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An oligodeoxynucleotide capable of lessening acute lung inflammatory injury in mice infected by influenza virus
Biochemical and Biophysical Research Communications 415 (2011) 342–347
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
An oligodeoxynucleotide capable of lessening acute lung inflammatory injury in mice infected by influenza virus Mingli Fang a, Min Wan a, Sheng Guo a, Ran Sun c, Ming Yang b, TieSuo Zhao b, Youyou Yan b, Yongsheng Zhang a, Wenhui Huang a, Xiuli Wu a, Yongli Yu b, Liying Wang a,⇑, Shucheng Hua d,⇑ a
Department of Molecular Biology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China Tissue Bank of China-Japan Union Hospital, Jilin University, Changchun 130021, China d Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun 130021, China b c
a r t i c l e
i n f o
Article history: Received 7 October 2011 Available online 18 October 2011 Keywords: Influenza virus Infection Acute lung inflammatory injury Oligodeoxynucleotide Therapy
a b s t r a c t Infection of influenza virus could induce acute lung inflammatory injury (ALII) that was at least partially caused by excessive innate immune responses. To study whether down-regulating Toll-like receptor (TLR)-mediated innate immune response could lessen influenza virus-induced ALII, a microsatellite DNA mimicking oligodeoxynucleotide (MS ODN), named as SAT05f capable of inhibiting TLR7/9-activation in vitro, was used to treat mice infected with FM1 virus. In parallel, two MS ODNs confirmed with less or no in vitro activities, named as MS19 and MS33, were used as controls. Unexpectedly, SAT05f failed to lessen ALII in the mice, whereas MS19 significantly inhibited the weight loss and displayed dramatic effect on lessening the ALII by reducing consolidation, hemorrhage, intra-alveolar edema and neutrophils infiltration in lungs of the mice. Meanwhile, MS19 could decrease the mortality of influenza virus infected mice and down-regulate TNF-a production in their lungs. The data suggest that MS19 might display its therapeutic role on ALII induced by influenza virus by reducing over-production of TNF-a. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Influenza is a highly infectious respiratory disease caused by influenza virus, posing a great global infectious disease threat. In the 20th century, three influenza pandemics emerged and killed tens of millions of people [1,2]. Among the patients, most deaths occurred in young people, previously healthy adults or children [3]. Analysis showed that one of the main reasons for the death was due to acute respiratory distress syndrome (ARDS) caused by influenza virus-induced inflammatory injury [4]. In recent years, over-activated innate immune response has been found attributable to the development of the influenza virus-induced lung inflammatory injury. Upon infection, influenza virus derived single stranded RNA and double stranded RNA, generated as intermediate molecules, are sensed by toll-like receptors (TLRs), initiating activation of the innate immune cells, such as macrophages and dentritic cells (DC) [5]. The activated cells produce type I interferon and other cytokines to combat the virus ⇑ Corresponding authors. Address: Department of Respiratory Medicine, The First Hospital, Jilin University, Changchun 130021, China (S. Hua), Department of Molecular biology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China (L. Wang). Fax: +86 431 85647872. E-mail addresses: [email protected] (L. Wang), [email protected] (S. Hua). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.10.062
[6]. However, if infection persists, innate immune cells could be over-activated and produce excessive pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1), and interleukin-6 (IL-6), undesirably [7,8]. The over-produced cytokines, as found in basic and clinical studies, contribute to acute lung inflammatory injury (ALII), leading to a fatal outcome [9]. As happened in human infection of highly pathogenic H5N1 avian influenza virus, a high mortality (about 60%) was mainly attributed to the virus induced ALII and the development of ARDS [10]. Obviously, it is required to develop agents to curb the over-reaction and the over-produced cytokines for saving life of the patients infected with influenza virus, especially those highly pathogenic ones. Recently, efforts have been made to develop agents capable of down-regulating the over-whelming innate immune response and inhibiting the over-production of pro-inflammatory cytokines caused by influenza virus. As reported, interference with TNF-a signaling pathway could decrease ALII in mice infected with influenza virus [11]. The involvement of TNF-a in ALII was partially supported by the data that mice lacking TNF-a and IL-1 receptors displayed less lung inflammation and delayed in onset of death following infection with a highly virulent H5N1 Virus [12]. Antagonizing the receptor of monocyte chemoattractant protein-1 (MCP-1) could alleviate ALII, and consequently reduced mortality of mice infected with influenza virus, by inhibiting the migration
M. Fang et al. / Biochemical and Biophysical Research Communications 415 (2011) 342–347
of inflammatory cells to the lungs [13]. Furthermore, erythromycin, an antibiotic with potent anti-inflammatory effects, was found to improve the survival of mice infected with influenza virus [14]. In addition to, DNA molecules from diverse sources have been tested for their potentials in limiting the over-activation of innate immune cells. Initially, synthetic deoxyguanosine oligomers were identified able to inhibit IFN-c production induced by bacterial DNA [15]. Further on, more oligonucleotides (ODNs) were proved with immunosuppressive activities, as exemplified by A151 capable of rescuing mice from endotoxic shock by blocking LPS-induced production of IFN-c and IL-12 [16], a guanosine (G)-rich ODN effective in blocking the production of TNF-a and IL-12p40 in mice with lethal shock [17], and IRS954 functional in reducing the severity of clinical symptoms in lupus-prone mice by antagonizing activation of TLR7 and TLR9 [18]. In our previous study, SAT05f, designed with reference of the sequence of human microsatellite DNA (MS DNA), was shown able to rescue mice from lethal shock by down-regulating TLR7/9 activation [19]. In this study, to test whether the MS ODN could lessen ALII in mice infected by influenza virus, SAT05f was used to treat the mice infected with FM1 virus. In parallel, two MS ODNs, named as MS19 and MS33, were used as controls because compared with SAT05f, MS19 inhibited TNF-a production from murine macrophages induced by CpG ODN, a TLR9 agonist, with much less potency and both of MS19 and MS33 failed to induce CpG ODN stimulated proliferation of murine splenocyte in vitro. Unexpectedly, it was found that MS19 displayed a significant role in lessening ALII in FM1 virus infected mice while SAT05f could not. 2. Materials and methods 2.1. Oligodeoxynucleotides Nuclease-resistant phosphorothioate-modified ODNs were synthesized in Takara Biotechnology Company (Dalian, China). The following ODNs were used in this study: SAT05f (50 -CCTCCTCC TCCTCCTCCTCCTCCT-30 ) [19], MS19 (50 -AAAGAAAGAAAGAAAGAA AGAAAG-30 ) [20], MS33 (50 -AAAAAGAAAAAGAAAAAGAAAAAG-30 ) and CpG 1826 (50 -TCCATGACGTTCCTGACGTT-30 ). All ODNs were diluted in PBS buffer and had no detectable endotoxin (Limulus amebocyte lysate assay, Associates of Cape Cod, Inc.).
