N-Acetylcysteine : An Old Drug With Variable Anti-Influenza Properties

N-acetylcysteine (NAC) is a mucolytic drug commonly used as an adjuvant therapy in patients with respiratory conditions associated with excessive mucus production. NAC also has anti-oxidant activities that proved useful in the management of oxidative stress. These anti-oxidant capacities of NAC are mostly indirect, via a pro-glutathione effect where NAC provides L-cysteine residues required for glutathione synthesis. This activity is thought to be the basis for the protective effect of NAC administration in influenza patients and in mouse models of the disease. NAC was shown to limit lung inflammation, damage associated with the virus, and viral growth, at least in vitro. However, the anti-viral activity was highly variable depending on the influenza A strain. The reasons for these inter-strain variations are still unknown, but might be related to the level of nuclear factor-κB activation required for the virus to achieve its infectious cycle.


Introduction
N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine (molecular formula: HSCH2CH(NHCOCH3)CO2H; Figure 1A), has been used for decades as an adjuvant therapy for several respiratory conditions, thanks to its mucolytic properties (1).NAC also has anti-oxidant and proglutathione (pro-GSH) activities that support its therapeutic use in patients with oxidative stress (2).Its main clinical applications include the treatment of acetaminophen (paracetamol) overdose, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, contrast-induced nephropathy and influenza pneumonia (3,4).NAC was further described as a chelating agent, notably of methylmercury, a glutamate precursor and a methyl donor allowing the conversion of homocysteine to methionine (5)(6)(7).As a consequence, NAC administration has been Key points  N-acetylcysteine (NAC) has been reported to reduce symptoms of influenza. Administration of NAC is thought to have contributed to the successful treatment of patients infected with H1N1. Conversely, some studies have shown a null effect of NAC on certain strains of influenza virus. The effect of NAC on influenza A virus is strain dependent. Susceptibility of influenza viruses to NAC cannot be considered as universal. Anti-influenza property of NAC should be interpreted in terms of strains.
Although NAC has been considered as extremely safe, administration of acetylcysteine has been associated with several adverse reactions (9).The most significant adverse reaction of NAC resembles anaphylaxis and has been classified as anaphylactoid reaction.Less common reactions that were reported consisted of electrocardiogram abnormalities, status epilepticus and a syndrome similar to serum sickness (9).

NAC: a mucolytic agent used since the early 1960s
The first clinical application of NAC was related to its mucolytic properties (1).NAC is a thiol compound, with a free sulfhydryl group (Figure 1A).Thanks to its sulfhydryl moiety; NAC is able to break down disulfide bonds in mucus glycoproteins, which accounts for its "mucolytic" capacities (23,24) (Figure 1B).These reactions of thioldisulfide interchange may take place spontaneously or be catalyzed by thiol transferases (25,26).Mostly used as an adjuvant therapy in patients with cystic fibrosis and chronic obstructive pulmonary diseases (24,27), NAC has also been suggested as a supportive treatment for influenza pneumonia (11).Recent evidence suggests that a large part of the positive impact of NAC in the treatment of these conditions is actually related to direct and indirect anti-oxidant effects (23).

Anti-oxidant and immunomodulatory activities
A large proportion of clinical signs and lesions associated with respiratory conditions as diverse as asthma, cystic fibrosis or viral pneumonia is related to the inflammatory reaction (23).In particular, inflammation is critical in the pathogenesis of lower respiratory tract viral infections (28,29).The main effectors of the tissue damage associated with influenza infection are cytokines (the so-called cytokinic storm) and reactive oxygen species (ROS), mostly associated with neutrophils (28).NAC has emerged as a potent and safe anti-oxidant and immunomodulatory drug in the case of influenza pneumonia (11,(13)(14)(15)(16)(17)(18)(19)(20)22).
The anti-oxidant effect of NAC is thought to be dual: direct and indirect (23).NAC, as a thiol compound, is a reducing agent and thus endowed with anti-oxidant properties.However, this direct effect is believed to be negligible at classical in vivo concentrations when compared to its indirect, pro-GSH activity (23).GSH (γglutamyl-cysteinyl-glycine) is a highly efficient intracellular tripeptide antioxidant, which limits the impact of oxidative stress on lipids, proteins and nucleic acids.NAC, after deacetylation, provides reduced L-cysteine, which is required for the synthesis of GSH (23) (Figure 2).The immunomodulatory properties of NAC, observed both in vitro and in vivo, are thought to be related to the inhibition of the activation of oxidantsensitive pathways such as those involving nuclear factor (NF)-κB and mitogen-activated protein kinase p38 (14,19).Levels of cytokines/chemokines after influenza infection were significantly reduced by NAC treatment in vivo (14,19,22).NAC treatment also decreased the macrophages, lymphocytes and neutrophils infiltration and myeloperoxidase activity in infected lungs (14,22).Furthermore, NAC was shown to increase the proliferation of influenza-specific lymphocytes and the effector function of cytotoxic lymphocytes (10).
The interplay between ROS and the NF-κB pathway is complex (30).ROS can modulate the activation of the NF-κB pathway by acting on several factors, located in the cell cytoplasm or in the nucleus, resulting in an either activation or inhibition of the pathway (30).Although out of the scope of the present review, it is worth mentioning here that most of the pro-NF-κB activities of ROS are thought to be related to effects on cytoplasmic factors, mainly the activation of IκB (inhibitor of κB) kinases or alternative phosphorylation of IκBα (30).This is the most likely mechanism by which NAC reduces the activation of NF-κB pathway.

