H1N1 Influenza Virus Induces Narcolepsy-Like Sleep Disruption and Targets Sleep–Wake Regulatory Neurons in Mice

H1N1 influenza virus induces narcolepsy-like sleep 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 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

Significance arcolepsy is a rare, lifelong sleep disorder associated with Nloss of neurons expressing the neuropeptide orexin/hypocretin (Orx/Hcrt), which reside in the lateral hypothalamic area Influenza A virus infections are risk factors for narcolepsy, a (LH) (1, 2). This disorder is characterized by many symptoms of disease in which autoimmunity has been implicated. We tested disturbed sleep, dominated by sleep–wake instability and frag- experimentally whether influenza virus infections could be mentation and, in narcolepsy type 1, by cataplexy (3–5). Narcolepsy causally related to narcolepsy. We found that mice infected is strongly associated with the HLA DQB1*06:02 haplotype, and with a H1N1 influenza A virus strain developed over time more weakly with polymorphisms in the genes encoding TNF-α sleep–wake changes described in murine models of narcolepsy and TNF receptor II (6), as well as the T-cell receptor-α chain (7) and narcolepsy patients. In the brain, the virus infected orexin/ and P2RY11 (8). The genes encoding the HLA histocompatibil- hypocretin-producing neurons, which are destroyed in human ity system in humans are located in the MHC region, which narcolepsy, and other cells in the distributed sleep–wake-regu- is associated with more diseases, mainly of infectious or autoim- lating neuronal network. The findings, obtained in mice lacking mune nature, than any other region of the genome (9). Because an adaptive autoimmune response, thus provide new avenues narcolepsy is primarily related to a single allele, the class II for research on infection-related mechanisms in narcolepsy. HLA-DQB1*06:02 haplotype, an autoimmune mechanism, has been hypothesized for the loss of Orx/Hcrt neurons, although 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., antigens involved have not been identified (3 6). In light of the 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- high rate of discordance of narcolepsy among monozygotic twins, lyzed data; and H.K., T.G.M.H., M.B., and K.K. wrote the paper. critical roles of environmental triggering factors have also been Reviewers: A.A., University of Bern; D.G.-D., INSERM; F.H., People’s Hospital Peking Uni- proposed (5, 10). Additional risk alleles in both MHC class I and versity; and T.S.K., SRI International. II have also been identified, which might influence adaptive The authors declare no conflict of interest. immune response and virus clearance. The association of HLA See Commentary on page 476. 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.

