H1N1 influenza virus induces narcolepsy-like sleep PNAS PLUS disruption and targets sleep–wake regulatory neurons in mice Chiara Tesorieroa,b,1, Alina Coditac,1, Ming-Dong Zhanga,d,1, Andrij Cherninskye, Håkan Karlssona, Gigliola Grassi-Zucconib, Giuseppe Bertinib, Tibor Harkanyd,f, Karl Ljungbergg, Peter Liljeströmg, Tomas G. M. Hökfelta,2, Marina Bentivogliob, and Krister Kristenssona,2 aDepartment of Neuroscience, Karolinska Institutet, Stockholm SE-17177, Sweden; bDepartment of Neurological and Movement Sciences, University of Verona, Verona 37134, Italy; cSection of Neurogeriatrics, Department of Neurobiology, Care Sciences, and Society, Karolinska Institutet, Huddinge 14157, Sweden; dDivision of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm SE-17177, Sweden; eDepartment of Brain Physiology, Institute of Biology of Taras Shevchenko National University, Kiev 01601, Ukraine; fDepartment of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna A-1090, Austria; and gDepartment of Microbiology, Tumor, and Cell Biology, Karolinska Institutet, Stockholm SE-17177, Sweden Contributed by Tomas G. M. Hökfelt, October 31, 2015 (sent for review July 16, 2015; reviewed by Antoine Adamantidis, Daniel Gonzalez-Dunia, Fang Han, and Thomas S. Kilduff) An increased incidence in the sleep-disorder narcolepsy has been On the one hand, association with one of the influenza vaccines associated with the 2009–2010 pandemic of H1N1 influenza virus administered during the 2009–2010 influenza A H1N1 virus in China and with mass vaccination campaigns against influenza pandemic has been suggested (14, 15). On the other hand, in- during the pandemic in Finland and Sweden. Pathogenetic mech- fluenza virus infections have previously been reported to repre- anisms of narcolepsy have so far mainly focused on autoimmunity. sent risk factors for narcolepsy (16) and seasonal onset of this We here tested an alternative working hypothesis involving a direct disorder did indeed increase following the 2009–2010 pandemic role of influenza virus infection in the pathogenesis of narcolepsy in in China, where there was no concurrent vaccination campaign susceptible subjects. We show that infection with H1N1 influenza (17). In Europe, relatively few patients with narcolepsy reported NEUROSCIENCE virus in mice that lack B and T cells (Recombinant activating gene influenza-like symptoms preceding the illness (18). However, 1-deficient mice) can lead to narcoleptic-like sleep–wake fragmenta- serological studies have suggested a high rate of mild or asymp- tion and sleep structure alterations. Interestingly, the infection tomatic infections during the 2009 pandemic (19). The lack of targeted brainstem and hypothalamic neurons, including orexin/ laboratory verification of previous influenza exposure in most hypocretin-producing neurons that regulate sleep–wake stability reported cases of narcolepsy precludes conclusions on a causa- and are affected in narcolepsy. Because changes occurred in the tive role for influenza virus in narcolepsy. absence of adaptive autoimmune responses, the findings show Influenza virus infections have, however, been associated with that brain infections with H1N1 virus have the potential to cause a number of functional disturbances in the nervous system. per se narcoleptic-like sleep disruption. These could be secondary to respiratory tract infections. For example, daytime somnolence is common as part of sickness influenza A virus | lateral hypothalamus | orexin | locus coeruleus | behavior caused by the release of proinflammatory cytokines, noradrenaline such as IL-1β and TNF-α (20). An exaggerated cytokine release, arcolepsy is a rare, lifelong sleep disorder associated with Nloss of neurons expressing the neuropeptide orexin/hypo- Significance cretin (Orx/Hcrt), which reside in the lateral hypothalamic area (LH) (1, 2). This disorder is characterized by many symptoms of Influenza A virus infections are risk factors for narcolepsy, a disturbed sleep, dominated by sleep–wake instability and frag- disease in which autoimmunity has been implicated. We tested mentation and, in narcolepsy type 1, by cataplexy (3–5). Narcolepsy experimentally whether influenza virus infections could be is strongly associated with the HLA DQB1*06:02 haplotype, and causally related to narcolepsy. We found that mice infected more weakly with polymorphisms in the genes encoding TNF-α with a H1N1 influenza A virus strain developed over time and TNF receptor II (6), as well as the T-cell receptor-α chain (7) sleep–wake changes described in murine models of narcolepsy and P2RY11 (8). The genes encoding the HLA histocompatibil- and narcolepsy patients. In the brain, the virus infected orexin/ ity system in humans are located in the MHC region, which hypocretin-producing neurons, which are destroyed in human is associated with more diseases, mainly of infectious or autoim- narcolepsy, and other cells in the distributed sleep–wake-regu- mune nature, than any other region of the genome (9). Because lating neuronal network. The findings, obtained in mice lacking narcolepsy is primarily related to a single allele, the class II an adaptive autoimmune response, thus provide new avenues HLA-DQB1*06:02 haplotype, an autoimmune mechanism, has for research on infection-relatedmechanismsinnarcolepsy. been hypothesized for the loss of Orx/Hcrt neurons, although the antigens involved have not been identified (3–6). In light of the Author contributions: T.G.M.H., M.B., and K.K. designed research; C.T., A. Codita, and M.