Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system

Journal of Autoimmunity xxx (2016) 1e10

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Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system Qingyun Chen, Yinping Liu, Aizhen Lu, Ke Ni, Zheng Xiang, Kun Wen, Wenwei Tu* Department of Paediatrics & Adolescent Medicine, University of Hong Kong, Hong Kong, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 August 2016 Received in revised form 12 October 2016 Accepted 18 October 2016 Available online xxx

Multiple sclerosis starts with increased migration of auto-reactive lymphocytes across the blood-brain barrier, resulting in persistent neurodegeneration. Clinical and epidemiological studies indicated upper respiratory viral infections are associated with clinical exacerbation of multiple sclerosis. However, so far there is no any direct evidence to support it. Using the experimental autoimmune encephalomyelitis mice as the model for multiple sclerosis, we demonstrated that mice experienced with influenza virus infection were unable to recover from experimental autoimmune encephalomyelitis with a long-term exacerbation. The exacerbated disease was due to more type I T cells, such as CD45highCD4þCD44high, CD45highCD4þCCR5þ, CD45high IFNgþCD4þ, MOG35-55-specific IFNgþCD4þ and influenza virus-specific IFNgþCD4þ T cells, infiltrating central nervous system in mice with prior influenza virus infection. Influenza virus infection created a notable inflammatory environment in lung and mediastinal lymph node after influenza virus inoculation, suggesting the lung may constitute an inflammatory niche in which auto-aggressive T cells gain the capacity to enter CNS. Indeed, the early stage of EAE disease was accompanied by increased CCR5þCD4þ, CXCR3þCD4þ T cell and MOG35-55 specific CD4þ T cells localized in the lung in influenza virus-infected mice. CCL5/CCR5 might mediate the infiltration of type I T cells into CNS during the disease development after influenza infection. Administration of CCR5 antagonist could significantly attenuate the exacerbated disease. Our study provided the evidence that the prior influenza virus infection may promote the type I T cells infiltration into the CNS, and subsequently cause a long-term exacerbation of experimental autoimmune encephalomyelitis. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Influenza EAE Spinal cord Lung Type I T cells

1. Introduction Multiple sclerosis (MS), a chronic autoimmune disease of central nervous system (CNS), is the most prevalent disabling neurological disease among young adults [1,2]. Although its pathogenesis has been well studied during recent years, the mechanism underlying the initiation and progression of MS is not well understood. A large body of clinical and epidemiological evidence indicates that virus infection is an important factor in the induction and progression of MS [3]. Approximately one-third of all MS relapses have been associated with infections caused by various transmissible and

Abbreviations: MS, multiple sclerosis; EAE, Experiment autoimmune encephalomyelitis; CNS, central nervous system; MLN, mediastinal lymph node; MNCs, mononuclear cells; DCs, dendritic cells. * Corresponding author. E-mail address: [email protected] (W. Tu).

endogenous pathogens [4]. Among these viral infections, upper respiratory infections have also been demonstrated to be significantly associated with clinical exacerbation of multiple sclerosis [5]. Influenza A virus infection in the general population are temporally associated with a higher frequency of MS relapses [4], and influenza virus-caused pneumonia also contributes to the death of MS patients [2]. In addition, the risk of experiencing a MS relapse was six times greater after influenza illness than that after influenza vaccination [4,6]. However, the underlying mechanism remains unknown. Experiment autoimmune encephalomyelitis (EAE) is the bestknown animal model for MS, which can be induced by immunization with myelin-derived antigens in the adjuvant [1]. It is characterized by a progressive ascending clinical paralysis followed partial symptom recovery in MOG35-55-induced EAE in C57BL/6 mice [7]. Immunohistological analysis of mononuclear cell infiltration in CNS has revealed that myeloid cells and myelin-specific inflammatory

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Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006

