rho= 0.779, P < 0.001. (b)

ur

IL-1β vs. LDH: = 0.793, P < 0.001. (c) HMGB1 vs. LDH: = 0836, P < 0.001. (d) 3D

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correlation of IL-1β, HMGB1, and LDH in infectious effusion. The oblique lines in (a), (b), and (c) indicate the regression line of each plot. Linear regression: IL-1vs.HMGB1:R2 = 0.546, P = 0.002; IL-1 vs. LDH R2 = 0.667, P < 0.001; HMGB1 vs. LDH: R2 = 0.864, P < 0.001. Arrows indicate the three empyema cases (see Table 1).

30

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31

Tables

Table 1. Demographic data and etiologies of effusion samples from patients

Variable

Value

Male

20

Female

20

69.0

(57.0,78.3)

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Median Age, years

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Gender (n)

Diagnosis (n)

10

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- Transudate

- Exudate

na

- Infectious effusion

10

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Congestive heart failure

15

8

Empyema

3

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ur

Para pneumonic effusion

30

4

Tuberculosis

- Malignant effusion

15

Lung cancer

11

Metastatic cancer

4

32

Malignant mesothelioma of peritoneum

1

Breast cancer

2

Colon cancer

1

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Patients’ ages are expressed as median with interquartile range (25%, 75%).

33

f oo

Table 2. Demographic and laboratory data of the pleural effusions of the patient cohort (40 patients)

Transudative

Infectious

pr

Variable

Median age (years)

3&7

6&5

Pr

Male/female (n)

e-

Parapneumonic & Empyema

70.0 (66.8, 80.0)

52.0(43.5, 78.5)

Malignant

P-values

Tuberculosis

4&0

7&8

73.5(68.3, 76.5)

69.0(57.5, 76.0)

0.246

Protein (g/dL)

na l

Pleural effusions

2.1 (1.9,2.5)

3.6 (3.4,4.1)

3.4 (3.2, 3.8)

4.4 (4.0, 4.7)

< 0.001*

145.5 (123.8,184.5)

74.0 (31.5, 118.5)

102.5 (97.0, 105.3)

112.0 (98.5, 142.5)

0.025*

RBC (× 109/L)

725.0 (143.8,900.0)

1600.0 (725.0, 10000.0)

2159.0 (1777.5, 2661.0)

3800.0 (2095.0,10000.0)

0.034*

WBC (× 109/L)

210.0 (126.8,330.0)

1600.0 (610.0, 6000.0)

985.0 (723.0, 1252.5)

850.0 (290.0,1071.0)

0.003*

53.0 (31.5,76.0)

35.0 (8.0, 58.0)

92.5 (90.1, 93.8)

73.0 (29, 81.5)

0.007*

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Glucose (mg/dL)

Lymphocyte (%)

34

9.0 (5.5, 18.5)

7900.0 (7200.0, 9200.0)

10100.0 (6850.0, 13600.0)

Blood

WBC (× 109/L)*

f

39.0 (21.0, 43.0)

2.0 (1.8, 2.8)

1.0 (1.0, 6.0)

<0.001*

5.6 (4.5, 7.0)

25.0 (17.5, 67.0)

0.002*

9750.0 (5050.0, 14225.0)

7200.0 (6300.0, 9450.0)

oo

30.0 (20.0, 84.0)

pr

Others (%)

4.0 (2.3, 12.8)

e-

Neutrophil (%)

0.668

Pr

All the data were obtained from the pleural effusion samples, except for the blood WBC. The data is expressed as the median with an interquartile range

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(25%, 75%). Data analysis was carried out using Kruskal-Wallis one-way analysis.

*There are statistically significant differences of these variables expressed in transudative, infectious (parapneumonic and empyema, tuberculosis) and

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malignant pleural effusions.

35

Table 3. Concentration of IL-1β, HMGB1, HO-1, and LDH in transudative, infectious, and malignant pleural effusions

Categories

IL-1β (pg/mL)

HMGB1 (ng/mL)

HO-1 (ng/mL)

LDH (IU/mL)

11.7 (11.0, 13.6)

15.0 (14.3, 19.6)

10.0 (4.3, 13.7)

95.0 (74.8, 118.5)

Infectious

51.0 (24.2, 85.0)

35.1 (22.5, 96.9)

21.1 (10.9, 36.1)

866.0 (378.0, 1173.5)

Malignant

11.5 (10.9, 14.9)

29.6 (21.0, 40.0)

17.8 (9.5, 27.8)

P < 0.001

P = 0.011

P = 0.021

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P-value

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Transudative

235.0 (151.0, 446.0)

P < 0.001

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The data is expressed as the median with an interquartile range (25%, 75%). Data analysis was

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carried out using Kruskal-Wallis one-way analysis.

36

Table 4. Binary logistic regression model of IL-1β, HMGB1, HO-1, and LDH for malignancy analysis in exudative pleural effusions B

S.E.

Wald

df

Sig.

Odds

95% C.I. for odds

ratio

ratio

Lower

Upper

-0.343

0.157

4.740

1

0.029

0.710

0.522

0.966

HMGB1

0.129

0.116

1.247

1

0.264

1.138

0.907

1.427

HO-1

0.016

0.058

0.080

1

0.778

1.016

0.908

1.138

LDH

-0.003

0.003

1.196

1

0.264

0.997

0.990

1.003

Constant

4.177

2.851

2.146

1

0.143

-p

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IL-1β

re

65.140

Only IL-1β is statistically significant in the analysis of the four proteins in malignant effusions.

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Increasing IL-1β was significantly associated with a decreased cancer risk. OR (odds ratio) =

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0.71, 95% C.I. (0.522-0.966), P = 0.029. B: intercept; S.E.: standard error; df: degrees of

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freedom; Sig.: p-value.

37

Table 5. Binary logistic regression model of IL-1β, HMGB1, HO-1, and LDH for malignancy analysis in exudative pleural effusions

B

S.E.

Wald

df

Sig.

Odds

95% C.I. for odds

ratio

ratio

Upper

ro of

Lower

-0.343

0.157

4.740

1

0.029

0.710

0.522

0.966

HMGB1

0.129

0.116

1.247

1

0.264

1.138

0.907

1.427

HO-1

0.016

0.058

0.080

1

0.778

1.016

0.908

1.138

LDH

-0.003

0.003

1.196

1

0.264

0.997

0.990

1.003

Constant

4.177

2.851

2.146

re

-p

IL-1β

0.143

65.140

lP

1

Only IL-1β is statistically significant in the analysis of the four proteins in malignant effusions.

na

Increasing IL-1β was significantly associated with a decreased cancer risk. OR (odds ratio) =

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0.71, 95% C.I. (0.522-0.966), P = 0.029. B: intercept; S.E.: standard error; df: degrees of

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freedom; Sig.: p-value.

38