|Year : 2022 | Volume
| Issue : 1 | Page : 39-45
Blood monocyte subtypes in patients with pulmonary tuberculosis infection
Tamer G El-Rab Attia1, Mona M Abdelmeguid1, Ali S Mohammed1, Hamada K.S Fayed2
1 Clinical Pathology Department, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
2 Chest Diseases Department, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
|Date of Submission||29-Aug-2020|
|Date of Decision||03-Aug-2021|
|Date of Acceptance||24-Aug-2021|
|Date of Web Publication||4-Mar-2022|
MBBCh Tamer G El-Rab Attia
Clinical Pathology Department, El Attar Street, Besary, Assiut 71511
Source of Support: None, Conflict of Interest: None
Background and aim Monocytes are the primary target for Mycobacterium tuberculosis infection. Important alterations in the proportions of circulating monocyte subpopulations were found in patients with active tuberculosis (aTB). Our aim was to investigate the peripheral blood monocyte subsets in patients with active pulmonary tuberculosis (aPTB) and to evaluate their role in treatment response.
Patients and methods A case–control study included 30 patients with aPTB and 30 healthy controls. Laboratory investigations include sputum examination, tuberculin test, and nucleic acid amplification test using GeneXpert MTB/RIF assay on sputum sample for patients only to confirm the diagnosis of aPTB. For all participants; complete blood count, erythrocyte-sedimentation rate, HIV antibodies, and flow cytometry analysis of peripheral blood monocyte subsets were done using surface markers CD14 phycoerythrin cyanin 7 and CD16 fluoroisothiocyanate for identification of monocyte subsets. Flow cytometry analysis was repeated 2 months after the start of treatment in aTB patients only.
Results Classical monocytes were decreased while intermediate, and nonclassical monocytes were increased in aTB patients before treatment more than after 2 months of treatment and control group (P≤0.0001 for each). There was no significant difference in the three monocyte subsets between PTB patients after treatment and healthy controls. Also, the classical monocytes decreased while intermediate, and nonclassical monocytes increased in aTB patients with positive tuberculin test more than healthy controls with positive tuberculin test (P<0.0001 for each).
Conclusion The expansion of CD16+ monocytes was reversed after treatment with anti-TB drugs and could be used to follow up tuberculous patients for treatment effectiveness.
Keywords: Active pulmonary tuberculosis, CD14, CD16, flow cytometry, tuberculin skin test
|How to cite this article:|
El-Rab Attia TG, Abdelmeguid MM, Mohammed AS, Fayed HK. Blood monocyte subtypes in patients with pulmonary tuberculosis infection. Al-Azhar Assiut Med J 2022;20:39-45
|How to cite this URL:|
El-Rab Attia TG, Abdelmeguid MM, Mohammed AS, Fayed HK. Blood monocyte subtypes in patients with pulmonary tuberculosis infection. Al-Azhar Assiut Med J [serial online] 2022 [cited 2022 Jun 29];20:39-45. Available from: http://www.azmj.eg.net/text.asp?2022/20/1/39/339066
| Introduction|| |
Tuberculosis (TB) remains a major public health problem. In 2016, it caused 1.67 million deaths worldwide. However, the number of people infected with Mycobacterium tuberculosis was estimated to reach to three billion of individuals . It is caused by the pathogenic bacterium M. tuberculosis, which unlike most disease-causing pathogens, persists in humans because of its highly evolved ability to evade and subvert the host immunity .
Although the human immune system can control the infection, it does not invariably lead to sterilization . Mycobacteria escaping the early intracellular destruction can multiply and interrupt the macrophage, and then the released chemokines can attract the monocytes and other inflammatory cells to the lung. These inflammatory monocytes will differentiate into macrophages, which ingest the mycobacteria but not destroy them .
Monocytes are the primary target for infection with M. tuberculosis and their innate ability to treat mycobacteria defines the early progression of infection. The aggregation of macrophages and their precursors, circulating monocytes, is an essential feature of host response to mycobacteria and the development of granuloma at infectious foci . Important alterations in the proportions of circulating monocytes and dendritic cell subpopulations were found in patients with active tuberculosis (aTB). The hallmark of patients with aTB is the blood tolerogenic and proapoptotic monocytes that could play a major role in downregulating the adaptive immune responses during the disease process .