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CD4 T cells and, in some models, B cells constitute the recruited cell population and contribute to neurological deficits [8,9]. It is known that chemokine and chemokine receptors may determine the migration of immune cells into CNS in EAE disease [9]. Numerous chemokines such as CCL2, CCL3, CCL5, CXCL9 and CXCL10 have been detected within the CNS in MS patients and their active plaque lesions [10e12]. These chemokines also play important roles in attracting circulating leukocytes to acute demyelinating lesions [13,14]. CCL5 is a chemoattractant for both T cells and macrophages and could be a key pro-inflammatory factor in the pathogenesis of MS [10]. For example, samples of cerebral spinal fluid isolated from MS patients undergoing clinical relapse contain significantly higher levels of CCL5 compared to control populations [12]. Accumulating evidence also suggests that CXCL9, CXCL10 and their receptor CXCR3 play important roles in attracting encephalitogenic T cells into CNS in both EAE and MS [15e17]. In the helper T (Th) cell differentiation process, CD4 T cells acquire the ability to produce sets of cytokines and to express chemokine receptors. Type I helper T (Th1) cells preferentially express CCR5 and CXCR3, Type 2 helper T (Th2) cells express CCR4, and Th17 cells express CCR6 [18,19]. Both IFNg-producing Th1 and IL17-producing Th17 cells are implicated to cause inflammatory pathology in the CNS [20]. Although it is clear that activated T cells cross the impaired blood-brain barrier (BBB) in EAE through various chemokines and chemokine receptors, the role of chemokines and chemokine receptors in EAE disease after influenza virus infection is still not clear. Respiratory virus infections such as influenza virus infection is common among MS patients [21]. In the prospective studies, a strong correlation was found between upper respiratory virus infection and MS relapses, and revealed that the respiratory virus infections were accompanied or followed by exacerbations [2,6,21e24]. However, so far there is no any direct evidence to show such effect of upper respiratory virus infection on MS relapse. It also remains unknown how the respiratory virus infection such as influenza virus can affect the subsequent autoimmune response in CNS, especially when the acute respiratory infection is totally recovered clinically. In this study, using completely recovered mice after 50 days of influenza PR8 virus infection (post-flu mice), we explored a previously unknown mechanism whereby influenza A virus infection exacerbates EAE disease by promoting type I T cells infiltrating into CNS.

days later, the mice that infected with PR8 virus were completely recovered without any clinical symptom (post-flu mice), and then used for construction of EAE model. Both post-flu mice and control mice were received a subcutaneous injection of 150 mg MOG35-55 peptide in incomplete Freund's adjuvant containing 4 mg/ml of mycobacterium tuberculosis. On days 0 and 2 after immunization, mice received 400 ng pertussis toxin intravenously in 200 ml sterile PBS. For TAK-779 administration, mice were injected s.c. with 150 mg of TAK-779 (dissolved in 5% mannitol solution) in a volume of 100 ml every other day after MOG35-55 immunization. Individual animal was assessed daily for symptoms of EAE and scored. 2.3. Mononuclear cell isolation and flow cytometry Mononuclear cells (MNCs) were isolated from the spinal cord, lung tissue, mediastinal lymph node (MLN), spleen, and inguinal lymph nodes in EAE mice perfused with PBS. The spinal cords and lung tissue were incubated with 0.5 mg/ml collagenase at 37  C for 30 min, and MNCs were isolated using 30/70% Percoll gradients. Cell viability was assessed by trypan blue exclusion. For intracellular staining, MNCs isolated from spinal cord and lung were stimulated with MOG35-55 peptide (20 mg/ml) for 6 h with the addition of BFA (10 mg/ml) during the last 4 h of incubation. The intracellular staining was performed as described before. For PR8 virus-specific T cell detection, splenocytes were isolated from WT C57BL/6 mice, and CD3þ T cells were depleted by CD3ε MicroBeads kit (Miltenyi Biotec). The CD3-T cell-depleted splenocytes were stimulated with PR8 virus (MOI ¼ 5) overnight, and co-cultured with MNCs isolated from spinal cord for 8 h. BFA was added during the last 5 h of incubation. The intracellular staining of IFN-g was performed as we described before [25,26]. 2.4. DC purification and functional analysis in vitro Dendritic cells (DCs) were isolated from lung tissue of post-flu and control mice at the indicated time by anti-CD11c micro-beads (Miltenyi Biotec). CD4þ T cells that isolated from the inguinal lymph nodes in EAE mice during acute phase were labeled with CFSE. CFSElabeled CD4þ T cells were then cultured for 72 h in 96-well roundbottomed plates with DCs from lung tissue of the control or PR8 virus-infected mice at different ratios in the presence of MOG35-55 peptide (10 mg/ml). T cells proliferation was examined by FACS.

