Delayed Antiviral Plus Immunomodulator Treatment Still Reduces Mortality in Mice Infected by High Inoculum of Influenza A/H5N1 Virus

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Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus

Bo-Jian Zheng*†‡, Kwok-Wah Chan§, Yong-Ping Lin†, Guang-Yu Zhao†, Chris Chan†, Hao-Jie Zhang†, Hong-Lin Chen*†‡, Samson S. Y. Wong*†‡, Susanna K. P. Lau*†‡, Patrick C. Y. Woo*†‡, Kwok-Hung Chan*†‡, Dong-Yan Jin¶, and Kwok-Yung Yuen*†‡ʈ

*State Key Laboratory of Emerging Infectious Diseases, †Department of Microbiology, ‡Research Centre of Infection and Immunology, and Departments of §Pathology and ¶Biochemistry, University of Hong Kong, Pok Fu Lam, Hong Kong

Edited by Tak Wah Mak, University of Toronto, Toronto, ON, Canada, and approved April 7, 2008 (received for review December 20, 2007) The mortality of human infection by influenza A/H5N1 virus can cyclooxygenase-2 (COX-2) inhibitor celecoxib and mesalazine exceed 80%. The high mortality and its poor response to the could be effective in reducing mortality. To imitate the clinical neuraminidase inhibitor oseltamivir have been attributed to un- situation, we delayed combination therapy for 48 h after chal- controlled virus-induced cytokine storm. We challenged BALB/c lenging the BALB/c mice with an inoculum of 1,000 LD50 of a mice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival, highly virulent influenza A virus, human isolate A/Vietnam/ body weight, histopathology, inflammatory markers, viral loads, T 1194/04. Our results demonstrate that combination therapy lymphocyte counts, and neutralizing antibody response were doc- consisting of an inhibitor of the viral neuraminidase (zanamvir) umented in infected mice treated individually or in combination and two inhibitors of inflammation (celecoxib and mesalazine) with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate greatly increased the survival rate of mice infected with a highly the real-life scenario, treatment was initiated at 48 h after viral pathogenic strain of influenza A/H5N1 virus. challenge. There were significant improvements in survival rate survival time (P < 0.02), and inflammatory markers (P < Results ,(0.02 ؍ P) MICROBIOLOGY 0.01) in the group treated with a triple combination of zanamivir, All mice survived with early institution of i.p. zanamivir treat- celecoxib, and mesalazine when compared with zanamivir alone. ment (Fig. 1A). The survival rate of mice was decreased to 13.3% Zanamivir with or without immunomodulators reduced viral load (2/15) if the treatment with zanamivir was delayed for 48 h, to a similar extent. Insignificant prolongation of survival was although the mean survival time was prolonged to 10.7 Ϯ 1.6 observed when individual agents were used alone. Significantly Ϯ ؉ ؉ days compared with 6.6 1.6 days in controls (Fig. 1B). As higher levels of CD4 and CD8 T lymphocytes and less pulmonary expected, all PBS-treated controls died, whereas all mice on inflammation were also found in the group receiving triple ther- immunomodulators alone died with a trend toward increased apy. Zanamivir alone reduced viral load but not inflammation and mean survival time to Ϸ8.5 days for mice given celecoxib or mortality. The survival benefits of adding celecoxib and mesalazine mesalazine and Ϸ9.5 days for those given both celecoxib and to zanamivir could be caused by their synergistic effects in reducing mesalazine, but only Ϸ7.5 days for those given gemfibrozil alone cytokine dysfunction and preventing apoptosis. Combinations of a or both celecoxib and gemfibrozil. Therefore, we did not select neuraminidase inhibitor with these immunomodulators should be gemfibrozil for further study. Single use of any of these immu- considered in randomized controlled treatment trials of patients nomodulators did not confer survival benefit. However, when suffering from H5N1 infection. zanamivir was combined with both immunomodulators, the survival rate increased to 53.3% (8/15) (P ϭ 0.02) and the mean zanamivir ͉ celecoxib ͉ mesalazine survival time increased to 13.3 days (P ϭ 0.0179) compared with zanamivir alone (survival rate 13.3% and survival time 8.4 days). he mortality of patients suffering from pneumonia and The body weight of all infected mice steadily decreased to a Tmultiorgan involvement caused by influenza A/H5N1 virus minimum at day 11 and then increased again for those that (H5N1) varies between 45% and 81% since earlier reports (1, 2). survived (Fig. 1C). Subsequent use of oseltamivir has not reduced the mortality Significant decrease (Ͼ2.5 logs) of viral titers in tracheal- associated with this virus. The unsatisfactory outcome of its pulmonary lavage (TPL) by TCID50 or copies of viral RNA treatment was attributed to either deficiencies in antiviral ad- genomes in lung tissues by real-time quantitative RT-PCR was ministration or the induction of a severe cytokine storm (3). The found in groups treated by zanamivir with or without immuno- poor response to oseltamivir can also be the result of delayed modulators at days 6 and 8 postchallenge (Fig. 2). Levels of initiation of treatment because of the nonspecific initial mani- inflammatory markers IL-6, IFN-␥, TNF-␣, macrophage inflam- festations of H5N1 infection, high initial viral load, poor oral matory protein 1 (MIP-1), and leukotriene assayed by enzyme bioavailability of oseltamivir in the seriously ill, lack of i.v. immunoassays were significantly higher in TPL obtained from preparations of oseltamivir, and the emergence of resistance during therapy (4, 5). Attempts to use antiinflammatory doses of

