Differential expression and predictive value of monocyte scavenger receptor CD163 in populations with different tuberculosis infection statuses
Monocytes are the predominant innate immune cells at the early stage of Mycobacterium tuberculosis (M. tb) infection as the host defense against intracellular pathogens. Understanding the profile of different monocyte subpopulations and the dynamics of monocyte-related biomarkers may be useful for the diagnosis and prognosis of tuberculosis.
We enrolled 129 individuals comprising patients with pulmonary tuberculosis (PTB) (n = 39), tuberculous pleurisy (TBP) (n = 28), malignant pleural effusion (MPE) (n = 21), latent tuberculosis infection (LTBI) (n = 20), and healthy controls (HC) (n = 21). Surface expression of CD14, CD16, and CD163 on monocytes was detected using flow cytometry. In addition, soluble CD163 (sCD163) was determined by enzyme linked immunosorbent assay.
Higher frequency of CD14+CD16+ (15.7% vs 7.8%, P < 0.0001) and CD14−CD16+ (5.3% vs 2.5%, P = 0.0011) monocytes and a decreased percentage of CD14+CD16− (51.0% vs 70.4%, P = 0.0110) cells was observed in PTB patients than in HCs. Moreover, PTB patients displayed a higher frequency of CD163+ cells in CD16+ monocytes than those in the HC group (40.4% vs 11.3%, P < 0.0001). The level of sCD163 was elevated in TBP patients and was higher in pleural effusion than in plasma (2116.0 ng/ml vs 1236.0 ng/ml, P < 0.0001). sCD163 levels in pleural effusion and plasma could be used to distinguish TBP from MPE patients (cut-off values: 1950.0 and 934.7 ng/ml, respectively; AUCs: 0.8418 and 0.8136, respectively). Importantly, plasma sCD163 levels in TBP patients decreased significantly after anti-TB treatment.
Higher expression of membrane and soluble CD163 in active tuberculosis patients might provide insights regarding the pathogenesis of tuberculosis, and sCD163 may be a novel biomarker to distinguish TBP from MPE and to predict disease severity.
KeywordsTuberculosis Monocyte subpopulations CD163 Innate immunity Disease severity
Enzyme linked immunosorbent assay
Interferon-γ release assay
Latent tuberculosis infection
- M. tb
Malignant pleural effusion
Peripheral blood mononuclear cells
Mycobacterium tuberculosis (M. tb) invades the host and activates immune responses including innate and adaptive immunity, and causes tuberculosis (TB), which remains a global public health concerns . Monocytes/macrophages, as the first line of defense, are critical for host immunity against M. tb infection [2, 3, 4]. Human monocytes are classified into three major subpopulations based on the expression of markers CD14 and CD16: CD14+CD16− (classical), CD14+CD16+ (intermediate), and CD14−CD16+ (non-classical) monocytes [5, 6, 7]. The three monocyte subsets represent different stages of macrophage differentiation and play different roles in M. tb infection . Classical monocytes account for the majority of total subsets; they differentiate into pro-inflammatory M1 macrophages (classical activated) that are permissive to M. tb infection in vitro and produce several pro-inflammatory cytokines . However, both non-classical and intermediate monocytes are considered precursors of anti-inflammatory M2 macrophages (alternative activated) in different disease conditions .
Increasing evidence suggests that switching the M1/M2 phenotype influences the clinical outcome of host infection with M. tb [2, 3, 9, 10]. The early stage of anti-TB immune responses is predominated by M1 macrophages, which are characterized by high production of iNOS and IFN-γ, with the function of killing most M. tb and restricting the replication of the remainder. Nevertheless, M2 macrophages are poorly microbicidal and play an immunomodulatory role . Thus, the shift from M1 to M2 polarization during M. tb infection might be a microbial strategy to escape immune attack and cause disease progression.
CD163, a scavenger receptor that serves as an M2 macrophage phenotype marker, is also expressed on monocytes, and binds to haptoglobin-hemoglobin complexes, mediating their endocytosis . In the context of TB, expansion of CD16+CD163+MerTK+ monocytes contribute negatively to the host defense against M. tb by a low ratio of pro−/anti-inflammatory cytokine production and a poor capacity to activate T cells. Moreover, CD163 and MerTK act as M2-like macrophage activation markers, which are characterized by pathogen permissivity and immunomodulatory activity . Indeed, the soluble form of CD163 (sCD163) from monocyte activation, exists in plasma and is correlated with TB disease severity. In this study, we explored the expression of scavenger receptor CD163 on monocyte subsets in populations with different tuberculosis infection statuses including active tuberculosis, latent tuberculosis infection and non-infection, detected the sCD163 levels in plasma and pleural effusions, further assessed the value of sCD163 in diagnosing tuberculosis and in predicting the disease severity and treatment outcome.
