Differed IL-1 Beta Response between Active TB and LTBI Cases by Ex Vivo Stimulation of Human Monocyte-Derived Macrophage with TB-Specific Antigen

Background The difference of macrophage-specific interleukin-1 beta (IL-1b) response between latent tuberculosis infection (LTBI) and active tuberculosis (TB) remains less studied. Method We performed this prospective study and recruited active TB patients, contacts with LTBI, and uninfected contacts. The gene and protein expression of human monocyte-derived macrophage (hMDM) after ex vivo stimulation by early secretory antigenic target-6KD (ESAT-6) and tuberculin purified protein derivatives (PPD) was studied by real-time PCR and flow cytometry. The effect of caspase-1 inhibitor was also studied. Result The IL-1b gene expression after 6 hr ESAT-6 1 μg/ml stimulation was different among active TB patients (n = 12), LTBI cases (n = 12), and uninfected contacts (n = 23) (log fold change: 0.98 ± 1.26 vs. 2.20 ± 0.96 vs. 2.20 ± 0.96, P = 0.013). The IL-1b gene expression at 24 hours was higher than that at 6 hours in LTBI cases (n = 4) and uninfected contacts (n = 6). After 24 hr ESAT-6 1 μg/ml stimulation, the percentage of IL-1b-expressed hMDM was borderline lower in the active TB patients (n = 9) than in the LTBI cases (n = 10) (14.0 ± 11.2% vs. 31.6 ± 22.5%, P = 0.065). Compared with ESAT-6 1 μg/ml stimulation but without the addition of caspase-1 inhibitor (CasI) (55.6 ± 16.3%), the percentage of IL-1b-positive hMDMs decreased after addition of CasI (50 μg/ml CasI: 49.8 ± 18.2%, P = 0.078; 100 μg/ml CasI: 46.6 ± 20.8%, P = 0.030; 150 μg/ml CasI: 33.7 ± 15.5%, P = 0.016). Conclusions This study revealed that macrophage-specific IL-1b response differed among different stages of Mycobacterium tuberculosis infection. The role of IL-1b and inflammasome in the process of LTBI progressing to active TB warrants further investigation.


Introduction
Tuberculosis (TB) remains an important global infectious disease with an estimated 10.4 million new cases and 1.4 million deaths in 2015 [1]. The World Health Organization (WHO) has set its goal in eradicating TB as a public health problem, aiming to achieve 50% reduction and 90% reduction in TB incidence by 2025 and 2035, respectively [2]. Latent tuberculosis infection (LTBI), which was underestimated to affect one-third of the overall human population, also emerged as an important target for better TB infection control [3]. To achieve the global goal to significantly reduce TB disease burden, a better understanding of the pathophysiology underlying the progression of LTBI to active TB was urgently needed.
Macrophages are primary host cells against Mycobacterium tuberculosis (M. tb) infection and are responsible for the regulation of various cytokines [4]. Macrophages are also involved in the initial infection process of M. tb, including being infected and granuloma formation response [5]. Macrophages are also the main cells for inflammasome expression, which were crucial for triggering the inflammation process and defending against intracellular pathogens [6,7]. Due to sitting in a central position for controlling and preventing M. tb disease, macrophage and inflammasome functions have been intensely investigated [5]. Interleukin-1 beta (IL-1b) stands as the end product of inflammasome activation and plays a pleiotropic role in resistance to M. tb infection [4,8].
LTBI refers to the status where viable M. tb is contained in a human and does not lead to clinical symptoms [9]. At the same time, persons with LTBI were at ineligible risk for progressing to active TB [3]. It would be important to explore the association between M. tb-induced macrophagedependent host immune responses, particularly IL-1b and tumor necrosis factor-alpha (TNFa), and M. tb disease status. While abundant studies may exist in the literature regarding the role of IL-1b and TB, few studies have focused on the differential gene expression and regulation of macrophagerelated IL-1b between LTBI and active TB. Most of them were either cell-line or animal studies, and only few studies have used human peripheral blood monocytes for experiment. We, therefore, conducted this study and investigated the macrophage inflammasomal responses with and without the caspase-1 inhibitor by gene and protein expression after ex vivo stimulation with TB antigens using real-time PCR and flow cytometry.

Materials and Methods
2.1. Study Design and Duration. This prospective study was conducted in the National Taiwan University Hospital from August 2015 to July 2017. All experiments were performed in accordance with relevant guidelines and regulations.

