Telomere shortening correlates with immune dysregulation and severity in pulmonary tuberculosis

Abstract

Background

Telomere shortening is suspected to influence the severity of inflammation and tissue damage in tuberculosis. This study aims to analyze the association between telomere length and inflammatory cytokines in tuberculosis patients.

Methods

The study was conducted at Persahabatan National Respiratory Referral Hospital and Jakarta Islamic Hospital Cempaka Putih between October 2024 and March 2025. Adult tuberculosis patients who met the criteria were enrolled. Relative Telomere Length (RTL), Tumor Necrosis Factor α (TNF-α), and Interleukin 10 (IL-10) levels were measured from peripheral blood mononuclear cells using Quantikine Enzyme-Linked Immunosorbent Assay (ELISA) kits, and chest X-rays (CXR) were graded into minimal or extensive disease to assess severity.

Results

A total of 64 tuberculosis patients were enrolled, with a mean age of 41.36 ± 16.23 years, and the majority of subjects were male (68%). The analysis revealed significant differences in Body Mass Index (BMI) (p = 0.01), RTL (p = 0.03), and TNF-α (p = 0.02) among the tuberculosis patients and healthy subjects. A negative correlation was observed between RTL and TNF-α (p = 0.04), whereas no significant association was found with IL-10 (p > 0.05). Patients with extensive radiographic lesion had significantly shorter telomeres than those with minimal lesion (p = 0.021).

Conclusion

Telomere shortening is closely associated with the pro-inflammatory response in tuberculosis, as reflected by its significant association with elevated TNF-α levels and higher extensive disease severity based on radiographic findings.

Introduction

Telomere shortening is a pivotal mechanism driving cellular senescence and immune dysfunction [1]. Critically short telomeres induce a senescence-associated secretory phenotype (SASP), characterized by the release of pro-inflammatory cytokines, chemokines, and proteases that sustain local inflammation and promote systemic immune dysregulation [2–4]. The inflammatory processes influenced by telomere alterations can be observed in several chronic and age-related diseases, including idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease [5]. In these conditions, telomere attrition correlates with increased circulating inflammatory mediators such as Tumor Necrosis Factor α (TNF-α), Interferon γ (IFN-γ), and Interleukin 6 (IL-6), suggesting that telomere shortening not only reflects cellular aging but also perpetuates inflammation [5].

Tuberculosis (TB) is one of the leading causes of mortality worldwide and is recognized as a chronic inflammatory disease [6]. Infection with Mycobacterium tuberculosis (Mtb) activates a cascade of immune responses including T helper 1 (Th1) cells that release various cytokines to enhance inflammation [7]. Th1 is in charge of producing inflammatory cytokines such as IFN-γ, TNF-α, and IL-6 [6, 7]. TNF-α is an important factor in granuloma formation by inducing macrophages to release chemokines that guide cells to infection sites and prevent migration. The absence of TNF-α can hinder granuloma integrity, thus causing widespread dissemination of Mtb. On the other hand, IL-10 is known to inhibit inflammation during Mtb infection by surpassing the production of pro-inflammatory cytokines and chemokines that contribute towards granuloma formation [8]. IL-10 is able to block antigen presentation, which leads to diminished T cell responses. Although Mtb growth can increase due to insufficient inflammation, the anti-inflammatory response allows tissue damage prevention. Therefore, the balance between pro-inflammatory and anti-inflammatory response is crucial to kill Mtb, while limiting lung tissue injury. The balance between these opposing pathways influences disease progression and tissue injury, and emerging evidence suggests that telomere shortening may further amplify inflammatory damage, linking host cellular aging with TB immunopathogenesis [5].

Telomere alterations in immune cells can impair cellular function and cytokine production, which influence the inflammatory response to infection. However, the contribution of telomere dysfunction as a molecular link between chronic immune activation and disease severity remains limited. This study aimed to investigate the association between telomere length and inflammatory cytokines (TNF-α and IL-10) in patients with tuberculosis. Accordingly, telomere length is examined to understand the mechanistic connection to chronic inflammation.

Method

Study population

This cross-sectional study was conducted at Persahabatan National Respiratory Referral Hospital and Jakarta Islamic Hospital Cempaka Putih between October 2024 and March 2025. All participants provided written informed consent. The study protocol was approved by the Ethics Committees of both hospitals (No. 0125/KEPK-RSUPP/05/2024 and No. 20/EC/KEP/07/2024) and complied with institutional and international ethical standards.

