Confronting the next influenza pandemic with anti-inflammatory and immunomodulatory agents: why they are needed and how they might work
Recent studies suggest the host response determines the outcome to severe influenza virus infection
Given the overwhelming need for alternatives to vaccination and antiviral treatment, agents that improve the host response to influenza virus infection must be considered.11,14,15,31 Although most influenza scientists doubt this approach will work, several studies suggest it might be effective.
In a study of mice massively infected with H5N1 influenza virus (1000 LD50), treatment with zanamivir begun 48 hours after infection reduced lung virus titers but led to little improvement in survival.32 However, when two immunomodulatory agents (celecoxib and mesalazine) were added, virus titers remained much the same but survival improved significantly. Unfortunately, the investigators failed to include a group of mice that were treated with celecoxib and mesalazine alone. If they had measured survival rates and virus titers in the two groups (two immunomodulators with and without an antiviral agent), they could have determined whether the antiviral agent was necessary for improving survival.
A commentary that accompanied this study emphasized that co-administration of the two anti-inflammatory and immunomodulatory agents along with an antiviral agent was essential.33 Improved survival was ascribed to inhibition of cyclooxygenase (COX)-2, but it is unlikely that it was due to COX-2 inhibition alone. The cell signaling pathways involved in the regulation of COX-2 expression are complex.34 In other models of acute lung injury, COX-2 inhibition actually impairs resolution of pulmonary inflammation, probably because it prevents the up-regulation of pro-resolution factors such as lipoxin A4.35 Moreover, mesalamine is not simply a COX-2 inhibitor; it is primarily a peroxisome proliferator activator receptor (PPAR)γ agonist36 (see below).
Another report has compared survival rates of mice sequentially infected with influenza virus and S. pneumoniae who were treated with either a cell wall-active antibiotic (ampicillin) or one of two macrolides known to inhibit bacterial protein synthesis (clindamycin or azithromycin).37 Ampicillin-treated mice had lower survival rates, presumably because of increased inflammation caused by the lysis of bacterial cell walls. The better survival of macrolide-treated animals 'appeared to be mediated by decreased inflammation as manifested by lower levels of inflammatory cells and pro-inflammatory cytokines.37 However, the greater survival of macrolide-treated mice was probably due to more than inhibition of pneumococcal protein synthesis. Macrolides have well-documented anti-inflammatory and immunomodulatory effects that improve the host response to a wide variety of non-infectious as well as infectious conditions.38–41
A masterly study by Imai et al. has defined common set of major cell signaling events in acute lung injury due to different causes.42 It is well known that in mice, intra-tracheal instillation of acid or lipopolysaccharide (LPS) reliably induces severe acute lung injury. Imai et al. showed that both of these insults activated pulmonary macrophages. This led to oxidative stress and the formation of reactive oxygen species (ROS). ROS in turn generated large amounts of oxidized phospholipids (OxPLs) derived mostly from cellular debris. OxPLs then triggered the production of pro-inflammatory cytokines [e.g., interleukin (IL)-6] via a TLR4/TRIF/TRAF6/nuclear factor (NF)-kappaB signaling cascade (Figure 2).
What is important about this study is that the same degree of acute lung injury and the same cell signaling cascade was observed following intra-tracheal instillation of inactivated (not live) H5N1 virus.42 The same pattern was seen in human peripheral blood mononuclear cells when they were exposed to inactivated H5N1 virus. The pulmonary lesions in mice were histologically identical to those seen in fatal cases of H5N1 influenza. None of these changes was seen with inactivated H1N1 virus.
Among the many factors contributing to pulmonary defenses, several investigators have shown that heme-oxygenase (HO)-1 directly affects the initiation of the TLR4 signaling cascade described above.43–47 HO-1 is a stress responsive enzyme that degrades heme to carbon monoxide, biliverdin and iron. The location of TLRs within cells determines their signaling effects.46 Carbon monoxide derived from endogenous HO-1 activity limits ROS-induced TLR4 signaling by inhibiting the relocalization of TLR4 from the cytoplasm to lipid rafts on macrophage cell membranes.43 This is one of several ways in which HO-1 contributes to pulmonary host defenses and improves survival in a wide range of pathologic disorders.47
The host response to acute lung injury due to any cause is far too complex to be captured in a single study.48,49 The same can be said about the host response in sepsis.50–53 Early and late (e.g. high molecular group box 1 (HMGB1)) mediators of inflammation, the balance between inflammatory and anti-inflammatory (e.g., lipoxin A4) factors, the contributions of innate and adaptive immunity (especially late immunosuppression50,53) (see below), disorders of the complement and coagulation systems (and their interactions), autonomic system involvement (cholinergic anti-inflammatory pathway), endocrine and metabolic dysfunction and disturbances in energy homeostasis all affect outcome in these conditions. Maintaining or restoring a balanced host response seems to be the key to recovery.
