Proinflammatory Cytokine Responses in Extra-Respiratory Tissues During Severe Influenza

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1 The Journal of Infectious Diseases BRIEF REPORT Proinflammatory Cytokine Responses in Extra-Respiratory Tissues During Severe Influenza Kirsty R. Short, 1,2 Rebecca Veeris, 1 Lonneke M. Leijten, 1 Judith M. van den Brand, 1 Victor L. Jong, 1,3 Koert Stittelaar, 4 Ab D. M. E. Osterhaus, 1,5 Arno Andeweg, 1 and Debby van Riel 1 1 Department of Viroscience, Erasmus University Medical Center, Rotterdam, The Netherlands; 2 School of Biomedical Sciences, University of Queensland, Brisbane, Australia; 3 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, and 4 Viroclinics Biosciences BV, Rotterdam, The Netherlands; and 5 Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Hannover, Germany Severe influenza is often associated with disease manifestations outside the respiratory tract. While proinflammatory cytokines can be detected in the lungs and blood of infected patients, the role of extra-respiratory organs in the production of proinflammatory cytokines is unknown. Here, we show that both 2009 pandemic influenza A () virus and highly pathogenic avian influenza A () virus induce expression of tumor necrosis factor α, interleukin-6, and interleukin-8 in the respiratory tract and central nervous system. In addition, virus induced cytokines in the heart, pancreas, spleen, liver, and jejunum. Together, these data suggest that extra-respiratory tissues contribute to systemic cytokine responses, which may increase the severity of influenza. Keywords. cytokines; cytokine storm; extra-respiratory responses; ; ; influenza; pathogenesis; systemic disease. Severe influenza virus infections are typically associated with a dysregulated proinflammatory cytokine response in the lungs and blood of infected individuals, a condition often referred to as a cytokine storm [1]. Compared to seasonal influenza A () viruses, the highly pathogenic avian influenza A (HPAI) virus is a more potent inducer of proinflammatory pulmonary cytokines in vitro and in vivo [2, 3]. In addition to causing a pronounced inflammatory response in the lungs and blood, severe influenza virus infections are associated with a wide range of extra-respiratory complications, including central nervous system and cardiovascular disease [4 6]. These extra-respiratory tract complications can be severe, and lead to multiorgan distress syndrome with fatal consequences [4]. Despite the involvement of extra-respiratory Received 6 April 2017; editorial decision 30 May 2017; accepted 8 June 2017; published online June 16, Correspondence: D. van Riel, PhD, Department of Viroscience, Erasmus MC, Wytemaweg 80, 3015CN Rotterdam, The Netherlands (d.vanriel@erasmusmc.nl). The Journal of Infectious Diseases 2017;216: The Author Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, journals.permissions@oup.com. DOI: /infdis/jix281 organs during severe influenza virus infections, no study to date has comprehensively examined the production of proinflammatory cytokines by these organs during the acute phase of infection. However, in mice, proinflammatory cytokines can be found in the central nervous system after influenza virus infection [7], and hypoxia-inducible factor-1 (a known regulator of the inflammatory response) is upregulated in the extra-respiratory tissues of macaques infected with influenza virus [8]. These studies suggest that extra-respiratory tissues respond to an influenza virus infection in the respiratory tract and contribute to a systemic proinflammatory cytokine response. Among laboratory animals, ferrets are considered to be the gold standard for studying human influenza, even though in-depth studies are limited by the lack of available ferret-specific reagents. In a previous study, we have shown that HPAI infection in ferrets resulted in higher morbidity and mortality compared to infection with 2009 pandemic virus (p) [9]. In the present study, we sought to investigate if these differences in morbidity and mortality were associated with differences in extra-respiratory cytokine expression. Therefore, we assessed the expression of tumor necrosis factor α (), interleukin-6 (), and interleukin-8 (), which are known to play important roles in influenza virus pathogenesis in different organs of