In total, 129 individuals were enrolled in this study including patients with pulmonary TB (PTB) (n = 39), tuberculous pleurisy (TBP) (n = 28), malignant pleural effusion (MPE) (n = 21), latent tuberculosis infection (LTBI) (n = 20), and healthy controls (HC) (n = 21). All the patients were recruited from Wuxi Fifth People’s Hospital, Zhuji People’s Hospital, and Fudan University Affiliated Huashan Hospital from 2011 to 2018. Populations with LTBI and HC were recruited from the relatives of PTB patients and the volunteers of Huashan Hospital during the same period.
This study was approved by the Ethics committee of Huashan Hospital, Fudan University. Written informed consent was obtained from all the participants.
PTB patients were diagnosed based on identification of M. tb in the sputum or bronchoscopy. TBP patients were diagnosed according to the following criteria: 1) pleural biopsy; 2) acid-fast bacilli (AFB) smear or culture positive in pleural effusion (PE); 3) AFB smear or culture positive in sputum; 4) combination with clinical symptoms, radiological results, and effective anti-TB treatment upon lack of etiology evidence. MPE patients without tuberculosis infection were enrolled as controls, diagnosed based on either histopathology in pleural tissue or cytology in PE. Populations with LTBI and HC were interferon-γ release assay (IGRA)-positive and -negative, respectively. In addition, they had no evidence of active tuberculosis infection.
All enrolled participants were free of HIV infection, autoimmune disease or other chronic infections (i.e., chronic HBV/HCV infection). Furthermore, they were not undergoing immune-modulating treatment.
Interferon-γ release assay (IGRA)
In this study, the T-cell-based enzyme-linked immunospot assay for tuberculosis (T-SPOT.TB) (Oxford Immunote Ltd., Oxford, UK) was performed as IGRA according to the manufacturer’s instructions. The positive results were analyzed as described previously .
Cell surface staining and flow cytometry
Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density-gradient centrifugation from 10 ml venous blood samples. For phenotyping of monocyte subsets, one million fresh PBMCs were surface stained with monoclonal anti-human CD14-APC (61D3, eBioscience), CD16-FITC (3G8, Biolegend) and CD163-BV421 (GHI/61, Biolegend) at room temperature in the dark for 15 min, and were washed twice in PBS containing 2% FBS. Stained samples were detected on a Beckman Moflo flow cytometer. According to the expression of CD14 and CD16, three major monocyte subsets in PTB patients and controls were analyzed as classical (CD14+CD16−), intermediate (CD14+CD16+), and non-classical (CD14−CD16+) monocytes. Furthermore, expression of the scavenger receptor CD163 on monocytes subsets was evaluated. Data were analyzed using Flowjo 10 (Tree Star, Inc. Ashland, OR).
Detection of soluble CD163 levels using enzyme linked immunosorbent assay (ELISA)
Levels of soluble (s)CD163 were assessed in plasma and pleural effusion (PE) samples stored at − 80 °C using an ELISA kit (DC1630, R&D systems) according to the manufacture’s protocol. Absorbance was immediately determined at 450 nm and 570 nm on a microplate reader, and was corrected by subtracting the readings at 570 nm from the readings at 450 nm. The minimum detectable dose was 0.177 ng/ml.
Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc. La Jolla, CA) and MultiExperiment Viewer (MeV) 4.9 (Dana-Farber Cancer Institute, Boston, USA). Two different groups were compared using an unpaired t test or Mann-Whitney U test (when the variances were significantly different). Categorical variables were compared using the χ2 test or Fisher’s exact test, as appropriate. Receiver operating characteristics (ROC) curve analysis was performed to evaluate the diagnostic performance of sCD163 levels in plasma or the PE for differential diagnosis of PTB and controls, or TBP and MPE. Comparisons of plasma sCD163 levels in unpaired pre- and post-treatment groups were performed using unpaired Mann-Whitney U test. Statistical significance was referred as P < 0.05.