Little is known about the molecular events that characterize the overall response of influenza patients who progress to multi-organ failure and death. Nonetheless, no virus replication occurred in the study of Imai et al.,42 so under these experimental conditions, antiviral treatment would not have affected the outcome.* These findings call into question the claim that antiviral agents are essential if treatment of H5N1 or any other severe influenza virus infection is to be effective.19 They also suggest that agents capable of interrupting one or more of the steps in the cell signaling cascade demonstrated by Imai et al. might reduce the severity of the acute lung injury seen in H5N1 and pandemic influenza, and in doing so prevent or reverse multi-organ failure and improve survival.
Anti-inflammatory and immunomodulatory agents might be effective for treatment and prophylaxis of H5N1 and pandemic influenza
Several lines of experimental and clinical evidence suggest that three classes of drugs – statins, PPARα agonists (fibrates), and PPARγ agonists (glitazones) might individually or in combination prevent H5N1-associated acute lung injury [reviewed in Refs (11,14,15,31)]. Each of these groups of agents (as well as several others) has been shown to inhibit the cell signaling pathways set in motion by inactivated H5N1 virus (Table 1; DS Fedson, unpublished data).42
Statins [hydroxymethyl glutaryl - coenzyme A (HMG- CoA) reductase inhibitors] are taken every day by millions of patients with cardiovascular diseases to lower their low density lipoprotein (LDL) cholesterol levels. These agents also have anti-inflammatory (pleiotropic) effects,54 and investigators are studying their use in treating patients with sepsis55 and pneumonia. Several retrospective studies have suggested that prescriptions for statins are associated with an approximately 50% reduction in pneumonia hospitalizations and deaths [reviewed in Refs (14,15)]. A preliminary report of a randomized controlled trial of statin treatment in 67 ICU pneumonia patients showed that hospital mortality was reduced by 51%.56 Thus far, no reports have been published showing beneficial effects of statins in cell culture or animal influenza virus infections, although lack of benefit has been mentioned in one report.5
Investigators often observe a lack of correlation between the results of treatments in experimental animals and in humans.49,57 The evidence for statin benefit in humans with sepsis and pneumonia justifies further studies, including randomized controlled trials. These studies must pay close attention to the pharmacokinetics of each agent; early evidence indicates that acute blood levels of atorvastatin might be much higher in patients with severe acute illness than they are in normal subjects.58 If statins prove to be effective against pneumonia, they might be similarly effective against H5N1 and pandemic influenza.
Many investigators believe that fibrates – PPARα agonists that lower cholesterol levels – and glitazones – PPARγ agonists used to increase insulin sensitivity in diabetic patients – could also be used to treat acute lung injury.14,15,59 Like statins, these agents have anti-inflammatory and immunomodulatory activities.59,60 Moreover, there is considerable molecular cross-talk between statins, fibrates, and glitazones, and the pleiotropic effects of statins are achieved because of their interactions with PPARs.61,62 In experimental studies, the cell signaling effects of statins and PPAR agonists (both α and γ) can be additive.63,64 Likewise, in patients with cardiovascular diseases, the effects of therapy on biomarkers of disease are greater with combination than with single agent treatment. Given many years of use in clinical practice, the safety profile for each group of agents is well established.14
An important study published in 2007 showed that in H2N2 influenza virus-infected mice, treatment with a common PPARα agonist (gemfibrozil) reduced mortality by 54%.65 Some have criticized this study because pulmonary virus titers were not measured. Nonetheless, it was structured like a randomized controlled trial of an acute treatment; gemfibrozil was started 4 days following infection when mice were beginning to show signs of clinical illness. Moreover, the investigators used an unambiguous end-point (death) and like a clinical trial they chose a sample size (96) that gave them statistically significant results.
