Abstract
Tuberculosis (TB) is a disease instigated by Mycobacterium tuberculosis. Peripheral blood monocytes represent highly efficient effector cells of innate immunity against TB. Little is known about monocyte subsets and their potential involvement in the development of M. tuberculosis drug resistance in patients with TB. This study was conducted to investigate alterations in monocyte subsets, CD163 expression on monocytes, and its serum level in patients without and with rifampicin resistance TB (RR-TB) and healthy controls. A total of 164 patients with TB (84 without RR-TB and 80 patients with RR-TB) and 85 healthy controls were enrolled in this study. The percentages of various monocyte subsets and surface expression of CD163 on monocytes were quantitatively determined using flow cytometry. The serum level of CD163 was determined by commercially available ELISA kits. Decreased frequency of classical monocytes was detected in patients with RR-TB. Non-classical monocytes were decreased in patients without RR-TB; however, intermediate monocytes were raised in patients with RR-TB. The serum level of CD163 was decreased in patients of RR-TB that showsed a positive correlation with the frequency of CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes. It is concluded that decreased classical monocytes and sCD163 in patients with RR-TB could be an indicator of drug resistance.
Keywords: Mycobacterium tuberculosis, Monocyte subsets, Drug resistance, Immune response, Classical monocytes
Introduction
Tuberculosis (TB) is a global public health problem and is the leading cause of death worldwide. In 2016, worldwide TB resulted in 1.3 million deaths and 6.3 million new cases [1]. Pakistan ranks fifth among high-burden TB countries and accounts for 61% of the TB load in the WHO Eastern Mediterranean Region. Pakistan is estimated as the fourth highest prevalence country of multidrug-resistant TB (MDR-TB) globally [2] that is a significant public health and therapeutic problem that may rapidly worsen as the human immunodeficiency virus epidemic spreads [3]. Both innate and adaptive immune responses are required to prevent the establishment of the disease. The cellular immune response determines whether an infection is arrested at the latent stage or progresses to active TB [4]. Peripheral blood monocytes represent highly efficient effector cells of innate immunity against TB. Monocytes and macrophages are the primary targets of Mycobacterium tuberculosis, and their innate capacity to control M. tuberculosis determines an early progression of the infection. During active TB disease, the monocyte number expands in the peripheral blood [1]. Monocytes can be divided into subpopulations based on the expression level of membrane antigens i.e. CD14 (receptor for bacterial LPS) and CD16 (Fc gamma RIII) [5]. In 2010, Nomenclature Committee of the International Union of Immunological Societies suggested three monocyte subsets: classical (CD14++CD16–), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) [6].
The three monocyte subsets vary in size, trafficking, and their ability to differentiate in response to stimulation with immunomodulating agents (e.g. cytokines) and microbial substances. Classical monocytes which are a major population in peripheral blood (~85%) produce IL-1, IL-12, and tumor necrosis factor (TNF)-α. Non-classical/intermediate monocytes are up to 15% of peripheral blood monocytes, but their numbers may be increased in TB [5]. They produce a large amount of IL-10 and are mostly involved in immunoregulation [7]. Non-classical monocytes actively defend the luminal side of the vascular endothelium during inflammation [8].
Numerous studies have established that variations in the profile of monocyte subsets during M. tuberculosis infection show bacterial persistence [9–11]. Classical monocytes are the dominant innate immune cells against TB that contribute to the restriction of M. tuberculosis growth by rapid migration to infection sites and high production of reactive oxygen species (ROS) [11]. Mycobacterium tuberculosis infection has been shown to induce expansion of peripheral blood non-classical/intermediate monocytes which extemporaneously undergo late apoptosis [12]. Intermediate monocytes exhibit higher phagocytic activity and lower antigen presentation compared with the non-classical monocytes and are a major source of the immunosuppressive cytokine IL-10 [13].
