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Cytokines and H5N1: Still No Clear Answers

By Eric Toner, M.D., July 30, 2007

The Cytokine Storm Hypothesis

Ever since the first outbreak of highly pathogenic H5N1 in humans occurred in 1997 in Hong Kong, it has been speculated that the severity of illness and the high fatality rate associated with this infection may be attributed to a dysregulation of the immune system involving excessive levels of pro-inflammatory cytokines (“cytokine storm”). This hypothesis is supported by several lines of evidence:

  1. Elevated levels of cytokines and chemokines are present in infected humans and animals. 1

  2. In vitro laboratory studies have demonstrated that H5N1 infection caused significantly greater expression of some cytokines than H1N1 or H3N2
    infections. 2

  3. The constellation of acute respiratory distress syndrome (ARDS), lymphopenia, hemophagocytosis and multi-organ failure characteristic of H5N1 disease in humans has been previously associated with cytokine dysregulation. 3

A similar role for a “cytokine storm” has been proposed for the H1N1 strain that caused the 1918 pandemic that was also characterized by severe morbidity and high mortality. 4 According to the proponents of this hypothesis, it is not the virus that kills, but rather the host’s overactive immune response. This line of reasoning has been applied to explain the unusually high fatality rate in young, otherwise healthy people who were infected during the 1918 pandemic and more recently with H5N1. Because young people generally have  more vigorous immune systems, it is reasoned that they would be more likely to mount a “cytokine storm.”

Immune Modulation Therapy

As a result of this hypothesis, there has been much interest in using “immune modulators” (drugs that suppress the immune response) to treat H5N1 infections. Among those proposed are “statins,” which are cholesterol lowering drugs that have also been shown to have some anti-inflammatory properties. Statins are widely available and relatively inexpensive. 5 No studies have yet been published examining the benefit of statins in treating influenza.

However, the notion of immune suppression or modulation in influenza is controversial. Some prominent influenza experts have countered that the high levels of cytokines may be an essential part of the host’s defense against the virus and that suppressing the immune response might allow unchecked viral replication and result in an even worse outcome. 6

Two Recent Papers

Two papers have just been published that may influence this debate. Each is from a leading influenza research laboratory. In one, Szretter and colleagues7 report on a series of experiments carried out in Jacqueline Katz’s laboratory at the CDC’s Influenza Branch. In the other, Salomon and colleagues8 report on work from Robert Webster’s laboratory at St. Jude’s Children’s Research Hospital.

In both studies, transgenic mice with discreet deficiencies in one of several cytokine pathways were infected with highly pathogenic H5N1 and compared with controls that had normal immune systems. Results from both indicated there was no significant difference in mortality between the two groups. In other words, knocking out a single cytokine pathway did not prevent death from H5N1. Webster concludes that these results refute the “cytokine storm” hypothesis. Others have noted that since there are many overlapping cytokine pathways, eliminating just one at a time may not really modulate the dysregulated immune response.9

Several details of each study are interesting, particularly because, as Webster and colleagues note, “cytokines’ multiple functions potentially allow them to affect host survival both positively and negatively.” 8  In one arm of the Katz experiments, when mice deficient in the interleukin-1 (IL-1) receptor were infected with a mildly pathogenic form of H5N1 (without the E627K substitution of the PB2 gene that is associated with increased viral replication and pathogenicity in mammals), they actually had higher viral titers, more extensive inflammation, and worse mortality than the controls. This suggests that IL-1 is important in eradicating the virus in mice and that, at least in this particular setting, the deleterious effect of the lack of this clearance function outweighs any potential anti-inflammatory benefit.

In the study by the Katz group, lack of tissue necrosis factor receptor-1 (TNFR1) reduced the amount of weight lost by the infected mice without affecting their survival. Likewise, infected mice that were administered neutralizing anti-TNF-alpha sera lost less weight, but had no difference in viral titers or mortality when compared with controls. This suggests that TNF-alpha contributes to the severity of the illness but not to viral replication or ultimate outcome. On the other hand, lack of interleukin-6 (IL-6) or macrophage inflammatory protein one alpha (MIP-1 alpha) did not affect either weight loss or survival.

In the studies led by Dr. Webster, mice deficient in TNF-alpha, TNFR1, IL-6 or chemokine ligand 2 (CCL2) had no significant increase in survival over control mice when challenged with lethal doses of highly pathogenic H5N1. Mice treated with glucocorticoids lost less weight and had better survival rates, although these results were not significant (p values were not reported and the number of animals in the study was small). 

Conclusions

In summary, it is clear that the levels of some cytokines are significantly elevated in the setting of H5N1 infection, which may contribute to some of the symptoms associated with the disease. On the other hand, some of these substances may play a significant role in eradicating the infection. One has to wonder whether blocking cytokine pathways would have a beneficial effect if co-administered with an antiviral drug to block viral replication. In these two recent studies, eliminating one cytokine pathway at a time did not improve survival. It should be noted that, in both studies, very high viral inocula (1000 x mouse ID50) were administered. Whether the outcome would be the same at lower doses is not known. In addition, care must be taken when extrapolating results from mice for human application. Clearly, much more research is needed before conclusions about immune modulation therapy can be drawn. Unfortunately, these 2 new studies do not provide clear answers as to whether immune modulation therapy is likely to be either safe or effective for treating human H5N1 infections.

References

  1. de Jong MD, Simmons CP, Thanh TT, et al. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat. Med 2006; 12:1203-1207.
  2. Cheung C Y,  Poon LL, Lau AS, et al. Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 2002; 360:1831-1837.
  3. Chan  MCW, Cheung  CY, Chui WH. Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respiratory Research 2005, 6:135. Available at:http://respiratory-research.com/content/6/1/135. Accessed July 23, 2007
  4. Kobasa D, Jones SM, Shinya K, et al. Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 2007;445: 319-323.
  5. Fedson D. Pandemic influenza: a potential role for statins in treatment and prophylaxis. Clinical Infectious Diseases 2006;43:199–205.
  6. Toner E. CBN Report: New Research on Antiviral Treatment of Influenza. Clinicians Biosecurity Network. October 13, 2006. Available at: http://www.upmc-cbn.org/report_archive/2006/10_October_2006/cbnreport_101306.html. Accessed July 23, 2007.
  7. Szretter KJ, Gangappa S, Lu X, et al. Role of host cytokine responses in the pathogenesis of avian H5N1 influenza viruses in mice.  2007; 81: 2736–2744. Available at: http://jvi.asm.org/cgi/content/full/81/6/2736. Accessed July 23, 2007.
  8. Salomon R, Hoffmann E, Webster R. Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection. Proc. Natl Acad  Sci 2007;104:12479-12481. Available at: http://www.pnas.org/cgi/content/abstract/0705289104v1. Accessed July 23, 2007.
  9. Schnirring L. Study: Inhibiting cytokine response might not reverse H5N1 infections. CIDRAP News. July 16, 2007. Available online at http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/news/jul1607cytokine.html. Accessed July 23, 2007

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