| Abstract|| |
Background and Aims: The host immune system plays an important role in the pathogenesis and defense mechanism of Mycobacterium tuberculosis (Mtb). This study aimed to explore the different changes in the immune system between smear-negative pulmonary tuberculosis (PTB) and smear-positive PTB patients. Materials and Methods: A total of 85 active PTB patients and 50 healthy adults were enrolled. The participants were divided into smear-negative PTB, smear-positive PTB, and control groups. Chest computed tomography (CT) and lymphocyte subgroup counts in peripheral blood were measured in all participants. Results: There were higher numbers of CD4 + T-cells, NK cells, and pulmonary cavities in the smear-positive PTB group, whereas the numbers of B-ells were significantly increased in the smear-negative PTB group. Conclusions: Smear-negative PTB showed fewer pulmonary cavities, mild inflammatory response, lower numbers of immune cells, and higher numbers of B- cells.
Keywords: Lymphocyte subsets, mycobacterium tuberculosis, pulmonary tuberculosis, smear-negative, smear-positive
|How to cite this article:|
Yang X, Feng H. Changes of lymphocyte subsets in smear-negative pulmonary tuberculosis. Indian J Pathol Microbiol 2023;66:321-6
| Introduction|| |
Pulmonary tuberculosis (PTB) is caused by Mycobacterium tuberculosis (Mtb). The main methods of bacteriological examination are smear acid-fast staining test, Mtb in smear culture, and X-pert detection technology, which can help to confirm the diagnosis of PTB., However, there are many smear-negative PTB patients, who have active PTB but are negative by bacteriological tests as smear acid-fast bacilli (AFB) test and culture. As diagnosis of PTB involves a medical history, symptoms, chest X-ray, chest computed tomography (CT), result of purified protein derivative (TB-PPD) or interferon gamma release assay (IGRA), and outcome of experimental treatment, it is difficult to diagnose smear-negative PTB.
As host immune status is an important factor affecting the outcome after Mtb infection, this study was designed to further explore the differences in immune responses between smear-negative and smear-positive PTB patients.
| Materials and Methods|| |
Between August 2018 and August 2019, a total of 85 active PTB cases admitted to the Nanjing Jiangbei hospital (Nanjing, China) were included in this study as the PTB group. The median age of the patients was 45.6 ± 18.8 years (range: 17–91 y), with 24 females (28.2%) and 61 males (71.8%), including 37 patients in the smear-positive PTB (SPPTB) group, and 48 patients in the smear-negative PTB (SNPTB) group. Patients with smear-negative PTB were diagnosed based on history, symptoms, chest CT, and test results of TB-PPD or IGRA The smear-positive PTB cases were diagnosed if at least one of the following test results was positive: smear AFB test, Mtb in smear culture, and X-pert detection technology. The diagnosis was based on the People's Republic of China Health Industry Standard-Diagnosis of Tuberculosis (WS 288-2017). Fifty healthy individuals were enrolled as normal controls (Control group) and 50 patients with community-acquired pneumonia (CAP) were enrolled as the positive control (CAP group) [Table 1].
The exclusion criteria were as follows: (1) positive pregnancy test in females, (2) received immunomodulatory treatment within 6 months, (3) coinfected with human immunodeficiency virus, (4) accompanied by thyroid dysfunction, autoimmune diseases, or psychiatric conditions.
Peripheral blood samples (5 mL) and smear samples (3 mL) were taken from all the study participants. The numbers of lymphocyte subsets were detected by flow cytometry (Beckman Coulter, American). Smear acid-fast staining test, Mtb in smear culture, and X-pert test were conducted on all smear specimens. C-reactive protein (CRP) was detected by enzyme-linked immunosorbent assay (ELISA) (DENUO, China).
The results were reported as means ± SD. Statistical comparisons were made between two groups using independent samples t-test. The one-way ANOVA method was used for multiple comparisons. Data analysis was performed using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL). The confidence interval was 95%.
| Results|| |
Baseline characteristics of the groups
There was no significant difference in average age among the PTB, CAP, and control groups. Results of chest CT showed that pulmonary cavities were found in 32 (37.6%) PTB cases, including 27 (73%) patients with smear-positive, and 5 (10.4%) patients with smear-negative PTB. The cavity ratio and the proportion of multiple cavities were significantly higher in the smear-positive PTB group [Table 1].