The relative distribution of monocyte subsets may change in response to several circumstances, and a deviation from normal may lead to a diseased state. Liu et al. (2019) documented an increased frequency of intermediate and non-classical monocytes in patients with TB whereas a decreased classical monocytes population was observed in the same study. Similarly, another study has suggested that CD16+ monocytes (non-classical/intermediate monocytes) are increased in TB infection, and inappropriate expansion of this subset may increase disease severity [12]. Sanchez et al. (2006) [9] proposed that the expansion of non-classical/intermediate monocytes is caused by microbial or host components and is reversed with anti-TB treatment. Another study by Balboa et al. (2015) [11] established that classical monocytes confer anti–M. tuberculosis immune responses during TB infection by infiltration to the infection site and the production of ROS.
CD163 is a hemoglobin scavenger receptor that is specifically expressed on activated monocyte subsets and plays a significant role in immunological homeostasis [14]. It directly contributes to defense against pathogens via phagocytosis leading to pathogen destruction [15, 16], indicating that CD163 mediates both anti-inflammatory and antimicrobial responses. The soluble form of CD163 has a biological value as an interesting candidate marker for inflammation, sepsis, and immunological disorders. It is considered a prognostic marker, which can be used for monitoring these diseases. It has been identified that CD163 expression is not associated with other inflammation parameters such as C reactive protein [15]. The upregulation of CD163 by cytokines and glucocorticoids leads to anti-inflammatory responses that attenuate inflammation through two mechanisms. The first reveals CD163 as a soluble component, exhibiting cytokine-guided functions. The second shows that CD163 acts as a scavenger of the haptoglobin/hemoglobin complex, preventing hemoglobin intoxication and the adverse effects of the free hem. Oxidative stress and damage to cells are also avoided by this process [16]. Lastrucci et al. (2015) [17] reported that CD163 expression is linked with immunomodulatory activity in tuberculosis. They also reported that the soluble form of CD163 positively correlated with TB disease severity and could be a potential biomarker for TB diagnosis, disease severity, and treatment efficacy.
Mycobacterium tuberculosis can influence the monocyte differentiation resulting in immune tolerance. Macrophage/monocyte immune tolerance is considered an immune escape mechanism for M. tuberculosis [18]. Activated classical monocytes are the dominant innate immune cells against TB that contribute to the restriction of M. tuberculosis growth by rapid migration to infection sites and high production of reactive oxygen species (ROS) [19]. Decreased classical monocytes along with decreased expression of CD163 may lead to the persistence and dissemination of M. tuberculosis infection. In this study, we sought to elucidate the frequency of different monocyte subsets and CD163 expression on monocyte in subjects without and with drug-resistant TB, and healthy controls. We hypothesized that one potential mechanism for developing M. tuberculosis drug resistance could be decrease in activated classical monocytes resulting in immune evasion and drug tolerance.
Materials and subjects
The project proposal was approved by the university’s ethical review committee. The study with human blood samples was carried out in accordance with the Declaration of Helsinki (2013). Patients were recruited from a tertiary care chest hospital in Lahore after taking written consent. The patient’s diagnostic criteria were based on the identification of M. tuberculosis in sputum along with positive clinical symptoms (fever, weight loss, and cough) and positive radiological findings. The M. tuberculosis infection was further confirmed by using Xpert MTB/RIF Assay (Cepheid, Sunnyvale, USA) which simultaneously detects Mycobacterium tuberculosis complex (MTBC) and resistance to rifampicin (rif) by nucleic acid amplification. The sample size (80) was calculated by the OpenEpi calculator [20] using the data obtained from Liu et al (2019) [19]. A structured questionnaire was designed to obtain participants’ relevant data. Using this questionnaire, blood samples were collected from a total of 249 TB patients and healthy controls who were given consent to enroll in this study. They were assigned to three groups. Group I had 85 unrelated age- and gender-matched healthy volunteers who neither had history nor signs and symptoms of active TB infection or any other disease. All the healthy controls had received Mycobacterium bovis Bacille Calmette–Guerin (BCG) vaccination in their childhood. Group II had 84 pulmonary TB patients who were admitted to the hospital for the start of standard anti-TB treatment and did not have resistance to anti–Mycobacterium tuberculosis drugs detected by Xpert MTB/RIF assay. Group III comprised of 80 rifampicin-resistant pulmonary TB patients (RR-TB) detected by Xpert MTB/RIF assay who were admitted to the MDR-TB ward for the start of standard treatment for drug-resistant M. tuberculosis infection. All the subjects were recruited before or within the first week of anti-TB treatment. None of the patients had HIV or any other chronic disorder e.g., malignancy and genetic or autoimmune disease. Furthermore, participants in the study were not undergoing any immune-modulating treatment. The baseline characteristics of the study participants are summarized in Table 1.
