2.4.2. Non-Steroidal Anti-Inflammatory Drugs (NSAID) and Leukotriene Inhibitors

A series of mouse studies have shown beneficial effects of non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, diclofenac and ibuprofen when used alone or in combination with common antibiotics in *M. tuberculosis*-infected mice. The main mechanism of action seems to be inhibition of prostaglandin synthesis via inhibition of cyclooxygenase 1 and 2. Prostaglandins are known drivers of tissue damaging inflammation. It is important to note that diclofenac was shown to possess growth inhibitory effects on the bacterium itself in addition to its anti-inflammatory properties. The substances have been extensively discussed elsewhere [78]. NSAID Clinical trials initiated recently are listed in Table 1. Another category of anti-inflammatory drugs is represented by leukotriene receptor antagonists, such as zafirlukast, which was reported to have anti-mycobacterial activity in vitro and cause alterations in the transcription profile in *M. tuberculosis* [79]. The potential of these drugs in HDT against TB deserves deeper investigation given the role for leukotriene A(4) hydrolase (LTA4H) demonstrated by Tobin and colleagues in animal models of infection [80,81].

## 2.4.3. Necrosis

Necrotic host cell death is a highly dynamic research field increasingly linked to the release of pro-inflammatory cytokines. A better understanding of the mechanisms of *M. tuberculosis* induced cell death may provide additional starting points for HDTs. Most studies have been focusing on cell death in macrophages, however, necrosis of other cell types such as neutrophils seems to play a pivotal and additive role in *M. tuberculosis* pathogenicity. *M. tuberculosis* released by necrotic neutrophils displays improved survival and growth once phagocytosed by adjacent macrophages [82]. Neutrophil necrotic cell death is driven by reactive oxygen species (ROS) which can be abrogated by ROS inhibitors. In addition, ROS and nitric oxide (NO) have been found to show antimicrobial activity and to modulate neutrophil recruitment to the granuloma [83]. While ROS seems to increase cytokine production and to inhibit inflammasome activation, NO shows a regulatory effect on macrophages with increased expression of hypoxia-inducible factor 1 alpha (HIF-1α) and repression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [84,85]. A recent study highlights a role for ferroptotic cell death in TB. Ferroptosis is a type of regulated necrosis induced by accumulation of free iron and toxic lipid peroxides which seems to be mediated by decreased levels of glutathione peroxidase-4 (Gpx4) upon *M. tuberculosis* infection in vitro. Intraperitoneal treatment of *M. tuberculosis* infected mice with ferrostatin, a ferroptosis inhibitor resulted in reduced lung pathology and decreased bacterial load [86]. In addition to ferroptosis, efferocytosis (the physiological process of removing apoptotic cells by macrophages) is an anti-bacterial mechanism that seems to play a relevant role in TB as well [87]. Indeed, efferocytosis of apoptotic neutrophils was shown to improve control of *M. tuberculosis* in an in vitro model of HIV-*M. tuberculosis* macrophage co-infection [88,89].

## 2.4.4. TNF and TNF-Mediated Signaling

Further downstream of intracellular mediators or regulators of cell death, inflammation and cytokine release, there are more direct targets amenable to therapeutic interventions. These include the cytokines themselves. Biologicals targeting TNFα such as infliximab and adalimumab (monoclonal antibodies) or etanercept (TNF receptor fusion protein) may be used to limit exacerbated pathology and improve antibiotic activity. These substances are restricted for use in combination with antibiotics (adjuvant treatment) since TNFα is essential for protective immunity and granuloma integrity. Monotherapy with infliximab and other anti-TNF antibodies led to reactivation of latent TB [90]. However, when combined with anti-TB drugs, TNF neutralization enhanced *M. tuberculosis* clearance and reduced lung pathology [91]. A clinical study performed with adjuvant etanercept in patients

with pulmonary TB and HIV showed a trend towards improved outcome when the TNF blocker was added to the antibiotic regimen [92].

TNF signaling is also the main target of other HDT candidates such as thalidomide, phosphodiesterase inhibitors or Janus kinase (JAK) inhibitors. Thalidomide has potent anti-inflammatory properties which led to successful application of the drug in cases where anti-TB or HIV treatment triggered hyperinflammatory syndromes such as paradoxical reactions or immune reconstitution syndrome (IRIS). A general application for adjuvant treatment approaches may be hampered due to side effects as seen in a study performed with children suffering from TB meningitis [93]. Phosphodiesterase (PDE) inhibitors seem to be more promising for broad application in TB patients. PDEs degrade cyclic AMP (cAMP), a second messenger negatively regulating TNF levels. Decreased levels of cAMP stimulate TNFα secretion, thus making PDE inhibitors interesting HDT targets. Among the five PDE subtypes, targeting PDE4 seems to be the most promising option in TB with adjuvant use of inhibitors leading to improved outcome in several animal models [94,95]. A phase II clinical trial with the PDE4 inhibitor CC-11050 is ongoing (Table 1).