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well) were cultured with CpG 1826 (1 lg/ml) alone or with SAT05f (8 lg/ml) or MS19 (8 lg/ml) or MS33 (8 lg/ml) for 48 h. The cells proliferation was determined by MTT assay [21] and expressed as mean OD570 value ± standard deviations (SD) of triplicate wells. 2.5. Animal experiments The mouse model of influenza virus-induced acute lung inflammatory injury (ALII) was established by intranasal inoculation of 10 LD50 of FM1 virus (50 ll) to BALB/c mice anaesthetized intraperitoneally (i.p.) with 2% pentobarbital sodium (50 ll). Mice sham-inoculated with sterile PBS were used as normal control (NC). To study the effect of MS ODN on the morbidity, mortality and acute lung injury (ALI) caused by influenza virus, the model mice were injected i.p. with PBS, SAT05f (25 lg per mouse) or MS19 (25 lg per mouse) or MS33 (25 lg per mouse) on day 2 and 4 post infection, respectively. The weight of mice was measured daily and their survival was recorded. At the end of experiments, lungs of the mice were isolated and fixed for histopathological examination. 2.6. Histological analysis of acute lung injury The lungs of the mice were fixed in 10% neutral buffered formalin and processed routinely. Paraffin sections, 5–10 lm thick, were stained with hematoxylin and eosin and then examined under microscopy in a blinded manner. Pathological scores of the tissue were determined based on the criteria: 0, no pneumonia; 1, mild interstitial pneumonia (<25% of the lung); 2, moderate interstitial pneumonia (25–50% of the lung); 3, severe interstitial pneumonia (>50% of the lung). The neutrophils in the sections were counted by observing 400 per high field and the average was obtained from the neutrophils in five separated fields of the sections from each mouse [22]. 2.7. Viral titer assay The viral titer in mouse lung homogenate was measured by hemagglutination (HA) assay [23].
2.2. Influenza virus and cells
2.8. TNF-a bioassay
FM1 virus, a mouse-adapted strain A/FM/1/47 virus (H1N1), was provided by Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (Beijing, China). The FM1 virus was stored in aliquots at 70 °C. RAW264.7 cells (murine macrophage-like cell line) and L929 cells from American Type Culture Collection (ATCC), were cultured at 37 °C in a 5% CO2 humidified incubator and maintained in complete RPMI 1640 medium (GIBCO).
TNF-a activity in the lung homogenate or supernatants was measured by an L929 cytotoxicity bioassay as described [24]. Briefly, L929 cells (2 104 cells/well) were cultured in a 96-well plate in complete RPMI 1640 medium for 24 h. Then, the culture medium was replaced with the test sample in doubling dilutions with complete RPMI 1640 medium in the present of 1 lg/ml Actinomycin D. The cells were cultured for another 24 h. Cytotoxicity of the cells was determined by MTT assay. TNF-a activity was expressed as the percentage of the L929 cytotoxicity, calculated by following formula: TNF-a activity = [(OD value of medium wells OD value of sample wells)/OD value of medium wells] 100%. The mean ± SD of the percentage of L929 cytotoxicity was calculated with the OD value from triplicate wells.
2.3. Mice Eight-week-old specific pathogen-free female BALB/c mice (20 ± 2 g) were obtained from the Experimental Animal Center, Jilin University (Changchun, China). The mice were maintained at 22 ± 2 °C with a 12-h light/dark cycle, and had free access to food and water for experiments in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.4. Proliferation assay of mouse splenocytes The spleens of BALB/c mice were minced and grinded to prepare for single cell suspension. Typically, the cells (6 105 cells/per
2.9. Statistical analysis The statistical significance of differences was determined using the ANOVA test. Survivals of mice were compared by using the Kaplan–Meier test. Differences were considered statistically significant for p < 0.05. Statistical analyses were performed using SPSS software.
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3. Results 3.1. The inhibitory effect of MS ODN on TLR9 mediated activation in innate immune response in vitro To develop MS ODNs capable of reducing the detrimental ALII caused by influenza virus infection, SAT05f, MS19 and MS33, three synthesized ODNs designed based on the sequence of human microsatellite (MS) DNA in our previous study, were selected as candidate agents. Firstly, we tested the inhibitory effect of MS ODNs on TLR9 activation in a mouse spleen cells proliferation assay. The result showed that SAT05f was capable of inhibiting CpG 1826-induced proliferation of mouse spleen cells significantly (p < 0.01) while MS19 and MS33 failed to (Fig. 1A). Next, to determine whether MS ODNs could block the production of TNF-a, a pro-inflammatory cytokine involved in lung inflammatory injury caused by influenza virus infection [25], we detected the inhibitory effect of MS ODNs on blocking TNF-a production from RAW264.7 cells induced by CpG 1826. RAW264.7 cells were incubated in the medium containing CpG 1826 alone or with SAT05f, MS19 or MS33 for 8 h, and then the supernatants were harvested for assaying TNF-a activity. The results showed that SAT05f could significantly inhibit the TNF-a production from RAW264.7 cells triggered by CpG 1826 (p < 0.01). Notably, compared with SAT05f, MS19 displayed a weaker inhibition on the TNF-a production (p = 0.047) and MS33 failed to (Fig. 1B). Upon the results, SAT05f, MS19 and MS33 were selected to conduct in vivo studies in a mouse model of ALII induced by influenza virus. 3.2. The effect of MS ODN on the morbidity, mortality, and lung inflammatory injury of mice infected with influenza virus In FM1 virus infected mice, we studied the effect of MS ODN on the morbidity, mortality and ALII caused by influenza virus. On day 0, mice were inoculated intranasally with 10 LD50 of FM1 virus. On days 2 and 4 post infection, the model mice were injected i.p. with PBS, SAT05f, MS19 or MS33, respectively, and then observed and weighted daily. As shown in Fig. 2A, MS19 significantly inhibited the weight loss in the model mice at day 7 and 8, while SAT05f and MS33 as well as PBS failed to. From day 9 post infection, the body weight of the mice treated with MS19 was gradually recovered towards the normal. Whereas the body weight of the mice
Fig. 2. Effect of MS ODN on body weight loss and survival of mice infected with influenza virus. (A) Body weight curves. The mean and standard deviation from the mice per group are shown. ⁄P < 0.05 vs. PBS group. (B) Survival curves.