Table 1.
Baseline characteristics of the study participants
| Variables | Group I (healthy controls) (n = 85) |
Group II (patients without RR-TB) (n = 84) |
Group III (patients with RR-TB) (n = 80) |
|
|---|---|---|---|---|
| Age (mean ± SD) | 34 ± 12 | 39.99 ± 16 | 32.93 ± 14 | |
| Male gender n (%) | 53 (62.4) | 35 (41.7) | 30 (37.5) | |
| Smoking history n (%) | 04 (4.7) | 20 (23.8) | 06 (07.5) | |
| Family history of TB n (%) | 05 (5.9) | 20 (23.8) | 08 (10.0) | |
|
Sputum AFB smear n (%) |
Negative | NA | 07 (8.3) | 49 (61.3) |
| 1+ | 33 (39.3) | 13 (16.3) | ||
| 2+ | 31 (36.9) | 17 (21.3) | ||
| 3+ | 13 (15.5) | 01 (1.3) | ||
|
Radiology (positive X-ray) n (%) |
NA | 29 (34.5) | 38 (47) | |
| Productive cough n (%) | NA | 53 (63) | 51 (63.8) | |
| Fever n (%) | NA | 52 (61.9) | 52 (65) | |
| Wight loss n (%) | NA | 32 (38.1) | 30 (37.5) | |
| Chest pain n (%) | NA | 31 (36) | 25 (31.3) | |
| Hemoptysis n (%) | NA | 09 (10.7) | 06 (7.5) | |
No AFB per 100 fields = negative, 1–99 AFB per 100 fields = 1+, 1–10 AFB per field = 2+, >10 AFB per field = 3+
NA not applicable
Monocyte staining and flow cytometric analysis
The peripheral blood leucocytes from freshly collected EDTA blood were stained by direct immunofluorescence lyse-wash method [21–23] using anti-CD14-FITC (clone REA599), anti-CD45-PerCP (clone 5B1), anti-CD16-PE (clone REA423), and anti-CD163-APC (clone REA812) antibodies (Miltenyi Biotec Bergisch Gladbach, Germany). Mouse isotype antibodies were used as background staining controls. Flow cytometric analysis was carried out using FACSCalibur (BD Biosciences, USA), and samples were acquired and analyzed with CellQuest Pro software. Fifty thousand events were acquired for each sample, and forward and side scatter were used to gate the monocyte population which was further analyzed for different monocyte subset populations. Monocyte subsets and CD163 expression were determined by using the gating strategy defined by Loon et al. (2012) [24] and Li et al. (2018) [7] (Fig. 1).
Fig. 1.
The dot plots of representative data from one healthy control illustrate the analysis method for the identification of monocyte subpopulations in peripheral blood following three-color staining. a Monocyte population was gated (region R1) using FSC and SSC plot. b Next, the monocytes accumulated in the R1 were analyzed for the staining of monocyte subpopulations. We used dot plots of CD14 FITC versus CD16 PE. The dot plot shows classical (CD14++CD16–), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) monocytes. The expression of CD163 (c, d) was assessed in classical and intermediate monocyte subpopulations. Representative isotype control dot plot (e). FSC, forward scatter; SSC, side scatter
Measurement of human sCD163 levels
The serum level of CD163 was determined by using a commercially available human sCD163 ELISA kit (Elabscience Biotechnology Inc., USA) according to the manufacturer’s instructions. The concentration of sCD163 was calculated by using an automated microplate reader (model 680, Bio-Rad, USA).
