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研究目标 Bats have an enhanced innate immune signature while simultaneously having reduced systemic inflammation (to both sterile and pathogenic stimuli) [1-6]. Their role as a natural reservoir is important for zoonotic viruses, however, the focus of this research proposal is on the use of comparative biology to study issues in disease ecology, metabolism and aging, with their respective links to immunology. To modulate the increasing incidence of metabolic disorders and complications arising from aging populations, studies on “healthy” populations are underway. Bats, the only flying mammal, are unique from terrestrial mammals. Through studying a natural, healthy host, absent in obvious metabolic diseases and long-lived with no obvious signs of aging, I will identify clues to regulating these phenomena in humans. How dampened inflammation links to metabolic diseases and reduced aging in bats will be examined. These aspects are inherently linked to the basal immunological state of bats and this has a consequence on their pathogen burden and potential to spread diseases to other animals. A metabolic focus to bat inflammation Multiple components of the inflammasome signaling pathway, particularly the metabolic/pathogen stress sensor NLRP3 [1], have reduced functional capacity in bats compared to human (Fig. 1). Several dsDNA sensors are deleted from bat genomes, along with dampened functional activity of the cGAS/STING pathway. [2,4] to actively suppress danger signals (DAMPs). The excessive metabolic-stress induced by powered-flight in fruit bats [7], their fruit-based diet [8,9] and limited inflammation in response to stress [3] are all adaptations to cope with their high energy demands, I hypothesize that bats have a unique ability to cope with metabolic stress and can efficiently remove potentially hazardous by-products such as ROS or oxidized metabolites. A recent study revealed limited association of oxidative DNA mutations with aging in bats [10] and our own results show limited ROS in the sera of adult bats (Fig.1).
Figure 1 ROS activity in sera of adult animals. Promega ROS-Glo Luciferase activity exhibiting relative light units/ml of sera from P. alecto bats (n=3D12) and Cynomolgus macaques (n=3D3). Very little free radicals are detected in the Sera of bats compared to mammals and expected values from humans. I will induce metabolic stress in cell lines and primary cultures through modifiers of glycolysis & TCA metabolism (e.g. 2-DG/D-Galactose, AMPK-induction/inhibition, nutrient deprivation and various inhibitors of mitochondrial function) [11-29]. I will couple cellular work with in vivo bat studies and induce metabolic stress through sugar/lipid supplementation (20%-High Fat Diet (HFD) [30] or 20%-High-Sugar Diet (HSD)) [31,32] in their nectar-pollen diet mix or via the same chemical modifiers of mitochondrial function (long-term in a colony or short-term in captured bats). A similar HFD has been used on fruit bats to accelerate weight gain in premature/orphan bat pups. 2-DG injection in Pteropus bats in vivo induces a similar inflammation to that observed in mice (Fig 2) [11] suggesting glucose metabolism is important in controlling inflammation. Gene expression profiles can then be examined in vitro and in vivo using custom NanoString probesets &/or NGS, along with functional physiological readouts (ROS/SOD activity, glucose-homeostasis, fatty acid accumulation, mitochondrial respiration, UPR response) [33-45] and compared to known pathways from human/mouse models to identify novel mechanisms/targets for tolerance/clearance of metabolic stress products relevant to bat/human physiology. This can be used as a model for comparing the response of bats to infection (e.g. PRV3M, MERS-CoV) with/without stress. Profiling aging in bats for unique anti-aging pathways compared to known human pathways.
Bats, in general, are long-lived, with several species having “exceptional longevity” relative to body size. In captivity, several live up to 30-40 years, including Pteropus alecto and Eonycteris spelaea. Wild bats, however, exhibit minimal signs of aging [10,46-48]. Similar to middle-aged humans, young bats express high Shelterin genes, telomere maintenance genes and minimal expression of TERT (Fig. 3) [47]. This hints that bats may utilize similar genes as humans to minimize aging. I will characterize aging in bats at the cellular level via primary cell lines and iPSC cells. The ‘aging’ gene expression profile (NGS/methylation) and functional status (SOD/CAT, oxidation, SASP) of these cells will be examined across passage time, with and without cell stress [49-55].
Figure 2: Inflammatory markers after 2-DG injection in P. alecto bats by Nanostring. Heatmap of Nanostring counts for PBMC’s and primary splenocytes isolated from P. alecto bats (n=3D3 per group) injected with saline or 200 mg/kg of 2-DG ~1 hr post-feeding and left overnight (~14 hrs). Average ratiometric fold-change to control is displayed from -4 (Green) to 4 (Red) for inflammatory genes (cytokines, ISGs and immune markers). Genetic variation within young/old primary somatic cells from bats born in captivity, with a defined age, will be compared with blood samples collected across age groups (pups, juveniles, sub-adults, young adults and of adults >3 years). These in vivo known samples will track aging across growth/maturation of bat pups. Interesting findings may be further evalsuated for potential therapeutic potential in mice/humans. How aging may affect the response to various pathogens can then be interrogated.