corticosteroids to control excessive inflammation were associ- Author contributions: B.-J.Z. and K.-Y.Y. designed research; B.-J.Z., K.-W.C., Y.-P.L., G.-Y.Z., ated with severe side effects without any improvement in sur- C.C., H.-J.Z., and H.-L.C. performed research; K.-W.C., S.S.Y.W., S.K.P.L., P.C.Y.W., K.-H.C., vival (6). Moreover, cytokine and chemokine knockout mice or D.-Y.J., and K.-Y.Y. analyzed data; and B.-J.Z., S.S.Y.W., S.K.P.L., P.C.Y.W., and K.-Y.Y. wrote steroid-treated wild-type mice did not have survival advantage the paper. over wild-type mice after viral challenge (7). This paradox can The authors declare no conﬂict of interest. be explained if both a high initial viral load and the commen- This article is a PNAS Direct Submission. surate degree of excessive inflammation are important in the ʈTo whom correspondence may be addressed. E-mail: [email protected]. pathogenesis and outcome of H5N1 infection. Here, we tested This article contains supporting information online at www.pnas.org/cgi/content/full/ the hypothesis that the combination of a parenterally adminis- 0711942105/DCSupplemental. tered neuraminidase inhibitor, zanamivir, together with the © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711942105 PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 ͉ 8091–8096 Downloaded by guest on September 26, 2021 Z Z C A 100% M 1000000 Z+C+M G <0.05 80% C+M 100000 <0.01 PBS C+G 60% PBS 10000 40% 1000

20% 100 Undetectable Survival rates

0% Viral titres +SD 10 1 3 5 7 9 11 13 15 17 19 1 Days post-challenge 468 Z Days post-challenge 100% Z+C Z+M Z Z+C+M B 80% 1000000 <0.01 Z+C+M PBS <0.01 PBS 60% 100000 40% -actin 10000 20% >0.05

Survival rates 1000 0% 135791113151719 100 Days post-challenge 10 Viral copies/100 copies/100 Viral Z 1 Z+C 468 24 Z+M Z+C+M Days post-challenge 20 PBS Fig. 2. Detection of viral load in infected mice treated with zanamivir, 16 celecoxib, and mesalazine. (A) Titers of released virus in TPL collected at the indicated days from mice treated with zanamivir alone (Z), zanamivir/ 12 celecoxib/mesalazine (ZϩCϩM), and PBS, which were started at 48 h postchallenge, as measured by TCID50. The detection limit (undetectable) is 1:20. (B) Weight (g ±SD) 8 Viral RNA copies in lung tissue from the above mice were determined by 1 3 5 7 9 1113151719 real-time RT-PCR and normalized by ␤-actin. The P values between groups Days post-challenge ZϩCϩM and Z or PBS are indicated.