Statistical analysis
The data were analyzed using SPSS 26.0 (IBM Corporation, Armonk, New York, USA) and GraphPad Prism 8.4.3.686 software (GraphPad Software Inc., La Jolla, California, USA). Shapiro-Wilk and Kolmogorov-Smirnov tests were used to check the normality of data. Numeric data were expressed as mean ± SD, median, and interquartile range. Differences among multiple groups were compared with one-way ANOVA, Kruskal-Wallis, and post hoc Tukey’s test. Spearman’s correlation was used to evaluate the correlation between serum level of CD163 and frequency of CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes. A p-value of ≤ 0.05 was considered statistically significant.
Results
Frequency of peripheral blood monocyte subsets in TB patients and healthy controls
The differences in the percentages of various monocyte subsets i.e., classical (CD14++CD16–), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) monocytes in controls and patients without/with RR-TB were analyzed using flow cytometry. A significantly decreased frequency of circulating classical monocytes was detected in RR-TB patients (median = 51.78, IQR = 95.00–21.55) compared with healthy controls (median = 75.64, IQR = 90.61–57.00) (p = 0.019), and it was significantly decreased compared with patients without RR-TB (median = 79.50, IQR = 92.06–23.92) (p = 0.005); however, there was no statically significant difference between healthy controls and patients without RR-TB (p = 1.00).
A significantly higher peripheral intermediate monocyte frequency was observed in patients with RR-TB (median = 30.64, IQR = 08.29–42.73) relative to patients without RR-TB (median = 13.00, IQR = 08.24–14.75) (p < 0.0001) and was equally elevated compared with healthy controls (median = 20.0, IQR = 15.0–23.75), although the difference was not statically significant (p = 0.2).
A significantly decreased frequency of circulating non-classical monocyte was detected in patients without RR-TB (median = 2.00, IQR = 3.37–1.15) compared with healthy controls (median = 2.67, IQR = 4.94–2.25) (p = 0.0001); however, there was no statistically significant difference between healthy controls and patients with RR-TB (p = 0.08) (Table 2) (Fig. 2).
Table 2.
Peripheral blood monocyte subsets in TB patients and healthy controls
| Variables | Group I (controls) n = 85 |
Group II (TB patients without rifampicin resistance) (n = 84) |
Group III (TB patients with rifampicin resistance) (n = 80) |
p-value |
|---|---|---|---|---|
| CD14++ CD16− classical monocytes (%) |
75.64 (90.61–57.00) |
79.50 (92.06–23.92) |
51.78 (95.00–21.55) |
0.011* 1.002 0.0193* 0.0054* |
| CD14++ CD16+ intermediate monocytes (%) |
20.00 (15.00–23.75) |
13.00 (8.24–14.75) |
30.64 (8.29–42.73) |
<0.00011* <0.00012* 0.263 <0.00014* |
| CD14+ CD16++ non classical monocyte (%) |
2.67 (4.94–2.25) |
2.00 (3.37–1.15) |
2.64 (3.46–1.82) |
<0.00011* <0.00012* 0.0883 0.1124 |
Data are median (interquartile ranges) or real case number
NA not applicable
*Statistically significant p ≤ 0. 05
1Comparison among the three groups
2Comparison between group I and group II
3Comparison between group I and group III
4Comparison between group II and group III
Fig. 2.