E368–E377 | PNAS | Published online December 14, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1521463112 Downloaded by guest on September 30, 2021 such as IL-1β and TNF-α (20). An exaggerated cytokine release, ings as well as sleep structure alterations (5). These include PNAS PLUS “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 SEE COMMENTARY − − − − 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. S3C), and fourth weeks postinfection (Fig. 2C). No significant Results 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 postinfluenza 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. In partic- duced gain in weight. For ethical reasons, all mice were killed ular, the number of state episodes (Fig. 2C) and of sleep–wake before reaching 15% weight loss (Fig. 1). Until then, the animals transitions was increased, and the time spent in each state epi- did not show any visible sign of sickness behavior. sode was decreased (Fig. 2C), with reduced mean REM sleep Wake and sleep states were recorded before (baseline) and latency (Fig. 2C). This resulted in a marked fragmentation of during the infection using electroencephalography (EEG), sleep in the fourth week postinfection, with rapid cycling be- accelerometric recording of body movements, as well as elec- tween state episodes during the light phase (Fig. 2B). In addition, tromyography (EMG). Sleep includes two sleep states, slow wave SOREM episodes (Fig. 2A and SI Appendix, Fig. S1), not seen in sleep (SWS) and rapid eye movement (REM) sleep, and normal the saline-treated controls, appeared in the fourth week post- sleep consists of cycles of SWS epochs followed by REM sleep infection (Fig. 2B). Video-recording was not possible because of episodes. Sleep and wake, characterized by behavioral quies- requirements of the facilities for infected animals, but behavioral cence or activity, respectively, are defined by distinct neuro- immobility during SOREM episodes was confirmed in the infected physiological parameters (Fig. 2A). In particular, REM sleep is mice by accelerometric recording (SI Appendix,Fig.S1). characterized by EEG traces similar to wake but with loss of During the dark phase, when nocturnal rodents are mostly skeletal muscle tone (Fig. 2A and SI Appendix, Fig. S1). awake (Fig. 2B and SI Appendix,Fig.S3B), no significant changes Narcolepsy is characterized in humans by sleep–wake frag- were found in the second and third weeks postinfection (SI mentation, with daytime sleep episodes and nocturnal awaken- Appendix, Fig. S3 G–J). During the fourth week postinfection, NEUROSCIENCE the total time spent in wakefulness was decreased and that spent in REM sleep was increased (Fig. 2C). In addition, the number of state episodes (Fig. 2C) and sleep–wake transitions was markedly increased in the infected mice, in which the mean duration of wake episodes decreased (Fig. 2C). This resulted in the frequent intrusion of sleep episodes into wakefulness in the infected mice (Fig. 2B). Furthermore, SOREM episodes occurred among the infected mice also during the dark phase (Fig. 2B). Importantly, the percentage of body weight changes was not significantly correlated with the total number of sleep–wake transitions [r = −0.551, P (two tailed) > 0.05] observed in the − − Fig. 1. Experimental time line and survival rate of Rag1 / mice following infected group during the fourth week postinfection. intranasal infection with the WSN/33 strain of influenza A virus. The EEG/ EMG/actimetry recording system was set-up and baseline recordings were Spectral Power Analysis. No significant changes in the spectral performed. At time 0 (the day after baseline recordings), one group of mice power of the EEG traces were found over time for any of the (n = 13, including 6 mice used for EEG/EMG/actimetry recording) was in- sleep–wake phases in the saline-exposed control group. No dif- fected with the WSN/33 virus and a control group was exposed to saline. ferences in the spectral power of any bandwidth for wakefulness, EEG/EMG/actimetry recordings were analyzed in the second (w2), third (w3), SWS, and REM sleep were seen in the infected mice compared and fourth (w4) weeks postinfection. Some infected mice started to show with saline-treated controls. In the infected mice a significant reduced body weight in the fourth week postinfection (with the exception θ of one mouse that was killed at 17 d after infection) and were then killed, increase in the spectral power of the SWS -band was instead together with time-matched controls, in the following weeks, at the time of observed in the fourth week postinfection during both the light 15% weight loss at the most. Note that “survival” at the y axis is from killing and dark phases (SI Appendix,Fig.S4). These findings are similar because of reduced gain in or loss of body weight, and not from death at a to those observed in a conditional ablation model of Orx/Hcrt terminal state. deficiency (33).

Tesoriero et al. PNAS | Published online December 14, 2015 | E369 Downloaded by guest on September 30, 2021 Fig. 2. Changes in the sleep–wake pattern induced by influenza virus infection. (A) EEG and EMG traces (1-s epochs) of wakefulness (W), SWS, REM sleep, and a SOREM episode recorded in an infected mouse (the arrowhead points to REM sleep onset after wake). (B) Representative 4-h hypnograms during the light and dark phases in saline-treated control mice (n = 5) and infected mice (n = 6) at 4-wk posttreatment (Zeitgeber time, ZT, 0 corresponds to the lights-on time). In both the early light and dark phases the hypnograms from a representative infected mouse show marked sleep–wake fragmentation and SOREM episodes (S, indicated by arrowheads). (C) Quantitative data of vigilance state parameters in the light and dark phases are presented (Mann–Whitney test: *P < 0.05, **P < 0.01). In the light phase the percentage of time spent in each state does not differ significantly between groups, whereas significant changes are found in the infected mice in other parameters: increase of the number of W and SWS episodes, decrease of the mean duration of sleep–wake states and of the mean REM latency. In the dark phase the percentage of time spent in W decreases significantly in the infected mice, whereas the percentage of time spent in REM sleep increases. In addition, the number of state episodes increased and the mean duration of W episodes decreased.