Fig. 1. Survival times, survival rates, and body weight for infected mice treated with zanamivir, celecoxib, mesalazine, and gemfibrozil. (A) Shown is protein in the cytoplasm of pulmonary alveolar epithelial cells the survival rate and time of the mice (five mice per group) treated with (Fig. 4C). Tissue damage was primarily confined to lung tissues. zanamivir (Z), celecoxib (C), mesalazine (M), gemfibrozil (G), celecoxib/ However, there was mild perivascular mononuclear cell infiltra- mesalazine (CϩM), celecoxib/gemfibrozil (CϩG), and PBS (control) at 4 h postchallenge. (B) Survival time and rate of the mice (10–15 mice per group) tion in the cerebral cortex from the mice treated with zanamivir treated with zanamivir (Z), zanamivir/celecoxib (ZϩC), zanamivir/mesalazine alone but not in those from mice treated by both zanamivir and (ZϩM), zanamivir/celecoxib/mesalazine (ZϩCϩM), and PBS at 48 h postchal- immunomodulators, whereas focal dense mononuclear cell in- lenge were monitored for 21 days. (C) Body weights of the mice were moni- filtration in the cerebral cortex was observed in brain tissues tored for 21 days (survival mice) or until death. taken from the untreated mice (Fig. S2A). Reactive lymphoid cells that appeared paler in staining were found in spleens obtained from zanamivir-treated and PBS control mice, in which the mice treated with zanamivir alone and controls than those Ͻ reactive lymphoid cells were present along with frequent apo- treated by triple therapy (P 0.01 or 0.05) or uninfected normal ptotic bodies with prominent nuclear fragmentation, but not in mice (Fig. 3). However, IL-1 levels were only slightly lower in those collected from mice treated with zanamivir and immuno- Ͼ those treated with zanamivir alone and controls (P 0.05), modulators (Fig. S2B). Nevertheless, no significant pathologic whereas prostaglandin E2 (PGE2) levels were found to be changes or tissue damages could be detected in liver (Fig. S2C) significantly higher in the samples collected at day 8 postchal- and kidney (Fig. S2D) from all mice. lenge from the group receiving triple therapy (Fig. 3). As As shown in Fig. S3A, 12 surviving mice with undetectable expected, their serum cytokine and chemokine changes were viral load in lung tissues at day 21 after viral challenge also had similar to those of their TPL [supporting information (SI) Fig. ϩ ϩ a neutralizing antibody titer of 80. Western blot confirmed that S1]. Furthermore, levels of both CD4 and CD8 T lymphocytes the neutralizing antibody reacted specifically with baculovirus- were significantly higher in the blood taken at days 6 and/or 8 expressed nucleoprotein and hemagglutinin of H5N1 (Fig. S3B). from the mice given triple therapy than those taken from Interestingly, two surviving mice treated with triple therapy still zanamivir-treated and PBS control mice (Fig. 4A). had a detectable low viral load and a neutralizing antibody titer The degree of lung damage as evidenced by increased albumin of 40. Compared with the zanamivir-treated group whose levels in the TPL (Fig. 3) and higher elastase activity in the TPL TCID50 titer in the TPL was below our detectable limit, the triple 2 2 (Fig. S1) was significantly lower in groups treated by the therapy group had a TCID50 titer of 5.1 ϫ 10 Ϯ 4.9 ϫ 10 , which combination therapy compared with the groups treated by was still 2.5 log below the titer of 2.7 ϫ 105 Ϯ 2.0 ϫ 105 in the zanamivir alone (P Ͻ 0.01) or PBS (P Ͻ 0.03). Histopathologic PBS control group (Fig. 2A). This finding is not completely examination further showed that the alveolar damage and in- unexpected because the immunomodulators may still have some terstitial inflammatory infiltration in mice treated by the com- degree of immunosuppression that is not clinically apparent. bination were much less severe than those treated by zanamivir Consistent with these findings, these two mice [zanamivir ϩ alone (Fig. 4B). Immunohistochemical staining with antiinflu- celecoxib ϩ mesalazine (2)], together with the surviving mouse enza nucleoprotein mAb demonstrated strong expression of this from the zanamivir-treated group, also had inflammatory infil-

8092 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711942105 Zheng et al. Downloaded by guest on September 26, 2021 Z Z Z CD3+/CD4+ CD3+/CD8+ Z IL-1 IL-6 Z+C+M >0.05 Z+C+M Z+C+M A Z+C+M 1000000 > PBS 5000 5000 1500 0.05 >0.05 PBS PBS PBS <0.03 Normal <0.01 >0.05 >0.05 >0.05 >0.05 4000 <0.03 <0.02 Normal 4000 10000 >0.05 0.04 0.03 1000 >0.05 0.01 0.04 3000 3000 2000 0.01 <0.01 100 2000 500 pg/ml +SD pg/ml +SD 1000 1000 Nunber/10000 Nunber/10000 cell counts +SD counts cell Number/10000

1 0 cell counts +SD 0 0 468 468 468 468 Days post-challenge Days post-challenge Days post-challenge Days post-challenge IFN- Z TNF- Z Z+C+M Z+C+M 4000 PBS 500 <0.01 <0.01 PBS Z Z+C+M PBS Normal <0.01 Normal <0.01 B 3000 400 <0.01 Normal <0.01 300 2000 200

pg/ml +SD 1000 pg/ml +SD 100 Day 4 0 0 468 468 Days post-challenge Days post-challenge Z Z PGE2 Z+C+M MIP-1 Z+C+M PBS 70 > PBS 500 0.05 <0.01 Normal 60 <0.01 Normal < >0.05 400 Day 6 50 0.05 <0.05 <0.01 <0.01 40 <0.05 300 30 200 20 pg/ml +SD ng/ml +SD 10 100 0 0 468 468 Days post-challenge Days post-challenge Day 8 Leukotriene Z Albumin Z Z+C+M Z+C+M 800 <0.01 600 <0.05 PBS PBS 500 <0.01 <0.01 <0.01 <0.01 Normal Normal 600 400