Monocyte subset profiles in controls, patients without/with RR-TB. a Representative monocyte-gated dot plots showing the profile of monocyte subsets based on CD14 and CD16 expression in controls and patients without/with RR-TB. b Percentages of CD14++CD16− (below), CD14++CD16+ (center), and CD14+CD16++ (top). Monocytes were compared among the controls and patients without/with RR-TB. Data are expressed as median with IQR and are analyzed using the Kruskal-Wallis Test. RR-TB, rifampicin-resistant tuberculosis; IQR, interquartile range; statistically significant p ≤ 0.05
Lower CD163 expression on CD14++CD16– and CD14++ CD16+ monocytes in the RR-TB patients
A significantly decreased frequency of CD14++CD16–CD163+ monocytes was detected in RR-TB patients (median = 1.63, IQR = 9.54–0.53) compared with healthy controls (median = 44.22, IQR = 85.71–14.88) (p < 0.0001), and patients without RR-TB (median = 49.94, IQR = 94.35–15.41) (p < 0.0001). There was no statistically significant difference between patients without RR-TB and healthy controls (p = 1.00). Significantly decreased frequency of CD14++CD16+CD163+ monocyte was detected in RR-TB patients (median = 3.71, IQR = 8.30–1.17) compared with healthy controls (median = 48.64, IQR = 88.14–8.93) (p < 0.0001), and patients without RR-TB (median = 42.70, IQR = 94.80–16.77) (p < 0.0001). There was no statistically significant difference between patients without RR-TB and healthy controls (p = 0.261) (Table 3) (Fig. 3).
Table 3.
Serum level and expression of CD163 on monocytes in TB patients and controls
| Variables | Group I (controls) n = 85 |
Group II (TB patients without rifampicin resistance) (n = 84) |
Group III (TB patients with rifampicin resistance) (n = 80) |
p-value |
|---|---|---|---|---|
| CD14++ CD16− CD163++ monocytes (%) |
44.22 (85.71–14.88) |
49.94 (94.35–15.41) |
1.63 (9.54–0.53) |
<0.00011 1.002 <0.00013 <0.00014 |
| CD14++ CD16+ CD163++ monocytes (%) |
48.64 (88.14–8.93) |
42.70 (94.80–16.77) |
3.71 (8.3050–1.1775) |
<0.00011 0.2612 <0.00013 <0.00014 |
|
sCD163 (ng/ml) |
95.37 (97.29–91.35) |
86.5680 (90.01–81.93) |
87.87 (92.06–87.87) |
<0.00011 <0.00012 <0.00013 0.44 |
Data are median (interquartile ranges) or real case number
NA not applicable
*Statistically significant p ≤ 0. 05
1Comparison among the three groups
2Comparison between group I and group II
3Comparison between group I and group III
4Comparison between group II and group III
Fig. 3.
CD163 expression on monocyte subsets in controls, patients without/with RR-TB. a Representative dot plots showing the expression of CD163 on CD14++CD16– monocytes (top). b Representative dot plots showing the expression of CD163 on CD14++CD16+ monocytes (center). c Comparison of CD14++CD16–CD163+ monocyte among three groups. d Comparison of CD14++CD16+CD163+ monocyte among three groups. Data are expressed as median with IQR and are analyzed using the Kruskal-Wallis Test. RR-TB, rifampicin-resistant tuberculosis; IQR, interquartile range; statistically significant p = ≤0.05
Decreased serum level of CD163 in patients without and with RR-TB
A statistically significant decreased serum level of CD163 was detected in patients with RR-TB (median = 87.87 (ng/ml), IQR = 92.06–87.87) and patients without RR-TB (median = 86.56 (ng/ml), IQR = 90.01–81.93) compared with healthy controls (median = 95.37 (ng/ml), IQR = 97.29–91.35) (p < 0.0001). There was no statistically significant difference between patients without and with RR-TB (0.44) (Table 3) (Fig. 4).
Fig. 4.