Innate-Immune Responses. Mediators of the innate-immune re- Highly neurovirulent avian strains of influenza A virus can in- sponse are released by the host during the infection to limit viral vade the brain in ferrets and mice along the trigeminal and olfac- spread. These mediators can also affect sleep and wakefulness (20). tory pathways following intranasal instillation (24). In line with this, During the progression of the infection (17–39 d postinfection), viral antigens were seen in a few trigeminal ganglia neurons and brains from the infected animals harbored significantly increased were highly expressed in the dorsal trigeminal root central to the levels of transcripts encoding TNF-α,IL-1β,andIFN-β, but not of periphery-brain border (SI Appendix,Fig.S6C–E). In the olfactory that encoding inducible nitric oxide synthase, which is involved in bulb (OB), the virus spread from the olfactory nerve layer to the SWS recovery after sleep deprivation (34) (SI Appendix,Fig.S5). deeper layers as revealed at early and late stages (Fig. 3A). Notably, the infection was restricted to areas connected to the olfactory and Influenza Virus Targets Brain Areas Involved in Sleep–Wake Regulation. trigeminal systems and there was no further spread into the neo- Cells targeted by the virus were investigated by immunohisto- cortex or hippocampus, except in rare instances. chemistry. Importantly, viral antigens were not detected in the Overall the viral invasion displayed a distinct pattern in the lung at 28–29 d postinfection (SI Appendix, Fig. S6 A and B), brain, although with a considerable interindividual variability indicating that the effect on vigilance states was not secondary to in viral antigen load also in the OB (Fig. 3B). The rostro-caudal a pulmonary infection. spread was also mostly bilateral, with exception of the LH,

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Fig. 3. Viral antigen distribution in the OB and the LH. (A) At an early stage (14 d postinfection) after nasal infection, viral antigens are detected in in the olfactory nerve layer (ON; green color in adjacent Cresyl violet-stained section). (Scale bars: 200 μm; Inset is 20 μm.) At a late stage (28 d postinfection), viral antigens extensively label neurons and glia in all layers except the granular layer (GrO), as shown at three different levels (adjacent sections stained with Cresyl violet). Right sagittal drawing shows the viral spread. Pink color reflects severity of infection, and the dashed green arrow indicates the spreading pathway. (Scale bars, 1 mm.) (B)Overviewofthemice analyzed for occurrence of viral antigens in the OB and the LH. Viral antigens are present in the LH of all mice with abundant OB infection as well as in one mouse with spread of virus into the ventricles, but not in the mice showing no or only a few infected cells in the OB (SI Appendix,Fig.S9)(mouse#6).(C) Summary of virus-infected area in the LH. The dashed lines in the Inset show the size of the infected area for six mice. Mouse #1 and #6 were subjected to EEG recoding. Mouse #1 and #2 showed a clearlossofOrx/Hcrtneurons.3V,3rdventricle;EPl, external plexiform layer; f, fornix; Gl, glomerular layer; IPl, internal plexiform layer; Mi,mitralcelllayer;opt,optictract.

where, however, interindividual variations in viral load seemed innervation mainly targets the glomerular layer (GL) and granule NEUROSCIENCE to depend on the degree of infection of the OBs (Figs. 3C and 4 cell layer (GCL) of the OB (SI Appendix,Fig.S7A–E), whereas the and SI Appendix, Fig. S11). MCH mainly projects to the GCL (SI Appendix,Fig.S7F–J). Cresyl Caudal to the OB many, often intensely labeled cell groups violet-stained adjacent sections revealed cell loss only in the heavily encompassed both neurons and glia (Fig. 4 and SI Appendix, Fig. infected area (mainly Orx/Hcrt neurons) but not in the marginal S6 F–I). These were located, inter alia, in the nucleus of the zone (mainly MCH neurons), whereas contralateral, noninfected vertical and horizontal limbs of the diagonal band, piriform neurons appeared normal (Fig. 5 E–G). Triple-labeling by coincu- cortex, cortical amygdaloid area, LH, ventral tuberomammillary bation with two additional antibodies, one against glutamic acid nucleus (TMN), lateral mammillary nucleus, posterior hypotha- decarboxylase 67 (GAD67) to reveal GABAergic elements and one lamic nucleus, ventral tegmental area (VTA), dorsal and median against the vesicular glutamate transporter type 2 (VGLUT2) to raphe nuclei (DRN/MRN), locus coeruleus (LC), and sur- identify glutamatergic elements, indicated that the staining of nerve rounding nuclei, as well as trigeminal and facial nerve territories endings in the neuropil was well preserved on both sides (Fig. 5H). along the ventro-lateral aspects of the pons and medulla (Fig. 4). Moreover, microglia gradually increased from the periphery to the Viral antigens were not detected in the ventro-lateral preoptic heavily infected center of the lesions, exhibiting a series of pheno- nucleus or the suprachiasmatic nucleus. typic changes suggesting activation (Fig. 5I and SI Appendix,Fig.S8 We next investigated the chemical phenotype of the neurons A–C). Astrocytes also showed features of activation in the periphery targeted by the virus. Combinations of double-labeling showed of the heavily infected area, in particular (Fig. 5J and SI Appendix, that both Orx/Hcrt neurons and neurons containing melanin- Fig. S8E). Viral antigens were seen within astrocytes, but rarely in concentrating hormone (MCH) were infected (Fig. 5 A–D). This microglia (SI Appendix,Fig.S8D and E). finding is of particular interest in view of the well-established Similar infection and double-staining patterns could also be de- involvement of both these peptidergic cell types in sleep–wake tected in many other nuclei/areas involved in sleep–wakefulness + regulation (35). There was a reduction in numbers of Orx/Hcrt regulation: (i) cholinergic ventral forebrain neurons (Fig. 6 A neurons (56 ± 19% loss) (Fig. 5A) in the LH of the infected side, and B); (ii) histaminergic neurons in the TMN (Fig. 6 C and D); whereas the MCH-immunolabeled neurons appeared preserved (iii) the transition zone between the VTA and the most medial (3 ± 8% loss) (Fig. 5C). This is probably because the Orx/Hcrt aspects of the substantia nigra, overlapping with dopamine