ferrets infected with the p or HPAI virus. MATERIALS AND METHODS Ferrets Tissues from ferrets inoculated with p (A/Netherlands/ 602/2009), HPAI virus (A/Indonesia/5/2005), or Madin- Darby canine kidney cell lysate (mock) were obtained from a previous study [9] at 1 and 3 days postinfection (dpi). All samples collected during necropsy were immediately stored at 80 C. Samples were thawed once for homogenization for virus titrations. Tissues for histology were immediately fixed in formalin and processed 2 4 weeks later into paraffin. For the present study, 4 ferrets per time point were selected for ribonucleic acid (RNA) extraction (using frozen tissue homogenates) and in situ hybridization (using paraffin blocks) [9]. RNA Extraction and Complementary DNA Synthesis RNA was extracted from the nasal turbinates, lung, olfactory bulb, cerebrum, spleen, liver, heart, kidney, pancreas, and jejunum homogenates using the High Pure RNA Isolation Kit (Roche LifeScience) and further purified using the Micro RNA Isolation Kit (Qiagen, Hilden, Germany) following manufacturer s instructions. Complementary DNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen) and random primers. BRIEF REPORT JID 2017:216 (1 October) 829

2 Quantitative Polymerase Chain Reaction Quantitative polymerase chain reaction (qpcr) for,, and was performed using the Universal PCR Master Mix (Applied Biosystems). The respective primer/probe combinations are shown in Supplementary Table 1 [10]. The specificity of primer combinations was confirmed by Sanger sequencing of generated amplicons (data not shown). Reactions were performed on a 7500 Real-Time PCR System (Applied Biosystems). The delta cycle time (ΔCt; Ct gene of interest: Ct glyceraldehyde 3-phosphate dehydrogenase) were used to calculate the fold induction of individual cytokines. Fold inductions over mock-inoculated ferrets (log-fold changes) were calculated as the difference between the mean log expression values. In Situ Hybridization RNAScope RNA probes were custom designed by Advanced Cell Diagnostics (Hayward, CA) for ferret,, and. In tissues in which we observed a significant cytokine induction by qpcr, the cell type in which cytokine RNA was expressed was determined in tissue sections that were cut a maximum of 48 hours before the in situ hybridization procedure. Serial tissue sections were stained for histopathological analysis by hematoxylin and eosin. Cell types were identified by a European College of Veterinary Pathologist board-certified veterinary pathologist on morphological characteristics. The specificity of the probes was verified using a positive control probe ubiquitin C (UBC) and a negative control probe dihydrodipicolinate reductase (DapB-C4). Statistical Analysis Log-fold changes were calculated and differential expression analysis was performed using empirical Bayes linear models implemented in the R/Bioconductor software package limma. We controlled for multiple testing at a 5% false discovery rate using a Benjamini Hochberg procedure. RESULTS Increased Expression of,, and in the Respiratory Tract of p and Virus-Inoculated Ferrets In p virus inoculated ferrets, there was no significant upregulation of,, and in the nasal turbinates relative to mock-infected ferrets (Figure 1A). In the lungs,,, and were significantly upregulated at 1 dpi, and at 3 dpi (Figure 1A). In situ hybridization showed that in the lungs was predominantly produced by macrophage-like cells and alveolar and bronchial epithelial cells; was predominantly produced by epithelial cells of the bronchus, bronchioles, and alveoli; and was predominantly expressed in neutrophils. In HPAI virus inoculated ferrets, only was significantly upregulated at 3 dpi in the nasal turbinates (Figure 1A). In the lungs,,, and were significantly upregulated at 1 and 3 dpi (Figure 1A). was predominantly expressed in macrophage-like cells and few alveolar epithelial cells at 1 dpi, and in macrophage-like cells and bronchial epithelial cells at 3 dpi. was predominantly expressed in epithelial cells of the bronchus, bronchioles, and alveoli, and was predominantly expressed in neutrophils (Supplementary Figure 1). In general, cytokine expression in the lungs colocalized more with histological lesions (as described by [9]) than with expression of influenza virus antigen. Increased Expression of,, and