Comparison of serum level of CD163 (sCD163) among three groups. Data are expressed as median with IQR and are analyzed using the Kruskal-Wallis Test. RR-TB, rifampicin-resistant tuberculosis; IQR, interquartile range; statistically significant p = ≤0.05
Correlation of serum CD163 level with the percentage of CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes
Correlation analysis of serum CD163 level was carried out with the percentage of CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes among the three groups. Positive statistically significant correlation was detected between the serum level of CD163 and CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes (r = 0.22, p = 0.04 and r = 0.22, p = 0.04, respectively) in RR-TB patients, whereas weak negative correlation (statistically insignificant) was observed in patients without RR-TB (r = −0.09, p = 0.40 and r = –0.16, p = 0.13, respectively). However, weak positive correlation (statistically insignificant) was detected in healthy controls for CD14++CD16−CD163+ and CD14++CD16+CD163+ monocytes (r = 0.19, p = 0.07 and r = 0.2, p = 0.054, respectively) (Fig. 5).
Fig. 5.
Correlation of serum level of CD163 with the percentage of CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes. a–c Correlation of serum CD163 with the percentage of CD14++CD16–CD163+ in controls, patients without RR-TB, and patients with RR-TB, respectively. d–f Correlation of serum CD163 with the percentage of CD14++CD16+CD163+ in controls, patients without RR-TB, and patients with RR-TB, respectively. (–) Negative correlation. Statistically significant = p ≤ 0. 05
Discussion
In the current study, a statistically significant decrease in the percentage of classical monocytes was detected in patients with RR-TB as compared with healthy controls and patients without RR-TB. To our knowledge, this is the first report showing a decreased percentage of classical monocytes in patients with RR-TB. Decreased classical monocytes in patients with drug-resistant M. tuberculosis infection could be an important finding as monocyte recruitment is essential for anti-tuberculous host immune defense. Classical monocytes are highly sensitive and reactive to pathogen-derived molecules, immediate infiltration of classical monocytes to the infection site, and the production of reactive oxygen species (ROS) results in a reduction of bacterial growth, and hence, they can respond quickly to microbial stimuli to inhibit pathogens at early stages of infection [25, 26]. Therefore, reduction of classical monocytes may have contributed to the development of drug resistance by prolonged exposure of M. tuberculosis to the drug without sufficient immune response; hence, it is suggested to conduct functional studies involving patients with drug-resistant TB to evaluate possible mechanisms involved in the development of M. tuberculosis drug resistance.
In the current study, there was no statistically significant difference in the percentage of classical monocytes between healthy controls and patients without RR-TB, which is consistent with the finding of Morais-papini et al. (2017) [27] who reported no difference in the percentage of classical monocytes between healthy controls and TB patients. However, the current study is not in agreement with Castaño, García, and Rojas (2011) [12] who reported an increased percentage of classical monocyte in TB patients. This discrepancy could be due to the study individuals who were before or within the first 2 weeks of anti-TB treatment whereas the current study recruited active TB patients, as some reports suggest changes in monocyte populations during anti-TB treatment [9].
In the current study, non-classical monocytes (also called patrolling monocytes) were decreased in patients without and with RR-TB as compared with healthy controls. This finding is in agreement with Morais-papini et al. (2017) [27] who reported a conspicuous reduction in circulating non-classical monocytes in TB patients and increased activation of this subset assessed by the upregulation of HLA-DR expression. Morais-papini et al. (2017) [27] suggested that this decrease in the percentage of circulating non-classical monocytes in TB patients is due to the recruitment of these cells to the site of infection which is highly influenced by the cell composition and chemokine/cytokine milieu [28]. However, the current study is not in agreement with Castaño, García, and Rojas (2011) who observed an increase in non-classical monocytes during TB infection. Similarly, the current study is inconsistent with Balboa et al. (2011) [10] who reported increased non-classical monocytes in TB patients. It is worth mentioning that the abovementioned studies only included pulmonary TB patients without knowing the status of M. tuberculosis drug resistance, whereas in the current study, monocyte subsets in patients without and with RR-TB were included, and there was no statistically significant difference between the two groups. Apparently, the percentage of non-classical monocytes was raised in patients with RR-TB, strengthening the hypothesis of Balboa et al. (2015) [11] who reported that CD16+ monocyte subpopulations can contribute to the infection. CD16+ monocyte subpopulations are somewhat resistant (unresponsive) to microbial growth and present decreased nitric oxide production capacity compared with CD16− monocytes. These monocyte subsets are involved in promoting bacterial resilience by inducing a minimal level of respiratory burst and are unresponsive at the early stages of infection due to the lack of chemokine receptors (CCR2), which are necessary for the migration of this subset to the site of infection [25].