400 300 MICROBIOLOGY 200 200 C pg/ml +SD

pg/ml +SD 100 0 0 468 468 Day 6 Days post-challenge Days post-challenge

Fig. 3. Detection of proinflammatory cytokines, chemokines, prostaglan- dins, and albumin in TPL. Concentrations of IL-1, IL-6, IFN-␥, IFN-␣, MIP-1, PGE2, leukotrienes, and albumin in TPL collected from mice treated with zanamivir Fig. 4. T lymphocyte counts in peripheral blood and histopathologic changes ϩ ϩ ϩ ϩ alone (Z), zanamivir/celecoxib/mesalazine (ZϩCϩM), untreated control (PBS), in lungs. (A) Numbers of CD3 /CD4 and CD3 /CD4 T lymphocytes in 10,000 and uninfected (normal) mice at the indicated days were determined by ELISA blood cells taken from the mice in the indicated days were counted by flow and compared between different groups. Their P values are shown. cytometry, and the P values between zanamivir alone (Z), zanamivir/celecoxib/ mesalazine (ZϩCϩM), and PBS groups are shown. (B) Histopathologic changes in mouse lung tissues collected at the indicated days postinfection are shown. trate in their alveoli on histologic examination, whereas no Representative histologic sections of the lung tissues from these mice and significant inflammation was observed in the other surviving uninfected mice (Normal) were stained with H&E (original magnification: ϫ mice [zanamivir ϩ celecoxib, zanamivirϩ mesalazine, and zana- 100). Inflammatory infiltrate and alveolar damage are seen as thickening of ϩ ϩ the alveolar septum with obliteration of some alveolar spaces at this magni- mivir celecoxib mesalazine (6)], which was similar to those fication. (C) Viral infection in lung of the mice was further demonstrated by found in normal mice (Fig. 4B and Fig. S3C). immunohistochemical staining. Positivity is indicated by brown staining in the cytoplasm. Representative histologic sections of the lung tissues taken from Discussion these mice and uninfected mice (Normal) at the indicated day were stained There is an urgent need to find an effective treatment strategy with an antiinfluenza NP mAb (Original magnification: ϫ400.) against H5N1 infection in humans because of the substantial mortality associated with this virus. Although oseltamivir is highly effective in mouse models, humans treated with this drug vir in sick mice and the known risk of emergence of oseltamivir still exhibited high fatality, which can be attributed to delayed resistance during therapy, i.p. zanamivir was used. However, as initiation of therapy. Many antiviral treatment studies of mouse in the case of oseltamivir, 87% of the mice died when the Ϸ models infected by H5N1 used an inoculum of 10 LD50 of zanamivir treatment was delayed for 48 h, although the survival H5N1. Good treatment results were obtained if the antiviral was time was insignificantly prolonged. Our animal model provided started 4 h before, soon after, or within 36 h after inoculation (8, an ideal situation for testing combination therapy with immu- 9). Only a few studies showed good results when the antiviral nomodulators that had no antiviral effects or any significant treatment was started after 36 h. However, in those series, either a low viral inoculum was used or a duck H5N1 virus adapted to effect on survival if used alone. mice was used instead of a human virus for inoculation (10–12). Our study showed that even if the viral replication had been Thus the pathophysiologic status of the infected mice in those suppressed in the mice treated with antiviral, levels of cytokines studies could be quite different from the real clinical situation and chemokines were still similar to the untreated mice, which when patients often do not enter the hospital until 2–4 days after were significantly higher than those in the mice receiving com- the onset of symptoms, when the viral load in respiratory bination therapy. This finding suggests that once the viral secretions is already high. The high inoculum and delayed infection has triggered the cytokine storm, even if viral replica- therapy in the presently reported mouse model provided a more tion is suppressed by antiviral therapy, the proinflammatory realistic simulation for testing various forms of therapy. To avoid cytokines and chemokines will continue to drive the immuno- the confounding effects of poor oral bioavailability of oseltami- pathologic progression. Previous studies showed that antiinflam-