Tesoriero et al. PNAS | Published online December 14, 2015 | E371 Downloaded by guest on September 30, 2021 Fig. 4. Overview of distribution of viral antigens in the brain (mouse #1). (A–F) Infection is seen bilaterally in the piriform cortex (Pir), the vertical (VBD) and horizontal (HDB) limb of the diagonal band nucleus, the nucleus of the lateral olfactory tract (LOT), and cortical amygdaloid area (CxA); (G–I) the lateral hypothalamic area (LH, essentially unilateral); note lack of labeling of Pir or entorhinal cortices at posterior levels. (J–P) Infection is also seen in the ventral tubero-mammillary nucleus (VTM), lateral mammillary nucleus (LMN), posterior hypothalamic area (PH), ventral tegmental area (VTA), dorsal and median raphe nuclei (DR/MnR), pontine reticular nucleus (oral, PnO), locus coeruleus (LC), and surrounding nuclei: for example, the lateral dorsal tegmental nucleus (LDTg), mesencephalic trigeminal nucleus (Me5). The overlaying ependymal layer is also infected. (N–S) Caudally the infection essentially forms two bilateral columns along the ventro-lateral aspects of pons and medulla, associated with the trigeminal (5N) and facial nerve (7N) territories, extending into adjacent nuclei, such as the intermediate (IRt) and parvicellular nuclei at the pontine and medullary levels. (T) In addition, the nucleus raphe pallidus (RPa) and area postrema (AP) are labeled. ac, anterior commissure; Aq, aqueduct; CPu, caudate-putamen; LR4V, lateral recess of the fourth ventricle; LV, lateral ventricle; m5, motor root of the trigeminal nerve; MeA, medial amygdaloid nucleus; PAG, periaqueductal gray. (Scale bar, 2 mm.)

(tyrosine hydroxylase-positive) neurons (Fig. 6 E and F); (iv) DRN/ In one mouse, viral spread into the ventricles occurred, probably MRN neurons overlapping with serotonergic neurons (Fig. 6 G originating from infection in the OB extending into the olfactory and H); and (v) LC and adjacent areas overlapping with nor- part of the ventricles (Fig. 3B and SI Appendix, Figs. S10 and S11). adrenergic neurons (Fig. 6I). Activated astrocytes and microglia Importantly, the LH on one side was infected (SI Appendix,Fig. were abundant in these areas (SI Appendix, Fig. S9). S11 G and H), and a considerable number of Orx/Hcrt and MCH In the LC, degeneration of noradrenergic neurons occurred in neurons was virus-labeled, especially the former neurons (SI Ap- – the heavily infected area, surrounded by morphologically preserved pendix,Fig.S12A J). There was no evidence for neuron loss in this – neurons (Fig. 6 I and J). Similarly to the LH, cell loss was seen case (SI Appendix,Fig.S12K M), suggesting a relatively early-stage viral attack. The LC showed a similar degree of changes and in- in Cresyl violet counter-stained adjacent sections (Fig. 6 K–N). fected neurons were seen also in other areas of sleep–wake regu- GABAergic and glutamatergic nerve endings were preserved also lating network, including the DRN/MRN and TMN. in the LC, even within the heavily infected area (Fig. 6 O and P). Notably, the viral infection extended in most mice outside the LC Discussion “core” into the pontine central gray (Barrington’s nucleus, lateral In this study we show that the H1N1 influenza A virus causes al- − − dorsotegmental nucleus), as well as the adjacent large neuronal cell terations in the sleep–wake pattern in Rag1 / mice and targets bodies of the mesencephalic trigeminal nucleus (Fig. 6 O–T). multiple, distinct neuronal populations belonging to the distributed