treated with SAT05f and MS33 as well as PBS was not recovered until the end of experiment. As shown in Fig. 2B, the mortality rates of the mice treated with MS33 or PBS or SAT05f or MS19 were 100%, 87.5%, 62.5% or 37.5%, respectively, indicating the more potential of MS19 on inhibiting lung inflammation responses. At the end of experiments, their lungs of the mice were removed for gross observation and histopathological examination. As shown in Fig. 3A, the lungs of the mice treated with PBS, MS33 and SAT05f displayed edema, consolidation and profuse hemorrhage. Unexpectedly, MS19 could almost completely prohibit the development of the changes. Histopathologically, mice treated with PBS, MS33 and SAT05f showed thickened and congested alveolar walls, intra-alveolar edema, and numerous infiltrated neutrophils in their lung tissues. In contrast, mice treated
Fig. 1. The inhibitory effect of MS ODN on TLR9 mediated activation in innate immune response in vitro. (A) Effect of MS ODN on CpG ODN induced proliferation of mouse splenocyte. (B) Effect of MS ODN on CpG ODN induced TNF-a production from RAW264.7 cells. Each bar represents mean value ± SD. ⁄⁄p < 0.01 vs. CpG 1826, ⁄p < 0.01 vs. CpG 1826.
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Fig. 3. Inhibitory role of MS ODN on ALII of mice infected with influenza virus. The images of gross and histopathological change of lung from a representative animal in each group are shown. (A) Macroscopic appearance of mouse lungs. (B) Pathological changes of lung tissues (H&E stain, Original magnifications: top row, 40; bottom row, 400). The arrow indicated the infiltrated neutrophilis. (C) The average number of infiltrated neutrophilis per high power field on H&E-stained lung tissue sections. (400 high power field). (D) Pathological scores. Each bar represents the mean for each group ± SD (n = 3, ⁄p < 0.05 vs. PBS group).
with MS19 showed no or much less the changes in their lung tissues (Fig. 3B). Further quantification of neutrophils per high field showed that the neutrophils of lung tissues in the model mice treated with MS19 were significantly less than that in mice treated with PBS (p 0:05). No obvious difference of the infiltrated neutrophils was observed among the mice treated with PBS, MS33 or SAT05f (P 0:05) (Fig. 3C). The pathological score of the lung tissues in MS19-treated mice was lower than that in mice treated with PBS, MS33 and SAT05f (P 0:05) (Fig. 3D). The results demonstrate that MS19 can lessen lung injury of the mice infected with influenza virus. 3.3. The effect of MS19 on the production of TNF-a and amplification of influenza virus in lung tissue of mice infected with FM1 virus To find how MS19 inhibited the development of ALII caused by FM1 virus, we detected the production of TNF-a and replication of influenza virus in lung tissues of the mice infected with FM1 virus. The mice were treated with PBS or MS19 on day 1 and 3 post infection and sacrificed on day 2 and 4, respectively. Their lungs were used to prepare for homogenates for analyzing TNF-a biological activity and FM1 virus replication. As shown in Fig. 4A, on day 2 post infection, the activity of TNF-a in the lung homogenate of mice treated with PBS was two-fold higher than that of the normal mice. Comparatively, the activity of TNF-a in the mice treated with MS19 was obviously lower than that in the model mice treated with PBS, suggesting that MS19 could inhibit TNF-a production induced by FM1 virus. Upon that the FM1 virus titer reached its peak in lungs of the model mice, we detected the virus titers in the
homogenates collected on day 4 after infection. As shown in Fig. 4B, FM1 virus titers in lung homogenates of the mice treated with MS19 was within the same range as that of the homogenates from the mice treated with PBS, suggesting that MS19 mediated inhibition on the development of ALII induced by FM1 virus was unrelated to the direct action of MS19 on replication of FM1 virus. 4. Discussion In this study, we found that MS19 displayed dramatic effect on lessening the influenza virus induced-ALII in mice with a decreased production of TNF-a, a pro-inflammatory cytokine, in their lungs. The data indicate that TNF-a plays a role in the injury and MS19 could lessen the injury by inhibiting TNF-a production. The inhibition could be correlated with the reduced infiltration of neutrophils because our data showed that much less neutrophils were infiltrated in the lung tissues of the mice treated with MS19, in response to influenza virus challenge. It is established that neutrophils, as sources of TNF-a, are contributors to lung damage induced by influenza virus. Evidence from other studies in humans or mice supports the TNF-a involvement in the influenza virus induced ALI by showing that the level of circulating TNF-a in the serum of the patient infected with H1N1 influenza virus was directly correlated with degree of illness [26] and that the strain of influenza virus in 1918 induced high level of TNF-a in fatally infected mice [27]. The TNF-a involvement was also showed by our observation that the TNF-a in the lung of the mice infected with FM1 virus rose rapidly on day 2 post infection and then maintained at a high level until the mice died (data not shown). However, it is
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Fig. 4. Effect of MS19 on TNF-a production and FM1 virus load in lung homogenates from mice infected with influenza virus. (A) Activity of TNF-a. (B) Virus titer. Each symbol represents the value from one mouse. The horizontal line represents the mean value ± SD for each group.