Inclusion criteria were patients aged > 18 years with newly diagnosed, drug-sensitive pulmonary tuberculosis. Diagnosis was based Indonesia Tuberculosis Guideline [9] with the criteria (1) bacteriological confirmation of Mycobacterium tuberculosis in sputum samples or if negative (2) Chest X-Ray (CXR) appearance supporting TB lesions (e.g. upper lobe infiltrates, consolidations, or thick-walled cavities) supported with TB typical symptoms and no improvement despite empirical antibiotic treatment. Exclusion criteria are (1) HIV infection, (2) Chronic lung disease other than pulmonary tuberculosis, (3) Radiological evidence of destroyed lung, (4) autoimmune or other immunodeficiency disorders. As comparison, a little number of healthy subjects were recruited with no history of tuberculosis, smoking, or other comorbidities.

Study design

Participants who met the criteria were enrolled. Demographic data, including age and gender, were recorded. Clinical information was documented using a standardized case report form, which included symptoms such as persistent cough, fever, malaise, weight loss, night sweats, hemoptysis, chest pain, and reduced appetite. Comorbidities, including diabetes, hypertension, dyslipidemia, and smoking history, were also recorded. Body weight and height were measured to calculate body mass index (BMI). Peripheral venous blood was collected to measure telomere length and inflammatory cytokines (TNF-α and IL-10). Baseline chest X-rays (CXR) were obtained before initiation of anti-tuberculosis treatment (ATT).

Chest X-Ray severity

An initial CXR was obtained and graded into two categories to assess disease severity [10]. (1) Minimal defined as lesions of mild to moderate density in one or both lungs, confined above the chondrosternal junction bilaterally and the T4–T5 vertebrae, without evidence of cavitation. (2) Extensive defined as lesions of moderate to severe density in one or both lungs, which could be accompanied by cavitation. These criteria were adapted from the US National Tuberculosis and Respiratory Disease Association [11]. Two pulmonologists independently assessed the CXRs, and discrepancies were resolved by consensus to ensure consistent interpretation.

Inflammatory cytokines measurement

The levels of TNF-α and IL-10 were measured using the enzyme-linked immunosorbent assay (ELISA). Supernatants were collected from peripheral blood mononuclear cells (PBMCs) of participants before initiation of ATT. Quantification was performed with R&D Systems Quantikine ELISA kits (TNF-α, catalog no. DTA00D; IL-10, catalog no. D1000B) according to the manufacturer’s instructions.

Telomere length measurement

Leukocyte telomere length was measured using a flow cytometry–based fluorescence in situ hybridization (flow-FISH) assay with the Telomere PNA Kit/FITC (Agilent/Dako, K5327), following the manufacturer’s instructions and established protocols. Briefly, peripheral blood leukocytes were isolated from EDTA samples, hybridized with a fluorescein-labeled peptide nucleic acid (PNA) probe targeting telomeric repeats, and analyzed by flow cytometry (BD FACSLyric or equivalent) within the G0/G1 gate, using the 1301 cell line as an internal reference standard. Relative telomere length (RTL) was calculated as the ratio of telomere fluorescence intensity of sample cells to that of 1301 cells after background subtraction and DNA content normalization, enabling inter-sample comparability. Negative controls (no-probe and unstained) were included for each run, and all samples were processed in duplicate, with experiments repeated if intra-assay variation exceeded 10–12%. Data interpretation followed the kit manual, with relative telomere length (RTL) calculated using the formula:

RTL = ((Mean FL1 of Sample with Probe – Mean FL1 of Sample without Probe) × DNA Index of Control × 100) ÷ ((Mean FL1 of Control with Probe – Mean FL1 of Control without Probe) × DNA Index of Sample).

Statistical analysis

Descriptive statistics were used to summarize baseline characteristics, with numerical variables presented as mean ± standard deviation for normally distributed data or as median (minimum–maximum) for non-normally distributed data, while categorical variables were expressed as frequencies and percentages. Correlation analysis with Spearman’s were performed between telomere length and age. Bivariate analysis was performed using the independent t-test or Mann–Whitney U test for continuous variables and the Chi-square test for categorical variables, according to data distribution. Significant result with p-value < 0.05 were subsequently included in multivariate models to determine independent predictors. All statistical analyses were performed using SPSS version 30.