Zheng et al. PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 ͉ 8093 Downloaded by guest on September 26, 2021 matory dose of steroid was ineffective in mice in the absence of protective because infected IL-1 receptor knockout mice showed antiviral treatment (7) and was associated with significant side increased morbidity, mortality, lung viral titer, and inflamma- effects in human infected by the H5N1 virus without improving tory infiltrate when infected with a low-lethality HK/486 virus survival (5, 6). Therefore other immunomodulators have to be (19). In this study, mice treated by triple therapy had improved considered. survival without significant suppression of IL-1 in TPL despite Because COX-2 knockout mice had significantly better survival the use of a hypervirulent virus. after challenge with mouse-adapted influenza A H3N2 virus than H5N1-infected patients who succumbed often had persistently wild-type BALB/c mice (13), i.p. celecoxib was chosen in this study. high levels of serum proinflammatory cytokines and chemokines Sulfasalazine and related compounds such as mesalazine and (3, 5). Therefore, the pathogenesis was initially attributed to 5-aminosalicylic acid are also chosen in this study because they are virus-induced cytokine storm. However, studies with knockout highly active in alimentary tract epithelial cells and are commonly mice deficient in TNF-␣, TNF receptor 1, TNF receptor 2, IL-6, used in the treatment of inflammatory bowel diseases. They have chemokine (C-C motif) ligand 2, MIP-1␣, and IL-1R (7) did not diverse effects on the immune system including inhibition of confer better survival after viral challenge when antivirals were lipoxygenase and COX pathways, which decrease proinflammatory not given. Moreover, recent studies showed that the levels of cytokines and eicosanoids, and therefore decrease the activation of serum proinflammatory cytokines and chemokines correlated inflammatory cells such as macrophages and neutrophils. In addi- closely with the viral load (5). Therefore, the pathogenesis tion, sulfasalazine and 5-aminosalicylic acid inhibit NF-␬B activa- should involve the interplay between a rising viral load and the tion and promote the synthesis of phosphatidic acid. Both actions resulting proinflammatory response. An effective therapy inhibit the potent stimulatory effects of ceramides on apoptosis (14, should consist of combinations of an effective antiviral agent and 15). The combined actions of mesalazine (the effective moiety of immunomodulatory agents to control viral load and cytokine sulfasalazine) and celecoxib have synergistic effects in counteract- storm, respectively. This scenario is especially true if patients ing virus-induced cytokine dysregulation and apoptosis. Both drugs present late in the course of influenza, when local and systemic are relatively inexpensive, currently used in humans, not known to proinflammatory cascade are fully activated. cause immunosuppression, and relatively free from adverse drug Postmortem examination of patients who succumbed to H5N1 interactions or major side effects with short-term use. infection often showed severe lymphopenia and lymphoid atro- The main target of action of the fibrates such as gemfibrozil phy or necrosis in the spleen and other lymphoid tissues (1, 3). is peroxisome proliferators-activated receptor ␣ (PPAR␣). Our study also showed that both CD4ϩ and CD8ϩ T lymphocytes PPARs are members of the nuclear receptor superfamily that were significantly decreased in antiviral-treated and untreated affects the lipid and glucose metabolism and inflammatory mice during disease progression. However, unlike the use of the responses. PPAR␣ activation inhibits NF-␬B, COX-2 activity, steroid or other immunosuppressants, the use of celecoxib and and production of proinflammatory cytokines such as IL-6 and mesalazine with zanamivir maintains significantly higher levels ϩ ϩ TNF-␣ (16). Therefore, activation of the PPAR␣ by gemfibrozil of CD4 and CD8 T lymphocytes at days 6 and 8 postchallenge. is expected to damp down the excessive inflammatory response. Histopathologic examination also showed that reactive lymphoid Budd et al. (17) demonstrated that gemfibrozil improved survival cells with frequent apoptotic bodies were found in spleens of mice infected by influenza A/H2N2 virus from 26% (controls) obtained from zanamivir-treated and untreated mice, but were to 52% (treated). However, no improved survival was noted infrequent in spleens from mice treated with zanamivir and when the hypervirulent H5N1 virus was used in this study. This immunomodulators. Thus, the antiapoptotic effects of celecoxib discrepancy could be related to the different pathophysiology plus mesalazine may play a role in averting this type of damage. between H2N2 and H5N1 viruses or the relatively weak agonistic Our results provide a sound theoretical and experimental basis activities of gemfibrozil on PPAR␣. for further studies on the role of triple therapy. An antiviral and The association between higher levels of PGE2 and mice the combined use of celecoxib and mesalazine may cause survival is compatible with the known immunologic profiles of synergistic reduction in proinflammatory cytokines, chemo- human and experimental H5N1 infection. Among other cytokines, and leukotrienes via different pathways. These inhibitory kines and chemokines, severe H5N1 infections are associated activities, together with the antiapoptotic activities of the amino- with increased RANTES and MIP-1, and their synthesis is salicylates, reduce cell death and tissue damage in the host (20). inhibited by PGE2. Our triple therapy also showed a reduction Apoptosis in pulmonary alveoli and lymphoid tissues leading to in MIP-1 levels without suppressing PGE2. PGE2 has antiin- lymphopenia are prominent pathological features in patients flammatory and antiapoptotic properties, both of which may dying of H5N1 infection. The concomitant use of an effective play a beneficial role in preventing excessive tissue and cellular antiviral is essential, not only to limit the extent of viral repli- damage. Previous reports showed that COX-2Ϫ/Ϫ knockout mice cation (which drives the cytokine dysfunction) from natural had a significantly lower mortality, lesser degree of inflamma- infection, but also to counteract the possible increase in viral tory cell infiltrates in the lungs, and lower levels of proinflam- load after COX-2 inhibition. We suggest that H5N1 avian matory cytokines (TNF␣, IL-1␤, IFN-␥, IL-6) in the TPL as influenza could be treated with an effective antiviral like i.v. compared with wild-type and/or COX-1Ϫ/Ϫ knockout mice after zanamivir, in conjunction with immunomodulating drugs like infection by influenza A/H3N2 virus (13). But the PGE2 levels celocoxib and mesalazine to control the symptoms associated in the TPL and the viral load in the lungs were significantly with cytokine storm. Triple therapy offers some hope for higher in COX-2Ϫ/Ϫ mice. Our findings of lower leukotrienes and surviving the devastating consequences associated with a pan- higher PGE2 levels in the TPL in mice treated by combination demic influenza outbreak. therapy is compatible with the above findings. Although mesalazine or celecoxib have not been shown to cause immuno- Materials and Methods suppression, two surviving mice after triple therapy still had a Animal Model and Viral Challenge. BALB/c female mice, 5–7 weeks old, were low, but detectable, viral load and a low level of neutralizing kept in biosafety level-3 housing and given access to standard pellet feed and water ad libitum. All experimental protocols followed the standard operating antibody. This finding is not unexpected because the same procedures of the approved biosafety level-3 animal facilities and were ap- immunologic factors causing tissue damage during the mounting proved by the Animal Ethics Committee (21). Aliquots of stocks of inﬂuenza A of the immune response may also be critical for viral clearance virus strain A/Vietnam/1194/04 were grown in embryonated eggs. Virus- (18). The combination of mesalazine and celecoxib may cause containing allantoic ﬂuid was harvested and stored in aliquots at Ϫ70°C. The mild subclinical immunosuppression. IL-1 was speculated to be LD50 was determined in mice after serial dilution of the stock. One thousand