worthwhile to note that the inhibition of TNF-a might not be translated to the protection of mice from influenza virus infection. In influenza virus infected mice, TNF-a depletion with anti-TNF antibody reduced pulmonary recruitment of inflammatory cells, cytokine production by T cells and the severity of illness without preventing virus clearance [28]. The similar phenomenon was also observed in our study that MS19 could reduce the ALII by inhibition of the production of TNF-a, but failed to inhibit the replication of influenza virus, which might be correlated with the inability of MS19 to prolong the survival time of the mice. The inability was verified by gene deletion studies, showing that TNF-a deficient mice displayed the similar mortality to wild-type animals when infected with H5N1 influenza virus [29]. It is known that TLR7 is expressed in lung plasmacytoid dendritic cells (pDCs) in mice and influenza virus is a natural ligand of TLR7 [30]. TLR7 activation in pDCs plays a cell-specific role against influenza virus through MyD88-dependent IFN-a induction. However, MyD88-deficient mice can still produce type I IFN, control viral replication, and recover from the infection, suggesting that other pathways can compensate for TLR7 initiated signaling pathway during influenza virus infection [31]. Recently, it has been found that influenza virus can be recognized by retinoic acid inducible gene I (RIG-I) and nucleotide binding oligomerization domain (NOD)-like receptors (NLRs). The recognition leads to activation of inflammasome, resulting in the processing and release of cytokines, such as the pro-inflammatory cytokines interleukin (IL)-1b and IL-18, causing lung inflammation [32]. Based on these, we speculate that MS19 might display its inhibitory activity on lung inflammation and injuries caused by influenza virus in a TLR7 independent way. The speculation is supported by our unpublished data that MS19 fails to inhibit TNF-a production from TLR7 expressing murine macrophage-like cell (RAW264.7 cells) induced by the FM1 influenza virus. In addition to TLR7, TLR9 is also expressed in bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells in mice [33]. Because of lacking evidence for direct contributions of TLR9 activation to influenza virus caused ALII, it is hardly to correlate TLR9 activation with the MS19 mediated alleviation on ALII of mice infected with influenza virus. However, our data might reveal that TLR9 activation could involve in the development of ALII and MS19 could alleviate the ALII by antagonizing TLR9 activation, at least partially. As showed in this study, MS19 could reduce the CpG ODN (TLR9 agonist) induced TNF-a from RAW macrophages in vitro, though the effect was significantly weaker than that displayed by SAT05f, a TLR9 antagonist. Considering that influenza virus cannot be directly sensed by TLR9 in lung and influenza virus infection can lead to cell death in bronchial/bronchiolar epithelial cells, alveolar cells and lymphoid cells in mice [34], we may speculate that MS19 could block the TLR9 activation induced by
the released nucleic acids from the dead cells, to some extent, because the released damage-associated molecular patterns including endogenous nucleic acids from the dead cells can be sensed by TLR9, resulting in acute inflammation in mouse model, characterized by rapid influx of neutrophils [35]. Compatibly, MS19 did reduce the neutrophil infiltration in lung of the mice infected with influenza virus. Furthermore, it is worthy of note that MS19, compared with SAT05f, might posses superior pharmacokinetic qualities that make it easier to penetrate into the lung and persist there longer, therefore displaying obvious inhibition on the development of ALII in mice infected with influenza virus. To address this, further delicate analysis is required to study the pharmacokinetic behaviors of MS19 in lung of the mice. Acknowledgments This works were supported by National Nature Scientific Foundation of China (No. 31070800 and 81172843). References [1] N.P. Johnson, J. Mueller, Updating the accounts: global mortality of the 1918– 1920 Spanish influenza pandemic, Bull. Hist. Med. 76 (2002) 105–115. [2] Y. Kawaoka, S. Krauss, R.G. Webster, Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics, Virology 194 (1993) 781–788. [3] L. Magali, C. Fabrice, Comparative age distribution of influenza morbidity and mortality during seasonal influenza epidemics and the 2009 H1N1 pandemic, BMC Infectious Diseases 10 (2010) 162–167. [4] R. Perez-Padilla, D. de la Rosa-Zamboni, S. Ponce de Leon, et al., Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico, N. Engl. J. Med. 361 (2009) 680–689. [5] S.J. Gibson, J.M. Lindh, T.R. Riter, et al., Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod, Cell Immunol. 218 (2002) 74–86. [6] A.P. Carine, T. Giorgio, Production of type I interferons: plasmacytoid dendritic cells and beyond, J. Exp. Med. 202 (2005) 461–465. [7] L.A. Perrone, J.K. Plowden, A. García-Sastre, et al., H5N1 and 1918 pandemic Influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice, PLoS Pathog. 4 (2008). [8] D. Us, Cytokine storm in avian influenza, Mikrobiyol. Bul. 42 (2008) 365–380. [9] H. Kawashima, S. Go, Y. Kashiwagi, et al., Cytokine profiles of suction pulmonary secretions from children infected with pandemic influenza A (H1N1) 2009, Crit. Care 14 (2010) 411. [10] A.N. Abdel-Ghafar, T. Chotpitayasunondh, Z.C. Gao, et al., Update on avian influenza A (H5N1) virus infection in humans, N. Engl. J. Med. 358 (2008) 261– 273. [11] A. Srikiatkhachorn, J. Chintapalli, J. Liu, et al., Interference with intraepithelial TNF-a signaling inhibits CD8 (+) T-cell-mediated lung injury in influenza infection, Viral Immunol. 23 (2010) 639–645. [12] L.A. Perrone, K.J. Szretter, J.M. Katz, et al., Mice lacking both TNF-a and IL-1 receptors exhibit reduced lung inflammation and delay in onset of death following infection with a highly virulent H5N1 virus, J. Infect. Dis. 202 (2010) 1161–1170. [13] K.L. Lin, S. Sweeney, B.D. Kang, et al., CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection, J. Immunol. 186 (2011) 508–515.