in the Central Nervous System of p and Virus-Inoculated Ferrets In p-inoculated ferrets, was significantly upregulated in the cerebrum at 1 dpi (Figure 1B). At 3 dpi, was significantly upregulated in the cerebrum, and,, and were significantly upregulated in the olfactory bulb (Figure 1B). Only a few cells of undetermined origin were positive for the different cytokines by in situ hybridization. In HPAI virus inoculated ferrets, was significantly upregulated at 1 dpi in the cerebrum, and,, and were upregulated in the cerebrum and olfactory bulb at 3 dpi (Figure 1B). In situ hybridization showed that was expressed in a few small neurons or microglial cells in the brain stem at 1 dpi and 3 dpi; was expressed in a few small neurons or microglial cells and meningeal cells of undetermined cell type at 3 dpi; and was expressed in a few endothelial cells in the brain stem, and in a moderate number of cells in the olfactory bulb, cerebellum, and meninges at 3 dpi (Supplementary Figure 2). Histological lesions were not observed in the central nervous system (CNS) [9]. Expression of,, and in Tissues Outside the Respiratory Tract and CNS of and Virus-Inoculated Ferrets In p virus inoculated ferrets, there was no significant upregulation of the selected cytokines in tissues outside the respiratory tract and CNS (Figure 1C). However, at least 1 cytokine was downregulated in tissues of the spleen, liver, and kidney at 1 and/or 3 dpi (Figure 1C). Ferrets inoculated with HPAI virus showed a marked upregulation of 1 or more of the selected cytokines in the spleen, liver, heart, pancreas, and jejunum (Figure 1C). In the spleen, was expressed in macrophage-like cells at 1 dpi, and in megakaryocytes and a few neutrophils at 3 dpi (Supplementary Figure 3). In the liver, was expressed in Kupffer cells or fibroblasts and small cells, most likely endothelial cells at 1 dpi (Supplementary Figure 3). In the heart, was expressed in a few cardiomyocytes at 1 dpi; was focally expressed in cardiomyocytes and macrophage-like cells at 1 dpi, and in endocardial cells, endothelial cells, fibroblasts, and some cardiomyocytes at 3 dpi; was expressed in neutrophils and endocardial cells at 1 and 3 dpi (Supplementary Figure 4). In the pancreas, a few bile duct epithelial cells expressed at 1 dpi; was expressed 830 JID 2017:216 (1 October) BRIEF REPORT

3 A Nasal turbinates Day 1 Day 1 Color Key Lung Log-fold Change B Olfactory bulb Day 1 Day Cerebrum C Spleen Day 1 Day Liver Heart Kidney Pancreas Jejunum Figure 1. Proinflammatory cytokine expression in the respiratory organs (A), CNS (B), and extra-respiratory (C) organs of p or HPAI virus-infected ferrets at 1 and 3 days postinfection. Heat maps show gene expression relative to mock-infected ferrets. A P value for a specific gene and time point indicates the statistical significance. Abbreviations: CNS, central nervous system; HPAI, highly pathogenic avian influenza A; IL, interleukin; p, 2009 pandemic virus;, tumor necrosis factor α. in scattered endothelial cells and fibroblasts at 1 dpi, and in endothelial cells, fibroblasts, and a few acinar cells at 3 dpi; was expressed in a few endothelial cells, fibroblasts, bile duct cells, and acinar cells at 1 dpi, and in a few endothelial cells and acinar cells 3 dpi (Supplementary Figure 5). In general, cytokine expression did not colocalize with histological lesions, except in the heart, where and were associated with mild histological lesions, such as the degeneration and loss of striation of cardiomyocytes. Extra-Respiratory Cytokine Production Correlates with the Presence of Infectious Virus in p-infected Ferrets In the original study, we showed that active virus replication, as measured by immunohistochemistry, was restricted to the respiratory tract [9]. In contrast, infectious virus could be isolated from multiple extra-respiratory tissues (Supplementary Table 2). Therefore, the association between extra-respiratory cytokine expression and the presence of infectious virus was determined. Unfortunately, this analysis was not possible on virus infected ferrets due to the presence of infectious virus in all extra-respiratory organs. However, in p virus infected ferrets, the presence or absence of infectious virus was significantly associated with the expression of the selected cytokines (point Biserial correlation; P =.02 [Day 1]; P =.043 []). DISCUSSION Here, we provide the first evidence that,, and are upregulated in extra-respiratory tissues of ferrets early after infection with p or HPAI (Figure 2). Specifically, while inoculation with p or HPAI induced cytokine expression in the CNS, HPAI inoculation induced a significant upregulation of the selected cytokines in the spleen, liver, heart, pancreas, and jejunum. In contrast, p virus infection triggered a significant downregulation of proinflammatory cytokines in the spleen, liver, and kidney. At present, the precise trigger of extra-respiratory cytokine production remains unclear. These data may reflect a systemic BRIEF REPORT JID 2017:216 (1 October) 831

4 CNS Legend Liver Endothelial cell Neuron/microglia Cardiomyocyte Macrophage Acinar/duct cell Megakaryocyte Day 1 Spleen Neutrophil Epithelial cell Active virus replication * Upregulated by qpcr but not detected by ISH Nose Jejunum Lung Pancreas Heart Figure 2. Schematic representation of cytokine induction in different organs and cells in HPAI virus-infected ferrets at 1 and 3 days postinfection. Active virus replication refers to the detection of viral antigen by immunohistochemistry. Original content provider for ferret image: CDC. Original image available at: home.asp, image number Abbreviations: CNS, central nervous system;, avian influenza A; HPAI, highly pathogenic avian influenza A; IL, interleukin; ISH, in situ hybridization; qpcr, quantitative polymerase chain reaction;, tumor necrosis factor α. response to cytokines produced within the respiratory tract, which enter the circulation early after infection. It is also possible that extra-respiratory cytokine production is the result of local virus replication, even though we were not able to detect virus antigens in these extra-respiratory tissues by immunohistochemistry. However, it is interesting to note that extra-respiratory cytokine expression in p virus infected ferrets was associated with the presence of infectious virus. It is therefore tempting to speculate that during p influenza virus infection, the virus spreads to extra-respiratory organs but fails to replicate efficiently in extra-respiratory organs. However, the mere presence of virus might be sufficient to induce a proinflammatory response. The role of these extra-respiratory cytokines for disease severity remain to be determined, but it is striking to note that -infected ferrets, which suffered from severe disease, had more pronounced extra-respiratory cytokine production than p-infected ferrets [9]. Both and virus infection triggered cytokine responses in the CNS, and while extra-respiratory cytokine responses were often not associated with histological lesions, both and are able to induce neuronal apoptosis [11]. Although influenza viruses can enter the CNS and cause local damage [12], these data suggest that CNS disease could also be the result of immune-mediated damage [6, 13]. For example, TNF is known to increase the permeability of the blood-brain barrier and induce cell death [14], and production in the CNS has been associated with seizures [15]. Taken together, these data reveal that extra-respiratory tissues, in particular the CNS, actively contribute to proinflammatory 832 JID 2017:216 (1 October) BRIEF REPORT cytokine responses during severe p and fatal HPAI virus infections. These data indicate an association between disease severity and extra-respiratory proinflammatory cytokine expression, raising the intriguing possibility that systemic proinflammatory cytokine response may increase the severity of influenza. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. The authors thank Peter van Run for excellent technical assistance and Thijs Kuiken for critical reading of the manuscript. Financial support. This work was supported by a National Health and Medical Research Council (NHMRC) CJ Martin postdoctoral fellowship to K. R. S. ( ) and by a fellowship from the Netherlands Organization for Scientific Research (contract ) and a fellowship from the Erasmus University Medical Center Foundation to D. v. R. This work was further supported by the European Union Seventh Framework Programme for Research and Technological Development project ANTIcipating the Global Onset of Novel Epidemics (ANTIGONE; contract ).

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