Result

A total of 64 tuberculosis patients were included in this study (Table 1), with a mean age of 41.36 ± 16.23 years and majority male subjects (68.7%). Common comorbidities included diabetes mellitus (20.3%), hypertension (12.5%), and dyslipidemia (1.6%), and nearly half of the patients reported a history of smoking (43.8%). Clinically, the most frequently reported symptoms were loss of appetite (71.9%), malaise (67.2%), and cough (65.5%), followed by weight loss (62.5%), fever (43.8%), chest pain (34.4%), night sweats (29.7%), and hemoptysis (26.6%). The mean body mass index (BMI) was 20.93 ± 4.85 kg/m². Inpatient care was required for 71%, and mycobacterial sputum positivity was observed in 46% of cases. Chest radiography revealed extensive lesions in the majority of patients (76.6%), while the remaining cases demonstrated minimal lesions. The mean RTL in the study population was 6.83 ± 1.36. Inflammatory cytokine examination showed mean serum TNF-α and IL-10 concentrations of 17.12 ± 7.61 pg/mL and 5.87 ± 3.00 pg/mL, respectively. A significant negative correlation was observed between RTL and age (R = − 0.36, p = 0.003) (Fig. 1), indicating shorter telomere length with increasing age.

Table 1.

Baseline characteristics of study participants

Characteristics	tuberculosis patients (n = 64)
Age	41.36 ± 16.23
Gender
Male	44 (68.7)
Female	20 (31.3)
Comorbidities
Diabetes	13 (20.3)
Hypertension	8 (12.5)
Dyslipidemia	1 (1.6)
Symptoms
Cough	42 (65.5)
Fever	28 (43.8)
Malaise	43 (67.2)
Weight Loss	40 (62.5)
Night Sweat	19 (29.7)
Hemoptysis	17 (26.6)
Chest Pain	22 (34.4)
Appetite Loss	46 (71.9)
Smoking	28 (43.8)
BMI	20.93 ± 4.85
Inpatient	46 (71.9)
Mycobacterial Sputum	30 (46.9)
Chest X-ray
Minimal	15 (23.4)
Extensive	49 (76.6)

BMI Body mass index, RTL Relative telomere length, TNF-α Tumor necrosis factor-alpha, IL-10 Interleukin-10. numeric variable: mean ± standard deviation; categoric variable: frequency (percentage)

Fig. 1.

Relative telomere length (RTL) demonstrated a negative correlation with age (R = −0.36) with a borderline p value of 0.003. This association was considered weak. Therefore, age was not included as an adjustment variable in subsequent analyses. RTL: relative telomere length. Correlation coefficient (R) calculated using Spearman’s rank correlation test due to non-normal data distribution

The level of both cytokines were grouped using their respective median values (TNF-α: 14.7 pg/mL; IL-10: 4.8 pg/mL) for further analysis. The analysis shows RTL significantly shorter in patients with higher TNF-α levels (p = 0.04) (Fig. 2A). In contrast, no significant difference in RTL was observed when participants were stratified according to IL-10 levels (p = 0.76) (Fig. 2B). Neither TNF-α nor IL-10 levels were significantly associated with the occurrence of diabetes mellitus (p > 0.05). Additionally, patients presenting with extensive chest X-ray lesions exhibited significantly shorter RTL compared with those showing minimal radiographic involvement (p = 0.021) (Fig. 2C).

Fig. 2.

Leukocyte relative telomere length (RTL) associations with inflammatory and radiographic parameters: (A) RTL showed a significant negative correlation with TNF-α (p = 0.04), (B) but no association with IL-10 (p > 0.05). (C) Patients with extensive CXR involvement exhibited significantly shorter RTL compared to those with minimal disease (p = 0.021). RTL: relative telomere length; TNF-α: tumor necrosis factor-alpha; IL-10; CXR: Chest X-ray. Statistical analysis was performed using the Mann–Whitney test for group comparisons. Ap-value < 0.05 was considered statistically significant.

Reference values of normal telomere length in the Indonesian population are currently unavailable. Therefore, healthy individuals were included for comparison, although the number of subjects was limited. This healthy subjects had a mean age of 28.25 ± 4.65 years and consisted of three males and five females, with an average BMI of 25.24 ± 3.56 kg/m². Compared with tuberculosis patients, healthy subjects exhibited lower levels of inflammatory cytokines. Nearly all cytokine concentrations in the healthy group were below the median levels observed in the tuberculosis cohort. However, correlation analyses revealed no significant association between RTL and both inflammatory cytokine in the healthy subjects (p > 0.05).

