Neutrophil-Mediated Immunopathology and Matrix Metalloproteinases in Central Nervous System - Tuberculosis

Abstract

Tuberculosis (TB) remains one of the leading infectious killers in the world, infecting approximately a quarter of the world's population with the causative organism Mycobacterium tuberculosis (M. tb). Central nervous system tuberculosis (CNS-TB) is the most severe form of TB, with high mortality and residual neurological sequelae even with effective TB treatment. In CNS-TB, recruited neutrophils infiltrate into the brain to carry out its antimicrobial functions of degranulation, phagocytosis and NETosis. However, neutrophils also mediate inflammation, tissue destruction and immunopathology in the CNS. Neutrophils release key mediators including matrix metalloproteinase (MMPs) which degrade brain extracellular matrix (ECM), tumor necrosis factor (TNF)-α which may drive inflammation, reactive oxygen species (ROS) that drive cellular necrosis and neutrophil extracellular traps (NETs), interacting with platelets to form thrombi that may lead to ischemic stroke. Host-directed therapies (HDTs) targeting these key mediators are potentially exciting, but currently remain of unproven effectiveness. This article reviews the key role of neutrophils and neutrophil-derived mediators in driving CNS-TB immunopathology.

Keywords: central nervous system tuberculosis; host-directed therapy; matrix metalloproteinases; neutrophils; stroke; tuberculosis.

Figure 1

The blood-brain-barrier structure and cellular composition. The BBB is a highly complex structure, made up of brain microvascular endothelial cells, pericytes, astrocytes and a non-cellular component – the basal lamina. Tight junctions between brain endothelial cells maintain the integrity and permeability of brain microvessels. Both the endothelial cells and pericytes are enclosed by, and contribute to the perivascular extracellular matrix (basal lamina 1, BL1), which is different in composition from the extracellular matrix of the glial end feet (BL2) bounding the brain parenchyma. Figure created with Biorender.com.

Figure 2

Neutrophil mediators in CNS-TB immunopathology. Neutrophils secrete cytokines including IL-1β to enhance neutrophil swarming and recruitment, and TNF-α to promote neutrophil necrosis, granuloma formation and thrombosis leading to ischemia stroke and brain tissue damage. Neutrophils also form NETs containing destructive enzymes which can damage the brain tissue. The release of MMP-9 degrades the extracellular matrix (ECM) resulting in BBB breakdown, leukocytes influx and eventually chronic cerebral inflammation. The ROS production also mediates BBB breakdown and drives neutrophil necrosis. 5-LO: 5-lipoxygenase; AA, arachidonic acid; COX-2, cyclooxygenase-2; CtG, cathepsin G; DC, dendritic cell; LTB4, leukotriene B4; MMP, matrix metalloproteinase; MPO, myeloperoxidase; M. tb, Mycobacterium tuberculosis; NE, neutrophil elastase; NETs, neutrophil extracellular traps; PGE2, prostaglandin E2; PR3, proteinase 3; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α. Illustration created with Biorender.com.

Figure 3

Cell fate of M. tb-infected neutrophils determine protection or pathology in TB. Left: Virulent M. tb induces neutrophil necrosis in a reactive oxygen species (ROS)- and ESAT-6 secretion system 1 (ESX-1)-dependent manner, resulting in the release of bioactive molecules that damage surrounding host tissue. Removal of necrotic neutrophils by macrophages drive them into necrosis with subsequent release of virulent M. tb to infect more host cells, thus resulting in a vicious cycle. Right: When the M.tb lacks a functional ESX-1 type 7 secretion system, or the production of ROS by neutrophils is inhibited, neutrophils undergo the default apoptosis instead. Cross-presentation of mycobacterial antigens to dendritic cells result in naïve T cell activation and differentiation into protective Th1 cells that produce interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). IFN-γ-induced nitric oxide (NO) production limits inflammation by inhibiting neutrophil recruitment, thereby reducing immunopathology. IL-1β, interleukin-1β; M. tb, Mycobacterium tuberculosis; NOS2, nitric oxide synthase 2. Illustration created with Biorender.com.