More recently, Aldridge et al. studied the effects of treatment with pioglitazone (a PPARγ agonist) in influenza-infected mice.66 They found that a subset of dendritic cells (DCs) known as tumor necrosis factor (TNF)α/inducible nitric oxide synthase DCs (TipDCs) accumulated with high frequency in the lungs of mice infected with highly pathogenic PR8 virus. TipDCs are known to recruit CCR2-positive mononuclear cells from the bone marrow and traffic them to sites of pulmonary infection. CCR2-deficient mice are generally more susceptible to non-viral infections, but CCR2-positive monocyte-derived cells have been shown to be a major cause of the immunopathology of influenza.67 Aldridge et al. speculated that pioglitazone suppression of CCL2 (the pro-inflammatory ligand for CCR2) would reduce the number of CCR2-positive mononuclear cells and increase protection. The results showed that with 3 days of pre-treatment, mortality fell from 92% to 50%. However, they also found that TipDCs increased the frequency of virus-specific CD8+ T-cells in the later stages of infection. As CD8+ T-cells are critical for influenza virus clearance, TipDCs appeared to induce a protective response. Yet, protection was not reflected in pulmonary virus titers; they were the same in control and pioglitazone-treated animals. Thus, although pioglitazone was able to 'tip the balance' in favor of protection,68 it must have done so through mechanisms that were independent of its effects on virus replication and clearance.
The study by Aldridge et al. was not designed to test whether pioglitazone could be used to treat an already established infection, unlike the gemfibrozil study discussed above.65 As such, the findings are similar to those obtained in the observational studies that have shown that patients already taking statins have reduced rates of pneumonia hospitalization and death (i.e., both act as prophylactic agents).14,15 Interestingly, if reducing the number of CCR2-positive mononuclear cells has any role to play in recovery from influenza, statins are known to suppress CCR2 gene expression and monocyte recruitment,69,70 and might have effects similar to those seen with pioglitazone.
Several other agents with anti-inflammatory and immunomodulatory or even antiviral activities should be considered for treatment and prophylaxis of H5N1 and pandemic influenza [reviewed in Ref. (14)]. For example, in cell culture chloroquine, a classic anti-malaria drug, impairs lysosomal acidification, preventing the release into the cytoplasm of viral nucleic acid from H3N2 and H1N1 but not H5N1 influenza viruses [discussed in Ref. (14)]. The many effects of catechins (found in green tea) and curcumin (turmeric in curry) on inflammation and the host response suggest that they too might be beneficial against influenza.
A potentially important but overlooked compound is resveratrol, a commonly available polyphenol found naturally in dark grapes and red wine. In a study of influenza PR8-infected mice, resveratrol treatment inhibited virus replication and reduced mortality by half.71 Resveratrol has statin-like effects on HMG-CoA,72 activates PPARα73 and PPARγ,74 and synergizes with statins in protecting against experimental myocardial infarction.75 The effects of resveratrol on ROS, TLR4, NF-kappaB, pro-inflammatory cytokines (e.g., TNFα, IL-6), and HO-1 are the same as those of statins, fibrates, and glitazones (see Table 1).76–78 In experimental Serratia marcescens pneumonia in rats, resveratrol has been shown to down-regulate NF-kappaB, TNFα, IL-6, and IL-1β, increase macrophage infiltration, decrease neutrophil infiltration, reduce the bacterial burden in the lung and improve survival.78
The report on the efficacy of resveratrol treatment of influenza in mice was published in 2005 by investigators who work outside the influenza scientific community.71 Remarkably, this important study has gone unnoticed by mainstream influenza scientists.
Other aspects of the host response might be affected by statins, fibrates and glitazones
The pathologic effects of influenza virus infection are mediated though several pathways, of which three might be targets of treatments that modify the host response.
Much attention has been given recently to the role of inflammasomes in the host response to influenza virus infection. Inflammasomes are multi-protein complexes that are responsible for the activation of caspase-1 that, in turn, generates two pro-inflammatory cytokines – IL-1β and IL-18.79 Among the three major groups of pattern recognition receptors – TLRs, retinoic acid inducible gene-I-like receptors and the Nod-like receptors (NLRs) – inflammasomes are part of the NLR family of receptors, and they participate in the innate and adaptive immune response. For influenza virus infection, the NLRP3 inflammasome seems to be important.
Two recent studies by Allen et al.80 and Thomas et al.81 have examined the responses of PR8-infected knockout mice deficient in caspase-1 or NLRP3. Compared with wild-type mice, mice with either deficiency had lower survival rates and reduced numbers of mononuclear cells and neutrophils in their lungs. Although the histological findings in the lungs of knockout mice in the two studies differed, it was clear from both studies that NLRP3 was protective. Both macrophages and epithelial cells were involved in early NLRP3 signaling, but compared with wild-type mice, much lower levels of IL-1β and IL-18 were found in the bronchoalveolar fluid of mice deficient in caspase-1 and NLRP3. Neither deficiency, however, had an appreciable effect on the adaptive immune response.80–82
These studies demonstrate the importance of NLRP3 signaling pathways in mounting a controlled inflammatory response to influenza virus infection.83 Moreover, the study by Thomas et al.81 showed that the NLRP3 inflammasome response could be triggered by intra-peritoneal administration of influenza viral RNA alone. In other words, virus replication was not required to trigger a protective inflammatory response.