Comparison between tuberculosis patients and healthy subjects revealed significant differences in several parameters (Table 2). Patients with tuberculosis had a significantly lower BMI (20.93 ± 4.85 vs. 25.24 ± 3.56 kg/m², p = 0.01), and shorter RTL (6.83 ± 1.36 vs. 8.28 ± 2.01, p = 0.03) compared to healthy subjects. Serum TNF-α concentrations were significantly higher in the tuberculosis group compared to healthy subjects (17.12 ± 7.61 vs. 11.80 ± 1.77 pg/mL, p = 0.02), whereas IL-10 levels did not differ significantly (5.87 ± 3.00 vs. 4.46 ± 1.41, p = 0.17).

Table 2.

Comparison of relative telomere length and inflammatory cytokines between tuberculosis patients and healthy subjects

Characteristics	Healthy subject (n = 8)	Tuberculosis subject (n = 64)	P value
RTL	8.28 ± 2.01	6.83 ± 1.36	0.03◆
TNF-α (pg/mL)	11.80 ± 1.77	17.12 ± 7.61	0.02◆
IL-10 (pg/mL)	4.46 ± 1.41	5.87 ± 3.00	0.17◆

RTL Relative telomere lengt, TNF-α Tumor necrosis factor-alpha, IL-10 Interleukin-10

Numeric variable: mean ± standard deviation; statistical analysis: ^◆man-whitney

p-value < 0.05 was considered statistically significant

Discussion

This study demonstrates that telomere shortening in tuberculosis is associated with pro-inflammatory responses and disease severity. We observed that higher TNF-α levels were significantly associated with shorter telomere length, whereas IL-10 showed no such association. In addition, patients with more extensive pulmonary lesions on CXR exhibited greater telomere shortening compared to those with minimal disease. These findings suggest that inflammation drives telomere attrition in tuberculosis, potentially linking immunopathology to cellular senescence and disease progression.

Telomere shortening is widely acknowledged as a hallmark of biological aging and cellular senescence [12]. Telomeres progressively decrease in length through successive cell divisions and oxidative damage, ultimately leading to replicative arrest, the Hayflick limit, and impaired tissue regeneration [13]. Consequently, telomere length has emerged as a well-established biomarker of biological aging, consistently showing an inverse relationship with chronological age in both human and animal studies [2]. Consistent with this concept, Ye et al. [14], in a large meta-analysis spanning individuals aged 0–112 years, demonstrated a robust inverse relationship between telomere length and chronological age. Similarly, Devrajani et al. [15] reported a strong negative correlation between telomere length and age in healthy populations. In line with these findings, the present study observed a significant inverse association between age and telomere length (R = − 0.36, p = 0.003). However, contrasting evidence has also been reported. Park et al. [16], in a cohort of adolescent and young adult cancer survivors, found that telomere length was not significantly associated with chronological age but was instead strongly influenced by clinical stressors, including hematopoietic stem cell transplantation and chronic health conditions. These observations suggest that telomere attrition and inflammatory processes may evolve independently rather than in parallel [3, 17].

Telomere length has been increasingly recognized as a molecular marker that interacts with the balance of pro-inflammatory and anti-inflammatory pathways [5, 18]. In our study, telomere length showed a significant association with TNF-α, but not with IL-10, suggesting that telomere attrition may be more tightly linked to Th1-driven pro-inflammatory responses than to Th2-mediated immunoregulation [19, 20]. TNF-α is a hallmark cytokine of the Th1 pathway, playing a critical role in activating macrophages and maintaining granuloma integrity in tuberculosis [21, 22]. In contrast, IL-10 represents an anti-inflammatory cytokine often produced by Th2 and regulatory T cells, which functions to dampen excessive immune activation [23, 24]. However, the underlying inflammatory pathways linking telomere dynamics with Th1 activation and TNF-α signaling remain unclear.

Previous studies have reported that a Th1/Th2 imbalance is central to tuberculosis pathogenesis, with reduced Th1 responses and heightened Th2 activity contributing to immune evasion and disease progression [20, 25]. Cytokines such as IFN-γ, IL-2, and TNF-α are typical of Th1-mediated cellular immunity, whereas IL-4, IL-5, and IL-10 characterize the Th2 profile that promotes humoral responses and immune suppression [21, 26, 27]. The selective correlation of telomere length with TNF-α, but not IL-10, may therefore reflect a preferential link between telomere dynamics and pro-inflammatory Th1 signaling, where persistent activation accelerates telomere shortening [5].