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An oligodeoxynucleotide capable of lessening acute lung inflammatory injury in mice infected by influenza virus Mingli Fang a, Min Wan a, Sheng Guo a, Ran Sun c, Ming Yang b, TieSuo Zhao b, Youyou Yan b, Yongsheng Zhang a, Wenhui Huang a, Xiuli Wu a, Yongli Yu b, Liying Wang a,⇑, Shucheng Hua d,⇑ a
Department of Molecular Biology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China Tissue Bank of China-Japan Union Hospital, Jilin University, Changchun 130021, China d Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun 130021, China b c
a r t i c l e
i n f o
Article history: Received 7 October 2011 Available online 18 October 2011 Keywords: Influenza virus Infection Acute lung inflammatory injury Oligodeoxynucleotide Therapy
a b s t r a c t Infection of influenza virus could induce acute lung inflammatory injury (ALII) that was at least partially caused by excessive innate immune responses. To study whether down-regulating Toll-like receptor (TLR)-mediated innate immune response could lessen influenza virus-induced ALII, a microsatellite DNA mimicking oligodeoxynucleotide (MS ODN), named as SAT05f capable of inhibiting TLR7/9-activation in vitro, was used to treat mice infected with FM1 virus. In parallel, two MS ODNs confirmed with less or no in vitro activities, named as MS19 and MS33, were used as controls. Unexpectedly, SAT05f failed to lessen ALII in the mice, whereas MS19 significantly inhibited the weight loss and displayed dramatic effect on lessening the ALII by reducing consolidation, hemorrhage, intra-alveolar edema and neutrophils infiltration in lungs of the mice. Meanwhile, MS19 could decrease the mortality of influenza virus infected mice and down-regulate TNF-a production in their lungs. The data suggest that MS19 might display its therapeutic role on ALII induced by influenza virus by reducing over-production of TNF-a. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Influenza is a highly infectious respiratory disease caused by influenza virus, posing a great global infectious disease threat. In the 20th century, three influenza pandemics emerged and killed tens of millions of people [1,2]. Among the patients, most deaths occurred in young people, previously healthy adults or children [3]. Analysis showed that one of the main reasons for the death was due to acute respiratory distress syndrome (ARDS) caused by influenza virus-induced inflammatory injury [4]. In recent years, over-activated innate immune response has been found attributable to the development of the influenza virus-induced lung inflammatory injury. Upon infection, influenza virus derived single stranded RNA and double stranded RNA, generated as intermediate molecules, are sensed by toll-like receptors (TLRs), initiating activation of the innate immune cells, such as macrophages and dentritic cells (DC) [5]. The activated cells produce type I interferon and other cytokines to combat the virus ⇑ Corresponding authors. Address: Department of Respiratory Medicine, The First Hospital, Jilin University, Changchun 130021, China (S. Hua), Department of Molecular biology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China (L. Wang). Fax: +86 431 85647872. E-mail addresses: [email protected] (L. Wang), [email protected] (S. Hua). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.10.062
[6]. However, if infection persists, innate immune cells could be over-activated and produce excessive pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1), and interleukin-6 (IL-6), undesirably [7,8]. The over-produced cytokines, as found in basic and clinical studies, contribute to acute lung inflammatory injury (ALII), leading to a fatal outcome [9]. As happened in human infection of highly pathogenic H5N1 avian influenza virus, a high mortality (about 60%) was mainly attributed to the virus induced ALII and the development of ARDS [10]. Obviously, it is required to develop agents to curb the over-reaction and the over-produced cytokines for saving life of the patients infected with influenza virus, especially those highly pathogenic ones. Recently, efforts have been made to develop agents capable of down-regulating the over-whelming innate immune response and inhibiting the over-production of pro-inflammatory cytokines caused by influenza virus. As reported, interference with TNF-a signaling pathway could decrease ALII in mice infected with influenza virus [11]. The involvement of TNF-a in ALII was partially supported by the data that mice lacking TNF-a and IL-1 receptors displayed less lung inflammation and delayed in onset of death following infection with a highly virulent H5N1 Virus [12]. Antagonizing the receptor of monocyte chemoattractant protein-1 (MCP-1) could alleviate ALII, and consequently reduced mortality of mice infected with influenza virus, by inhibiting the migration
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of inflammatory cells to the lungs [13]. Furthermore, erythromycin, an antibiotic with potent anti-inflammatory effects, was found to improve the survival of mice infected with influenza virus [14]. In addition to, DNA molecules from diverse sources have been tested for their potentials in limiting the over-activation of innate immune cells. Initially, synthetic deoxyguanosine oligomers were identified able to inhibit IFN-c production induced by bacterial DNA [15]. Further on, more oligonucleotides (ODNs) were proved with immunosuppressive activities, as exemplified by A151 capable of rescuing mice from endotoxic shock by blocking LPS-induced production of IFN-c and IL-12 [16], a guanosine (G)-rich ODN effective in blocking the production of TNF-a and IL-12p40 in mice with lethal shock [17], and IRS954 functional in reducing the severity of clinical symptoms in lupus-prone mice by antagonizing activation of TLR7 and TLR9 [18]. In our previous study, SAT05f, designed with reference of the sequence of human microsatellite DNA (MS DNA), was shown able to rescue mice from lethal shock by down-regulating TLR7/9 activation [19]. In this study, to test whether the MS ODN could lessen ALII in mice infected by influenza virus, SAT05f was used to treat the mice infected with FM1 virus. In parallel, two MS ODNs, named as MS19 and MS33, were used as controls because compared with SAT05f, MS19 inhibited TNF-a production from murine macrophages induced by CpG ODN, a TLR9 agonist, with much less potency and both of MS19 and MS33 failed to induce CpG ODN stimulated proliferation of murine splenocyte in vitro. Unexpectedly, it was found that MS19 displayed a significant role in lessening ALII in FM1 virus infected mice while SAT05f could not. 2. Materials and methods 2.1. Oligodeoxynucleotides Nuclease-resistant phosphorothioate-modified ODNs were synthesized in Takara Biotechnology Company (Dalian, China). The following ODNs were used in this study: SAT05f (50 -CCTCCTCC TCCTCCTCCTCCTCCT-30 ) [19], MS19 (50 -AAAGAAAGAAAGAAAGAA AGAAAG-30 ) [20], MS33 (50 -AAAAAGAAAAAGAAAAAGAAAAAG-30 ) and CpG 1826 (50 -TCCATGACGTTCCTGACGTT-30 ). All ODNs were diluted in PBS buffer and had no detectable endotoxin (Limulus amebocyte lysate assay, Associates of Cape Cod, Inc.).