Intermediate monocytes (CD14++CD16+) are another important subset of monocytes comprise about 2–8% of circulating monocytes. Their functions include the production of reactive oxygen species (ROS), antigen presentation, participation in the proliferation and stimulation of T cells, inflammatory responses, and angiogenesis [25]. In the current study, a statistically significant increase in intermediate monocytes in patients with RR-TB relative to healthy controls has been observed. To the best of our knowledge, this is the first study that reports the increased frequency of intermediate monocyte in RR-TB patients. It is in agreement with Castaño, García, and Rojas (2011) [12] who reported an increased percentage of intermediate monocytes in TB patients as compared with healthy controls; however, the status of drug susceptibility testing of TB patients in this study was unknown. This increases in the proportion of intermediate monocytes that may act as a host environmental factor for M. tuberculosis drug resistance [29]. It can be explained by the fact that classical monocytes produce IL-10 following M. tuberculosis infection, resulting in a higher frequency of intermediate monocytes. Other factors such as antigenic components of M. tuberculosis (secreted and membrane-bound) and the host environment (antigen presentation) also favor the expansion of these cells [25]. Antigenic products secreted by the mycobacteria or the infected macrophages can reach the circulation and promote the expansion of intermediate monocytes in circulation, which migrate into the affected tissues and participate in the granuloma formation. In addition, M. tuberculosis infection also induces delayed apoptosis of intermediate monocytes that facilitate the increased frequency in the circulation [12]. The interaction of intermediate monocytes with M. tuberculosis is important for the secretion of TNF-α at the site of infection as it has been associated with the control of M. tuberculosis infection. However, if these monocytes are continuously recruited into lung, the excess of TNF-α can intensify the proinflammatory response that would be detrimental to infected lung tissue and the prognosis of the tuberculosis by triggering spontaneous gene mutations in M. tuberculosis by oxidative stress [12, 29]. Oxidative stress is a substantial mutagenic influence encountered by M. tuberculosis in the host. Macrophages, as an antibacterial defense mechanism, produce ROS and reactive nitrogen intermediates (RNI). These reactive species interact with nucleotides, resulting in chemical modifications that can lead to base mispairing and DNA damage. These gene mutations may render the bacteria-resistant to the most commonly used anti-TB drugs [29, 30], but to understand this phenomenon, further studies are needed.
In this study, the expression of CD163 on classical monocytes was significantly decreased in patients with RR-TB relative to healthy controls and patients without RR-TB. Similarly, it was also significantly reduced on intermediate monocytes in patients with RR-TB. To the best of our knowledge, this study represents the first report that reduced expression of CD163 on classical and intermediate monocytes was detected in patients with drug-resistant M. tuberculosis infection; however, there was no significant difference in the expression of CD163 between healthy controls and patients without RR-TB. Taken together, these results suggest decreased expression of CD163 on classical and intermediate monocytes or decreased frequency of circulating CD14++CD16+CD163+ and CD14++CD16–CD163+ monocytes in patients with drug-resistant M. tuberculosis infection indicating the possible involvement of these cells in the development of drug-resistance in M. tuberculosis; however, further studies are needed to investigate the underline mechanism. To understand these decreases in CD14++CD16+CD163+ and CD14++CD16–CD163+ monocytes in patients with drug-resistant M. tuberculosis infection, the serum level of CD163 (sCD163) was investigated in all three groups. It was observed that the serum level of CD163 was significantly decreased in patients with RR-TB as well as in patients without RR-TB; however, in both TB groups, the level of sCD163 was comparable. Further, the sCD163 was correlated with the percentages of CD14++CD16+CD163+ and CD14++CD16–CD163+ monocytes, and a significant positive correlation was detected in patients with RR-TB. However, no correlation was detected in healthy controls and patients without RR-TB; hence, these results also support the hypothesis that decreased CD14++CD16+CD163+ and CD14++CD16−CD163+ monocytes may have a role in the development of M. tuberculosis drug resistance in TB patients. To date, no study is available on patients without and with RR-TB to compare these results for the percentage of CD14++CD16+CD163+ and CD14++CD16–CD163+ monocytes; however, there are some previous reports, e.g., in the context of HIV infection [31] percentage of CD14++CD16+CD163+ monocytes was significantly increased while Li et al. (2018) [7] suggested decreased CD163 on classical monocytes during the acute stage of hemorrhagic fever with renal syndrome compared with the controls [7]. In the current study, less severe patients were recruited, indicated by the baseline characteristics provided in Table 1, which may have been the explanation for similar profiles of patients without RR-TB and healthy control groups. This can also be explained by the fact that the degree of expression of sCd163 shows a direct association with disease severity (TB), the load of the sputum AFB, and lung involvement (cavitation) [19].
