Functional variations of the TLR4 gene in association with chronic obstructive pulmonary disease and pulmonary tuberculosis

Both chronic obstructive pulmonary disease (COPD) and tuberculosis (TB) primarily affect the lungs and are major causes of morbidity and mortality worldwide [1]. COPD is characterized by persistent airflow limitation because of various combinations of obstructive bronchiolitis and emphysema [2, 3]. The airflow limitation is usually not completely reversible and is associated with an abnormal inflammatory response of the lungs exposed to noxious particles and gases [4]. In a current nationwide, cross-sectional survey, the estimated overall prevalence of COPD in China was 13.6% among adults over 40 years old [5]. The etiology of COPD is complicated, and tobacco smoking is considered one of the foremost risk factors [2‐4, 6‐8]. Other factors, such as pulmonary tuberculosis (PTB), air pollution and low body mass index (BMI), may also contribute to COPD [6, 7]. TB is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB) infection, of which PTB is the most common type [9]. In 2017, there was an estimation of 10 million new cases and 1.3 million deaths due to TB worldwide, and the number of new TB cases in China was approximately 889 thousand, making it one of the top 30 countries with high TB burden (www.​who.​int).

COPD and TB have common risk factors, such as tobacco smoking, malnutrition, low socioeconomic status and dysregulation of host defense functions [1, 10]. Both COPD and TB are characterized by an abnormal innate immune response of alveolar macrophages exposed to both cigarette smoke and Toll-like receptor (TLR) stimulation [11, 12]. Since the first discovery of TLR4 in humans in late 1997, 13 types of TLRs (TLR1-TLR13) have been described in mammals [13]. As an important pattern recognition receptor in the natural immune system, TLR4 serves to discern pathogen-associated molecular pattern (PAMP) molecules or endogenous damage-associated molecular pattern (DAMP) molecules [14, 15]. TLR4 can trigger inflammatory responses by inducing the synthesis and release of proinflammatory cytokines and chemokines. Single nucleotide polymorphisms (SNPs) among various TLR genes have been identified, and their association with susceptibility/resistance to certain infections and other inflammatory diseases has been reported [13]. The interrelationship between COPD and TB is very complex [16]. Growing evidence appears to point towards a bidirectional relationship between these two lung diseases where each may act as an independent risk factor for the other [17]. It appears that the susceptibility of an individual to develop COPD and active TB involves a complex interaction between genetic and environmental factors [16].

Previous studies have reported genetic polymorphisms of TLR4 in association with the risk of COPD and PTB [18, 19], but these two diseases were analyzed independently, and the function of genetic variations was not explored in these studies. In the present study, we compared the effects of genetic variations of the TLR4 gene on the susceptibility to COPD and PTB in a Chinese Han population. Furthermore, we constructed dual-luciferase reporter vectors expressing different genotypes and transfected them into human HEK 293 T cell lines to analyze the potential function of SNPs on the promoter activity of the TLR4 gene.

COPD is emerging as the third largest cause of human mortality worldwide, and there is growing evidence for comorbidity between COPD and TB, the leading cause of death globally due to respiratory infection [17]. COPD and TB share a number of common risk factors, including innate immunity-related genetic factors. In the present study, we compared the role of genetic variations of the TLR4 gene in the susceptibility to COPD and PTB. We observed that the genetic polymorphisms of rs10759932 and rs2737190 were significantly associated with both COPD and PTB, but with inverse effects. Dual-luciferase reporter vectors suggested the potential function of these two SNPs regulating the transcription activity of the TLR4 gene.