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well) were cultured with CpG 1826 (1 lg/ml) alone or with SAT05f (8 lg/ml) or MS19 (8 lg/ml) or MS33 (8 lg/ml) for 48 h. The cells proliferation was determined by MTT assay [21] and expressed as mean OD570 value ± standard deviations (SD) of triplicate wells. 2.5. Animal experiments The mouse model of influenza virus-induced acute lung inflammatory injury (ALII) was established by intranasal inoculation of 10 LD50 of FM1 virus (50 ll) to BALB/c mice anaesthetized intraperitoneally (i.p.) with 2% pentobarbital sodium (50 ll). Mice sham-inoculated with sterile PBS were used as normal control (NC). To study the effect of MS ODN on the morbidity, mortality and acute lung injury (ALI) caused by influenza virus, the model mice were injected i.p. with PBS, SAT05f (25 lg per mouse) or MS19 (25 lg per mouse) or MS33 (25 lg per mouse) on day 2 and 4 post infection, respectively. The weight of mice was measured daily and their survival was recorded. At the end of experiments, lungs of the mice were isolated and fixed for histopathological examination. 2.6. Histological analysis of acute lung injury The lungs of the mice were fixed in 10% neutral buffered formalin and processed routinely. Paraffin sections, 5–10 lm thick, were stained with hematoxylin and eosin and then examined under microscopy in a blinded manner. Pathological scores of the tissue were determined based on the criteria: 0, no pneumonia; 1, mild interstitial pneumonia (<25% of the lung); 2, moderate interstitial pneumonia (25–50% of the lung); 3, severe interstitial pneumonia (>50% of the lung). The neutrophils in the sections were counted by observing 400 per high field and the average was obtained from the neutrophils in five separated fields of the sections from each mouse [22]. 2.7. Viral titer assay The viral titer in mouse lung homogenate was measured by hemagglutination (HA) assay [23].
2.2. Influenza virus and cells
2.8. TNF-a bioassay
FM1 virus, a mouse-adapted strain A/FM/1/47 virus (H1N1), was provided by Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (Beijing, China). The FM1 virus was stored in aliquots at 70 °C. RAW264.7 cells (murine macrophage-like cell line) and L929 cells from American Type Culture Collection (ATCC), were cultured at 37 °C in a 5% CO2 humidified incubator and maintained in complete RPMI 1640 medium (GIBCO).
TNF-a activity in the lung homogenate or supernatants was measured by an L929 cytotoxicity bioassay as described [24]. Briefly, L929 cells (2 104 cells/well) were cultured in a 96-well plate in complete RPMI 1640 medium for 24 h. Then, the culture medium was replaced with the test sample in doubling dilutions with complete RPMI 1640 medium in the present of 1 lg/ml Actinomycin D. The cells were cultured for another 24 h. Cytotoxicity of the cells was determined by MTT assay. TNF-a activity was expressed as the percentage of the L929 cytotoxicity, calculated by following formula: TNF-a activity = [(OD value of medium wells OD value of sample wells)/OD value of medium wells] 100%. The mean ± SD of the percentage of L929 cytotoxicity was calculated with the OD value from triplicate wells.
2.3. Mice Eight-week-old specific pathogen-free female BALB/c mice (20 ± 2 g) were obtained from the Experimental Animal Center, Jilin University (Changchun, China). The mice were maintained at 22 ± 2 °C with a 12-h light/dark cycle, and had free access to food and water for experiments in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.4. Proliferation assay of mouse splenocytes The spleens of BALB/c mice were minced and grinded to prepare for single cell suspension. Typically, the cells (6 105 cells/per
2.9. Statistical analysis The statistical significance of differences was determined using the ANOVA test. Survivals of mice were compared by using the Kaplan–Meier test. Differences were considered statistically significant for p < 0.05. Statistical analyses were performed using SPSS software.
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3. Results 3.1. The inhibitory effect of MS ODN on TLR9 mediated activation in innate immune response in vitro To develop MS ODNs capable of reducing the detrimental ALII caused by influenza virus infection, SAT05f, MS19 and MS33, three synthesized ODNs designed based on the sequence of human microsatellite (MS) DNA in our previous study, were selected as candidate agents. Firstly, we tested the inhibitory effect of MS ODNs on TLR9 activation in a mouse spleen cells proliferation assay. The result showed that SAT05f was capable of inhibiting CpG 1826-induced proliferation of mouse spleen cells significantly (p < 0.01) while MS19 and MS33 failed to (Fig. 1A). Next, to determine whether MS ODNs could block the production of TNF-a, a pro-inflammatory cytokine involved in lung inflammatory injury caused by influenza virus infection [25], we detected the inhibitory effect of MS ODNs on blocking TNF-a production from RAW264.7 cells induced by CpG 1826. RAW264.7 cells were incubated in the medium containing CpG 1826 alone or with SAT05f, MS19 or MS33 for 8 h, and then the supernatants were harvested for assaying TNF-a activity. The results showed that SAT05f could significantly inhibit the TNF-a production from RAW264.7 cells triggered by CpG 1826 (p < 0.01). Notably, compared with SAT05f, MS19 displayed a weaker inhibition on the TNF-a production (p = 0.047) and MS33 failed to (Fig. 1B). Upon the results, SAT05f, MS19 and MS33 were selected to conduct in vivo studies in a mouse model of ALII induced by influenza virus. 3.2. The effect of MS ODN on the morbidity, mortality, and lung inflammatory injury of mice infected with influenza virus In FM1 virus infected mice, we studied the effect of MS ODN on the morbidity, mortality and ALII caused by influenza virus. On day 0, mice were inoculated intranasally with 10 LD50 of FM1 virus. On days 2 and 4 post infection, the model mice were injected i.p. with PBS, SAT05f, MS19 or MS33, respectively, and then observed and weighted daily. As shown in Fig. 2A, MS19 significantly inhibited the weight loss in the model mice at day 7 and 8, while SAT05f and MS33 as well as PBS failed to. From day 9 post infection, the body weight of the mice treated with MS19 was gradually recovered towards the normal. Whereas the body weight of the mice
Fig. 2. Effect of MS ODN on body weight loss and survival of mice infected with influenza virus. (A) Body weight curves. The mean and standard deviation from the mice per group are shown. ⁄P < 0.05 vs. PBS group. (B) Survival curves.