Clinical characteristics of participants
Clinical characteristics of enrolled individuals
ATB (n = 67)
MPE (n = 21)
LTBI (n = 20)
HC (n = 21)
PTB (n = 39)
TBP (n = 28)
Age, median (IQR)
History of BCG vaccination, n (%)
IGRA+, n (%)
Sputum AFB smear or culture positive, n (%)
>1+ (2+, 3+, 4+)
Cavity, n (%)
M. tb detection in pleural effusion
Confirmed TBP by pleural biopsy, n (%)
AFB smear or culture positive, n (%)
anti-TB therapy status at enrollment
Naive, n (%)
Days on anti-TB, median (IQR)
Increased percentage of circulating CD16+ monocytes and lack of classical (CD14+CD16−) monocytes in active tuberculosis patients
Higher expression of scavenger receptor CD163 on CD16+ monocytes in active tuberculosis patients
Soluble CD163 levels in plasma and pleural effusion and the value of sCD163 in diagnosing tuberculosis
Relationship between soluble CD163 levels in plasma and disease severity in patients with pulmonary tuberculosis
sCD163 levels in plasma decreased at different time points during anti-TB therapy
In this study, we compared three major monocytes subpopulations between PTB patients and HCs, and found an increased frequency of CD14+CD16+ and CD14−CD16+ monocytes and a decreased percentage of CD14+CD16− monocytes in patients with PTB. Currently, several studies have shown that changes in the profile of monocyte subsets during M. tb infection indicate bacterial persistence [14, 15, 16]. A previous study has similarly demonstrated that an increased proportion of CD14+CD16+ monocytes was associated with mortality in HIV-coinfected TB patients . M. tb infection has been shown to induce expansion of peripheral blood CD16+ monocytes spontaneously undergoing late apoptosis . However, in the context of TB, CD16− (classical) monocytes as the dominant innate immune cells against TB contribute to the restriction of M. tb growth by rapid migration to infection sites and high production of reactive oxygen species (ROS) . CD14+CD16+ monocytes exhibited higher phagocytic activity and lower antigen presentation compared to the CD14−CD16+ monocytes; additionally, this population was a major source of the immunosuppressive cytokine IL-10 . Therefore, according to the characteristics of the above different monocyte subsets, our results indicated that an increase in non-classical (CD14−CD16+) and intermediate (CD14+CD16+) monocytes plus a decrease in classical (CD14+CD16−) monocytes might lead to the dissemination of M. tb infection and be involved in the immunological pathogenesis of TB.
CD163 is considered a specific marker of M2 macrophages . We explored the expression of CD163 on different CD16+ monocytes subsets based on their CD14 expression. Surprisingly, scavenger receptor CD163 was mainly expressed on intermediate rather than non-classical monocytes, with a higher expression in PTB patients than that in HCs. Cougoule et al. also observed M2 markers including CD163 and CD206 augmented in the CD16+ subset compared to the CD16− population, but they did not detect the expression of M2 markers in intermediate monocytes . Indeed, they demonstrated that monocytes differentiated towards M2-like macrophages (CD16+CD163+MerTK+) depending on the IL-10/STAT3 signaling pathway in the context of TB, and that this phenotype rendered the host permissive to intracellular M. tb growth and an impaired ability to activate the Th1 immune response . Furthermore, CD163+ monocytes can secrete both pro- and anti-inflammatory cytokines such as TNF-α and IL-4 during Leishmania and hepatitis C infection, which might interfere in instructing T cells and inhibit the killing of intracellular pathogens . Thus, our findings indicated that high expression of CD163 on CD16+ monocytes in PTB patients might be involved in M. tb infection.
Additionally, sCD163 has been recently considered a novel soluble biomarker of monocyte/macrophage activation in pathological conditions [22, 23, 24]. Consistent with the membrane CD163 expression, our results show increased levels of sCD163 in ATB patients, which were particularly higher in PE than in plasma, suggesting that CD163+ monocytes might migrate to the site of infection in order to play an immunomodulatory role. This observation was consistent with the findings in patients with TB-associated immune reconstitution inflammatory syndrome (TB-IRIS), who exhibited high levels of sCD163 before anti-retroviral therapy (ART) and a worse prognosis . Our findings revealed sCD163 as a potential biomarker in the diagnosis of ATB with a high specificity and low sensitivity, allowing the distinction of TBP from MPE. With an optimal cut-off value of 1950.0 ng/ml and 934.7 ng/ml, respectively, the sCD163 levels in PE and plasma showed AUCs of 0.8418 and 0.8136 in the diagnosis of TBP, respectively. Regrettably, sCD163 levels in plasma could not differentiate ATB from LTBI, whereas they could be used to distinguish ATB from HC with an AUC of 0.7698. Similarly, ROC analysis was conducted in TB patients and healthy subjects, showing a AUC of 0.78, but the differences between tuberculous and malignant PE were not analyzed in this study . Indeed, many biomarkers such as adenosine deaminase (ADA), lactate dehydrogenase (LDH), and IGRA, have been investigated to distinguish TBP from MPE patients, with a large range variation in sensitivity and specificity [26, 27, 28, 29]. Based on our results, the sCD163 levels in PE and plasma could be a useful biomarker for the diagnosis of TBP.
