The recent controversial studies of man-made avian flu viruses caused a media storm, and brought new concerns to the potential of an avian influenza H5N1 virus pandemic, which has been pending since 19971,2. Although the estimated mortality rate of avian influenza A H5N1 virus infection in humans could be as high as 60%, the World Health Organization (WHO) phase of pandemic alert is currently set at 3, due to that there has not been human-to-human or community-level transmission (http://www.who.int/influenza/preparedness/pandemic/h5n1phase/en/index.html). However, the newly created H5N1 virus strains, which are genetically altered, are transmissible among ferrets, and thus may trigger a real pandemic that could potentially result in millions of deaths according to Science Insider3. While it is arguably a bit too late to debate whether regulations or mandatory reviews should be applied to these dual-use studies, in the matter of fact, these viruses that are probably among the most dangerous infectious agents known already exist. Therefore, a top priority at present is to find effective prophylactic or therapeutic agents that would help to control a pandemic of avian influenza A H5N1 viruses.
Previous reports have demonstrated that the high mortality in humans infected with avian influenza A H5N1 is partly due to acute lung injury or the resulting severe condition, acute respiratory distress syndrome (ARDS)4,5. There are few treatment choices for ARDS, aside from mechanical supporting equipment and empirical treatments. The use of cortisones is controversial.
We have recently discovered that avian influenza A H5N1 virus infection causes acute lung injury by inducing autophagic alveolar epithelial cell death6. Importantly, we found that autophagy inhibitors are effective in ameliorating murine acute lung injury induced by live avian influenza A H5N1 virus infections6. We thus hypothesize that if a drug that is currently in clinical use can act to inhibit autophagy, then such a drug might be a good candidate for treating H5N1 infections.
To test this, we have focused our efforts on chloroquine (CQ), as CQ is the only oral clinical drug that is known to be an autophagy inhibitor7. CQ, or N′-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine, was discovered in 1934 by Hans Andersag and his co-workers at Bayer Laboratories and was introduced into clinical practice in 1947 as a prophylactic treatment for malaria8. Currently, CQ and its hydroxyl form, HCQ, are used as anti-inflammatory agents for the treatment of rheumatoid arthritis, lupus erythematosus and amoebic hepatitis. More recently, CQ has been studied for its potential use as an enhancing agent in cancer therapies as well as novel antagonists to chemokine receptor CXCR4 in pancreatic cancer8,9.
We first tested whether CQ could inhibit cell death in the human lung carcinoma A549 cells infected with live avian influenza A H5N1 virus. The cell viability was improved both prophylactically and therapeutically in a dose-dependent manner with CQ treatments, and the efficacy of CQ was much higher than that of the potent autophagy inhibitor 3-methyladenine (3-MA) (Figure 1A). Next, we tested the potential therapeutic effect of CQ in a mouse model of live H5N1 infections. We found that when CQ was administered therapeutically at a dose equivalent to that for human clinical use, the survival rate of H5N1 virus-infected mice was improved dramatically (from 0% to 70% at day 8 post infection), and the body weight changes also showed a trend of improvement, but prophylactic application of CQ showed no protective effect (Figure 1B and 1C). We analyzed the lung histopathology in these mice and found fewer infiltrating leukocytes in the CQ therapeutic group, but not in the CQ prophylactic group (Figure 1D). Lung edema, as determined by the increased wet/dry weight ratio of lung tissue, was also significantly reduced by therapeutic CQ treatment, but not by prophylactic CQ treatment (Figure 1E). Taken together, our results demonstrate that CQ, a known autophagy inhibitor that is in clinical use, could efficiently ameliorate acute lung injury and dramatically improve the survival rate in mice infected with live avian influenza A H5N1 virus.
CQ acts to raise the lysosomal pH, leading to the inhibition of both the fusion of autophagosomes with lysosomes and lysosomal protein degradation7. As a lysosomotropic agent, CQ could prevent endosomal acidification, which might inhibit influenza virus endocytotic cell entry10,11. CQ was therefore proposed to be a candidate prophylactic agent for influenza virus. However, government-sponsored clinical trials have shown insufficient prophylactic effects against influenza infection12, which is consistent with our mouse results. CQ could also inhibit the innate immune responses through TLR signaling pathways and act as novel antagonists to chemokine receptor CXCR4 in pancreatic cancer9,13. We have shown that CQ treatment clearly inhibited the autophagy in mouse lung induced by avian influenza A H5N1 virus while the virus loads and proinflammatory cytokines were not significantly affected (Figure 1F, 1G, Supplementary information, Figure S1). Further studies are necessary to elucidate the precise molecular mechanisms by which CQ ameliorates murine acute lung injury induced by avian influenza A H5N1 virus.
This notwithstanding, our study strongly suggests that CQ (and potentially its derivatives) should be evaluated as a candidate drug for clinical treatment of H5N1-infected patients. Moreover, a systematic screening of common clinical drugs for potential autophagy inhibitors may lead to the identification of other novel treatments against avian flu.
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This work is supported by the Ministry of Science and Technology of China (2009CB522105), the National Natural Science Foundation of China (81230002), and the 111 Project (B08007). CJ is a Hsien Wu professor of Biochemistry.
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information, Figure S1
(A, B, C) Real time quantitativePCR analysis of IL-1β's, IL-6's and TNF-α's relative ratio to beta-actin in mice lung tissue receiving therapeutic treatment of CQ as described above. (PDF 68 kb)
Supplementary information, Data S1
Materials and Methods (PDF 99 kb)
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Yan, Y., Zou, Z., Sun, Y. et al. Anti-malaria drug chloroquine is highly effective in treating avian influenza A H5N1 virus infection in an animal model. Cell Res 23, 300–302 (2013). https://doi.org/10.1038/cr.2012.165
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