Figure 3 Tert/Shelterin gene expression and Telomere maintenance gene expression in Spleen. TERT/Shelterin gene expression from RNASeq transcriptomes in spleens of bats (E.s., n=3D2) and humans (H.s., n=3D4) displayed as log2FPKM (left panel). Average expression (log2FPKM) from spleen transcriptomes of 226 Telomere maintenance genes associated with longevity. Investigation of unique bat genes/pathways and their role in antiviral defense. I will utilize comparative genomics to identify key target genes and pathways related to both animal physiology and infection. I am currently analyzing data from two different bat species with multiple animals and up to 8 tissues for a cross-tissue and cross-species comparison of bat-specific genes involved in controlling their unique physiological behavior, or their unique immunological status. By comparison to published datasets, genes that play a key role in metabolic regulation, aging or pathogen tolerance and clearance will be uncovered. Several alternative PRR sensing pathways have already been identified and these along with new targets, will be interrogated. They can be further applied to other mammalian/human models (in vitro or in vivo) for translational validation. How these genes are triggered, (regulatory elements/diet/environmental cues) will be investigated and how they play into bats’ ecological niche will be investigated. Coupled to this, surveillance of pathogens (direct or via collaborators) will be applied (PCR, viral enrichment-NGS, phage-display antibody mapping PhIP-Seq) and examined for association to expression of these novel targets. The correlation (in various bat species) to pathogen burden will then be studied to elucidate the contribution of each gene to real-world disease burden and potential zoonotic spillover. Current data (serology >150 bats, NGS -34 & proteomics -15) will serve as the starting platform for this deep interrogation of basal immunity on bats. Targets will be compared to other mammalian groups by homology, evolutionary selection pressure and altered expression dynamics/ gene regulation networks to minimize potential targets and maximize research efforts. Pathway analysis for bat-specific regulators will be matched to promoter/transcription factor analysis. This should yield not only a clearer understanding of each target gene but if it is truly behaving differently compared to other mammals. Collectively, this will result in a well-defined list of target-pathways for possible therapeutic or genetic intervention for alleviation of disease, be it metabolic, age-related or that inflicted by pathogens. This will be coupled to status of disease burden to examine its role in disease ecology and potential zoonotic spillover.
工作思路
Establish new research team investigating host pathogen responses Establish a zoonotic surveillance program for pathogen detection Publish high impact papers on host-pathogen responses Establish a program or integrate with existing programs in comparative biology for the purpose of studies in inflammation and aging. Train multiple undergraduate/graduate students. Develop a teaching/educational platform for OneHealth and educated undergraduate/graduate students in this field Establish community ties and develop OneHealth education for a greater outreach to the general public/community.
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1) Matae Ahn, Danielle E. Anderson, Qian Zhang, Chee Wah Tan, Beng Lee Lim, Katarina Luko, Ming Wen, Wan Ni Chia, Shailendra Mani, Loo Chien Wang, Justin Han Jia Ng, Radoslaw M. Sobota, Charles-Antoine Dutertre, Florent Ginhoux, Zheng-Li Shi, Aaron T. Irving*, Lin-Fa Wang*., Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host., Nature Microbiology, 2019年5月, 4, 5, 789-799, SCI, 14.306, 2, 其他
2) Aaron T. Irving*, Pritisha Rozario, Pui-San Kong, Katarina Luko, Jefrey J. Gorman,Marcus L. Hastie, Wan Ni Chia, Shailendra Mani, Benjamin PY-H. Lee,Gavin J. D. Smith, Ian H. Mendenhall, H. Benjamin Larman, Stephen J. Elledge, Lin-Fa Wang*, Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia., Cellular and Molecular Life Sciences, 2019年7月on line, , , -, , , ,
3) Yok Teng Chionh, Jie Cui, Javier Koh, Ian H. Mendenhall, Justin H. J. Ng, Dolyce Low, Koji Itahana, Aaron T. Irving*, Lin-Fa Wang*, High basal heat-shock protein expression in bats confers resistance to cellular heat/oxidative stress, Cell Stress and Chaperones, 2019年7月, 24, 4, 835-849, SCI, 2.944, 0, 其他