8094 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711942105 Zheng et al. Downloaded by guest on September 26, 2021 Table 1. Treatment regimens containing zanamivir, celecoxib, mesalazine, and gemﬁbrozil used alone or in combination for infected mice Group Treatment regimen No.

1 3 mg zanamivir in PBS i.p. once every 12 h ϫ 8 days* 5 2 2 mg celecoxib in 10%DMSO/PBS i.p. once per day ϫ 8 days* 5 3 1 mg mesalazine in ddH2O i.p. once per day ϫ 8 days* 5 4 1 mg gemﬁbrozil in propylene glycol i.p. once per day ϫ 8 days* 5 5 2 mg celecoxib ϩ 1 mg mesalazine i.p. once per day ϫ 8 days* 5 6 2 mg celecoxib ϩ 1 mg gemﬁbrozil once per day ϫ 8 days* 5 7 PBS i.p. once per day ϫ 8 days* 5 8 3 mg zanamivir i.p. once every 12 h ϫ 6 days† 33‡ 9 3 mg zanamivir ϩ 2 mg celecoxib i.p. ϫ 6 days† 10 10 3 mg zanamivir ϩ 1 mg mesalazine i.p. ϫ 6 days† 10 11 3 mg zanamivir ϩ 2 mg celecoxib ϩ 1 mg mesalazine i.p. ϫ 6 days† 33‡ 12 PBS i.p. once per day ϫ 6 days 33‡

BALB/c mice (female, ages 5–7 weeks) were intranasally challenged with 1,000 LD50 of H5N1 virus strain A/Vietnam/1194/04. *The treatments started 4 h postchallenge. †The treatments started 2 days postchallenge. ‡ Experiments were conducted in triplicates of ﬁve mice for each group. Furthermore, six mice in each of these groups were killed on days 4, 6, and 8 postchallenge, while all survived mice were killed on day 21 postchallenge. Blood, TPL, lung, brain, kidney, liver, and spleen were collected from these mice.

LD50 were used for viral challenge in all of the experiments. Infection was Western Blot. Influenza A viral proteins NP from H5N1 strain A/Indonesia/5/ established by intranasal inoculation of mice anesthetized by isoflurane. 2005, HA1 from H5N1 strain A/Vietnam/1203/2004 (Immune Technology), and HA2 from strain A/Vietnam/1194/04, which was expressed in baculovirus Antiviral and Immunomodulatory Treatments. Antiviral and immunomodula- vector (BD Bioscience), were separated in 12% SDS/PAGE gel and then elec- MICROBIOLOGY tors were administered by the i.p. route using 0.5-ml, 29-gauge ultrafine troblotted onto PVDF membrane. The membranes were incubated with needle insulin syringes. The administered dosage for each agent followed mouse sera at 1/200 dilution, washed, and then incubated with HRP- protocols as described (18, 22–25). Control mice were given PBS i.p. on the conjugated anti-mouse IgG mAb at a dilution of 1/1,000 (Abcam). The blots same days (Table 1). Survival, body weight, and general conditions were were detected by the ECL Western blotting detection system (Amersham monitored for 21 days or until death. Biosciences). Experiments were conducted in duplicates or triplicates of five mice for each group of treated or control mice. Six mice in each of groups (groups 8, 11, and 12 in Table 1) were killed on days 4, 6, and 8 postchallenge, respectively. Flow Cytometry. Blood cells from the mice were stained with fluorescein- Blood, TPL, lung, brain, kidney, liver, and spleen tissue samples were collected labeled mAbs specific for mouse CD3, CD4, and CD8 (BD Pharmingen) and from these mice, normal uninfected mice, and the surviving mice for his- fixed with 4% p-formaldehyde overnight. The fixed blood cells were analyzed topathologic, immunologic, and virologic assays. by flow cytometry (FACSCaliber; BD) as described (21).