These findings might be relevant to those obtained in a study of patients with sepsis. Reductions in caspase-1 signaling were found in those with septic shock compared with other critically ill patients who were not in shock.84 Down regulation of caspase-1 signaling suggested that mononuclear cell dysfunction appeared in patients with more severe illness. Importantly, an experimental study of mitogen-activated mononuclear cells has shown that statins activate caspase-1 and increase IL-18 secretion, thus reversing mononuclear cell dysfunction.85 Whether statins and other agents (e.g., fibrates and glitazones) would produce the same response in influenza virus infections remains to be determined.
Apoptosis and autophagy
Almost all patients with seasonal and pandemic influenza survive, but for those who die there is little understanding of the factors responsible for their deaths. The emergence of H5N1 influenza and its high case fatality rate has focused attention on the 'cytokine storm' that accompanies infection.2–7 This is surely not the only factor and perhaps not even the main factor responsible. A better understanding of the probable pathogenesis of fatal influenza can gained from studies of fatal sepsis.50,53. Among sepsis patients who die, few die within the first few days. Most develop a sustained 'immunoparalysis' and die much later. Apoptosis (programmed cell death) is the central feature of this late stage of disease. With apoptosis, there is a profound decrease in the numbers of lymphocytes – B-cells and CD4+ T-cells, the critical effector cells of the adaptive immune response. DCs are also lost, compromising antigen presentation. Uptake of apoptotic cells by macrophages and DCs stimulates the release of anti-inflammatory cytokines (e.g., IL-10 and TGF-β) and induces immune suppression. Apoptotic cell death can follow extrinsic (caspase-8-mediated) or mitochondria-initiated (caspase-9-mediated) pathways, and both are involved in sepsis-induced lymphocyte depletion. Experimental studies show that caspase inhibition improves survival, but it has been difficult to develop caspase inhibitors suitable for clinical use.50,86
Autophagy is a cellular pathway that is central to cell preservation and turnover.87 It involves the self-digestion of proteins and cell organelles that are part of normal homeostatic cell function, but it also involves the response to stress (e.g., starvation, infection). The molecular interactions between autophagy and apoptosis are not well understood,87,88 but 'coordinated regulation of 'self-digestion' by autophagy and 'self-killing' by apoptosis may underlie diverse aspects of … disease pathogenesis'.87
Autophagy and apoptosis are features of influenza virus replication. That autophagy is involved is not surprising,89 as the virus must use the building blocks at hand to form new virus particles. Apoptosis also accompanies influenza virus infection. In cell cultures of human blood macrophages, the onset of apoptosis induced by H5N1 viruses is delayed compared with that for H1N1 viruses,90 suggesting that intracellular persistence of the H5N1 virus might have something to do with its pathologic effects. In other studies, H5N1 virus (but not H5N2 or H5N3 viruses) was shown to induce caspase-dependent apoptosis in porcine alveolar epithelial cells, although levels of virus replication for all three viruses were the same.91 The H5N1 NS1 protein has also been shown to cause caspase-dependent apoptosis in human lung epithelial cells.92
In a splendid study of murine influenza, the PB1-F2 protein of the 1918 influenza virus was shown to cause severe viral and secondary pneumococcal pneumonia.93 PBI-F2 is known to have no major effect on virus replication. Instead, by localizing to the inner and outer mitochondrial membranes, it disrupts mitochondrial morphology and dissipates mitochondrial energy potential, causing apoptosis and cell death. It is thought that apoptosis of immune cells prevents efficient maturation of the adaptive immune response, and that this explains its pathologic effects. Remarkably, the effects of the 1918 PB1-F2 protein can be produced by intranasal administration of only the C-terminal portion of the protein.93
In a limited study of two patients who died of H5N1 influenza, apoptosis was seen in alveolar epithelial cells and pulmonary leukocytes.94 Apoptotic lymphocytes were also found in the spleen but there was no evidence of virus replication, suggesting that unidentified host factors were responsible. Considered together, the findings from the studies discussed above indicate that apoptosis is not directly related to high levels of influenza virus replication. Instead, it is caused by poorly defined host factors that respond to the molecular features of the virus such as the PB1-F2 and NS-1 proteins and perhaps viral RNA81 and those of the host cells damaged by infection.