Monocyte phenotyping by flow cytometry has identified three different subtypes: classical (CD14++ CD16−), intermediate (CD14+ CD16+), and nonclassical (CD14+ CD16++) monocytes . The surface expression of human leukocyte antigen DR in CD14++ monocytes reflects the activation level of these cells. Immunosuppression is indicated by the reduction in the human leukocyte antigen DR expression levels .
This work is designed to investigate the peripheral blood monocyte subsets in patients with active pulmonary tuberculosis (aPTB) and to evaluate their role in treatment response.
| Patients and methods|| |
This case–control study included 30 patients with aTB and 30 age-matched and sex-matched healthy participants as a control group were enrolled in this study. They were recruited from the chest and internal medicine departments, Al-Azhar University Hospital, Assiut, Egypt. Patients diagnosed or suspected of having aPTB referred to Assiut Chest Hospital for confirming the diagnosis by nucleic acid amplification test (NAAT).
Patients with aPTB infection were included in the study. Patients with the following disorders were excluded from the study: any patient with a disease that affects the monocyte proportions or subtypes such as patients with chronic obstructive pulmonary disease, respiratory tract infection, bronchial asthma, and HIV infection.
Personal history, including smoking habits and symptoms of PTB infection (recent or previous cough, expectoration, night fever, night sweat, hemoptysis, chest pain, loss of appetite, and weight loss) was recorded.
General and local chest examination were done for each participant to record weight loss, cachexia, toxic look, temperature, pulse, blood pressure, and chest rhonchi. Also, radiological chest examination was done to show the type and the site of the lesion.
aPTB diagnosis was based on the presence of microbiological evidence (smear-positive, culture-positive, or NAAT-positive specimens) or clinical suspicion and satisfactory TB response.
Patients admitted to the chest hospital receive the antituberculous regimen as recommended by the National Tuberculosis Control Program of Egypt (NTP).
Laboratory investigations include complete blood count using hematology autoanalyzer (Micros 60–18 P, Horiba ABX micros 60/18 parameter, Paris, France), erythrocyte-sedimentation rate by Westergren’s method, sputum examination with Ziehl–Neelsen stain, tuberculin skin test, HIV antibodies by DIA PRO diagnostic biprobes Srl. HIV antibodies kits, and M. tuberculosis DNA by NAAT GeneXpert MTB/RIF Cepheid System, USA.
Nucleic acid amplification test
The NAAT was used to detect and confirm the diagnosis of aPTB infection in sputum samples from suspected patients.
Flow cytometry analysis of monocyte subsets
EDTA blood was used for the analysis of the monocyte subsets by flow cytometry using surface markers CD14− phycoerythrin cyanin 7 (Beckman Coulter A22331, Navios Beckham Coulter flow cytometer B47094, Indiana, USA), and CD16 fluoroisothiocyanate (Beckman Coulter IM247OU, Navios Beckham Coulter flow cytometer B47094, Indiana, USA).
Fifty microliters of the blood sample was stained with 5 µl of phycoerythrin cyanin 7-conjugated CD14 and fluoroisothiocyanate-conjugated CD16 for identification of monocyte subsets (classical, intermediate, and nonclassical). Incubation was carried out for 15 min at room temperature in the dark, after which red-blood cell lysis was done. Then the cells were washed once and were resuspended in phosphate buffer saline. The acquisition of data was performed on Navios flow cytometer using Beckman Coulter Navios Flow Cytometry. Forward-scatter and side-scatter histogram was used to define the monocyte populations. The classical monocytes are characterized by high-level expression of the CD14 cell surface receptor (CD14++ CD16− monocytes). The nonclassical monocytes show low-level expression of CD14 and additional coexpression of the CD16 receptor (CD14+ CD16++ monocytes). The intermediate monocyte shows high-level expression of CD14 and low-level expression of CD16 (CD14+ CD16+ monocytes).
All laboratory investigations were done to all participants at the beginning of the research. Flow cytometry analysis was repeated 2 months after the start of treatment in aPTB patients only.
This study was approved by the Al-Azhar Faculty of Medicine Ethical Committee and Committee of Publication Ethics, and verbal consent was obtained from all participants before sampling after explaining the idea and the benefits of the research. The study was conducted in accordance with Helsinki standards as revised in 2013.
