HMGB1 has a growing literature on its involvement in the pathogenesis of severe inflammatory states. Large amounts of HMGB1 have been detected in the serum of sepsis patients who succumbed to infection (35
). Similarly, during falciparum malaria levels of HMGB1 are higher in patients who do not recover (71
). However recent observations paint a more complex picture of the significance of elevated serum HMGB1 measurements. Sepsis patients with severe organ dysfunction had the lowest serum HMGB1 levels of all sepsis patients tested (72
), which probably points to a well-known side-effect of acute infection, immunosuppression. We have shown that despite plasma levels of HMGB1 being low at the time of peak mortality in mice infected with A/Japan/305/57 influenza virus (H2N2), HMGB1 may be elevated via its passive release in the lung at this time (73
). Therefore it would be important to first ascertain, using animal models of influenza, how levels of HMGB1, in the serum and/or lung, were associated with pathology and/or fatality. Our own work in this area continues, as does our focus on optimizing the way in which HMGB1 is measured for study. shows the likely sources and role of HMGB1 during infection with influenza virus.
Figure 1. Amplification and prolongation of inflammation by high mobility group box 1 protein (HMGB1) during influenza. Influenza virus-infected airway epithelial cells may passively release HMGB1 [see ref. (92) as an example], while alveolar macrophages, which (more ...)
Therapeutic interventions in sepsis models, such as administration of ethyl pyruvate (74
), an agent that limits HMGB1 release in vitro
, or administration of an anti-HMGB1 antibody (75
), save a significant proportion of mice, implicating the inhibition of HMGB1 release or activity as a therapeutic target during immune pathology. As previously mentioned, glycyrrhizin protects influenza virus (H2N2)-infected mice from fatality (33
) potentially via its ability to bind to, and inactivate, HMGB1 (34
). Therefore it would be important to establish whether or not therapeutic intervention with any of the agents discussed here reverse the fatality of severe influenza in animal models via effects on HMGB1 and/or other aspects of the host response.
However, endogenously produced molecules that dampen the effects of HMGB1 in the circulation are also elevated during severe disease states. Thrombomodulin, which binds to HMGB1 and prevents it binding to RAGE (76
) is elevated during severe malaria (77
), sepsis (78
) and influenza (80
). Paradoxically, it is this binding of HMGB1 to thrombomodulin that also prevents activated protein C generation (81
) and thus its ensuing anti-inflammatory activities (82
). Thus the balance between HMGB1 and thrombomodulin in vivo
is likely to be important (81
) and should also be determined as part of these studies.
A second, broader strategy could be to examine the traditional Chinese medicine formula discussed here, indeed, any other traditional formulas in their entirety, for anti-inflammatory activity in vitro. Cultured mouse macrophages could be stimulated with either commercially available recombinant influenza virus H5N1 haemagglutinin (HA) or LPS to induce production of pro-inflammatory cytokines, in the presence or absence of the formula, prepared as traditionally described (simmered in boiling water for 45–60 min). Cytokine assays to profile pro- and anti-inflammatory cytokines secreted over time into the culture supernatant could then be conducted, using a system such as multiplexing to examine many different cytokines in each sample (such as anti-inflammatory cytokines IL-4 and IL-10, and pro-inflammatory cytokines IL-1, IL-2, IL-6, IL-8, IL-17, IFN-γ, TNF and HMGB1). Given results which point towards levels of pro-inflammatory cytokines being reduced by treatment of the cells with the formula, the next step would be to conduct studies in mice infected with influenza virus (such as mouse-adapted PR8) and administered the formula orally, daily, adjusted according to body weight. Parameters measured in the in vivo experiments could initially include analysis of serum and lung cytokine levels, body weight and temperature, and even survival, providing researchers could interact with their institutional ethics committee to arrive at a mutually appropriate end-point for euthanasia for experiments of this nature.
Systematic reviews of the literature on clinical trials using traditional Chinese medicine in influenza patients have demonstrated the need for proper design, using randomized controlled trials and sufficiently large study groups to gain meaningful results (84
) as well as a more systematic approach to disease diagnosis (86
). Thus it would be informative to take the insights gained from results in mice into the clinic. Patients hospitalized with influenza could be recruited [see ref. (87
) for parameters relating to recruitment] to a randomized controlled trial to determine if treatment with a traditional Chinese medicine formula (for example, the formula previously discussed here) affected the course of disease. It would be prudent to begin treatment immediately, before laboratory confirmation of influenza virus infection. This is because symptoms are apparent at the time of increased serum cytokine levels (87
). Obviously patients testing negative to influenza virus would subsequently be excluded from analysis. The decoction would be prepared in the traditional manner (simmering in boiling water for 45–60 min) and administered to patients by hospital staff. The control treatment could simply be a flavored water solution that is stored in the same way as the decoction (at 4°C for not more than 1 week), and consumed daily in the same quantity as the decoction. Both solutions should be colored identically (with a food-grade dye, for example). It would be ideal to ensure the trial was conducted in a double-blind fashion so that neither the patient nor the staff knew which treatment was active. Previous studies have compared the incidence of influenza-like illness in healthcare workers using an herbal formula against those using no agent (88
) but results were questioned because workers given the herbal formula knew that they were taking the ‘active’ treatment. While these trials recruited thousands of patients, we suggest that patient numbers could be kept relatively small in the first instance (20 patients per group) if, in addition to patients being unaware of the activity of the treatment they are given, daily blood samples are taken to measure serum levels of cytokines, as for the in vitro
assays described above. One milliliter of blood would provide enough serum to analyze cytokine levels, and daily measurements on the same individual would allow for paired statistical analysis. The appropriate end-point would be convalescence, but obviously if symptoms worsened with treatment (or control) then standard interventions should immediately take place. These basic but necessary experiments could pave the way towards viable, low cost alternatives to planned influenza pandemic treatments— approximately $8 for a fortnight's treatment (88
). summarizes the possible inflammation-reducing effects of traditional Chinese medicines (e.g. A. sinensis
, S. miltiorrhiza
and glycyrrhizin), a natural agent (biochanin A), and pharmaceutical interventions (e.g. statins, fibrates), during influenza.
Figure 2. Potential mitigation of the action of HMGB1 and other pro-inflammatory cytokines by traditional Chinese medicines, a natural agent, and pharmaceutical drugs. The Chinese medicine glycyrrhizin, from liquorice root, binds to HMGB1 and inactivates its biological (more ...)