2. Materials and methods 2.5. RNA isolation, cDNA synthesis, and real-time PCR 2.1. Mice and reagents WT C57BL/6 mice were purchased from the Laboratory Animal Unit of Faculty of Medicine in Hong Kong University. All animal studies were approved and performed in compliance with the guidelines for the use of experimental animals by the Committee on the use of live animals in the teaching and research, the University of Hong Kong. Incomplete Freund's adjuvant (IFA) was purchased from Difco. MOG35-55 peptides (MEVGWYRSPFSRVVHLYRNGK) were purchased from Chinesepeptide Company, China. Pertussis toxin was purchased from List Biological Laboratories. The anti-mouse Abs CD4-FITC, CD45-Pacifc Blue, CD45-Alexa Fluor700, CD44-PE, CD8-APC, CCR5-PE, CXCR3-APC, CD11c-PEcy7, CD11bFITC, MHCII-Alexa Fluor700, CD80-APC, CD86-PE were purchased form Biolegend. TAK-779 was purchased from Sigma-Aldrich. QPCR primers were synthesized by Invitrogen Company.

RNA was extracted from mononuclear cells in spinal cords, lung tissue, MLN and spleens of perfused mice at indicated time points using RNeasy Mini kit (QIAGEN), and cDNA was synthesized from 500 ng RNA using PrimeScript™ RT Reagent Kit (Takara). Real-time PCR for amplifying genes was performed by using SYBR Green (Applied Biosystems). 2.6. Histopathology Spinal cords were removed from mice after PBS perfusion and fixed in 10% paraformaldehyde overnight. Paraffin-embedded 5 mm sections were stained with Luxol Fast Blue (American MasterTech Scientific), as well as Hematoxylin and Eosin (Sigma), slides were examined under light microscope. 2.7. Quantitation of cytokines and chemokines production

2.2. Infections and EAE induction C57BL/6 female mice were infected with PR8 virus (25ml, 103TCID50) or PBS (25ml) as control by intranasal administration. 50

Mononuclear cells were isolated from spinal cord and lung tissue at indicated time points and were stimulated with MOG35-55 peptide (10 mg/ml) in vitro. Supernatant were collected 24 h and

Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006

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48 h later. Cytokines and chemokines in the supernatant were measured by Luminex and LEGENDplex™ assay. 2.8. Statistical analysis The differences between experimental and control groups were analyzed using the Student t-test, except for comparing EAE clinical scores using the Mann-Whitney U test, with p < 0.05 being considered statistically significant. 3. Results 3.1. Mice with influenza virus infection exhibited an exacerbated EAE disease course To assess the role of influenza virus infection in EAE, C57BL/6 mice were infected with a sublethal dose of influenza A (H1N1) PR8 virus or treated with PBS intranasally. In general, most mice were recovered from influenza disease after two weeks of influenza virus infection. EAE was induced in the mice after 50 days of influenza virus infection (post-flu mice) or PBS treatment (control mice) (Fig. 1A). As shown in Fig. 1B, the time of acute EAE disease onset was similar in both groups. After the acute phase of EAE disease, the control mice had several times of relapse but eventually all the mice gradually recovered before 65 days post induction of EAE. In contrast, the post-flu mice were unable to recover from acute EAE disease and showed an extended and exacerbated disease course. Larger areas of demyelination indicated by loss of blue staining and vacuolation associated with MNCs infiltration were also present in the spinal cord of post-flu mice at day 30 post EAE induction compared with that in control mice (Fig. 1C). These results indicated that mice with previous experience of influenza virus infection suffered an exacerbated EAE disease. 3.2. MNCs infiltrate in the CNS were increased and prolonged in post-flu mice during EAE To investigate the composition of infiltrated immune cells,