-D.Z. performed research; K.L. and P.L. contributed new reagents/analytic tools; C.T., high rate of discordance of narcolepsy among monozygotic twins, A. Codita, M.-D.Z., A. Cherninsky, H.K., G.G.-Z., G.B., T.H., T.G.M.H., M.B., and K.K. ana- critical roles of environmental triggering factors have also been lyzed data; and H.K., T.G.M.H., M.B., and K.K. wrote the paper. proposed (5, 10). Additional risk alleles in both MHC class I and Reviewers: A.A., University of Bern; D.G.-D., INSERM; F.H., People’s Hospital Peking Uni- II have also been identified, which might influence adaptive versity; and T.S.K., SRI International. immune response and virus clearance. The association of HLA The authors declare no conflict of interest. alleles with narcolepsy is, thus, more complex than hitherto pre- 1C.T., A. Codita, and M.-D.Z. contributed equally to this work. sumed (11, 12). 2To whom correspondence may be addressed. Email: [email protected] or krister. Although the etiology of narcolepsy remains to be understood, [email protected]. an increased narcolepsy incidence in northern and western Eu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ropean countries since 2009 is being currently discussed (13). 1073/pnas.1521463112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1521463112 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 “cytokine storm” (21), may also be involved in the pathogenesis instability of REM sleep regulation, with the frequent occur- of the bilateral thalamic necrosis reported in Japanese children rence of so-called sleep-onset REM sleep (SOREM) episodes, in suffering from influenza infections (22). which the onset of REM sleep is preceded by wake instead of Certain strains of influenza A virus, especially avian strains, SWS (30). These alterations (Fig. 2A and SI Appendix, Fig. S1) can also cause primary lesions by invading the nervous system have also been reported in murine models of narcolepsy (31, 32). − − (23). After experimental intranasal instillation, such avian strains Sleep and wake states were analyzed in infected Rag1 / mice can spread by axonal transport to the brain along both the ol- at 2, 3, and 4 wk postinfection (in the fourth week, EEG was factory and trigeminal nerve pathways (24). analyzed 2–6 d before killing, when an initial loss of weight was In this context, the aim of the present study was to determine seen in only two of the six EEG-recorded mice) and compared whether H1N1 influenza A virus per se has the potential to cause with baseline recordings and with matched, saline-treated, control − − − − changes in the sleep pattern and to target neurons of the sleep– Rag1 / mice. Notably, in the control Rag1 / mice no significant wake-regulatory network (25–27). We here used mice with a changes were detected between the baseline EEG recordings and −/− targeted deletion of the Recombinant activating gene 1 (Rag1 ), those obtained at 2, 3, and 4 wk after saline treatment. This lacking B and T cells (28), and localized intranasal instillation of finding shows that the EEG recording implant and procedures did a mouse-neuroadapted H1N1 strain of influenza A virus, WSN/33. not cause sleep–wake changes over time per se (SI Appendix, Fig. These animals cannot mount an MHC-dependent adaptive im- S2). Several changes in the sleep–wake pattern were instead de- − − mune response that could clear the infection, and do not exhibit tected over time in the infected Rag1 / mice. lower respiratory tract infections upon the virus exposure (29), During the light phase, when nocturnal rodents sleep most of thus allowing studies on primary effects of the infection on the −/− the time (as shown in the control hypnograms in Fig. 2B and SI brain. Rag1 mice therefore provide a tool to disclose viral Appendix, Fig. S3A), no significant changes in the total time targets in individuals with an inefficient viral clearance, a con- spent in each state (wake, SWS, REM sleep) were recorded in − − dition recently suggested in narcoleptic patients (12). the infected Rag1 / mice in the second, third (SI Appendix, Fig. Results S3C), and fourth weeks postinfection (Fig. 2C). No significant alterations of the analyzed sleep and wake parameters were – Sleep Wake Changes. Mice were instilled intranasally with H1N1 found in the infected mice in the second and third weeks post- influenza A virus or saline. From the third to fourth week infection (SI Appendix, Fig. S3 D–F), whereas in the fourth week postinfection onwards, some infected mice started to show re- postinfection the sleep–wake pattern became altered.