In the current study, a decreased serum level of CD163 (sCD163) was observed in patients with and without RR-TB relative to controls. The current study is not in agreement with Suzuki et al. (2017) [32] who reported increased sCD163 level in patients with active pulmonary TB compared with controls, and this increased level was associated with increased mortality in patients with active TB. This discrepancy in results could be due to the inclusion of extrapulmonary TB patients with comorbidities (liver disease, cerebrovascular disease, neoplasm, etc.) in their study, whereas in the current study, only pulmonary TB patients without any comorbidity were recruited. Knudsen et al. (2005) [33] also reported an increased level of sCD163 in TB patients coinfected with HIV. This study also demonstrated that both TB and HIV status influenced serum CD163 levels, and high levels of sCD163 were associated with increased mortality in TB patients. Lastrucci et al. (2015) [17] proposed CD163 a potential biosignature and target for treatment. He described that, in the context of TB, human monocytes differentiate into a CD16+CD163+MerTK+pSTAT3+ phenotype which shows a high pathogen permissive protease-dependent motility and immunomodulatory activity that points to a detrimental role in host defense against TB. Importantly, this study also provides a direct correlation of abundance of CD163 positive non-classical monocytes with TB disease severity in humans and non-human primates and proposed presence of CD163 in sera as a potential biomarker to monitor disease progression and treatment efficacy. The findings of the current study suggest measurement of sCD163 levels that may be a useful prognostic and therapeutic tool in TB disease. However, further prospective studies are needed to evaluate the effect of monocyte CD163 expression on anti-TB treatment or the effect of anti-TB therapy on sCD163 levels to determine whether such measurements can be used to monitor treatment efficacy and predict the development of drug resistance.
Conclusion
The current study described changes in monocyte subsets frequency in TB patients; decreased frequency of classical and non-classical monocytes was detected in patients with drug-resistant and drug-sensitive M. tuberculosis infections, respectively; however, intermediate monocytes were increased in patients with drug-resistant M. tuberculosis infection. A significant decrease in the frequency of CD14++ CD16–CD163+ and CD14++CD16+CD163+ monocytes was observed in drug-resistant TB patients along with decreased serum CD163 level that also positively correlated with the frequency of CD14++CD16–CD163+ and CD14++CD16+CD163+ monocytes in drug-resistant TB patients.
It is concluded that decreased frequency of monocyte subsets and CD163 expression could be a predictor of M. tuberculosis drug resistance in TB patients. However, further studies are needed to identify the underlying mechanisms for the observed changes in monocyte subsets in patients with drug-resistant and drug-sensitive M. tuberculosis infections.
Funding
This work was supported by the University of Health Sciences Lahore, Pakistan.
Declarations
Ethics declaration
The study with human blood samples was approved by the university’s ethical review committee in accordance with the Declaration of Helsinki (2013).
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
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References
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