Locally-secreted interleukin-6 is related with radiological severity in smear-negative pulmonary tuberculosis

Highlights

• Pulmonary Mycobacterium tuberculosis infection induce expression of local IL-6 in smear-negative patients.

• TNF-α and IFN-γ were not detected in BALf of patients with smear-negative PTB, despite the use of highly efficient concentration through filtration columns and centrifugation.

• Level of IL-6 in BALf identified patients with bacterial ongoing infections from patients without pulmonary infection conditions.

• Locally-secreted IL-6 is related to tissue damage extension evaluated radiologically.

Abstract

Pulmonary tuberculosis (PTB) has been identified as a substantial public health threat and diagnostic challenge. A large proportion of patients exhibit negative smear tests despite active infection. The role of cytokines in the pathophysiology and clinical severity of PTB remains a controversial question. We evaluated the pattern of cytokines presents locally in patients with smear-negative PTB. Levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17 in bronchoalveolar lavage fluid (BALf) from patients with smear-negative PTB, as well as in those with other pulmonary diseases and controls, were performed by flow cytometry. ROC curve and a radiological severity scale were used to establish the potential diagnosis use and the relationship of the cytokine levels with disease severity, respectively. The levels of IL-6 were higher in the PTB (P = 0.0249) and pneumonia (P = 0.0047) groups compared to controls. Low to undetectable levels of TNF-α, IFN-γ, IL-2, IL-4, IL-10, and IL-17 were found in BALf, even after sample concentration using filtration columns and centrifugation. IL-6 levels measured in BALf could distinguish PTB patients or pneumonia patients from controls (AUC: 0.91, P = 0.002 and AUC: 0.86, P = 0.001, respectively), but not patients with PTB from those with pneumonia (AUC: 0.51, P = 0.86). IL-6 levels were related with the severity of PTB, as levels were higher in patients with higher radiological severity. These results confirm the importance of IL-6 in the immunopathology of smear-negative PTB.

Introduction

Pulmonary tuberculosis (PTB) remains a major public health concern due to its increasing incidence worldwide, developing drug resistance, and high mortality in low-income populations [1]. Tuberculosis is the world’s most lethal infectious disease, and one of the top ten causes of death globally [1].

Clinically, PTB can present as an asymptomatic, latent, or active infection, depending on the ability of the immune system to locally limit the spread of infection [2]. The immune response against Mycobacterium tuberculosis (MTB) is mediated by T lymphocytes (TL), which respond to phagocytic antigens presented by alveolar macrophages. In turn, the local secretion of interleukin (IL)-12p70 and other factors drives the differentiation of effector T helper 1 (Th1) cells in lung lymph nodes [3]. The Th1 response is characterized by the secretion of interferon gamma (IFN-γ), a cytokine that promotes the phagocytic response, production of reactive oxygen species (ROS), and antigen presentation, which contribute to the elimination of mycobacteria. TLs also secrete IL-2, which promotes their proliferation and chemotaxis [4]. In addition, macrophages, dendritic cells, and TLs secrete tumor necrosis factor alpha (TNF-α) and IL-6, which are implicated in the control of infection in its active phase [5]. TLs are involved in the recruitment of T and B lymphocytes to the pulmonary parenchyma for granuloma formation to further control and contain the infection. Thus, pulmonary MTB infection induces local cytokine expression [2], [5]. This immune response can be also associated with tissue damage in the pulmonary parenchyma due to necrosis or tissue fibrosis [6]. In vitro studies have reported increased levels of IFN-γ, IL-1β, TNF-α, and IL-6 after stimulating mononuclear cells with MTB [7]. Subsequent murine studies confirmed that TNF-α and IFN-γ play important roles in granuloma formation and thus in control of the infection [8].

Measurements taken from bronchoalveolar lavage fluid (BALf) better reflect the immune factors locally expressed in pulmonary tissues, as the immune response in the lung has shown been highly compartmentalized [9], [10]. Studies have explored the potential value of local immune response markers against MTB in its pathophysiology and diagnosis. Levels of IL-6 and TNF-α have been detected in BALf of patients with confirmed PTB, and these cytokines were preferentially expressed in the lung lesions [11]. Additionally, differential local expression of cytokines such as IL-6 and TNF-α discriminate patients with PTB from those with sarcoidosis, both diseases with characteristic granuloma formation [12]. Other models have used the secretion of IFN-γ, another immune marker released from T memory lymphocytes re-stimulated by MBT-associated peptides [13]. However, these assays are limited to measuring the memory response ex vivo, and do not permit the evaluation of the ongoing response that is naturally generated when the infection first occurs. Thus, they do not allow for differentiation between active and latent TB infections [14].

In this study, levels of seven locally secreted cytokines were analyzed in BALf from confirmed PTB patients, as well as from patients with other pulmonary pathologies and controls. All TB patients were smear-negative, as is the case with a large fraction of active PTB patients. This data was also evaluated with respect to diagnostic utility and its relation with radiological findings.