More importantly, we found a strong association between high sCD163 levels and TB disease severity in the present study. First, the increased levels of sCD163 in plasma were strongly linked to increased M. tb loads in sputum. Next, in the cavity (+) group, the proportion of high sCD163 (> 1260 ng/ml) expression (84.2%) was more than twice as large as that in the cavity (−) group (35.0%). Moreover, after effective anti-TB therapy, plasma sCD163 levels in patients with TBP were decreased significantly. A prospective cohort study followed up 113 verified TB patients, and demonstrated an association between high sCD163 levels (> 3950 ng/ml) and increased mortality . Actually, the serum levels of sCD163 in PTB patients were restored to normal levels after 12 months of anti-TB treatment . These findings suggest that sCD163 might be a predictive biomarker for TB prognosis.
Taken together, our study indicates an increased frequency of CD14+CD16+ and CD14−CD16+ monocytes and a decreased CD14+CD16− population in PTB patients. Both membrane and soluble CD163 are markers of monocyte/macrophage activation, which were increased in patients with ATB, especially in pleural effusions. Furthermore, sCD163 can be used to distinguish TBP from MPE patients with a high specificity. Importantly, there was a strong association between the high sCD163 levels and TB disease severity. We also followed up plasma samples of TBP patients and observed a significant decrease in plasma sCD163 after effective anti-TB treatment. Therefore, CD163 may offer a new insight in the diagnosis and prognosis of TB patients.
We express sincere thanks to all the enrolled participants of this study.
LS conceived and designed the study. QL performed the experiments and drafted the manuscript. QO, YG, QR and WZ contributed as clinicians. HC, YL and YX analyzed the data. All authors read and approved the final version of the manuscript.
The present study was financially supported by the National Natural Science Foundation of China (No. 81501359 and No. 81671553), Wuxi Science and Technology Development Fund Project (CSE31N1713) and Wuxi Health and Family Planning Committee Youth Project (Q201765). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics committee of Huashan Hospital, Fudan University. Written informed consent was obtained from all participants.
Consent for publication
The authors declare that they have no competing interests.
- 12.Lastrucci C, Benard A, Balboa L, Pingris K, Souriant S, Poincloux R, Al Saati T, Rasolofo V, Gonzalez-Montaner P, Inwentarz S, et al. Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16(+) monocyte population via the IL-10/STAT3 axis. Cell Res. 2015;25(12):1333–51.CrossRefGoogle Scholar
- 13.Shao L, Zhang W, Zhang S, Chen CY, Jiang W, Xu Y, Meng C, Weng X, Chen ZW. Potent immune responses of Ag-specific Vgamma2Vdelta2+ T cells and CD8+ T cells associated with latent stage of Mycobacterium tuberculosis coinfection in HIV-1-infected humans. AIDS. 2008;22(17):2241–50.CrossRefGoogle Scholar
- 15.Balboa L, Romero MM, Basile JI, Sabio y Garcia CA, Schierloh P, Yokobori N, Geffner L, Musella RM, Castagnino J, Abbate E, et al. Paradoxical role of CD16+CCR2+CCR5+ monocytes in tuberculosis: efficient APC in pleural effusion but also mark disease severity in blood. J Leukoc Biol. 2011;90(1):69–75.CrossRefGoogle Scholar
- 16.Balboa L, Barrios-Payan J, Gonzalez-Dominguez E, Lastrucci C, Lugo-Villarino G, Mata-Espinoza D, Schierloh P, Kviatcovsky D, Neyrolles O, Maridonneau-Parini I, et al. Diverging biological roles among human monocyte subsets in the context of tuberculosis infection. Clin Sci (Lond). 2015;129(4):319–30.CrossRefGoogle Scholar
- 25.Andrade BB, Singh A, Narendran G, Schechter ME, Nayak K, Subramanian S, Anbalagan S, Jensen SM, Porter BO, Antonelli LR, et al. Mycobacterial antigen driven activation of CD14++CD16- monocytes is a predictor of tuberculosis-associated immune reconstitution inflammatory syndrome. PLoS Pathog. 2014;10(10):e1004433.CrossRefGoogle Scholar
- 27.Blakiston M, Chiu W, Wong C, Morpeth S, Taylor S. Diagnostic performance of pleural fluid adenosine deaminase for tuberculous pleural effusion in a low-incidence setting. J Clin Microbiol. 2018;56(8). https://doi.org/10.1128/JCM.00258-18.
- 30.Knudsen TB, Gustafson P, Kronborg G, Kristiansen TB, Moestrup SK, Nielsen JO, Gomes V, Aaby P, Lisse I, Moller HJ, et al. Predictive value of soluble haemoglobin scavenger receptor CD163 serum levels for survival in verified tuberculosis patients. Clin Microbiol Infect. 2005;11(9):730–5.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.