Innate immune cells are the first to confront M. tuberculosis, significantly influencing the infection's progression. Dendritic cells (DCs) activate the adaptive immune response, while macrophages provide antimicrobial control and manage inflammation. The inflammatory response to M. tuberculosis can be a double-edged sword. Critical cytokines like TNF-alpha and IL-1 are necessary for protection, but excessive or insufficient levels can worsen the disease. Neutrophils, thought to control bacterial infections, also emerge as drivers of a harmful hyperinflammatory response. Understanding the balance between infection control and inflammation regulation is vital for developing effective host-targeted therapies [18, 19].

In this study, we utilized high-throughput Olink proteomics analysis technology to screen and identify precise and sensitive biomarkers for different subtypes of TB. Utilizing minimal sample requirements, we concurrently examined inflammatory proteins, identifying 47 proteins with significant expression among active TB, LTBI, and control healthy cases. Notably, the co-differentiated proteins were enriched in biological processes related to the proliferation and apoptosis of immune cells and chemokine-related activities. It is mainly enriched in the PPAR signaling pathway, IL-17 signaling pathway, primary immunodeficiency, and TNF signaling pathway. Identify the pathways and functions of co-differentiated proteins in TB subtypes to enhance our understanding of TB's clinical manifestations. These results indicate the importance of investigating how specific co-differentiated proteins may impact immune cell function, potentially decreasing inflammatory responses or enhancing the immune capabilities of patients for disease prevention and treatment.

LTBI screening is vital for identifying individuals at high risk of developing active tuberculosis, crucial for preventing disease progression and curbing its spread. It is essential in high-risk populations to reduce TB-related morbidity and mortality. Among these co-differentiated proteins, CXCL10 and TGF-alpha show potential as biomarkers for distinguishing between TB, LTBI, and the control healthy group. Recent observations show that CXCL10 is produced by antigen-presenting cells and activated macrophages during infection. This cytokine aids chemotaxis and leukocyte migration and may inhibit Mycobacterium tuberculosis replication [20,21,22]. CXCL10/IP-10 alone or combined with acute phase proteins or cytokines are proposed as markers of bacterial burden, LTB, and active TB discrimination [21, 23, 24]. Notably, CXCL10 levels significantly increase in patients with severe pulmonary tuberculosis. CXCL10, induced by interferon-gamma (IFN-gamma), is integral to the immune response against tuberculosis, as it aids in the recruitment of immune cells to the infection site and contributes to granuloma formation—a hallmark of TB pathology [24]. Previous research identified 24 parameters that were elevated exclusively in active TB, including TGF-alpha. Five parameters were increased in LTBI, specifically IL-5, IL-17F, IL-1, CCL20, and ICAM-1[25]. As observed, Influenza induces IL-8 and granulocyte–macrophage colony-stimulating factor (GM-CSF) secretion by human alveolar epithelial cells through recombinant human Hepatocyte growth factor (HGF)/c-Met and TGF-α/ epidermal growth factor receptor (EGFR) signaling [26]. The expression of TGF-alpha varies among individuals within a disease spectrum, and its expression trends still differ across various disease states. This indicates that the interaction between Mycobacterium tuberculosis and the host is heterogeneous and may depend on individual differences in innate immune responses.

Recently, research reported that chemokines CXCL10 and CXCL9 positively correlate and may serve as markers to distinguish between drug-resistant and drug-sensitive TB, enhancing disease stage differentiation [27]. In our study, the proteins CXCL10 and CXCL9 were not found to be distinct in R-TB and S-TB. CXCL10, a chemokine attracting immune cells to infection sites, is elevated in severe pulmonary disease patients’ serum and bronchoalveolar lavage fluid, positively correlating with disease severity [24, 28]. Additionally, Macrophages increase kynurenine (Kyn) production, activating the aryl hydrocarbon receptor (AhR). This activation upregulates cytokine signaling 3 (SOCS3) and inhibits the JAK-STAT1 pathway, reducing the secretion of the chemokines CXCL9 and CXCL10, vital for lung T-cell recruitment. In vivo mouse models show that knocking out AhR significantly enhances T-cell infiltration and activity, counteracting Mycobacterium tuberculosis-induced immunosuppression [29]. These results suggest that CXCL10 is associated with the progression of Mycobacterium tuberculosis infection and drug resistance through the regulation of T-cell immunity.

The co-expression of the protein IFN-gamma in LTBI and active TB indicates a significant increase in active TB and has substantial predictive value for active TB. Prior research has revealed that The CD5+ and CD10+ B cell subpopulations exhibit potential as biomarkers for distinguishing between LTBI and active TB. Specifically, LTBI is characterized by elevated levels of CD5+ B cells, which contribute to a cytokine-rich microenvironment containing IFN-gamma, IL-10, and IL-4. Conversely, active TB demonstrates an anti-inflammatory response only in stimulation with mycobacterial proteins or lipids [30]. CD5+ B cells may contribute to active TB progression via IFN-gamma. A recent study found that CD4+Foxp3+ cells have a time-dependent role in TB, with CCR4 crucially balancing IFN-gamma-mediated inflammation by managing these cells' influx and function. This suggests that targeting CD4+Foxp3+ cells or CCR4 could be a potential immunotherapy strategy, given TB's heterogeneity in immunocompetent adults [31]. Another protein, SCF, can differentiate between TB and LTBI. Various immune cells express the c-kit receptor and can be activated by SCF, contributing to fibrotic disorders. Eosinophils, for example, express c-kit and, when stimulated by SCF, produce pro-fibrotic cytokines like TGFβ and FGF, along with lipid mediators, proteases, and chemokines [32]. Integration of SCF and Leukotriene D4 (LTD4) signals may contribute to Mast cells (MCs) hyperplasia and hyper-reactivity during airway hyper-response and inflammation [33]. Its differential expression may indicate differences in inflammatory processes or the host’s attempt to control M. tuberculosis.