TNF-α is a central pro-inflammatory cytokine in tuberculosis pathogenesis, indispensable for granuloma maintenance yet al.so implicated in tissue injury when excessively produced [28]. Persistent TNF-α exposure induces oxidative stress, mitochondrial dysfunction, and DNA damage, all of which accelerate telomere attrition [29]. Importantly, telomere dysfunction itself can activate a senescence-associated secretory phenotype (SASP), characterized by increased TNF-α and other pro-inflammatory mediators, thereby establishing a self-perpetuating loop of inflammation and cellular aging [30]. In contrast, IL-10 levels showed no significant relationship with telomere length in our study. This divergence likely reflects their distinct positions in the inflammatory cascade. As a potent anti-inflammatory cytokine, IL-10 restrains immune activation by down-modulating macrophage and dendritic cell responses [31]. Because telomere shortening is primarily driven by cumulative pro-inflammatory oxidative stress, anti-inflammatory signals may not exert a parallel influence on telomere attrition. However, the lack of association between telomere length and IL-10 may be due to early-phase sampling, in which anti-inflammatory responses are not yet fully expressed.

Our finding that shorter telomeres were associated with more extensive radiographic lesions in TB patients is consistent with the notion that telomere attrition reflects the burden of inflammatory damage. This positive association supports the biological plausibility of our earlier observation that telomere length correlated with TNF-α, a key pro-inflammatory cytokine [3]. By contrast, IL-10 primarily modulates immune suppression, which may not translate into cumulative telomeric damage. Supporting this, Kumar et al. [32] demonstrated that TB patients with bilateral or cavitary disease exhibited significantly higher plasma levels of IFN-γ and TNF-α compared to those with unilateral or non-cavitary disease, suggesting that systemic inflammation is mirrored by radiographic severity. In line with this, our study confirmed that TNF-α and CXR severity were significantly correlated (p < 0.01).

The association between telomere shortening towards increased inflammatory responses contributes to the understanding of tuberculosis pathology [33]. This finding explains the mechanistic link that affects infection severity in tuberculosis. Understanding telomere biology can help clinicians to predict disease behaviour, which can be utilized along with available biomarkers. Future directions can explore host-directed therapies (HDTs) including targeting oxidative stress, immune dysregulation, or senescence-associated pathways on tuberculosis treatment [34, 35].

The cross-sectional design of this study limits the ability to draw causal inferences. This study evaluated only a limited number of cytokines supported by previous research while other potentially relevant cytokines, including IFN-γ, IL-1β, and IL-6, should be examined in future studies. This study also includes healthy subjects for comparison, but only limited subjects are included, which may influence the statistical power of the association towards tuberculosis subjects. In addition, Future research should employ longitudinal designs with larger TB populations and add more cytokines that correlate to TB pathology to validate these findings. Integrating telomere length into a potential disease predictor, alongside cytokines and imaging parameters, may provide more robust prognostic tools. Furthermore, interventional studies evaluating host-directed therapies or antioxidant strategies could help determine whether preserving telomere integrity translates into improved clinical outcomes for TB patients.

Conclusion

This study demonstrates telomere shortening association with pro-inflammatory response in tuberculosis, as evidenced by its significant increase TNF-α levels and extensive radiographic lesion. These observations indicate that telomere attrition is linked to heightened inflammatory activity in tuberculosis, which may contribute to greater disease severity. In contrast, no clear association was observed with IL-10, suggesting that telomere dynamics are more closely related to pro-inflammatory rather than anti-inflammatory pathways. Collectively, these findings highlight the potential role of telomere length in reflecting the inflammatory pathology of tuberculosis and its relevance to disease severity assessment.

Abbreviations

BMI

Body mass index

CXR

Chest X-Ray

ELISA

Enzyme-Linked Immunosorbent Assay

IFN

Interferon

IL

Interleukin

TB

Tuberculosis

TL

Telomere Length

TNF

Tumor Necrosis Factor

RTL

Relative Telomere Length

Authors’ contributions

FF, SLA, EB, MH, WHW conception of the work. FF, HW, GA data collection. FF, AK statistical analysis. FF, FIS interpretation of the data. FF, FIS drafting the manuscript All authors reviewed and validated the manuscript.

Funding

This study was supported by the Ministry of Education, Culture, Research, and Technology of Indonesia (Grant No. NKB-871/UN2.RST/HKP.05.00/2024) and by the Respiratory Programmatic Implementation and Research Institute (RPRI).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committees of Persahabatan National Respiratory Referral Hospital (No. 0125/KEPK-RSUPP/05/2024) and Jakarta Islamic Hospital Cempaka Putih (No. 20/EC/KEP/07/2024) that complied with institutional and international ethical standards.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.