Study
Population and Blood Sampling. We followed the definition of the study population and participant recruitment of Lee et al. [10]. We prospectively enrolled adult patients (age between 20 and 90 years) with culture-or histology-confirmed active TB (TB group). Close contacts of TB were defined as an exposure duration of 8 hours or more within one day or a cumulative duration of 40 hours or more without wearing adequate personal protective equipment for airborne transmission precautions. Close contacts of TB were then further screened for LTBI by using the QuantiFERON-TB Gold In-Tube (QFT) assay (Qiagen). If active TB was a concern, the contacts also received chest radiography and mycobacteriologic study (acid-fast smear and mycobacterial culture) from 3 sputum samples to exclude the possibility of active TB disease [11]. Uninfected contacts and LTBI contacts were considered for those with negative and positive QFT, respectively. After providing informed consent, they received peripheral blood sampling for further experiments.
The exclusion criteria were as follows: (1) human immunodeficiency virus infection, (2) bleeding tendency that increased the risk of blood sampling, and (3) life expectancies less than 6 months in TB patients or 2 years in household contacts.

QuantiFERON-TB Gold
In-Tube (QFT®). QFT was performed according to the manufacturer's instructions. The IFN-gamma level of the postreaction supernatant was then measured by an enzyme-linked immune-sorbent assay. Results were interpreted as positive, negative, or indeterminate accordingly [

Isolation and Differentiation of Peripheral Blood
Monocytes. We isolated human monocytes from PBMCs by using a CD14-positive selection system (MACS system, Miltenyi Biotec Inc.), followed by cultivation in RPMI-1640 medium supplemented with 10% FBS, 50 mM 2mercaptoethanol (Sigma-Aldrich, USA), and 50 ng/ml recombinant human macrophage colony-stimulating factor (R&D systems, USA) for 5 days to differentiate into macrophages. The culture medium was supplemented on the 3 rd day of differentiation. This process was also described previously [12].

Flow Cytometry for Assessing IL-1b and TNFa Protein
Expression. The hMDM was retrieved after 6 or 24 hours of in vitro stimulation in a medium containing 10% FBS and measured using flow cytometry (FACSVerse, BD Biosciences, USA). A protein transport inhibitor (GolgiStop™, BD Bioscience, USA) was added to the coculture. The retrieved cells were stained with anti-CD14-PerCP, anti-IL-1b-FITC, and anti-TNFa-PE. Data were analyzed using BD FACSuite V software (BD Biosciences, USA). We discriminated the cell population using forward scatter (FSC) and side scatter (SSC) and measured the percentage of IL-1b-and TNFa-expressed cells, respectively. We also calculated mean fluorescent intensity (MFI) in arithmetic means.

In Vitro Stimulation and the Addition of Caspase-1
Inhibitor. To assess the inflammasome response, the caspase-1 inhibitor (CasI) in three different concentrations was also added to hMDMs retrieved after 6 or 24 hours of in vitro stimulation. The percentage of IL-1b-and TNFaexpressed cells after in vitro stimulation and caspase-1 inhibition was also evaluated with flow cytometry.

Statistical Analysis. Nonparametric methods including the Kruskal-Wallis test, Mann-Whitney U test, and
Wilcoxon signed-rank test tests were used for gene and protein expression comparison. All data analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). A P < 0:05 on a two-sided test was considered statistically significant.