The level of CD4 + cells and the ratio of CD4+/CD8 + cells were significantly increased in the PTB group
The level of CD4 + cells was significantly higher in the PTB group than in the control and CAP groups [Table 1], [Figure 1]a, whereas the level of CD8 + cells showed no significant difference among the groups [Table 1], [Figure 1]b. The ratio of CD4+/CD8 + cells was significantly higher in the PTB group than in the control and CAP groups [Table 1], [Figure 1]c. Further subgroup analysis showed that the level of CD4 + cells was significantly increased in the smear-positive PTB group [Table 1], [Figure 2]a, but the level of CD8 + cells and the ratio of CD4+/CD8 + cells showed no significant differences among the groups [Table 1], [Figure 2]b and [Figure 2]c.
|Figure 1: The number of lymphocytes in the various groups. The number of CD4+ T-cells and the ratio of CD4+/CD8+ T cells were significantly higher in the PTB group than in the control and CAP groups (a,c). The number of CD8+ T-cells showed no difference among the groups (b). B-cells were significantly higher in the PTB and CAP groups (d). NK cells were decreased in the PTB group (e). CRP was increased in the CAP and PTB groups compared to the control group (f). Abbreviations: PTB (Pulmonary Tuberculosis), CAP (community-acquired pneumonia), NK (natural killer) cells, CRP (C-reactive protein)|
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|Figure 2: The number of lymphocyte subsets in the sub-groups. The number of CD4+ T-cells was significantly higher in the SPPTB group than in the other groups (a). The level of CD8+ T-cells and the ratio of CD4+/CD8+ T cells showed no difference among the groups (b,c). B-cells were significantly increased in the SNPTB and CAP groups (d). NK cells were decreased in the SPPTB group (e). CRP was increased in the CAP, SPPTB, and SNPTB groups as compared to the control group (f). Abbreviations: PTB (Pulmonary Tuberculosis), SPPTB (smear-positive Pulmonary Tuberculosis), SNPTB (smear-negative Pulmonary Tuberculosis), CAP (community-acquired pneumonia), NK (natural killer) cells, CRP (C-reactive protein)|
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The expression of B-cells was significantly increased in the PTB and CAP groups
CD3-CD19 + cells were selected as B-cells. The result showed that the level of B- cells was significantly increased in the PTB and CAP groups [Table 1], [Figure 1]d. Subgroup analysis showed that the expression of B-cells was significantly increased in the CAP and smear-negative PTB groups [Table 1], [Figure 2]d. To examine the selectivity of the numbers of B-cells in the smear-negative PTB group, the data were subjected to receiver operating characteristic (ROC) curve analysis, and the area under curve (AUC) value of B-cells was > 0.75 (0.755), with a sensitivity of 66.7% and specificity of 77.3% [Figure 3]a.
|Figure 3: ROC curve analysis of the lymphocyte subsets. AUC value of B-cells was 0.755, with sensitivity of 66.7% and specificity of 77.3% in the SNPTB group (a). The AUC value of CRP was >0.911, with sensitivity of 80.6% and specificity of 100% in the SPPTB group (b). The AUC value of CRP was 0.822, with sensitivity of 82% and specificity of 100% in the CAP group (c). Abbreviations: AUC (area under curve), CRP (C-reactive protein), SPPTB (smear-positive Pulmonary Tuberculosis), SNPTB (smear-negative Pulmonary Tuberculosis), CAP (community-acquired pneumonia)|
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The frequencies of NK cells were significantly decreased in the PTB group
CD3-CD56 + cells were selected as natural killer (NK) cells. The result showed that compared with the control and CAP groups, the level of NK cells was significantly lower in the PTB group [Table 1], [Figure 1]e. Subgroup analysis showed that the expression of NK cells was mainly decreased in the smear-positive PTB group [Table 1], [Figure 2]e.
The level of CRP was significantly increased in the CAP and PTB groups
The level of CRP was determined as an infection indicator. The result showed that CRP was elevated in the CAP and PTB groups [Table 1], [Figure 2]f. There were no significant differences between the smear-negative PTB, control, and smear-positive PTB groups [Table 1], [Figure 3]a. ROC curve analysis was used to examine the selectivity of CRP, and the AUC value of CRP in the smear-positive PTB group was > 0.75 (0.911), with high sensitivity (80.6%) and specificity (100%) [Figure 3]b. In the CAP group, the AUC value of CRP was also > 0.75 (0.822), with a sensitivity of 82% and specificity of 100% [Figure 3]c.
| Discussion|| |
Given the lack of specificity in clinical manifestations and chest CT changes in PTB, it is difficult to differentially diagnose smear-negative PTB from lung cancer, lung tuberosity, or other lung diseases.