Virologic Tests. Titers of released virus in TPL were determined by TCID50 as Histopathologic Analysis. The lung, brain, spleen, kidney, and liver tissues of described, whereas viral RNA in lung tissues was quantified by real-time challenged mice were immediately fixed in 10% buffered formalin and em- RT-PCR (26, 27). Briefly, total RNA in lysed lung tissues was extracted by using bedded in paraffin wax. Sections 4–6 ␮m in thickness were mounted on slides. a RNeasy Mini kit (Qiagen) and reverse-transcribed to cDNA by using applied Histopathologic changes were examined by H&E staining under a light mi- SuperScript II Reverse Transcriptase (Invitrogen). Viral nucleoprotein gene and croscope as described (3). internal control ␤-actin gene were measured by the SYBR Green Mx3000 Real-Time PCR System (Stratagene), using primers NP-forward, 5Ј-GAC CAG Immunohistochemical Assay. Lung sections were stained as described (3) by GAG TGG AGG AAA CA-3Ј; NP-reverse, 5Ј-CGG CCA TAA TGG TCA CTC TT-3Ј; ␤-actin-forward, 5Ј- CGT ACC ACT GGC ATC GTG AT-5Ј; and ␤-actin-reverse, using an antiinfluenza nucleoprotein mAb (HB65; ATCC) at 1:5,000 dilution, 5Ј-GTG TTG GCG TAC AGG TCT TTG-3Ј. goat anti-mouse IgG, H and L chain-specific biotin conjugate (Calbiochem) at 1:2,000 dilution, and streptavidin/peroxidase complex reagent (Vector ELISA. Proinflammatory cytokines and chemokines IL-1, IL-6, IFN-␥, TNF-␣ (BD Laboratories). Biosciences), PGE2, MIP-1␤ (R&D Systems), leukotriene (GE Healthcare), and lung injury indicator albumin (Bethyl Laboratories) in TPL and serum samples Statistical Analysis. Statistical analysis of survival time and rate were per- were tested by ELISA using the protocol as described (3) with modifications formed by the log rank Kaplan-Meier and ␹2 tests, respectively, whereas the according to the instructions of the kit suppliers. others were calculated by Student’s t test with Stata statistical software. Results were considered significant at P Յ 0.05. The Cox proportional hazards Elastase Activity Assay. Elastase activity in TPL was measured by the addition model was used to estimate hazard ratios. of the elastase-specific chromogenic substrate N-methoxysuccinyl-Ala-Ala- Pro-Val p-nitroanilide (Sigma) at a final concentration of 1 mM. After 30 min ACKNOWLEDGMENTS. This work is partly supported by the Providence at room temperature, the change in optical density at a wavelength of 405 nm Foundation Limited in memory of the late Dr. Lui Hac Minh, the Research was measured. Grant Council, the Hong Kong Special Administrative Region Research Fund for the Control of Infectious Diseases of the Health, Welfare, and Neutralization Assay. Neutralizing antibody levels in mice sera were deter- Food Bureau, the Hong Kong University Special Research Achievement mined by neutralization assay by using the same virus strain for challenge in Award, Croucher Senior Medical Research Fellowship 2006–2007, and The MDCK cells as described (3). Shaw Foundation.

1. Yuen KY, et al. (1998) Clinical features and rapid viral diagnosis of human disease 3. Peiris JS, et al. (2004) Re-emergence of fatal human influenza A subtype H5N1 disease. associated with avian influenza A H5N1 virus. Lancet 351:467–471. Lancet 363:617–669. 2. Beigel JH, et al. (2005) Writing Committee of the World Health Organization (WHO) 4. Wong SS, Yuen KY (2006) Avian influenza virus infections in humans. Chest 129:156–168. consultation on human influenza A/H5 avian influenza A (H5N1) infection in humans. 5. de Jong MD, et al. (2006) Fatal outcome of human influenza A (H5N1) is associated with N Engl J Med 353:1374–1385. high viral load and hypercytokinemia. Nat Med 12:1203–1207.