Data analysis was done using Statistical Package for Social Sciences (SPSS, version 25; SPSS Inc., Chicago, Illinois, USA). The Kolmogorov–Smirnov test was used for analyzing normality of distribution of continuous variables. Continuous data were compared by independent t test and expressed as mean and SD. χ2 test was used for categorical data that were expressed as number and percentage. Pearson’s correlation was used to test the correlation between two numeric variables in the same group. The level of significance was set at P value less than 0.05 (95% confidence interval). Sample size was calculated using the Epi info software considering the power of 80% and alpha error within 5%, giving the sample size of 30 patients being sufficient to detect the expected effect size.
| Results|| |
Demographic and laboratory data are listed in [Table 1]. The mean age of the patients and controls was around 44 years and they are of different occupations. Ninety percent of the patients with aPTB were males and 10% were females, while in the control group, males comprise 73.3% and females 26.7%. Most of the patients with TB were farmers (43.3%) and manual workers (43.3%), while most of healthy groups were professionals (60%) with a significant difference (P≤0.0001). The majority of TB patients were smokers (80%), while in the control group, the majority were nonsmokers (90%) with a significant difference (P≤0.0001).
|Table 1 Demographic, laboratory, and radiological characteristics of study participants|
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Only one of the aPTB patients had sputum smear-negative for acid-fast bacilli and all of them had positive tuberculin test and M. tuberculosis DNA positive by NAAT analysis. In the control group, 73.3% had tuberculin test positive with a significant difference compared with aPTB patients (P=0.005).
The hemoglobin levels were lower in the patients’ group than the control group (P<0.0001), while the other complete blood count parameters were higher in patients’ group than the controls, including total leukocyte count, neutrophils, monocytes, and monocyte/lymphocyte ratio (P<0.0001 for each), platelets (P=0.001), and lymphocytes (P=0.024). The erythrocyte-sedimentation rate levels were lower in the control group than the patient’s group (P<0.0001) ([Table 1]).
A chest radiograph was done to all patients to show the type and site of the lesion. About 63.3% of patients had a consolidation lesion, while 36.7% had cavitary. In 90% of patients, the lesion occupied the upper lobe of the lung, while in 10%, it was in the middle lobe ([Table 1]).
All patients (100%) had constitutional symptoms of aPTB, such as loss of appetite, cough, expectoration, weight loss, night sweat, and night fever. Chest pain and hemoptysis were found in only 56.7 and 23.3% of patients, respectively ([Figure 1]), classical (CD14++ CD16−), intermediate (CD14+ CD16+), and nonclassical (CD14+ CD16++).
Frequency of monocyte subsets among the studied groups
Monocyte populations were detected in the scatter histogram ([Figure 2]a), then the frequency of each subset was calculated from the monocyte population ([Figure 2]b,c).
|Figure 2 Flow cytometric analysis of monocyte subsets: (a) forward-scatter and side-scatter (FSC and SSC) gating to define the monocyte subtypes (green dots), (b) high percentages of intermediate and nonclassical monocyte subtypes in TB patients, (c) high percentages of classical monocyte subtypes in healthy controls. TB, tuberculosis.|
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The frequency of the classical lymphocytes (CD14++ CD16−) was lower, while the intermediate (CD14+ CD16+) and nonclassical lymphocytes (CD14+ CD16++) were higher in patients with aPTB before the start of anti-TB treatment than that after treatment and the healthy control (P<0.0001 for each). The three monocyte subset (classical, intermediate, and nonclassical) frequencies were nonsignificantly different between patients with TB 2 months after treatment with anti-TB drugs and the healthy control (P=0.672, 0.816, and 0.678, respectively) ([Table 2] and [Table 3]).
|Table 2 The frequencies of monocyte subsets in patients with active pulmonary tuberculosis before and 2 months after treatment and healthy controls|
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|Table 3 Frequency of monocyte subsets according to tuberculin skin test in tuberculin-positive healthy controls and active tuberculosis patients|
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| Discussion|| |
TB is a communicable disease, a major cause of morbidity, one of the top 10 causes of death worldwide, and is considered the main cause of death from a single infectious agent (ranking above HIV/AIDS) .
There are different types of diagnostic tests that are available for diagnosing TB, these tests are developing at a rapid pace and thus changing the landscape of diagnosing TB, such as radiographic, bacteriological, immunological, histopathological, and molecular .
Blood monocytes are an important source of antigen-presenting cells during chronic infections and play a major role in modulating immune responses .