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+ Fig. 5. Viral antigens in the LH. (A–G) The LH is strongly infected and many Orx/Hcrt neurons are lost (see, for example, dashed areas on left and right side) (A), as is also evident in the adjacent Cresyl violet-stained section (E and F). However, viral antigens are still found in some Orx/Hcrt+ neurons in the surrounding, marginal area (B). The intermingled MCH+ neurons do not appear overtly affected (C), even if some are infected (D). (H) Triple labeling of virus with GAD67 and VGLUT2 shows a distinct network of nerve endings within the heavily infected area, although less dense in the infected side (Insets). (I) Double labeling with Iba1 shows activated microglia in the area (marginal area between circle and dashed circle, as indicated by arrowhead) surrounding the heavily + infected area (for details, see SI Appendix, Fig. S8 A–C). (J) Astrocytes (GFAP ) are also activated but only in the marginal area (as labeled in I). Images in B and D were merged from z-stack (8-μm and 12-μm thick, respectively). (Scale bars: 500 μminA, C, H, I, J;20μminB and D;500μminE; 100 μminF and G.)

− − system of vigilance regulation. Because Rag1 / mice lack B inability to maintain consolidated wakefulness, which is a hallmark and T cells, the observed effects did not involve adaptive au- of human narcolepsy (30), described also in murine narcolepsy toimmune mechanisms. The study could, thus, disclose neuro- models (31, 33, 36, 37). Frequent sleep episodes interrupting nal populations targeted by the infection in the absence of an wakefulness, as documented in our study after influenza virus inefficient immune control. fection, occur in Orx/Hcrt-deficient mice, which also exhibit an increased sleep time during the dark phase (31). Sleep–Wake Alterations in the Infected Mice. During the fourth week In addition, a severe fragmentation of sleep, frequently postinfection, we observed changes in the sleep–wake pattern and interrupted by short episodes of wakefulness, was here observed features of sleep spectral power similar to those reported in murine in the infected mice during the light/rest phase. This is also an models of narcolepsy, represented by Orx/Hcrt- and Orx/Hcrt re- important element of the narcolepsy phenotype in humans (30), ceptor-deficient mice (31, 33, 36), indicating a fragmentation of although this phase is less affected than the dark/activity phase in vigilance states. Thus, during the dark/activity phase there was an murine models of narcolepsy (31, 36, 37). In the present study,

Tesoriero et al. PNAS | Published online December 14, 2015 | E373 Downloaded by guest on September 30, 2021 Fig. 6. Viral antigens in extrahypothalamic regions of the sleep-regulating network (arrowheads indicate colocalization). (A–D) Cholinergic neurons (choline acetyltransferase, ChAT) in the nucleus of the vertical limb of the diagonal band (VDB) (A and B) and histaminergic neurons (histidine decarboxylase, HDC) in the ventral tuberomammillary nucleus (VTM) (C and D). (E and F) Dopaminergic neurons (tyrosine hydroxylase, TH) in the VTA. (G and H) Serotoninergic (tryptophan decarboxylase, TPH) in the dorsal raphe (DR). Open arrowhead points to a nonserotoninergic neuron. (I–N) Noradrenergic neurons (TH) in the LC showing loss of TH compared with control. Adjacent Cresyl violet-stained section also shows neuronal loss (K–M) (see, for example, control in N). (O and P) Triple labeling (GAD67 plus VGLUT) shows that many nerve endings remain in the degenerated area (Insets). (Q–S) Higher magnification images show virus- targeted neurons in LC (R) and outside LC: lateral dorsal tegmental nucleus (LDTg, Q), Barrington nucleus (Bar) and mesencephalic trigeminal nucleus (Me5, S). (T) Cartoon shows projections from the LC to OB allowing retrograde transport to NA neurons in the LC and presumably followed by local spread to surrounding neurons. Images were merged from z-stack: 17 μm(A and H), 6 μm(D), 14 μm(F), 13 μm(Q–S), respectively. (Scale bars: 500 μminA, E, I , J, O, and P; 20 μminB, D, F, and Q–S; 100 μminC and G;20μminH; 500 μminK; 100 μminL–N;10μminInsets of O and P.)