TLR4 is a pattern recognition receptor that participates in both innate and adaptive immunity. TLR4 is located on the surface of monocytes and macrophages and transmits activation signals after lipopolysaccharide (LPS) recognition, induces the expression of proinflammatory cytokines, such as IL-1, myeloid differentiation factor 88 (MyD88) and TNF-α, and forms receptor complexes, resulting in the activation of NF-κB and its translocation into the nucleus, which mediates inflammatory responses and adaptive immune responses [21]. TLR4 plays an important role in lung structure maintenance and smoking-induced emphysema and stimulates low-grade activation of the innate immune system, which is required for lung structural stability [22‐24]. Previous studies have demonstrated that the expression of TLR4 on the surface of peripheral blood mononuclear cells and the concentrations of TNF-α, IL-6 and IL-8 in the serum of COPD patients were all significantly increased and positively correlated [25‐27]. TLR4 influences the expression of inflammatory cytokines and chemokines, such as TNF-α, IL-1, IL-6 and IL-8, and these cytokines and chemokines interact with each other, which in turn regulate the expression of TLR4 in the airway of patients with COPD [25‐27]. Speletas et al. investigated common TLR polymorphisms (TLR2-R753Q, TLR4-D299G and TLR4-T399I) in a group of heavy smokers (> 20 pack years). The presence of the TLR4-T399I polymorphism was associated with a 2.4-fold increased risk for COPD development, but not with the disease stage or frequency of exacerbations [28]. A study in Germany observed that the TLR4-D299G polymorphism was related to COPD [29]. In our study, five SNPs in the TLR4 gene were all significantly associated with the risk of COPD, indicating its critical role in the development of COPD in the Chinese population.

It is estimated that approximately one third of the global population has been infected with MTB, but only 5–10% of these latently infected individuals eventually develop active TB, which may be attributed to different individual immunity [30, 31]. When MTB invades the host, TLR4 can recognize LPS, triggering the adaptor molecule MyD88 and other signaling pathways and initiating innate host defense against MTB [32, 33]. A study with mice found that mice with TLR4 were all alive at week 15 after intranasal inoculation with an MTB suspension, whereas 7 of 12 TLR4-deficient mice died [34]. The bacterial load of TLR4-deficient mice was 3-fold higher than that of mice with TLR4, and the levels of cytokines in TLR4-deficient mice were significantly decreased [34]. In the present study, we genotyped five functional SNPs in the TLR4 gene, and only rs10759932 and rs2737190 were associated with PTB. Moreover, the effect of these two SNPs on the risk of COPD and PTB was inverse. This may be attributed to the dual role of the inflammatory response. On the one hand, inflammatory mediators directly or indirectly cause damage to tissues and cells. For example, the TLR4 signaling pathway mediates inflammation after ischemia-reperfusion and leads to tissue damage [35, 36]. On the other hand, it dilutes, kills and surrounds damage factors through inflammatory hyperemia and exudation reactions, and repairs and heals the damaged tissue through the regeneration of parenchymal and mesenchymal cells. Jiang et al. reported that the interaction between the endogenous matrix component hyaluronic acid and TLR4 can trigger an inflammatory response, maintain structural cellular integrity, and promote the recovery of tissue damage [37, 38].

In this study, we constructed dual-luciferase reporter vectors expressing different genotypes and transfected them into the human HEK 293 T cell line to explore the effects of SNPs on the TLR4 gene. Our results showed that rs10759932-C (vs. T) and rs2737190-G (vs. A) can decrease the promoter activity of the TLR4 gene. The mutations of these two loci not only weaken the capacity of the immune system to recognize MTB but also reduce the production and release of proinflammatory cytokines, resulting in an increased risk of PTB. In COPD, the excessive expression of proinflammatory cytokines damages lung tissue and cells beyond the tolerance of the body during the chronic inflammatory process, leading to various combinations of bronchitis, emphysema and pulmonary fibrosis, which in turn aggravate the condition of COPD patients. This may partly explain the reduced risk of COPD by mutations in these two loci.

There were several limitations in this study. First, we only analyzed five SNPs in the TLR4 gene, and other immune-related genes were not considered. Second, although the dual-luciferase reporter vectors were used to estimate the altered transcriptional activity of the TLR4 gene due to rs10759932-C and rs2737190-G, the affected transcription factors were unclear. The altered TLR4 protein and downstream cytokines need to be measured in future studies. Third, there is a complex interrelationship between COPD and PTB, one of which can serve as an independent risk factor for the other [16, 17]. We investigated the history of TB among COPD cases. However, we do not know the proportion of COPD among PTB cases because respiratory function tests were not routinely performed for suspected TB cases. Therefore, the concomitance of both diseases may have been an important confounding factor when we analyzed the effects of TLR4 SNPs. Fourth, we only explored the functional effects of TLR4 rs10759932 (− 1309 T/C) and rs2737190 (− 2272 A/G), which were related to both COPD and PTB. The effects of the other three SNPs on the transcriptional activity of TLR4 were unclear.