Monocyte-to-lymphocyte ratio was found to be raised in cases compared with controls and seems to be consistent with other researches in which this ratio was found to be predictive of TB infection ,. The results obtained in different prospective cohort studies indicated that an increased monocyte/lymphocyte ratio is associated with the risk of subsequent TB disease , possibly due to monocyte dysfunction as these changes were associated with an enrichment of interferon-associated transcripts in monocytes .
In this study, we found that the classical CD14++ CD16− monocytes were decreased, while the intermediate CD14+ CD16+ monocytes and nonclassical CD14+ CD16++ monocytes were increased in aPTB cases compared with controls. Several studies on aPTB had the same results ,,. The classical CD14++ CD16− monocytes have a highly phagocytic function and they are considered the main antimicrobial cells (scavenger cells) , so their reduction favors TB infection. It was assumed that the immediate infiltration of these cells to the site of infection and reactive oxygen species’ production leads to a reduction of bacterial growth . On the other hand, the intermediate CD14+ CD16+ monocytes have a proinflammatory action during the whole infection process and chronic inflammation that is caused mainly by the effect of tumor necrosis factors-alpha and interleukin-10 . Several studies also demonstrate that they have a possible role in T-cell activation, proliferation, and antigen presentation ,,,,,. Also, the increase in nonclassical CD14+ CD16++ monocytes in TB infection is due to their action in patrolling, sensing of nucleic acids, and viruses . This expansion of CD16+-circulating monocyte was also observed by others ,,.
Several authors already reported an increased proportion of circulating intermediate CD14+ CD16+ monocytes in patients with aPTB compared with patients with latent TBI and healthy controls ,, and is correlated with disease severity . The CD16+ monocytes probably promote microbial resilience . CD16+ monocytes are more prone to produce tumor necrosis factors-alpha, and to undergo cell death in response to M. tuberculosis infection . However, the findings of another study  do not support the previous research as they found higher proportions of classical CD14++ CD16− and intermediate CD14+ CD16+ monocytes. A possible explanation for these results may be the discrepancy in demographics regarding age or race and also discrepancy of risk factors such as occupations or any other environmental changes.On assessing the same patients in our study 2 months after treatment with the anti-TB drugs, we found that the changes in monocyte subsets were reversed reaching that of the healthy control. This finding was also reported by Sánchez et al. . The reversed expansion of CD16+ monocytes suggested that it was caused by microbial or host components and patients may be monitored for treatment effectiveness and compliance through assessment of these monocyte subsets .
The higher CD14++ CD16+ monocytes, and the lower CD14+ CD16+ and CD14+ CD16++ in our study, found in tuberculin-positive healthy individuals more than patients with aPTB, denote that these cells can protect these individuals against TB infection by innate mechanisms . This observation however needs more clarification as Castaño et al.  did not find this significant difference in their study.
Our study has some limitations, for example, the small sample size, as larger studies might be more powerful in detecting the effect size.
| Conclusion|| |
Classical monocytes were decreased, while intermediate and nonclassical monocytes were increased in patients with aPTB. This expansion of CD16+ monocytes was reversed after treatment with anti-TB drugs, accordingly, it could be used to follow up tuberculous patients for treatment effectiveness. Individuals with a positive tuberculin test had higher innate-immunity level than aPTB patients with positive tuberculin test. So, further studies to elucidate the role of these monocyte subsets in protection against TB infection are warranted.
Further studies with a large sample size are needed to confirm the increase of intermediate CD14+ CD16+ and nonclassical CD14− CD16++ monocyte subtypes in patients with aPTB in Egyptian people.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Flynn J, Chan J. Immune evasion by Mycobacterium tuberculosis: living with the enemy. Curr Opin Immunol 2003; 15:450–455.
Horsburgh C, Rubin E. Latent tuberculosis infection in the United States. New Engl J Med 2011; 364:1441–1448.
van Crevel R, Ottenhoff T, van der Meer J. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002; 15:294–309.
Gonzalez-Juarrero M, Shim T, Kipnis A, Junqueira-Kipnis A, Orme I. Dynamics of macrophage cell populations during murine pulmonary tuberculosis. J Immunol 2003; 171:3128–3135.
Dirix V, Corbière V, Wyndham-Thomas C, Selis E, Allard S, Hites M et al.
Blood tolerogenic monocytes and low proportions of dendritic cell subpopulations are hallmarks of human tuberculosis. J Leukoc Biol 2018; 103:945–954.
Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart D et al.
Nomenclature of monocytes and dendritic cells in blood. Blood 2010; 116:e74–e80.