4) Ming Wen, Justin H J Ng, Feng Zhu, Yok Teng Chionh, Wan Ni Chia, Ian H Mendenhall, Benjamin PY-H Lee, Aaron T Irving*, Lin-Fa Wang*, Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing., GigaScience, 2018年9月, 7, 10, 1-8, SCI, 7.441, 2, 其他
5) Peng Zhou, Yok Teng Chionh, Sergio Erdal Irac, Matae Ahn, Justin Han Jia Ng, Even Fossum, Bjarne Bogen, Florent Ginhoux, Aaron T Irving*, Charles-Antoine Dutertre*, Lin-Fa Wang, Unlocking bat immunology: establishment of Pteropus alecto bone marrow-derived dendritic cells and macrophages., Scientific Reports, 2016年12月, 6, 未知, 1-10, SCI, 4.847, 5, 其他
6) Julia María Martínez Gómez, Pravin Periasamy, Charles-Antoine Dutertre, Aaron T Irving, Justin Han Jia Ng, Gary Crameri, Michelle L Baker, Florent Ginhoux, Lin-Fa Wang, Sylvie Alonso, Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus Alecto., Scientific Reports, 2016年11月, 6, 未知, 1-13, SCI, 4.847, 6, 其他
7) Pravin Periasamy, Paul E. Hutchinson, Jinmiao Chen, Isabelle Bonne, Shahana Shereene Shahul Hameed, Pavithra Selvam, Ying Ying Hey, Katja Fink, Aaron T. Irving, Charles-Antoine Dutertre, Michelle Baker, Gary Crameri, Lin-Fa Wang and Sylvie Alonso, Studies on B Cells in the Fruit-Eating Black Flying Fox (Pteropus alecto), Frontiers in Immunology, 2019年3月, 10, 未知, 1-11, SCI, 5.789, 0, 其他
8) Matae Ahn, Jie Cui, Aaron T Irving, Lin-Fa Wang*, Unique loss of the PYHIN gene family in bats amongst mammals: implications for inflammasome sensing, Scientific Reports, 2016年2月, 6, 未知, 1-7, SCI, 4.847, 17, 其他
9) Anthony J Sadler, Fernando J Rossello, Liang Yu, James A Deane, Xiangliang Yuan, Die Wang, Aaron T Irving, Maria Kaparakis-Liaskos, Michael P Gantier, Hangjie Ying, Howard CH Yim, Elizabeth L Hartland, Amanda J Notini, Suzan de Boer, Stefan J White, Ashley Mansell, Jun-Ping Liu, D Neil Watkins, Steve Gerondakis, Bryan RG Williams, Dakang Xu, BTB-ZF transcriptional regulator PLZF modifies chromatin to restrain inflammatory signaling programs, Proceedings of the National Academy of Sciences, 2015年2月, 112, 5, 1535-1540, SCI, 10.285, 26, 其他
10) Anthony J Sadler, Bandar A Suliman, Liang Yu, Xiangliang Yuan, Die Wang, Aaron T Irving, Soroush T Sarvestani, Ashish Banerjee, Ashley S Mansell, Jun-Ping Liu, Steve Gerondakis, Bryan RG Williams, Dakang Xu, The acetyltransferase HAT1 moderates the NF-[kappa] B response by regulating the transcription factor PLZF, Nature Communications, 2015年4月, 6, 未知, 1-11, SCI, 12.001, 20, 其他
11) Ming S Liu, Die Wang, Hiroyuki Morimoto, Howard CH Yim, Aaron T Irving, Bryan RG Williams, Anthony J Sadler, Molecular dynamics reveal a novel kinase–substrate interface that regulates protein translation interaction reveal a regulating interface critical for activity, Journal of molecular cell biology, 2014年12月, 6, 6, 473-485, SCI, 7.597, 0, 其他
12) Qian Zhang, Lei-Ping Zeng, Peng Zhou, Aaron T Irving, Shang Li, Zheng-Li Shi, Lin-Fa Wang, IFNAR2-dependent gene expression profile induced by IFN-α in Pteropus alecto bat cells and impact of IFNAR2 knockout on virus infection, PloS one, 2017年8月, 12, 8, 1-13, SCI, 3.352, 3, 其他
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1) Learning from Bats: From Genomics to Controlling Viral Infection and Combating Cancer, 其他(), 纵向, National Research Foundation (NRF) of Singapore, 2012NRF-CRP001-056, 所在实验室主持的为220万新元/9,070,669新元, 2014年2月, 2019年4月, Prof. Linfa Wang, Prof. Florent Ginhoux, A. Prof. Sylvie Alonso, A. Prof. Koji Itahara, A. Prof. Manoj krishman, Dr. Aaron T Irving, Dr Charles-Antoine Dutertre, Dr Pravin Periasamy, Dr Yok-Teng Chionh, Dr Justin Ng, Dr Peng Zhou, Dr Jie Cui, Dr Yoko Itahara, Dr Ying Ying Hey, Dr Javier Koh, 以及学生等20人
2) Altered inflammation in response to pathogens via inflammasome signalling pathways in the fruit bat, 其他(), 纵向, Ministry of Health, Singapore, MH 095:003\030-0001, 199,700新元/199,700新元, 2016年3月, 2018年3月, Dr. Aaron T Irving, Dr. Radoslaw Sobota
3) Development of multiple serological platforms for differentiation of Zika and dengue virus infections, 其他(), 纵向, 2017 National Research Fund, ZRRF16006, 120,000新元/704,875新元, 2017年1月, 2019年1月, Prof. Linfa Wang, Prof. Lisa Ng, Prof. Leo Yee Sin, Dr. Aaron T Irving, Dr Danielle Andersen
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