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MNCs were isolated from spinal cords at day 15e25 (acute phase) and day 30e60 (relapse phase) post EAE induction. Post-flu mice had significantly increased percentages and numbers of CD4þCD44high and CD8þCD44high T cells at both acute and relapse phases compared with control mice (Fig. 2AeD). The percentages of CD11cþCD11bþ dendritic cells (DCs) were also significantly higher in post-flu mice than that in control mice at relapse phase of EAE disease (Fig. 2E, F). These infiltrated DCs in post-flu mice exhibited more mature phenotype as evidenced by the higher number of MHCII, CD80, and CD86 expressing CD11cþ cells than that in control mice (Fig. 2G). These results demonstrated that the previous experience with influenza virus infection contributed to the more immune cell infiltration into CNS during the disease course of EAE. 3.3. Higher type I proinflammatory cytokine expression in CNS in post-flu mice The interaction of CD44 and its ligands is essential for the generation of memory Th1 cells [27,28]. Using Q-PCR to analyze the cytokine gene expression in spinal cord MNCs, we found that postflu mice had elevated levels of IFN-g and TNF-a mRNA expression and comparable IL-17a expression compared to control mice (Fig. 3A, B). Intracellular staining demonstrated that CD4þ T cells from post-flu mice showed significantly higher secretion of IFN-g at both acute and relapse phases of EAE disease than that from control mice (Fig. 3C, D), whereas IFN-g secretion from CD8þ T cells was not altered between post-flu and control mice (data not shown). IL-17a was hardly detectable in CD4þ T cells after MOG35-55 stimulation in both post-flu and control mice (Fig. 3E, F), suggesting that IL-17a was not the key inflammatory cytokine that mediated the disease in our model. To further confirm these results, we cultured MNCs from spinal cord in vitro with MOG35-55 peptide to analyze cytokine production in culture supernatants. Type I cytokines such as IFN-g and TNF-a were markedly higher in CNS from post-flu mice than that from control mice (Fig. 3G). However, there were no significant changes between post-flu and control mice in other cytokines such as IL-17a, IL-1b, IL-6, IL-12p70, IL-22, IL-10 and IL-13 (Fig. 3G). We also cultured MNCs from inguinal lymph node and spleen at

Fig. 1. Mice with influenza virus infection exhibited an exacerbated EAE disease course. (A) Mice were infected with a sublethal dose of influenza A (H1N1) PR8 virus (post-flu mice) or treated with PBS (control mice) intranasally. After 50 days post infection, EAE was induced by immunization with MOG35-55 peptide in complete Freund's adjuvant. (B) The mice were monitored for signs of EAE daily. Data expressed as mean ± SEM for post-flu mice (n ¼ 29) and control group (n ¼ 34). (C) Representative of Luxol fast blue and HE staining for spinal cords at day 30 post immunization were shown. Demyelination is indicated by loss of blue staining and vacuolation associated with mononuclear cell infiltration. Scale bar, 200 mm.

Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006

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Fig. 2. Quantitation of the infiltrated immune cells in the spinal cords from post-flu and control mice at acute and relapse phases of EAE disease. (AeD) Total MNCs were isolated from spinal cords on day 15e25 after EAE induction (acute phase) and day 30e60 after EAE induction (relapse phase), and the percentage and number of CD45highCD44high CD4þ and CD8þ T cells were analyzed by FACS. (EeF) Total MNCs were isolated from the spinal cords on day 30e60 after EAE induction (relapse phase), and the percentage of CD45highCD11cþCD11bþ DCs were analyzed by FACS. (G) The numbers of MHCII-, CD80-, and CD86-expressing CD45highCD11cþCD11bþ DCs were analyzed by FACS. Data shown are representative of 6 independent experiments with 3e4 mice per group (means ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

