Influenza infection triggers disease in a genetic model PNAS PLUS of experimental autoimmune encephalomyelitis Stephen Blackmorea, Jessica Hernandeza, Michal Judaa, Emily Ryderb, Gregory G. Freunda,c, Rodney W. Johnsona,b,d, and Andrew J. Steelmana,b,d,1 aDepartment of Animal Sciences, University of Illinois Urbana–Champaign, Urbana, IL 61801; bNeuroscience Program, University of Illinois Urbana–Champaign, Urbana, IL 61801; cDepartment of Pathology, University of Illinois Urbana–Champaign, Urbana, IL 61801; and dDivision of Nutritional Sciences, University of Illinois Urbana–Champaign, Urbana, IL 61801 Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved June 13, 2017 (received for review December 13, 2016) Multiple sclerosis (MS) is an autoimmune disease of the central affect the progression of many neurological diseases including nervous system. Most MS patients experience periods of symptom Alzheimer’s disease (10, 11), Parkinson’s disease (12), and exacerbation (relapses) followed by periods of partial recovery multiple sclerosis (13–17). (remission). Interestingly, upper-respiratory viral infections increase We are interested in determining how upper-respiratory in- the risk for relapse. Here, we used an autoimmune-prone T-cell fection contributes to the progression of neurological dis- receptor transgenic mouse (2D2) and a mouse-adapted human eases including multiple sclerosis (MS), the most prominent influenza virus to test the hypothesis that upper-respiratory viral autoimmune-mediated demyelinating and neurodegenerating infection can cause glial activation, promote immune cell trafficking disease of the CNS. The majority of MS patients exhibit an os- to the CNS, and trigger disease. Specifically, we inoculated 2D2 mice cillating disease course that is characterized by relatively short with influenza A virus (Puerto Rico/8/34; PR8) and then monitored periods of neurological dysfunction followed by periods of re- them for symptoms of inflammatory demyelination. Clinical and mission, termed relapsing-remitting MS (18). Importantly, both histological experimental autoimmune encephalomyelitis was ob- relapse rate and intervals between relapses are predictors of served in ∼29% of infected 2D2 mice. To further understand how disability outcome and relapses contribute to the development of peripheral infection could contribute to disease onset, we inocu- permanent neurological dysfunction (19). Interestingly, within 5 lated wild-type C57BL/6 mice and measured transcriptomic alter- weeks of contracting an upper-respiratory infection an estimated INFLAMMATION ations occurring in the cerebellum and spinal cord and monitored 27–41% of patients will suffer disease exacerbation (13, 15, 16, IMMUNOLOGY AND immune cell surveillance of the CNS by flow cytometry. Infection 20–22). In addition, some reports suggest that relapses occurring caused temporal alterations in the transcriptome of both the cere- around the time of infection are associated with sustained neu- bellum and spinal cord that was consistent with glial activation and rological deficits, increased relapse severity, and an increased increased T-cell, monocyte, and neutrophil trafficking to the brain at number of gadolinium-enhancing lesions as measured by MRI day 8 post infection. Finally, Cxcl5 expression was up-regulated in (21). Picornaviruses have been identified as triggers of relapse the brains of influenza-infected mice and was elevated in cerebro- (13, 23), but infection with other viruses, including influenza A spinal fluid of MS patients during relapse compared with specimens virus, has also been associated with exacerbated disease (24). acquired during remission. Collectively, these data identify a mech- These data suggest that upper-respiratory infection with viral anism by which peripheral infection may exacerbate MS as well as pathogens can precipitate relapse, but the mechanisms remain other neurological diseases. poorly defined. In the case of MS patients, it is possible that upper-respiratory upper-respiratory viral infection | multiple sclerosis | neuroinflammation | infection-induced glial activation triggers immune surveillance immune cell surveillance | experimental autoimmune encephalomyelitis Significance he bidirectional connectivity between the brain and the im- Tmune system is well-established. It has been repeatedly Peripheral infections exacerbate symptoms of many neuro- demonstrated that systemic inflammation brought on by activa- logical diseases, including the most common autoimmune de- tion of toll-like receptors (TLRs) on cells in the periphery can myelinating disease of the central nervous system (CNS), trigger glial activation in mice and humans (1–3). Furthermore, multiple