In this study, the PD-L1(Programmed Death Ligand-1), EN-RAGE (Extracellular Newly Identified RAGE-Binding Protein), and CXCL10 (Chemokine C-X-C Ligand 10) have significant diagnostic value for severe pulmonary TB. PD-L1 is a protein present on the surface of many immune cells [34]. It interacts with its receptor, PD-1, to regulate the immune system and prevent excessive inflammation [35]. In severe pulmonary TB, the bacterial load is high, triggering the expression of PD-L1 on immune cells as an immune evasion mechanism [36]. The other protein, EN-RAGE, also known as S100A12, is highly expressed in monocytes and neutrophils, acting as a DAMP to interact with RAGE [37, 38]. It triggers signal transduction through the NF-κB and MAPK pathways, enhances the expression of ICAM-1, VCAM-1, NF-κB, and TNF-alpha, and directly activates endothelial cells, mononuclear phagocytes, and lymphocytes. This activation leads to the synthesis and secretion of proinflammatory cytokines, recruiting leukocytes to the inflammatory site, and, ultimately, the onset of inflammation [39]. It can activate monocytes through the TLR4 pathway, enhance monocyte migration, and upregulate proinflammatory factors such as IL-1β, IL-6, and IL-8 [40]. These findings suggest that they contribute to the struggle between the host and pathogen in both normal and inflammatory conditions, making them potential targets for disease prevention and treatment [41].

SLAMF1 (Signaling Lymphocytic Activation Molecule Family Member 1) protein can identify early or paucibacillary tuberculosis in Sputum Culture-Negative Pulmonary Tuberculosis patients, serving as an essential inflammatory factor that predicts negative pulmonary tuberculosis. SLAMF1 has been identified as a potential biomarker for Sputum Culture-Negative TB prediction due to its association with immune system functions and inflammatory response [42]. A previous study showed that SLAMF1 expression was reduced in active TB patients [43]. High SLAMF1 levels may indicate a normal immune response and lower TB risk. Therefore, SLAMF1 can predict sputum culture negative and positive TB status, a critical mechanism that needs further research.

The TRANCE (Tumor Necrosis Factor-Related Activation-Induced Cytokine) and IL-2RB (Interleukin-2 Receptor Beta) immune-related molecules [44, 45] exhibit significant predictive value for R-TB, with levels of TRANCE and IL-2RB being higher in R-TB than S-TB. Reports indicate that TRANCE can be a biomarker for diagnosing and assessing tuberculosis [46]. Previous research has shown that TRANCE expressed on activated T cells promotes the survival of dendritic cells and modulates T helper cell responses to viral infections [47]. Growing evidence has demonstrated that TRANCE is preferentially expressed on the surface of activated CD4+Th1 cells and dendritic cells through binding to RANK and enhances IFN-γ secretion via a p38-dependent pathway [48]. IL-2RB deficient patients demonstrated decreased Treg cell frequency, skewing toward memory T cells, and lymphocytic infiltration into multiple tissues [49, 50]. These molecules are integral to activating and regulating T-cell immunity, vital for controlling and eliminating mycobacterial infections.

In this research, creatinine is higher in R-TB, and D-Dimer is higher in S-TB,

Mainly correlated with the S-TB patients who were relatively older R-TB, and previous studies reported D-dimer levels, lactate dehydrogenase, C-reactive protein, erythrocyte sedimentation rate, and creatinine kinase were positively correlated with patient age [51]. Notably, A previous study reported that Urinary neopterin/creatinine ratios are significantly higher in patients with active tuberculosis than in patients with latent infection and may be a significant predictor of active tuberculosis in patients with M. tuberculosis infection [52]. Furthermore, Elderly tuberculosis patients are at high risk for thrombosis and renal injury. These conditions must be closely monitored during disease progression to prevent thrombosis and protect renal function.

The findings of this study have significant implications, especially in the context of potential advancements in TB diagnosis and treatment. The discovery of IL-2RB, TGF-alpha, and other proteins as potential biomarkers can transform the approach to TB diagnosis. These biomarkers offer the possibility of developing more accurate diagnostic tools to distinguish between active TB, LTBI, and control groups with heightened sensitivity and specificity. Furthermore, the predictive value of immune-related molecules like TRANCE and IL-2RB for multi-drug-resistant tuberculosis offers a promising avenue for the early identification of at-risk patients and developing strategies to combat drug resistance. This could be crucial in the fight against the rising global threat of multi-drug-resistant tuberculosis. The potential of proteins like SLAMF1 to distinguish between positive and negative could lead to earlier disease detection, allowing for interventions before the onset of symptoms. Understanding the role of these proteins in the immune response to TB could open new avenues for targeted therapies that enhance or modulate the immune system's reaction to the disease. Moreover, the study's findings could be instrumental in developing prognostic models that assess treatment outcomes by monitoring changes in biomarker levels. This could provide clinicians with valuable insights into the efficacy of treatments and disease progression.

This study is subject to several limitations. Firstly, additional validation with a larger sample size is necessary to elucidate the role of differential proteins in predicting tuberculosis across various disease states. Secondly, ongoing monitoring of specific proteins in patients with similar conditions is essential to understand the potential mechanisms of action of these proteins in the disease. Future research endeavors will address these limitations and advance the identification of early diagnostic biomarkers.

In summary, the importance of co-differentially expressed proteins in diagnosing the severity or subtype of TB lies in their correlation with immune responses and inflammatory processes during TB infection. Nevertheless, it is imperative to underscore the need for further research to validate their diagnostic value and establish standardized cut-off thresholds for their clinical application.