different disease phases ex vivo with MOG35-55 peptide stimulation, and found that there was no significant change of IFN-g and IL17-a secretion between post-flu and control mice (Fig. S1A-D). Similar results were also confirmed in mouse serum (Fig. S1E,F). Therefore, these results indicated that influenza virus infection selectively promoted IFN-g- and TNF-a-secreting type I T cells infiltration into CNS. 3.4. CCR5þCD4þ type I T cells were enriched in lung tissue and CNS in post-flu mice during EAE disease development To determine whether the chemokines and chemokine receptors can be selectively induced or regulated during EAE pathogenesis after influenza infection, gene expression profiles were analyzed by Q-PCR. Before EAE disease symptom, a significant increase of CCL5, but not CCL19, CCL21, CXCL9, CXCL10 and CXCL13 expression in MLN in post-flu mice was shown compared to that in control mice (Fig. S2A). A higher level of CCL5 transcript was also detected in the spinal cord in post-flu mice even before clinical EAE symptom was shown compared to that in control mice (Fig. S2B). Significantly more CCR5þCD4þ and CXCR3þCD4þ T cells in lung tissue, but not in MLN and spleen were found in post-flu mice than that in control mice before EAE symptom (Fig. 4AeC). CCL5, CXCL9 and CXCL10 secretions from lung MNCs were increased in post-flu

mice compared to that in control mice after MOG35-55 stimulation in vitro (Fig. 4D). These results indicated that more CCR5 and CXCR3 bearing CD4þ T cells were recruited into lung tissue in post-flu mice even before EAE disease symptom began to show. During the acute phase of EAE disease, we observed higher CCL5, CXCL9, CXCL10, CXCL11, CXCL13, CCR5 and CXCR3 transcript expressions, and elevated CCL5, CXCL10, and CXCL13 secretions in the spinal cord MNCs in post-flu mice than that in control mice (Fig. 4E, F). Consistent with the increased chemokine expression, more CCR5þCD4þ T cells were found to acumulate in spinal cord and lung tissue in post-flu mice than that in control mice during the acute phase of EAE disease (Fig. 4G). Interestingly, in the acute phase of EAE disease, there were significant decreases of CCL5 transcript and CCR5þCD4þ T cells in spleen MNCs in post-flu mice compared with that in control mice (Fig. 4G and Fig. S3A). One possible explanation for this observation is that type I T cells might leave from spleen and infiltrated into CNS in post-flu mice. In MLN, there is no significant difference between these two groups at acute phase (data not shown). During the later course of EAE disease (relapse phase), expression of CCL5 transcript (Fig. S3B) and the percentage of CCR5þCD4þ T cells (Fig. 4H) were still much higher in spinal cord in post-flu mice compared with that in control mice. Besides, elevated CCL5 secretion in spinal cord MNCs from post-flu mice could still be

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Fig. 3. Cytokine productions in MNCs isolated from the spinal cords in post-flu and control mice at acute and relapse phases of EAE disease. (AeB) Q-PCR analysis of IFN-g, TNF-a and IL-17a mRNA expressions in infiltrating MNCs. (CeF) MNCs isolated from spinal cord were stimulated with MOG35-55 (20 mg/ml) in the presence of brefeldin A for 6 h, percentages of infiltrating CD45highIFN-gþCD4þ (CeD) and CD45highIL-17aþCD4þ (EeF) T cells were analyzed by FACS. (G) MNCs isolated from spinal cord were stimulated with MOG35-55 (20 mg/ml) for 24 h, and cytokine productions in the supernatant were determined. Data shown are representative of 4e5 separate experiments with 3e4 mice per group (means ± SEM). ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001.