There have been no studies that report the effects of statins, fibrates, or glitazones on autophagy or apoptosis in acute lung injury due to any cause. However, the apoptosis observed in septic cardiomyopathy is reduced with statin treatment, with a corresponding improvement in cardiac function.95 Furthermore, in a rat model of hepatic ischemia/reperfusion injury, simvastatin pre-treatment reduced the amount of apoptosis, with an associated improvement in liver function.96
Studies of experimental and human sepsis97,98 have shown that mitochondrial dysfunction and the disruption of energy homeostasis could be responsible for much of the loss of pulmonary integrity and the multi-organ failure seen in acute lung injury. Thus, mitochondrial dysfunction could play a fundamental role in determining the outcome of H5N1 and pandemic influenza virus infection.
Mitochondrial dysfunction is responsible for oxidant-induced acute lung injury99,100 in a process that is regulated by peroxisome proliferator activator receptorγ co-activator (PGC)-1a.101,102 In mice with experimental bacterial sepsis103 and in critically ill patients,104 restoration of mitochondrial function clearly separates those who recover from those who die. Mitochondrial biogenesis can be restored by up-regulating HO-1105 and by glitazones.106 In a model of LPS-induced mitochondrial dysfunction in murine neutrophils, inhibition of mitochondrial respiratory complex 1 with metformin led to decreased activity of NF-kappaB and lower levels of pro-inflammatory cytokines.107 In whole animal studies, LPS-exposed mice treated with metformin showed an inhibition of mitochondrial respiratory complex 1 in the lungs and a reduction in the severity of acute lung injury.107 Glitazones and fibrates have the same down regulating effect on mitochondrial respiratory complex 1 as metformin.108,109
Mitochondrial dysfunction with diminished cardiac function is a late occurring event in the myocardial depression seen in sepsis, but these changes might actually be protective.110 By reducing energy expenditure when mitochondrial energy generation is compromised, a state analogous to hibernation is induced that maintains myocardial integrity until recovery sets in.
Unfortunately, there is little information on the effects of statins, fibrates, and glitazones on mitochondrial functioning in acutely ill patients, and it must be remembered that the toxic effects of each group of drugs are thought to be due to their effects on mitochondria.111 Nonetheless, in a clinical trial conducted in children with severe burn injury and mitochondrial dysfunction, mitochondrial biogenesis was restored by treatment with fenofibrate.112
Research on the host response should determine whether anti-inflammatory and immunomodulatory agents could be used to manage the next pandemic
In focusing on the structural characteristics of influenza viruses that are associated with receptor specificity, replication efficiency, virulence and transmissibility and on factors that affect virus-induced cell signaling and cytokine dysregulation,2–7 influenza scientists have largely ignored the system-wide effects of the disease (multi-organ failure) and have left unexplored differences in system-wide molecular pathophysiology that might explain the remarkably different mortality rates in children and young adults seen in the 1918 pandemic. The well-known ability of some species (e.g., guinea pigs113) to support high levels of influenza virus replication without developing illness must reflect intrinsic host factors that differ from those of other animals that develop severe disease. The molecular consequences of influenza's effects on cardiac function and on organs other than the lung are hardly known. The pulmonary infiltrates seen in patients with H5N1 influenza that have been attributed to local cytokine dysregulation, could well be due to the influx of pro-inflammatory factors generated in the liver114 or perhaps other organs.
The similarities in the clinical course of patients with 1918 and H5N1 influenza and that of patients with sepsis are striking. The median duration of illness from onset until death in 1918 and H5N1 influenza has been similar to that seen in sepsis.18,24 The time courses for the development of lymphocyte depletion and multi-organ failure are generally the same. Bacterial super-infection is often associated with late immunosuppression seen in patients with sepsis and in those with acute lung injury due to non-infectious conditions like severe trauma. Thus, it is reasonable to assume that many of the pneumonia deaths seen in the 1918 pandemic had a similar cause. It is also reasonable to assume that agents shown to be effective in treating one condition might also be effective in treating the other, as has already been suggested for statins in both sepsis55 and pneumonia.14,15 As noted recently by Hotchkiss et al., 'reengagement or preserving host immune function will be the next major advance in the management of patients with sepsis.53 The same could be said for the management of patients with severe and pandemic influenza.