treated with SAT05f and MS33 as well as PBS was not recovered until the end of experiment. As shown in Fig. 2B, the mortality rates of the mice treated with MS33 or PBS or SAT05f or MS19 were 100%, 87.5%, 62.5% or 37.5%, respectively, indicating the more potential of MS19 on inhibiting lung inflammation responses. At the end of experiments, their lungs of the mice were removed for gross observation and histopathological examination. As shown in Fig. 3A, the lungs of the mice treated with PBS, MS33 and SAT05f displayed edema, consolidation and profuse hemorrhage. Unexpectedly, MS19 could almost completely prohibit the development of the changes. Histopathologically, mice treated with PBS, MS33 and SAT05f showed thickened and congested alveolar walls, intra-alveolar edema, and numerous infiltrated neutrophils in their lung tissues. In contrast, mice treated
Fig. 1. The inhibitory effect of MS ODN on TLR9 mediated activation in innate immune response in vitro. (A) Effect of MS ODN on CpG ODN induced proliferation of mouse splenocyte. (B) Effect of MS ODN on CpG ODN induced TNF-a production from RAW264.7 cells. Each bar represents mean value ± SD. ⁄⁄p < 0.01 vs. CpG 1826, ⁄p < 0.01 vs. CpG 1826.
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Fig. 3. Inhibitory role of MS ODN on ALII of mice infected with influenza virus. The images of gross and histopathological change of lung from a representative animal in each group are shown. (A) Macroscopic appearance of mouse lungs. (B) Pathological changes of lung tissues (H&E stain, Original magnifications: top row, 40; bottom row, 400). The arrow indicated the infiltrated neutrophilis. (C) The average number of infiltrated neutrophilis per high power field on H&E-stained lung tissue sections. (400 high power field). (D) Pathological scores. Each bar represents the mean for each group ± SD (n = 3, ⁄p < 0.05 vs. PBS group).
with MS19 showed no or much less the changes in their lung tissues (Fig. 3B). Further quantification of neutrophils per high field showed that the neutrophils of lung tissues in the model mice treated with MS19 were significantly less than that in mice treated with PBS (p 0:05). No obvious difference of the infiltrated neutrophils was observed among the mice treated with PBS, MS33 or SAT05f (P 0:05) (Fig. 3C). The pathological score of the lung tissues in MS19-treated mice was lower than that in mice treated with PBS, MS33 and SAT05f (P 0:05) (Fig. 3D). The results demonstrate that MS19 can lessen lung injury of the mice infected with influenza virus. 3.3. The effect of MS19 on the production of TNF-a and amplification of influenza virus in lung tissue of mice infected with FM1 virus To find how MS19 inhibited the development of ALII caused by FM1 virus, we detected the production of TNF-a and replication of influenza virus in lung tissues of the mice infected with FM1 virus. The mice were treated with PBS or MS19 on day 1 and 3 post infection and sacrificed on day 2 and 4, respectively. Their lungs were used to prepare for homogenates for analyzing TNF-a biological activity and FM1 virus replication. As shown in Fig. 4A, on day 2 post infection, the activity of TNF-a in the lung homogenate of mice treated with PBS was two-fold higher than that of the normal mice. Comparatively, the activity of TNF-a in the mice treated with MS19 was obviously lower than that in the model mice treated with PBS, suggesting that MS19 could inhibit TNF-a production induced by FM1 virus. Upon that the FM1 virus titer reached its peak in lungs of the model mice, we detected the virus titers in the
homogenates collected on day 4 after infection. As shown in Fig. 4B, FM1 virus titers in lung homogenates of the mice treated with MS19 was within the same range as that of the homogenates from the mice treated with PBS, suggesting that MS19 mediated inhibition on the development of ALII induced by FM1 virus was unrelated to the direct action of MS19 on replication of FM1 virus. 4. Discussion In this study, we found that MS19 displayed dramatic effect on lessening the influenza virus induced-ALII in mice with a decreased production of TNF-a, a pro-inflammatory cytokine, in their lungs. The data indicate that TNF-a plays a role in the injury and MS19 could lessen the injury by inhibiting TNF-a production. The inhibition could be correlated with the reduced infiltration of neutrophils because our data showed that much less neutrophils were infiltrated in the lung tissues of the mice treated with MS19, in response to influenza virus challenge. It is established that neutrophils, as sources of TNF-a, are contributors to lung damage induced by influenza virus. Evidence from other studies in humans or mice supports the TNF-a involvement in the influenza virus induced ALI by showing that the level of circulating TNF-a in the serum of the patient infected with H1N1 influenza virus was directly correlated with degree of illness [26] and that the strain of influenza virus in 1918 induced high level of TNF-a in fatally infected mice [27]. The TNF-a involvement was also showed by our observation that the TNF-a in the lung of the mice infected with FM1 virus rose rapidly on day 2 post infection and then maintained at a high level until the mice died (data not shown). However, it is
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Fig. 4. Effect of MS19 on TNF-a production and FM1 virus load in lung homogenates from mice infected with influenza virus. (A) Activity of TNF-a. (B) Virus titer. Each symbol represents the value from one mouse. The horizontal line represents the mean value ± SD for each group.