measured during relapse phase (Fig. 4I). These data suggested that CCL5-CCR5 axis could dominantly mediate the infiltration of type I T cells into CNS during EAE disease development after influenza infection. 3.5. More MOG35-55 specific CD4þ T cells accumulation in lung tissue and MLN To investigate whether lung and draining lymph nodes in postflu mice may attract more MOG35-55-specific T cells, MNCs isolated from lung tissue and MLN before EAE symptom were treated with MOG35-55 peptide. Intracellular staining of IFN-gindicated that the IFN-gþCD4þ T cells were significantly higher in the lung and MLN in post-flu mice than those in control mice (Fig. 5 AeB and Fig. S2A). In contrast, no IFNgþCD4þ T cells in spinal cord before EAE symptom could be detected after MOG35-55 stimulation (data not shown). These data suggested that MOG35-55 specific IFNgþCD4þ T cells first

accumulated in lung tissue and MLN before entering the spinal cord, and post-flu mice have more of these cells in lung tissue and MLN than control mice. 3.6. More influenza virus-specific CD4þ T cells accumulation in spinal cords To investigate whether the infiltrated MNCs in spinal cords in the post-flu mice are reactive to influenza PR8 virus, influenza virus-specific T cells in MNCs from spinal cords were determined. MNCs isolated from spinal cords were cultured with the PR8 virustreated CD3-T cell-depleted splenocytes. IFN-g was detected by intracellular staining. As shown in Fig. 5CeD, significantly increased IFNgþCD4 T cells in spinal cords in post-flu mice versus control mice were observed, which indicated that the PR8 virusspecific T cells infiltrated into the spinal cords in post-flu mice. These results suggested that influenza virus-specific T cells also

Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006

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Fig. 4. CCR5þCD4þ type I T cells were enriched in lung and CNS in post-flu mice during EAE disease development. (A) MNCs were harvested from the lung in post-flu and control mice before EAE symptom (day 8e10), and the percentages of CD45þCCR5þCD4þ, CD45þCXCR3þCD4þ T cells were analyzed by FACS. (BeC) The percentages of CD45þCCR5þCD4þ, CD45þCXCR3þCD4þ T cells in lung, MLN and spleen before EAE disease symptom was shown. (D) Lung MNCs, isolated from mice before EAE symptom was shown, were stimulated with MOG35-55 (20 mg/ml) for 24 h, and chemokine in the supernatants were shown. (E) Chemokine and chemokine receptor gene expressions in MNCs isolated from spinal cord at acute phase in post-flu and control mice were shown. (F) MNCs harvested from spinal cords at the acute phase were stimulated with MOG35-55 (20 mg/ ml) for 24 h, chemokine productions in supernatant were shown. (G) MNCs harvested from spine, spleen and lung in post-flu and control mice at the acute phase of EAE, percentages of CD45highCCR5þCD4þ T cells were analyzed by FACS. (H) The percentages of CD45highCCR5þCD4þ T cells in spine at the relapse phase were shown. (I) MNCs harvested from the spinal cords at relapse phase were stimulated with MOG35-55 (20 mg/ml) for 24 h, chemokine productions in the supernatants were shown. Data shown are representative of 3e4 separate experiments with 3e4 mice per group (means ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

contribute to the exacerbated disease of EAE in post-flu mice. 3.7. Influenza virus infection affected the immune cell component in lung before EAE induction Even 50 days after influenza A virus inoculation (before EAE induction), the percentage of CD4þCD44high T cells in lung and draining MLN were significantly higher in post-flu mice than that in control mice (Fig. 6A). Compared to control mice, post-flu mice also had substantially greater percentage and number of CD11cþ DCs in the lung, particularly those of the mature subsets (MHCIIþ, CD80þ, and CD86þCD11cþ DCs) (Fig. 6B, C). Importantly, we further found that DCs from post-flu mice have higher capacity to stimulate CD4þ T cell proliferation than that from control mice (Fig. 6D, E). These data indicated that mice with previous influenza virus infection had more mature DCs and type I memory/effectors T cells resided in the

lung and draining MLN even before EAE induction. Therefore, he lung might serve as a location where autoreactive T cells become reactivated and gain the competence to enter the CNS [29]. 3.8. CCR5 antagonist decreased the severity of EAE disease in postflu mice Since CCL5-CCR5 axis may dominantly mediate the infiltration of type I T cells into CNS during EAE disease development after influenza infection (Fig. 4), we further determine the role of CCR5 in the EAE pathogenesis. The clinical disease courses in EAE control and post-flu mice were compared following the treatment with TAK-779, a non-peptide chemokine receptor antagonist specific for CCR5 [30,31]. As shown in Fig. 7, the administration of TAK-779 reduced the severity of disease, and minimized the difference in EAE score between control and post-flu mice, characterized by a