E374 | www.pnas.org/cgi/doi/10.1073/pnas.1521463112 Tesoriero et al. Downloaded by guest on September 30, 2021 the total amount of wakefulness, SWS, and REM sleep was not PNAS PLUS significantly altered in the infected mice during the light/rest phase, similarly to observations in Orx/Hcrt-deficient mice (31, 38). Therefore, importantly, in the infected mice there was no increase in the time spent in SWS, as it occurs in sickness behavior as a result of pulmonary influenza virus infections (20), and the increase in state transitions was not correlated to the sign of sickness represented by body weight loss. The sleep–wake pattern thus showed narcoleptic-like alterations, including SOREM episodes, indicating imbalances in the network of sleep–wake regulatory neurons. In murine models of SEE COMMENTARY narcolepsy these include behavioral arrests, providing evidence of cataplexy by video recording (32), which could not be performed here because of the experimental conditions. Using our accelerometer recording method, we could document behavioral immobility during SOREM episodes, “cataplexy-like” episodes, thereby strongly supporting the EEG evidence of sleep–wake changes that define human narcolepsy per se (39) and, as discussed above, have been repeatedly reported in murine narcolepsy due to deficient Orx/Hcrt signaling.

Influenza Virus Targets Olfactory Epithelium and Sleep–Wake Regulatory Neurons. Previous observations have shown that neurons in the olfactory epithelium can be targeted by influenza A virus in mice (29), ferrets (24), and humans (40), and that H5N1 Fig. 7. Schematic diagram of the hypothesized route of influenza virus infection and accumulation in CNS regions involved in sleep–wake regula- avian influenza strains in ferrets can spread along olfactory and tion. Airborne virus particles (Lower Left) can infect the animal through trigeminal nerves to the OBs and brainstem after intranasal indirect contact with the nasal cavity mucous membranes. Although the re- fection (24). In humans, influenza virus antigens in neurons and spiratory epithelium is the obvious entry point for systemic viral spread, the glia in the OBs and tracts, and the gyrus rectus have recently been olfactory epithelium provides direct access to the CNS (Upper Left enlarged found in an immune-compromised child (41). detail). Here, sensory cells (green) send axonal projections through the − − By using Rag1 / mice, which, as mentioned above, allow us to multiple bone discontinuities of the cribriform plate, and make contact in investigate primary effects of influenza A virus from the brain the glomerular layer of the OB with the dendrites of mitral cells. Viruses can (29), we could follow the spread of the virus to nerve cell groups be taken up by olfactory cells at the epithelial surface and anterogradely that project to the OB. Of particular interest, our study showed transported into the olfactory bulb, where, in the present study, they spread locally, including to the granule cell layer, during the third and fourth weeks the presence of influenza A virus antigens in hypothalamic postinfection. Viruses can also be taken up by afferent terminals (yellow and neurons, which produce Orx/Hcrt or MCH (42, 43) and are part orange) and be retrogradely transported along the axons of, for example, of the sleep–wake regulatory network. These peptidergic neu- Orx/Hcrt or MCH-expressing neurons located in the LH. The drawing also rons innervate the mitral and granule cell layers of the OB (44, shows influenza virus infection of the dorsal raphe (DR), locus coeruleus (LC), 45), and thus represent a target for retrograde axonal transport and the trigeminal nucleus (5n). of viruses along the olfactory pathway connectivity (46) (Fig. 7). In addition, infected neurons were found in the basal forebrain, TMN, VTA, DRN/MRN, and LC, which contain cholinergic, viral proteins such as the multifunctional nonstructural (NS1) histaminergic, dopaminergic, serotonergic, and noradrenergic protein (50) or the influenza nucleoprotein (NP) that accumu- neurons, respectively. These neuron groups, except the VTA, lates in dendritic spines to reduce excitatory synaptic activity NEUROSCIENCE have been shown to project directly to the OB (47, 48) and are (51). Interestingly, antibodies to the NP can cross-react with involved in the control of sleep and wakefulness (25–27). human hypocretin receptor 2 (15), which may further interfere In some of the infected mice the number of Orx/Hcrt-immu- with Orx/Hcrt signaling. More modest injury/dysfunction in the nolabeled neurons was markedly reduced on the infected side, – + multiple infected neuronal populations within the sleep wake whereas the part that only intermingled with MCH ones regulatory network may also play a role. In fact, little is known appeared better preserved. A possible differential susceptibility about the involvement of other than the Orx/Hcrt and MCH to the infection could reflect variations between neuronal pop- neurons in the sleep-wake regulating network in human narco- ulations with regard to viral uptake from their projection fields; lepsy (52). Furthermore, although systemic release of IL-1β and alternatively, the known high vulnerability of Orx/Hcrt neurons TNF-α is associated with hypersomnia, which was not here ob- (e.g., to nitric oxide-mediated S-nitrosation) could play a role served, a local release of such molecules into hypothalamic areas (49). Postmortem examination of brains of human narcoleptics by activated glia may disturb sleep–wake regulations (53). The showed number of Orx/Hcrt-immunolabeled neurons to be dramatically reduced, whereas MCH-immunolabeled neurons notion of noncytolytic effects on neurons by an infection is rel- were preserved (1, 2). However, it is difficult to determine to evant also in view of the finding that not all narcolepsy patients what extent our cell counts, showing differences between Orx/ have low Orx/Hcrt levels in the cerebrospinal fluid (14). Hcrt and MCH neurons in the infected mice, are relevant for the – above differences in the pathology of narcolepsy in humans. Influenza Virus Host Factor Interactions Involved in Neuroinvasion. In In conditional Hcrt knockout mice, about 95% of Orx/Hcrt general, the immune control of an infection in the nervous sys- neurons are lost before cataplexy appears (33), and such a tem is more efficient in peripheral ganglia (e.g., trigeminal marked cell loss was not seen in our study. Taken together, our ganglia) than in the CNS (46). Olfactory epithelium neurons are findings indicate that in the present paradigm the sleep pattern unique by bridging environmental exposure to pathogens with changes are not primarily because of cell death in a single cell CNS tissue in the OBs, pointing to the OB as a critical portal for group, such as the Orx/Hcrt neurons. Instead other mechanisms entry to the rest of the brain (Fig. 7). Because infections with in- could be involved, such as cellular dysfunctions resulting from fluenza A viruses are usually limited to the respiratory epithelium,