worthwhile to note that the inhibition of TNF-a might not be translated to the protection of mice from influenza virus infection. In influenza virus infected mice, TNF-a depletion with anti-TNF antibody reduced pulmonary recruitment of inflammatory cells, cytokine production by T cells and the severity of illness without preventing virus clearance [28]. The similar phenomenon was also observed in our study that MS19 could reduce the ALII by inhibition of the production of TNF-a, but failed to inhibit the replication of influenza virus, which might be correlated with the inability of MS19 to prolong the survival time of the mice. The inability was verified by gene deletion studies, showing that TNF-a deficient mice displayed the similar mortality to wild-type animals when infected with H5N1 influenza virus [29]. It is known that TLR7 is expressed in lung plasmacytoid dendritic cells (pDCs) in mice and influenza virus is a natural ligand of TLR7 [30]. TLR7 activation in pDCs plays a cell-specific role against influenza virus through MyD88-dependent IFN-a induction. However, MyD88-deficient mice can still produce type I IFN, control viral replication, and recover from the infection, suggesting that other pathways can compensate for TLR7 initiated signaling pathway during influenza virus infection [31]. Recently, it has been found that influenza virus can be recognized by retinoic acid inducible gene I (RIG-I) and nucleotide binding oligomerization domain (NOD)-like receptors (NLRs). The recognition leads to activation of inflammasome, resulting in the processing and release of cytokines, such as the pro-inflammatory cytokines interleukin (IL)-1b and IL-18, causing lung inflammation [32]. Based on these, we speculate that MS19 might display its inhibitory activity on lung inflammation and injuries caused by influenza virus in a TLR7 independent way. The speculation is supported by our unpublished data that MS19 fails to inhibit TNF-a production from TLR7 expressing murine macrophage-like cell (RAW264.7 cells) induced by the FM1 influenza virus. In addition to TLR7, TLR9 is also expressed in bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells in mice [33]. Because of lacking evidence for direct contributions of TLR9 activation to influenza virus caused ALII, it is hardly to correlate TLR9 activation with the MS19 mediated alleviation on ALII of mice infected with influenza virus. However, our data might reveal that TLR9 activation could involve in the development of ALII and MS19 could alleviate the ALII by antagonizing TLR9 activation, at least partially. As showed in this study, MS19 could reduce the CpG ODN (TLR9 agonist) induced TNF-a from RAW macrophages in vitro, though the effect was significantly weaker than that displayed by SAT05f, a TLR9 antagonist. Considering that influenza virus cannot be directly sensed by TLR9 in lung and influenza virus infection can lead to cell death in bronchial/bronchiolar epithelial cells, alveolar cells and lymphoid cells in mice [34], we may speculate that MS19 could block the TLR9 activation induced by
the released nucleic acids from the dead cells, to some extent, because the released damage-associated molecular patterns including endogenous nucleic acids from the dead cells can be sensed by TLR9, resulting in acute inflammation in mouse model, characterized by rapid influx of neutrophils [35]. Compatibly, MS19 did reduce the neutrophil infiltration in lung of the mice infected with influenza virus. Furthermore, it is worthy of note that MS19, compared with SAT05f, might posses superior pharmacokinetic qualities that make it easier to penetrate into the lung and persist there longer, therefore displaying obvious inhibition on the development of ALII in mice infected with influenza virus. To address this, further delicate analysis is required to study the pharmacokinetic behaviors of MS19 in lung of the mice. Acknowledgments This works were supported by National Nature Scientific Foundation of China (No. 31070800 and 81172843). References [1] N.P. Johnson, J. Mueller, Updating the accounts: global mortality of the 1918– 1920 Spanish influenza pandemic, Bull. Hist. Med. 76 (2002) 105–115. [2] Y. Kawaoka, S. Krauss, R.G. Webster, Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics, Virology 194 (1993) 781–788. [3] L. Magali, C. Fabrice, Comparative age distribution of influenza morbidity and mortality during seasonal influenza epidemics and the 2009 H1N1 pandemic, BMC Infectious Diseases 10 (2010) 162–167. [4] R. Perez-Padilla, D. de la Rosa-Zamboni, S. Ponce de Leon, et al., Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico, N. Engl. J. Med. 361 (2009) 680–689. [5] S.J. Gibson, J.M. Lindh, T.R. Riter, et al., Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod, Cell Immunol. 218 (2002) 74–86. [6] A.P. Carine, T. Giorgio, Production of type I interferons: plasmacytoid dendritic cells and beyond, J. Exp. Med. 202 (2005) 461–465. [7] L.A. Perrone, J.K. Plowden, A. García-Sastre, et al., H5N1 and 1918 pandemic Influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice, PLoS Pathog. 4 (2008). [8] D. Us, Cytokine storm in avian influenza, Mikrobiyol. Bul. 42 (2008) 365–380. [9] H. Kawashima, S. Go, Y. Kashiwagi, et al., Cytokine profiles of suction pulmonary secretions from children infected with pandemic influenza A (H1N1) 2009, Crit. Care 14 (2010) 411. [10] A.N. Abdel-Ghafar, T. Chotpitayasunondh, Z.C. Gao, et al., Update on avian influenza A (H5N1) virus infection in humans, N. Engl. J. Med. 358 (2008) 261– 273. [11] A. Srikiatkhachorn, J. Chintapalli, J. Liu, et al., Interference with intraepithelial TNF-a signaling inhibits CD8 (+) T-cell-mediated lung injury in influenza infection, Viral Immunol. 23 (2010) 639–645. [12] L.A. Perrone, K.J. Szretter, J.M. Katz, et al., Mice lacking both TNF-a and IL-1 receptors exhibit reduced lung inflammation and delay in onset of death following infection with a highly virulent H5N1 virus, J. Infect. Dis. 202 (2010) 1161–1170. [13] K.L. Lin, S. Sweeney, B.D. Kang, et al., CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection, J. Immunol. 186 (2011) 508–515.
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