Figure 4

Three types of NETosis induced by different stimuli. (A) Stimuli such as phorbol 12-myristate 13-acetate (PMA) induce suicidal NETosis after activating NADPH oxidase (NOX) to produce ROS. (B) Vital NETosis is induced within minutes by pathogens such as Staphylococcus aureus and Escherichia coli, through complement receptors (CR) and Toll-like receptors 2 and 4 (TLR2 and TLR4). This induces protein arginine deiminase 4 (PAD4) activation without the need for oxidants. Citrullination of histones allow chromatin to undergo decondensation and be dispersed in the form of NETs. NE and MPO translocate into the nucleus to promote further unfolding of chromatin. (C) ApoNETosis is induced by high-dose ultraviolet (UV) irradiation. This induces large amounts of mitochondrial ROS (mROS), caspase cascade activation, p38 activation, transcriptional firing and NETosis. Under apoNETosis conditions, although both apoptosis and NOX-independent NETosis occur simultaneously, NETotic events predominate apoptotic events. Unlike the other two types of NETosis, PAD4 is not activated and histones are not citrullinated. In addition, nuclear blebbing does not occur unlike classical apoptosis. Casp-3, caspase 3; CitH3, citrullinated H3; Cyt c, cytochrome c; MPO, myeloperoxidase; NE, neutrophil elastase; NETs, neutrophil extracellular traps; ROS, reactive oxygen species. Illustration created with Biorender.com.

Figure 5

Gelatinases MMP-2 and -9, collagenase MMP-1 and stromelysin MMP-3 contribute to BBB breakdown and brain tissue damage in CNS-TB. M. tb-infected monocytes interact with astrocytes and microglia in the brain to induce their secretion of MMP-9, -1 and -3 respectively, a process that is driven by pro-inflammatory mediators TNF-α and IL-1β. ① These MMPs degrade TJPs occludin and claudin-5 and ECM proteins of the basement membrane including type IV collagen, laminin, nidogen, perlecan and fibronectin. ② This BBB breakdown drives influx of plasma resulting in vasogenic edema and facilitates further influx of circulating inflammatory cells such as monocytes and neutrophils into the brain. ③ MMPs also degrade proteoglycans (aggrecan, versican, brevican), proteoglycan link proteins and tenascins found within the ECM of the brain parenchyma, thus resulting in ④ brain tissue destruction adding to the cerebral inflammatory response. MMP-9 secreted from neutrophils further compromise the BBB and exacerbate tissue damage. Illustration created with Biorender.com.

Figure 6

MMP-1, -2, -3, and -9 are expressed in human CNS-TB granulomas. Infiltrated mononuclear cells (MNCs) in the meninges were immunoreactive for MMP-2 and -9. In particular, MMP-9 was expressed in the perivascular leukocytes at necrotic vessel, contributing to BBB disruption (185). In the granuloma, MMP-9 was highly expressed around the area of caseous necrosis, unopposed by TIMP-1 (194). Astrocytes are the main CNS cellular source of MMP-9, compared with other sources including MNCs, neutrophils and microglia (184). Microglia-derived MMP-1 and -3 were found decreasing towards fibrosis peri-granuloma region (176). Illustration created with Biorender.com.

Figure 7

Neutrophil extracellular traps (NETs) promote thrombosis in brain microvessels. (A) Platelets interact with C3b and histones on NETs to stimulate the secretion of polyP which activates the extrinsic coagulation pathway. (B) Platelet-derived HMGB1 induces NETosis. (C) Multiple components of NETs induce coagulation either directly or by inhibiting the extrinsic coagulation pathway inhibitor. (D) NE is able to generate thrombin-derived immune modulatory peptides. (E) Fibrin fibres strengthened by NETs immobilize M. tb and are less prone to degradation by plasmin. Illustration created with Biorender.com.

Figure 8

Host-directed therapy in CNS-TB. Pro-coagulant thromboxane A2 causes thrombosis and subsequent formation of brain infarcts. It also increases the recruitment of neutrophils, which releases TNF-α and MMP-9, two major mediators contributing to brain immunopathology of TBM, including brain infarcts, hydrocephalus and tuberculoma. Other clinical features of TBM are cerebral salt wasting, seizures and raised intracranial pressure. Severe complications of TBM are stroke, neurological impairment and death. Aspirin is used to inhibit thromboxane A2, prevent brain infarcts and reduce stroke and mortality (245, 246). Thalidomide is TNF-α antagonist functions to reduce thrombosis and brain immunopathology in TBM (11). Corticosteroid dexamethasone can inhibit MMP-9 secretion and further reduce the consequent brain immunopathology (10, 247), while fludrocortisone can improve cerebral salt wasting (248). The use of steroid may therefore reduce neurological impairment and death. The anti-seizure phenytoin is taken to control seizures in TBM, while the diuretics acetazolamide and furosemide can be used to manage raised intracranial pressure in TBM (249). Illustration created with Biorender.com.