Tesoriero et al. PNAS | Published online December 14, 2015 | E375 Downloaded by guest on September 30, 2021 it is important to determine factors that may control spread into the olfactory route (40, 46), include mutant virus strains, which adjacent nervous tissue. could more selectively target Orx/Hcrt neurons and cause life- Viral and host factors contribute to the pathogenesis of CNS long sleep pattern changes in susceptible individuals. involvement in influenza infections (54). Viral factors reside in both the envelope and the NS1 proteins to promote the neuro- Concluding Remarks. Our observations show that Orx/Hcrt neurons tropism of certain influenza virus strains. For example, the sur- can be targeted by viruses that reach the OBs from the exposed face glycoprotein neuraminidase is a major neurovirulence olfactory epithelium. Infection of these and other neurons in the determinant of the presently used WSN/33 influenza virus strain sleep–wake regulatory network can lead to narcoleptic-like sleep– (55). Changes in two amino acids in the NS1 of an H5N1 strain wake disturbances in the absence of autoimmune mechanisms. can shift the tropism of the virus from the lung to the brain (56). Therefore, the adaptive-immune response, in addition to its pos- Interestingly, the NS1 protein can also regulate the host innate- tulated role in a causal involvement in sleep–wake disruption in immune response and thus affect viral spread and tissue reactions human narcolepsy, could modify the risk of viral neuroinvasion (50). Variants of genetically related isolates of the H1N1pdm virus during the course of an infection. can induce specific host gene-expression profiles that differ with Clinical studies aimed at linking a rare nervous system disorder regard to innate immune response genes (57, 58). like narcolepsy with infections are hampered not only by limita- In addition, acquired and genetic host factors affect the tions in measures of viral exposure in general, but also by the gap in pathogenesis or outcome of influenza A virus infections (54). knowledge of pathogenic properties of circulating virus strain Studies of host genes in mouse models have identified a number variants and of host-susceptibility factors. The present findings of loci associated with susceptibility or resistance to severe in- therefore prompt further experimental studies, complementary to fluenza disease (59). However, little is known regarding the the current search for autoimmune mechanisms, on the complex possible effects of human genetic variations, particularly in the interactions between variants of respiratory virus strains, host ge- HLA haplotypes, on the outcome of the infection with influenza netics, and other susceptibility factors in relation to sleep–wake virus strains (60). On the other hand, it is well-known that different class I and alterations, such as those occurring in narcolepsy. class II HLA haplotypes are associated with either mild or severe Materials and Methods outcomes of a range of other viral infections (61, 62). In addi- − − Animals. Female knockout mice (3- to 5-mo-old) for the Rag1 / (background tion, experimental studies indicate that more virulent mutant strain C57BL/6J) and wild-type mice were used. The experiments were ap- viruses escaping the adaptive-immune response can rapidly proved by the local ethics committee (Stockholms Norra Djurförsöksetiska evolve during serial passages in mice with identical genotypes. Nämnd, project N87/12). Such mutants do not exhibit increased virulence in other genotypes (63), demonstrating that subjects with one MHC haplotype Influenza A/WSN/33 Virus Infection. The mouse-neuroadapted influenza A can develop more severe outcomes than subjects with other (A/WSN/33) (1.4 × 105 plaque forming units) virus was intranasally in- haplotypes in the same population. It remains, however, to be fected (4–5 μL per mouse) (SI Appendix, Materials and Methods). investigated if escape mutants of influenza can evolve during epidemics in human carriers of certain HLA haplotypes. How- Surgery. Surgery