sclerosis (MS). We demonstrate that influenza viral upper-respiratory infection with influenza A (4, 5) and porcine infection of autoimmune-prone mice triggers clinical and his- reproductive and respiratory syndrome viruses up-regulate tological disease. We further show that influenza infection al- proinflammatory cytokines within the CNS and cause alter- ters the transcriptome of the central nervous system and ations to the microglial sensome (6). Under normal physio- facilitates immune cell trafficking to the brain. Finally, we logical circumstances, activation of glial cells by peripheral identified a specific chemokine that is upregulated in the CNS inflammatory processes likely facilitate functions that are es- during infection that is also increased in the cerebrospinal fluid sential for the maintenance of homeostasis. For example, type I of MS patients during relapse. These observations improve our IFN signaling to brain endothelial cells contributes to the understanding of how peripheral infection may act to exacer- development of sickness behaviors in rodents, which are im- bate neurological diseases such as multiple sclerosis. portant for combating infection (7). Furthermore, systemic Author contributions: A.J.S. designed research; S.B., J.H., M.J., E.R., G.G.F., and A.J.S. inflammation-induced glial activation promotes immune sur- performed research; G.G.F., R.W.J., and A.J.S. contributed new reagents/analytic tools; veillance of the CNS (2, 8). The function of this surveillance is S.B., J.H., M.J., E.R., and A.J.S. analyzed data; and S.B. and A.J.S. wrote the paper. not entirely clear, but may serve to protect against infection of The authors declare no conflict of interest. the CNS parenchyma and under certain circumstances facilitate This article is a PNAS Direct Submission. repair (9). Conversely, aberrant neuroinflammation contributes Data deposition: The sequence reported in this paper has been deposited in the NIH Gene to the pathogenesis of a myriad of neurological diseases and Expression Omnibus database (accession no. GSE96870). T-cell extravasation into the CNS is not always beneficial. In- 1To whom correspondence should be addressed. Email: [email protected]. triguingly, systemic inflammation brought on by pathogenic in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fection can exacerbate neuroinflammatory processes and may 1073/pnas.1620415114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620415114 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 of the CNS that may be detrimental. To model the effects of upper-respiratory infection on relapse and/or disease progres- sion in MS patients, we infected autoimmune-prone T-cell re- ceptor transgenic (2D2) mice with mouse-adapted human influenza A virus. This strategy was chosen because, although + ∼90% of CD4 T-cells in 2D2 mice express a T-cell receptor (TCR) with specificity for myelin oligodendrocyte glycopro- tein (MOG35–55) and neurofilament-m (NF-M18–30), few (4%) develop spontaneous classic experimental autoimmune en- cephalomyelitis (EAE) (25). Additionally, the presence of circu- lating autoreactive cells enabled us to bypass the need to polarize and activate T-cells before injection. Finally, influ- enza A virus can induce glial activation (4, 5) and has been associated with increased relapse risk in MS patients (22, 24, 26). Here, we report that upper-respiratory infection with influenza virus is capable of causing neurological disease in 2D2 mice. Furthermore, we found that viral infection alone caused temporal transcriptomic changes to the cerebellum and spinal cord that were in part mediated by IFN signaling. Finally, we demonstrate that infection caused immune cell surveillance of the CNS. Fig. 1. CNS pathology in saline- and influenza-infected 2D2 mice. (A and B) Transgenic 2D2 mice were inoculated with saline (n = 5) or influenza (n = 8; Results 0.7 HAU). After 28 d post infection, mice were euthanized. Brains and spinal cord sections were stained with H&E to identify inflammation and Luxol Fast Influenza A Infection Causes EAE Onset in 2D2 Mice. Transgenic + Blue and periodic acid Schiff (LFB/PAS) to identify demyelination. Sections 2D2 mice possess an inherent reduction in CD8 T-cells that were scanned by a rater blinded to condition. (A) Spinal cord lesion showing might impede virus-specific immune responses (25). Indeed, inflammation (Top) and demyelination (Bottom) in an influenza-infected following intranasal inoculation with a normally nonlethal dose mouse. (Right) Higher magnification of lesion is denoted by an asterisk. of influenza [1.0 hemagglutinating unit (HAU)], 2D2 mice lost (Scale bar is 50 μm.) (B) Representative cerebellar H&E (Top) and LFB/PAS