Anti-viral properties and variations between viral strains
Several studies addressed the question about the anti-viral activity of NAC against influenza A virus (Table 1).A dosedependent inhibition of highly pathogenic H5N1 influenza virus in vitro growth by NAC was demonstrated using two different strains of the virus: A/Thailand/1(Kan-1)/04 and A/Vietnam/1203/04 (14).The effect was maximal (48.9-fold reduction) at 24 hours post infection, with a 15 mM NAC concentration.
For the A/Port Chalmers/1/72 (H3N2) influenza strain, NAC treatment (10 mM) reduced the viral titers measured in vitro at 48 hours post infection about 8-fold when compared with untreated controls (19).
In similar conditions, the A/swine/Iowa/4/1976 (H1N1) strain appeared as more resistant to the inhibition by NAC, with a ~5.6-fold reduction of the viral titers at the highest NAC concentration (2.5 mg/ml or 15.3 mM) used by the authors (12).Moreover, with the same viral strain, administration of NAC at a high dosage (100 mg/kg per day) failed to protect mice after viral challenge (12).In similar experiments using the A/Puerto Rico/8/1934 (H1N1) strain, NAC alone only provided a very limited level of protection, but was much more efficient when combined with ribavirin or oseltamivir (13,15).The in vivo protective effect of NAC was more significant in a mouse model infected by the A/swine/HeBei/012/2008 (H9N2), but unfortunately the effect on viral growth in vitro has not been measured (22).ND: not determined.

References
Overall, the inhibition of viral growth and the in vivo protective effects of NAC appear as more pronounced for highly pathogenic strains (12,14).The lesions associated with the infection of mammals by a highly pathogenic versus a low pathogenic influenza A virus, even with a similar lethality, are extremely different (31).In both cases, diffuse alveolar damage is observed, but the typical target in the pathogenesis of highly pathogenic strains is the endothelium, leading to massive pulmonary edema and hemorrhages.In comparison, infections by low pathogenic strains are associated with a dense leukocytic infiltration of the lung parenchyma, which compromises gas exchanges, but with minimal damage to the alveolar epithelium (31).The endotheliotropism of highly pathogenic strains is believed to be linked to a differential affinity of the viral hemagglutinin for endothelial cells in vitro (32,33).But it appears that the activation of endothelial cells, more than their infection, is critical in the pathogenesis of H5N1 highly pathogenic influenza (33).Furthermore, the activation of endothelial cells is central in the development of the so-called cytokinic storm, typical of pathogenic influenza strains (34).In particular, a strong activation of the NF-κB pathway has been described as a critical step for the expression of genes typically overexpressed after H5N1 influenza infection, including cytokines and chemokines, with a much weaker activation in the case of low pathogenic strains (35).Moreover, pharmacological inhibition of the NF-κB pathway proved to be an efficient method to protect against lethal influenza challenges (14, 36).As a consequence, most of the anti-viral activity of NAC against influenza A virus is likely associated with its anti-NF-κB properties (14), together with the inhibition of the MAPK p38 pathway.The differences in terms of NF-κB activation between highly pathogenic and low pathogenic influenza strains (35) might be the explanation for the variable effects of NAC treatment on viral growth and host resistance between strains.
Through the inhibition of NF-κB activation, NAC likely has an additional, indirect effect on influenza growth via a downregulation of α2,6-linked sialic acid receptors expression on target cells, as demonstrated for the closely related drug carbocistein (37).Interestingly, in the same study, carbocistein did not affect the expression of α2,3-linked sialic acid receptors, which raises questions about the underlying mechanism (37).The potential impact of this observation on the variations in susceptibility between influenza strains and NAC treatment is unclear because highly pathogenic avian viruses, such as H5N1 strains, mostly target α2,3-linked sialic acid receptors, whose expression appears as unaffected by carbocistein and, supposedly, NAC (37).

Current clinical use of NAC in influenza patients and perspectives
High dose of NAC has been suggested and used since several years as an adjuvant treatment for influenza pneumonia in human patients (4,17,18,20).NAC was shown to relieve influenza symptomatology and to improve cell-mediated immunity (11).This protective activity was demonstrated for human H1N1 influenza strains, but it is likely that, as detailed above for mouse models, the therapeutic efficacy of NAC is strain dependent.Additional research is clearly needed to determine which human influenza strains are most responsive to NAC treatment to apply this medication only when relevant.Besides, even if NAC is considered a very safe drug, some severe adverse reactions have been reported (9), which raise the question of the risks of using high dose of the drug in patients infected by unresponsive influenza strains.Finally, because most of the anti-influenza activity of NAC is thought to be linked to its anti-NF-κB properties (14), it would be interesting to compare the clinical efficacy of NAC with that of more specific anti-NF-κB compounds.

Conclusions
NAC has been shown to be an interesting adjuvant therapy in the management of influenza patients, both through experimental infections of mouse models and through clinical evidence from the field.This is reinforced by the in vitro antiinfluenza effect of the drug reported by several studies.In spite of this, the level of clinical protection of NAC treatment alone appeared as weak or null in some models with a variation in its efficacy depending on the infecting viral strain.These differences might be related to the anti-NF-κB properties of NAC and the variations in terms of NF-κB activation between influenza strains.Additional effects of NAC, notably on the expression of some cellular receptors to the virus, might also be involved.Additional research is clearly needed to fully understand the mechanisms underlying the anti-viral effects of NAC and closely related molecules.

Figure 1 .
Figure 1.Structural formula of N-acetylcysteine (NAC; A).Disulfide bonds in proteins (P) can be disrupted by NAC in a two-step thiol-disulfide interchange reaction (B).Adapted from Samuni et al (26).

Figure 2 .
Figure 2. N-acetylcysteine (NAC) as a cysteine donor for glutathione synthesis.Most of the anti-oxidant activity of NAC is thought to be related to its pro-glutathione (GSH) properties.NAC serves as a donor of cysteine residues that are essential to GSH synthesis.Adapted Rushworth and Megson (23).