Döring M, Haufe S, Erbacher A, Müller I, Handgretinger R, Hofbeck M et al.
Surface HLA-DR expression in monocyte subpopulations during adverse events after hematopoietic stem cell transplantation. Blood 2011; 118:2161–2161.
Pai M, Schito M. Tuberculosis diagnostics in 2015: landscape, priorities, needs, and prospects. J Infect Dis 2015; 211(suppl_2):S21–S28.
Wang J, Yin Y, Wang X, Pei H, Kuai S, Gu L et al.
Ratio of monocytes to lymphocytes in peripheral blood in patients diagnosed with active tuberculosis. Braz J Infect Dis 2015; 19:125–131.
AsifBaig D. To assess the efficacyof acute phase reactants and monocyte: lymphocyte (M/L) ratio as a prognostic marker in anti-TB drug therapy. Int J Med Sci Clin Invent 2015; 2:9.
Naranbhai V, Hill A, Abdool Karim S, Naidoo K, Abdool Karim Q, Warimwe G et al.
Ratio of monocytes to lymphocytes in peripheral blood identifies adults at risk of incident tuberculosis among HIV-infected adults initiating antiretroviral therapy. J Infect Dis 2013; 209:500–509.
Naranbhai V, Fletcher H, Tanner R, O’Shea M, McShane H, Fairfax B et al.
Distinct transcriptional and anti-mycobacterial profiles of peripheral blood monocytes dependent on the ratio of monocytes: lymphocytes. EBioMedicine 2015; 2:1619–1626.
Liu Q, Ou Q, Chen H, Gao Y, Liu Y, Xu Y et al.
Differential expression and predictive value of monocyte scavenger receptor CD163 in populations with different tuberculosis infection statuses. BMC Infect Dis 2019; 19:1.
Aktas Cetin E, Pur Ozyigit L, Gelmez Y, Cakir E, Gedik A, Deniz G. CD163 levels, pro- and anti-inflammatory cytokine secretion of monocytes in children with pulmonary tuberculosis. Pediatr Pulmonol 2016; 52:675–683.
Castaño D, García L, Rojas M. Increased frequency and cell death of CD16+ monocytes with Mycobacterium tuberculosis infection. Tuberculosis 2011; 91:348–360.
Sampath P, Moideen K, Ranganathan U, Bethunaickan R. Monocyte subsets: phenotypes and function in tuberculosis infection. Front Immunol 2018; 9.
Balboa L, Barrios-Payan J, González-Domínguez E, Lastrucci C, Lugo-Villarino G, Mata-Espinoza D et al.
Diverging biological roles among human monocyte subsets in the context of tuberculosis infection. Clin Sci 2015; 129:319–330.
Heine G, Ulrich C, Seibert E, Seiler S, Marell J, Reichart B et al.
CD14++CD16+ monocytes but not total monocyte numbers predict cardiovascular events in dialysis patients. Kidney Int 2008; 73:622–629.
Lastrucci C, Bénard A, Balboa L, Pingris K, Souriant S, Poincloux R 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; 25:1333–1351.
Anbazhagan K, Duroux-Richard I, Jorgensen C, Apparailly F. Transcriptomic network support distinct roles of classical and non-classical monocytes in human. Int Rev Immunol 2014; 33:470–489.
Wong K, Yeap W, Tai J, Ong S, Dang T, Wong S. The three human monocyte subsets: implications for health and disease. Immunol Res 2012; 53:41–57.
Wong K, Tai J, Wong W, Han H, Sem X, Yeap W et al.
Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood 2011; 118:e16–e31.
Robbins C, Swirski F. The multiple roles of monocyte subsets in steady state and inflammation. Cell Mol Life Sci 2010; 67:2685–2693.
Cros J, Cagnard N, Woollard K, Patey N, Zhang S, Senechal B et al.
Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity 2010; 33:375–386.
Balboa L, Romero M, Basile J, Sabio Y, Garcia C, Schierloh P, Yokobori N 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; 90:69–75.
Barcelos W, Sathler-Avelar R, Martins-Filho O, Carvalho B, Guimarães T, Miranda S et al.
Natural killer cell subpopulations in putative resistant individuals and patients with active Mycobacterium tuberculosis infection. Scand J Immunol 2008; 68:92–102.
Sánchez M, García Y, Montes C, París S, Rojas M, Barrera L et al.
Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment. Microbes Infect 2006; 8:2492–2500.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]