Mtb is a parasitic bacterium. The host immune system plays an important role in the pathogenesis and defense mechanism of Mtb, and the main immune response is cell-mediated immunity (CMI). After infection, Mtb initially activates innate immunity, then natural receptors on the surface of macrophages recognize Mtb and produce phagolysosomes to finally clear the bacteria. During this process, the level of CD4 + T-cells increases in the peripheral blood of patients, which in turn increases the ratio of CD4+/CD8 + T cells, resulting in immune system disorders that contribute to disease progression.,,,
We found that CD4 + T-cells and the ratio of CD4+/CD8 + T cells were increased in the peripheral blood of smear-positive PTB patients. Moreover, there were significant differences in the levels of CD4 + T cells and the ratio of CD4+/CD8 + T-cells between smear-positive and smear-negative PTB patients. The expression of CD4 + T cells was associated with Mtb virulence, and highly virulent Mtb strains could stimulate CD4 + T-cell proliferation. In this study, patients with smear-positive PTB had a higher incidence of pulmonary cavities and multiple pulmonary cavities, which suggested that they may be infected with hypervirulent Mtb strains, and the immune response–related damage be the cause of the cavities. This also explained why the expression of CD4 + T-cells and the ratio of CD4+/CD8 + T cells was not significantly increased in patients with smear-negative PTB, which may be associated with weaker Mtb virulence.
The role of B-cells in immunity against PTB remains controversial. Some scholars believed that most of the studies on PTB patients examined B-cells isolated from peripheral blood and not inflamed Mtb-affected tissues, which led to inconsistent conclusions on B-cell levels in PTB. This study found that the expression of B-cells was higher in patients with smear-negative PTB. The possible reason is that after Mtb infection, B-cells proliferate and are accumulated at the site of pulmonary inflammation, where they secrete type I interferons to inhibit Mtb proliferation and delay the migration of neutrophils to the lesion, which leads to a negative result of bacteriological tests and low level of CD4 + T-cells. Smear-positive PTB may have insufficient B-cell function, leading to the neutrophil influx to the site of infection, thereby activating the immune response of T-cells, resulting in elevated levels of CD4 + T cells. ROC curve analysis showed that the AUC value of B-cells was above 0.75 (0.755), with a sensitivity of 66.7% and specificity of 77.3% in smear-negative PTB, which indicated that the increase in B-cells contributed to the diagnosis of patients with smear-negative PTB.
NK cells are the main effector cells of innate immunity, that possess potent cytolytic capacity without MHC restriction. After attachment of Mtb to the natural cytotoxicity receptor (NCR) NKp44 on NK cells, the cells can directly or indirectly control mycobacterial growth through cytotoxic mechanisms and macrophage activation. NK cells were previously thought to be functionally impaired during TB. This study proved that the expression of NK cells was significantly lower in patients with smear-positive PTB perhaps due to the dysfunction of NK cells, which led to a decline in their ability to inhibit the proliferation of Mtb that resulted in severe lung injury and positive result of bacteriological tests in smear.
CRP is considered an indicator of acute clinical disease, and its level is often related to the severity of the disease. A previous study revealed a lower median CRP in PTB compared to bacterial pneumonia, and our study supported this conclusion. We found that the level of CRP in PTB was much higher than the control group, but the CRP level of the smear-negative PTB group was comparable to the control group. As CRP is a component of the innate immune response, it suggests that the innate immunity factors in smear-positive PTB patients differ from the smear-negative patients, which results in the decrease of CRP production. ROC curve analysis showed that the AUC value of CRP in smear-positive PTB was above 0.75 (0.911), with high sensitivity (80.6%) and specificity (100%). However, the AUC value of CRP was also above 0.75 (0.822) in the CAP group, with a sensitivity of 82% and specificity of 100%, which indicated that the value of CRP could help to establish the diagnosis of PTB in smear-positive patients, but needed to be differentiated from CAP according to the result of smear bacteriological test.
| Conclusion|| |
Smear-negative PTB is easily confused with other pulmonary diseases and may convert to smear-positive PTB. The number and virulence of bacteria, host immunity, and other factors determine the outcome of the disease. This study demonstrated that the manifestation of the immune system in smear-negative PTB was different from smear-positive PTB and CAP, the inflammation in the infected lesion was limited, which decreased the T-cell response and innate immune activation. Moreover, smear-negative PTB showed fewer pulmonary cavities, mild inflammatory response, lower expression of immune cells, and high level of B cell-expression. These factors will facilitate the early diagnosis.
