Lipoarabinomannan as a Point-of-Care Assay for Diagnosis of Tuberculosis: How Far Are We to Use It?

Abstract

Tuberculosis (TB) is still a severe public health problem; the current diagnostic tests have limitations that delay treatment onset. Lipoarabinomannan (LAM) is a glycolipid that is a component of the cell wall of the bacillus Mycobacterium tuberculosis, the etiologic agent of TB. This glycolipid is excreted as a soluble form in urine. The World Health Organization has established that the design of new TB diagnostic methods is one of the priorities within the EndTB Strategy. LAM has been suggested as a biomarker to develop diagnostic tests based on its identification in urine, and it is one of the most prominent candidates to develop point-of-care diagnostic test because urine samples can be easily collected. Moreover, LAM can regulate the immune response in the host and can be found in the serum of TB patients, where it probably affects a wide variety of host cell populations, consequently influencing the quality of both innate and adaptive immune responses during TB infection. Here, we revised the evidence that supports that LAM could be used as a tool for the development of new point-of-care tests for TB diagnosis, and we discussed the mechanisms that could contribute to the low sensitivity of diagnostic testing.

Keywords: immune response; immunoregulation; lipoarabinomannan; point-of-care assay; tuberculosis.

Figure 1

Recent approaches in tuberculosis research. Up to 2019, there were 162,356 cites in the tuberculosis (TB) field. 159,109 (98%) were focused on mechanisms of pathogenicity (purple dots) and generation of new drugs for treatments (green dots; left y-axis). Whereas 3,247 (1.3%) cites were related with studies exploiting the biomarkers field (red dots). Studies of LAM as a potential exclusive marker in the diagnosis of TB first appeared in 1998 and have increased ever since. The data was generated in Web of Science.

Figure 2

Schematic organization of LAM in the Mycobacterial cell envelope. (A) The cell envelope of Mtb is characterized by four basic regions, from the inner to the outer: plasma membrane, periplasm, cell wall, and capsule. The plasma membrane has anchored PIMs which are the basic structure for LM, LAM, and manLAM. Other assembling proteins, such as EmbC, PonA1, or LDT2, are required for the elongation process of LAM. (B) LAM from virulent Mtb is characterized by a four-domain structure: the phosphatidyl-myoinositol is anchored to the plasma membrane and is the foundation for the mannan backbone, followed by the arabinofuranose backbone and the capping motifs of extra-mannoses, methylthio-D-xylose, or arabinofuranosides. The figure was created in affinity designer.

Figure 3

LAM-induced regulation of the innate immune cells. (A) Interaction of LAM with TLR-2 in human alveolar epithelial cells, the classical pathway of TLR activation is mediated by MyD88 and TIRAP phosphorylation. Downstream, IRAK and TRAF molecules are phosphorylated to induce p38 and ERK1/2 activation, which are involved in pro-IL-37 production. IL-37 maturation requires caspase 1 (casp-1), IL-37 forms a complex with Smad3 that is translocated into the nucleus to suppress the inflammatory response. (B) LAM is inserted into the lactosylceramide (LacCer)-enriched lipid rafts or it can be recognized by the CD11b/CD18 complex. When LAM is inserted in the cell membrane, the recruitment of hematopoietic cell kinase (Hck) by Lyn kinase is inhibited. Moreover, LAM regulates the phosphorylation of vav and, consequently, there is a downregulation of ras-related protein (Rab27a), limiting the traffic of azurophilic granules by GTP hydrolysis blockade. Thus, in neutrophils, LAM favors a negative regulation characterized by low production of myeloperoxidase (MPO), NADPH oxidase, LAMP3, and impaired intracellular calcium flux. (C) CD11b/CD18 expressed on the cell surface of eosinophils interact with LAM, blocking the TLR-2 mediated cell activation and consequently the inflammatory cytokine (TNF, IL-1Ra, IL-6, and IL-8) production is limited. The figure was created in BioRender.

Figure 4

LAM-induced regulation of the adaptative immune cells. (A) B cells express TLR-2 that recognizes LAM and activates MyD88-dependent pathway. The TLR-2 signaling cascade activates the PI3K pathway and the MAP kinase (MAPKs) pathway, the latter induces MEK1 and JNK phosphorylation and activation of AP-1. While the PI3K pathway activates NF-kB, both pathways lead to IL-10 production. Moreover, TLR-2/LAM complex induces polyubiquitylation of IKK-γ (subunit of NF-κB essential modulator) by K63 activity and, consequently, the Th1 immune response is affected. (B) The LAM insertion into lipid rafts in T cells blocks the activation process since phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck), linker for activation of T cells (LAT), Zeta-chain-associated protein kinase 70 (ZAP70), CD3-epsilon, and CD3-delta chains is inhibited. LAM also induces low expression of Akt and mTOR, whereas the levels of genes related to anergy in lymphocytes (GRAIL) increases, together limiting the T cell activation. Finally, there are also reports suggesting that LAM induces the limited phosphorylation of zeta chains that sequester endosomal vesicles and favor low expression of Usp9x and Otub1 enzymes, consequently limiting the differentiation to inflammatory phenotypes. The figure was created in BioRender.