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Fig. 5. MOG35-55 specific CD4þ T cells and PR8 virus-specific CD4þ T cells in post-flu and control mice. (AeB) MNCs were isolated from the lung and mediastinal lymph nodes (MLN) in control and post-flu mice before EAE symptom, and MOG35-55-specific IFNgþCD4þ T cells in MNCs from lung and MLN were analyzed and shown. (CeD) MNCs were isolated from spinal cords at acute phase in control and post-flu EAE mice, PR8 virus-specific IFNgþCD4 T cells in MNCs from spinal cord were analyzed and shown. Data shown are representative of 3e4 separate experiments with 4e6 mice per group (means ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

more complete recovery and attenuation of relapses in post-flu mice (Fig. 7). These data demonstrated that TAK-779 could significantly reduce the severity of EAE disease, suggesting a new therapeutic approach by using CCR5 antagonist to reduce EAE relapse. 4. Discussion A large body of clinical and epidemiological evidence indicates that upper respiratory infection is significantly associated with clinical exacerbation of MS [6,21,24,32,33]. However, so far there is no any direct evidence to support it. In this study, using EAE model, we have demonstrated that mice previously experienced with influenza virus infection were unable to recover from EAE with a long-term exacerbation. This, to our knowledge, is the first report to provide the direct evidence to show that upper respiratory virus infection, i.e. influenza virus, can exacerbate the disease of EAE. It is known that EAE-mediated neurological impairment and MS disease are usually related to leukocyte infiltrates and inflammatory lesions closely [34,35]. In our model, we demonstrated that type I T cells were the main pathogenic effector cells, characterized by more CD45highCD4þCD44high, CD45highCD4þCCR5þ, CD45high IFNgþCD4þ, MOG35-55-specific IFNgþCD4þ and PR8 virus-specific IFNgþCD4þ T cells in spinal cord in post-flu mice than that in control mice. Our results indicated that influenza viral infection may promote the infiltration of type I inflammatory cells into CNS. Influenza virus infection is known to induce type I immune responses, and type I responses are also prevalent in some autoimmune disorders [36,37]. Here we found that the PR8 virusspecific IFN-g secreting CD4þ T cells were detected in spinal cords in post-flu mice during EAE development, and the MNCs from

spinal cord in post-flu mice secreted significantly higher IFN-g and TNF-a than that in control mice. Indeed, Virus-induced type I cytokines can sustain a chronic state of inflammation by either mediating a continuous recruitment of activated T cells or directly inducing myelin breakdown, as demonstrated for TNF-a [38e41]. Molecular mimicry is an alternative mechanism by which a viral component can induce an immune cross-reaction with myelin proteins [3]. Several different viruses have been implicated in initiating autoimmunity in MS. For example, myelin-specific T cells have been demonstrated to be reactive to peptides from a group of viruses, such as Epstein-Barr virus (EBV), herpes simplex virus (HSV), and cytomegalovirus (CMV), influenza viruses, and papillomaviruses [32]. Give those previous finding, it seemed plausible that influenza PR8 virus has similar epitopes with MOG35-55 peptide. However, here using MOG35-55 peptide to stimulate PR8 virusinfected lung lymphocytes (before EAE induction), we did not find any alteration in cytokine production compared with mockinfected lung lymphocytes (data not shown), indicating that there are no over-lapping epitopes between PR8 virus and MOG35-55 peptide. Therefore, we suspected that influenza virus is not the sole triggering factor for EAE disease exacerbation. Indeed, other pathogens caused upper respiratory infection (URI) are well-known triggering factors for MS relapses [21]. Interestingly, in addition to the predictable inflammatory response that occurs in the CNS, we also found significant and wellcharacterized inflammatory cells changes in the lung tissue between the control and post-flu mice. Greater increase of type I phenotypic T cells and MOG35-55 specific CD4þ T cells were observed to accumulate in lung tissue in post-flu mice during EAE disease. According to previous literature, the intravenously

Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006

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Fig. 6. Influenza virus infection affected the immune cells components in lung before EAE induction. (A) Percentages of CD44highCD4þ T cells in MLN and lung in control and post-flu mice at day 50 post PR8 virus infection were shown. (B) Percentages and numbers of CD11cþ DCs in the lung in control and post-flu mice at day 50 post PR8 virus infection were shown. (C) Percentages of MHCIIþCD11cþ, CD86þCD11cþ, CD80þCD11cþ cells in lung in control and post-flu mice at day 50 post PR8 virus infection. (D) CFSE-labeled CD4þ T cells isolated from inguinal lymph node at acute phase in EAE mice were cultured with a different numbers of lung CD11cþ DCs from the lung in control or post-flu mice in the presence of MOG35-55 for 72 h, the proliferation of CD4þ T cells was examined by FACS. Data shown are representative of 3e4 separate experiments with 3e4 mice per group (means ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 7. CCR5 antagonist TAK-779 decreased the severity of EAE disease in post-flu mice. Mice were infected with a sublethal dose of influenza A (H1N1) PR8 virus (post-flu mice) or treated with PBS (control mice) intranasally. After 50 days post infection, EAE was induced by immunization with MOG35-55 peptide in complete Freund's adjuvant. Half of the control and post-flu mice were treated with TAK-779 (150 mg per mouse) every other day from day 0 post-immunization, the other half of the mice were treated with mannitol (TAK-779 dissolvent) as the control. All the mice were monitored for signs of EAE disease. Data shown are representative of 3 separate experiments with 4e5 mice per group (means ± SEM). ns, no significant differences.

transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissue [29]. After local stimulation in the lung, these cells strongly proliferate and gain the capacity to enter the CNS and induce paralytic disease [29]. Our data also indicated that the influenza virus infection could create a notable inflammatory environment in lung tissue and MLN even 50 days after influenza virus inoculation, characterized by an increase in CD4þCD44high effector T cell population and the increased costimulatory molecules and MHC class II expression on pulmonary DCs. We further found that the lung DCs in post-flu mice were more effective than those from control mice to induce CD4þ T cells proliferation. Therefore, we speculate that, after influenza virus infection, the lung may constitute a type I inflammatory niche in which CCR5þCD4þ T cells can strongly proliferate and gain the capacity to enter CNS. It is widely accepted that migration of autoimmune T cells into the inflammatory sites relies on the interaction between chemokine and chemokine receptor [42]. Previous data showed that CCR5 can express on autoimmune T cells and it is important for the infiltration of autoimmune T cells into the EAE lesions [43,44]. Here we found that significantly more CCL5 transcript expression in the

Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006

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CNS in post-flu mice than control mice over the whole course of EAE disease. The elevated CCR5þCD4þ T cells in lung and spinal cords were also found in post-flu mice compared with control mice, suggesting CCL5-CCR5 axis may dominantly mediate the infiltration of type I T cells into CNS during EAE disease development after influenza infection. Using CCR5 antagonist TAK-779, we further demonstrated that the administration of TAK-779 could reduce EAE exacerbation in post-flu mice, suggesting a new therapeutic strategy by using CCR5 antagonist to reduce EAE relapse. In summary, our data demonstrated that the prior influenza virus infection could promote the type I T cells infiltration into the CNS, and subsequently cause a long-term exacerbation of EAE disease. Conflict of interest disclosure The authors declare no conflicts of interest related to this study. Acknowledgments This work was supported in part by Theme-based Research Scheme (Project No. T11-705/14N), the General Research Fund (HKU 780113M, 17121214 and 17115015), Research Grants Council of the Hong Kong SAR, China.

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Appendix A. Supplementary data

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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jaut.2016.10.006.

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Please cite this article in press as: Q. Chen, et al., Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system, Journal of Autoimmunity (2016), http://dx.doi.org/10.1016/j.jaut.2016.10.006