The small sample population and short duration of follow-up were the limitations of this study. In this study, the immunomodulatory effects of Mtb are preliminarily discussed, but the exact mechanism remains unknown. Hence, we will further investigate the pathways related to the innate immune system and Mtb infection, and delineate the phenotypic and functional characterization of immune cells.
The study protocol was approved and monitored by the ethics committee of Nanjing Jiangbei Hospital (Nanjing, China), and written informed consent was obtained from the patients
Financial support and sponsorship
The project supported by the Nantong University Clinical Medicine Special Project (2019LY023); Nanjing Medical Science and Technique Development Foundation (QRX17098).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Higuchi K, Kawabe Y, Mitarai S, Yoshiyama T, Harada N, Mori T. Comparison of performance in two diagnostic methods for tuberculosis infection. Med Microbiol Immunol 2009;198:33-7.
Galal El-Din M, Sobh E, Adawy Z, Farghaly N. Diagnostic utility of gene X-pert in the diagnosis of tuberculous pleural effusion. Infect Dis (Lond) 2019;51:227-9.
Chaisson LH, Saraceni V, Cohn S, Seabrook D, Cavalcante SC, Chaisson RE, et al
. CD4 count stratification to guide tuberculosis preventive therapy for people living with HIV. AIDS 2020;34:139-47.
Hoft SG, Sallin MA, Kauffman KD, Sakai S, Ganusov VV, Barber DL. The rate of CD4 T cell entry into the lungs during Mycobacterium tuberculosis
infection is determined by partial and opposing effects of multiple chemokine receptors. Infect Immun 2019;87:1-15.
Liu X, Li F, Niu H, Ma L, Chen J, Zhang Y, et al
. IL-2 restores T-cell dysfunction induced by persistent Mycobacterium tuberculosis
antigen stimulation. Front Immunol 2019;10:1-12.
Silveira-Mattos PS, Barreto-Duarte B, Vasconcelos B, Fukutani KF, Vinhaes CL, Oliveira-De-Souza D, et al
. Differential expression of activation markers by Mycobacterium tuberculosis
-specific CD4+T-cell distinguishes extrapulmonary from pulmonary tuberculosis and latent infection. Clin Infect Dis 2020;71:1905-11.
Ramos-Martinez AG, Valtierra-Alvarado MA, Garcia-Hernandez MH, Hernandez-Pando R, Castañeda-Delgado JE, Cougoule C, et al
. Variability in the virulence of specific Mycobacterium tuberculosis
clinical isolates alters the capacity of human dendritic cells to signal for T cells. Mem Inst Oswaldo Cruz 2019;114:1-9.
Dyatlov AV, Apt AS, Linge IA. B lymphocytes in anti-mycobacterial immune responses: Pathogenesis or protection? Tuberculosis (Edinb) 2019;114:1-8.
Burbage M, Keppler SJ. Shaping the humoral immune response: Actin regulators modulate antigen presentation and influence B-T interactions. Mol Immunol 2018;101:370-6.
Liu CH, Liu H, Ge B. Innate immunity in tuberculosis: Host defense vs pathogen evasion. Cell Mol Immunol 2017;14:963-75.
Portevin D, Via LE, Eum S, Young D. Natural killer cells are recruited during pulmonary tuberculosis and their ex vivo
responses to mycobacteria vary between healthy human donors in association with KIR haplotype. Cell Microbiol 2012;14:1734-44.
Brown J, Clark K, Smith C, Hopwood J, Lynard O, Toolan M, et al
. Variation in C-reactive protein response according to host and mycobacterial characteristics in active tuberculosis. BMC Infect Dis 2016;16:265.
Infectious Diseases Section, Nanjing Pukou Central Hospital, Pukou Branch Hospital of Jiangsu Province Hospital, Nanjing 211800
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]