Zheng et al. PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 ͉ 8095 Downloaded by guest on September 26, 2021 6. Carter MJ (2007) A rationale for using steroids in the treatment of severe cases of H5N1 17. Budd A, et al. (2007) Increased survival after gemfibrozil treatment of severe mouse avian influenza. J Med Microbiol 56:875–883. influenza. Antimicrob Agents Chemother 51:2965–2968. 7. Salomon R, Hoffmann E, Webster RG (2007) Inhibition of the cytokine response does 18. La Gruta NL, Kedzierska K, Stambas J, Doherty PC (2007) A question of self-preservation: not protect against lethal H5N1 influenza infection. Proc Natl Acad Sci USA 104:12479– Immunopathology in influenza virus infection. Immunol Cell Biol 85:85–92. 12481. 19. Szretter KJ, et al. (2007) Role of host cytokine responses in the pathogenesis of avian 8. Leneva IA, Roberts N, Govorkova EA, Goloubeva OG, Webster RG (2000) The neur- H5N1 influenza viruses in mice. J Virol 81:2736–2744. aminidase inhibitor GS4104 (oseltamivir phosphate) is efficacious against A/Hong 20. Uiprasertkul M, et al. (2007) Apoptosis and pathogenesis of avian influenza A (H5N1) Kong/156/97 (H5N1) and A/Hong Kong/1074/99 (H9N2) influenza viruses. Antiviral Res virus in humans. Emerg Infect Dis 13:708–712. 48:101–115. 21. Du LY, et al. (2008) Intranasal vaccination of recombinant adeno-associated virus 9. Govorkova EA, Leneva IA, Goloubeva OG, Bush K, Webster R (2001) Comparison of encoding receptor-binding domain of severe acute respiratory syndrome coronavirus efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other (SARS-CoV) spike protein induces strong mucosal immune responses and provides avian influenza viruses. Antimicrob Agents Chemother 45:2723–2732. long-term protection against SARS-CoV infection. J Immunol 180:948–956. 10. Yen HL, Monto AS, Webster RG, Govorkova EA (2005) Virulence may determine the 22. Smith PW, et al. (1998) Dihydropyrancarboxamides related to zanamivir: A new series necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Viet- of inhibitors of influenza virus sialidases. 1. Discovery, synthesis, biological activity, and nam/1203/04 influenza virus in mice. J Infect Dis 192:665–672. structure-activity relationships of 4-guanidino- and 4-amino-4H-pyran-6-carboxam- 11. Sidwell RW, et al. (2007) Efficacy of orally administered T-705 on lethal avian influenza ides. J Med Chem 41:787–797. A (H5N1) virus infections in mice. Antimicrob Agents Chemother 51:845–851. 23. Ryan DM, Ticehurst J, Dempsey MH, Penn CR (1994) Inhibition of influenza virus 12. Simmons CP, et al. (2007) Prophylactic and therapeutic efficacy of human monoclonal replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N- acetylneura- antibodies against H5N1 influenza. PLoS Med 4:e178. minic acid) is consistent with extracellular activity of viral neuraminidase (sialidase). 13. Carey MA, et al. (2005) Contrasting effects of cyclooxygenase-1 (COX-1) and COX-2 Antimicrob Agents Chemother 38:2270–2275. deficiency on the host response to influenza A viral infection. J Immunol 175:6878– 24. Catalano A, et al. (2004) Preclinical evaluation of the nonsteroidal antiinflammatory 6884. agent celecoxib on malignant mesothelioma chemoprevention. Int J Cancer 109:322– 14. Nielsen OH, Munck LK (2007) Drug insight: Aminosalicylates for the treatment of IBD. 328. Nat Clin Pract Gastroenterol Hepatol 4:160–170. 25. Sudheer Kumar M, Unnikrishnan MK, Uma Devi P (2003) Effect of 5-aminosalicylic acid 15. Go´mez-Munõz A, O’Brien L, Salh B, Steinbrecher UP (2001) 5-Aminosalicylate stimu- on radiation-induced micronuclei in mouse bone marrow. Mutat Res 527:7–14. lates phospholipase D activity in macrophages. Biochim Biophys Acta 1533:110–118. 26. Li BJ, et al. (2005) Using siRNA in prophylactic and therapeutic regimens against SARS 16. Chinetti G, Fruchart JC, Staels B (2000) Peroxisome proliferator-activated receptors coronavirus in Rhesus macaque. Nat Med 11:944–951. (PPARs): Nuclear receptors at the crossroads between lipid metabolism and inflamma- 27. Zheng BJ, et al. (2005) Synthetic peptides outside the spike protein heptad repeat tion. Inflamm Res 49:497–505. regions as potent inhibitors of SARS-associated coronavirus. Antiviral Ther 10:393–403.

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