Discussion
By ex vivo stimulation of hMDMs from different stages of M. tb infection, we found that IL-1b was significantly highly expressed in the LTBI cases and uninfected contacts as compared to the active TB patients. On the contrary to TNFa, the expression of IL-1b was accentuated when the stimulation was prolonged from 6 hours to 24 hours. The flow cytometry study revealed similar findings, showing that the percentage of IL-1b-positive hMDMs was markedly elevated in LTBI cases, whereas the percentage of TNFa-positive hMDMs was not different between LTBI cases and active TB patients. Applying the caspase-1 inhibitor decreased the percentage of IL-1b-positive hMDMs. Our findings highlight the role of IL-1b and inflammasome in TB pathogenesis, especially in the progression of LTBI to active TB.
Most studies published regarding TB and IL-1b mainly focused on the comparison between active TB and uninfected or between pretreated and posttreated statuses [15]. Studies that have investigated the role of IL-1b among LTBI patients often measure the systemic IL-1b level [16,17]. Furthermore, most studies used a mouse model rather than peripheral blood monocyte-derived macrophages from human active TB and LTBI patients [4,18]. The uniqueness of our study, therefore, lies in studying macrophage-specific IL-1b expression at gene and protein levels and, more importantly, targeting active TB patients as well as LTB cases. Indeed, the focus of recent studies has been in exploring the interaction of macrophages and latent tuberculosis infection. For instance, a recent study showed that compared with active TB, antibodies production from latent tuberculosis leads to enhanced inflammasomal activation and macrophage killing of intracellular M. tb [19]. Given that LTBI infection is often the first step of developing active TB, elucidating LTBI pathophysiology is therefore important [20].
IL-1b is a double-edged sword in TB disease, with its antimicrobial effect as a protective role for disease development while prolonged expression could lead to progressive tissue damage [21]. In an animal model, IL-1 receptor knockout mice lose early pathogen control following aerogenic infection with M. tb [22]. Our study revealed that IL-1b gene expression after stimulation was upregulated in both TB patients and LTBI cases while the degree of elevation was more obvious in the latter group. Also, the percentage of IL-1b-positive macrophages also decrease substantially following 24 hr stimulation compared with the 6 hr stimulation in active TB patients. This finding could have two implications. First, the high IL-1b response by macrophages in the LTBI status may indicate activation of immunity to prevent development to active TB. The relatively low response among active TB patients may indicate a defective immunity response to TB bacilli and this would contribute to the development of active disease. Interestingly and contradictorily, a previous study has discovered a high IL-1b expressing genotype, which was associated with the development of active tuberculosis, higher disease severity, and poorer treatment outcome in TB patients [21]. On the other hand, the relatively low level of IL-1b response may also be a human regulatory mechanism to avoid further tissue inflammation and destruction [23][24][25]. Second, our study revealed that IL-1b could be the target for preventing progression from LTBI to active TB. Enhancing IL-1b activity may be an option to halt TB infection progression [26]. Whether use of IL-1b antagonists would increase the risk of LTBI progression to active TB also needs to be investigated [27,28].
The secretion of IL-1b by macrophages comes from two pathways, including the inflammasome and NF-κB pathways [4]. NF-κB was known to mediate and enhance the secretion of various cytokines, including IL-1b and TNFa [29]. In our study, TNFa secretion was not elevated and this may indicate that the higher response of IL-1b by macrophages was most likely through the pathway of inflammasomes. Furthermore, caspase-1 is an important inflammasome activator, and the findings of decreased IL-1b-positive hMDMs after addition of the caspase-1 inhibitor further supported the contribution of inflammasomes. Though our mRNA gene expression profile only revealed transient increase in expression of the NLRP3 pathway, our study still highlights the importance of inflammasomes in TB pathogenesis, through the effect of IL-1b. NLRP3 is an important inflammasome sensor, and study has shown that NLRP3 gene polymorphism is associated with increased IL-1b production and better M. tb con-trol [30]. The activation of NLRP3 by mycobacteria was more complex, and several components including ESAT-6 secretion system (ESX)-1, the never in mitosis A-related kinase 7 (Nek7), were considered important activators of NLRP3 [31,32]. The interaction and association between inflammasomes, activating components, and IL-1b production were less clear and remained to be studied.
Our study also has important implications for clinical practice. Due to limited performance of current biomarkers for monitoring TB treatment response and disease status, newer and more sophisticated biomarkers are needed [33]. Recently, Adekambi et al. found that host blood-based biomarkers including CD38, HLA-DR, and Ki-67 on M. tb-specific CD4 + T cells could help discriminate between active TB and LTBI and could be a biomarker for monitoring treatment response and cure [34]. Furthermore, combined analysis of M. tb-specific CD4 and CD8 T cell responses has also been proposed to be a powerful diagnostic tool for diagnosing TB [35]. Besides M. tb-specific T cell immune markers, studies targeting the ratio of the myeloid over lymphoid cells also highlight the importance of circulating monocytes as an important of biomarker for predicting development of active TB among contacts [36,37]. Also, antigen-specific IL-1b can be used as a biomarker for differential diagnosis of pulmonary TB and LTBI [38]. Interestingly, by measuring cytokines in supernatants generated from QFT tubes, higher IL-1b and TNFa levels were observed in active TB patients than in contacts and uninfected controls [38]. Additionally, our findings add knowledge to the current understanding of TB immunology and suggested that IL-1b-positive hMDM could be a novel immune cell signature biomarker differentiating between LTBI cases and active TB patients.
Our study also has limitations. Large and sufficient amount of blood specimens from participants were hard to collect in the clinical setting, and this may cause inconsistent number of cases measuring sequential change of IL-1b response. This, however, may reflect the distinguishing feature (using peripheral blood monocytes from active TB and LTBI patients) of our study. Second, different immunocompromised statuses can alter immune response and are not readily excluded in our study. Diabetes, for instance, is an important disease contributing to tuberculosis and can affect the measured parameters and is not among our exclusion criteria [39,40]. Third, we did not check genetic polymorphisms of targeted cytokines and inflammasomes [41,42]. The association between corresponding genetic polymorphisms and TB disease course was worth discussing and could be the perspective of future studies [43].
In conclusion, we found that macrophage-specific IL-1b response differed between LTBI cases and active TB patients after ex vivo TB-specific antigen stimulation, while response in LTBI was higher than active TB patients. In active TB patients, the IL-1b-positive human monocyte-derived macrophage after stimulation was decreased compared with LTBI cases. IL-1b gene expression was also decreased in active TB patients compared with LTBI cases. Our results suggested that IL-1b plays a role in the progression of LTBI patients, and further studies regarding mechanism and clinical implications were warranted.

Data Availability
The data will be made available from the corresponding author upon reasonable request.

Ethical Approval
Ethics approval was obtained from the Institutional Review Board of National Taiwan University Hospital (NTUH REC: 201512169RINA).

Consent
Informed consent was retrieved.

Disclosure
The funders had no role in the study design, data analysis, and manuscript writing.