was performed to implant wireless NeuroLogger microchips ever, because all postinfluenza narcolepsy patients in China were (NewBehavior, TSE) to record EEG as well as body movement (actimetry) via a HLA DQB1*06:02 carriers (17), it may be speculated that mu- built-in accelerometer (SI Appendix, Materials and Methods). tants adapted to this particular haplotype or a suboptimal response to influenza inherent to this haplotype contributed to the EEG Recording and Analysis. NeuroLoggers were configured using specialized development of disease in these individuals. This HLA class II software (CommSW, NewBehavior) and sampled at 199.8 Hz. Following haplotype is also the most common in Finland and Sweden, baseline recordings, performed 7 d after surgery, the mice were treated present in 15–30% of the populations of these countries. intranasally with saline or infected and recordings were analyzed every week (SI Appendix, Materials and Methods). Late-Onset Effects of Influenza Virus Infection on Brain Function. The −/− Quantitative Real-Time RT-PCR. The quantitative PCR reactions were perfected Rag1 mice developed signs of disease following influenza A with custom-designed primers on an ABI Prism 7000 real-time thermocycler virus infections after a relatively long latency period. This can (Applied Biosystems) (SI Appendix, Materials and Methods). Primers used in last for months (29), showing that in the absence of an adaptive- this study are listed in SI Appendix, Table S1. immune response the innate-immune response can delay, but not prevent, disease onset. Interestingly, a latency period of several Immunohistochemistry. For immunohistochemistry, 4% (wt/vol) paraformaldehyde- months between exposure to seasonal influenza and develop- fixed brains were frozen and sectioned on a cryostat. Sections were processed ment of narcolepsy has been reported in human patients in using the TSA Plus method (PerkinElmer) and indirect Coons procedure China (17). (SI Appendix, Materials and Methods). Antibodies used in this study are Although the influenza A virus is rapidly cleared from the listed in SI Appendix,TableS2. brain in wild-type mice, viral genes, notably the NS1-encoding gene, can persist in the brain for most of the lifespan in mice with Microscopy and Imaging. The material was examined with an LSM700 confocal defects in the adaptive-immune response (e.g., mice deficient in laser-scanning microscope (Zeiss), and images acquired from one airy unit the transporter associated with antigen processing and IFN-γ pinhole as previously described (SI Appendix, Materials and Methods). receptor) (29). It remains to be determined, however, whether such persistently infected mice develop sleep pattern changes. Statistics. Statistical analyses were performed with SPSS (IBM). In all analyses, statistical significance was set at P < 0.05 (SI Appendix, Materials and Methods). Studies of long-term effects of persistent infections on peptidergic/monoaminergic neuronal populations remain a challenge. ACKNOWLEDGMENTS. We thank Dr. S. Nakajima (The Institute of Public In this respect, intranasal infection with different viral strains or Health) for generous donation of the mouse-neuroadapted influenza A variants can show remarkable selectivity in the targeting of dif- (A/WSN/33) and the anti-influenza antibodies. This work was supported by ferent neuronal populations. For example, a mutant of vesicular grants from the Swedish Medical Products Agency (https://lakemedelsverket.se/) (to K.K., P.L., and T.G.M.H.); the Swedish Research Council (T.G.M.H. and T.H.); and stomatitis virus can target DRN neurons and cause lifelong the Karolinska Insitutet (T.G.M.H.). Laser-scanning microscopy was made avail- selective serotonin depletion in the brain (46). It is therefore able by the Center for Live Imaging of Cells (CLICK) at Karolinska Institutet, an possible that the several viruses that can invade the brain along imaging core facility supported by the Knut and Alice Wallenberg Foundation.

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