Latent virus infection upregulates CD40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS) by multiple groups working worldwide. Previously, we reported that when experimental autoimmune encephalomyelitis (EAE) was induced in mice latently infected with murine γ-herpesvirus 68 (γHV-68), the murine homolog to EBV, a disease more reminiscent of MS developed. Specifically, MS-like lesions developed in the brain that included equal numbers of IFN-γ producing CD4+ and CD8+ T cells and demyelination, none of which is observed in MOG induced EAE. Herein, we demonstrate that this enhanced disease was dependent on the γHV-68 latent life cycle and was associated with STAT1 and CD40 upregulation on uninfected dendritic cells. Importantly, we also show that, during viral latency, the frequency of regulatory T cells is reduced via a CD40 dependent mechanism and this contributes towards a strong T helper 1 response that resolves in severe EAE disease pathology. Latent γ-herpesvirus infection established a long-lasting impact that enhances subsequent adaptive autoimmune responses.

used to study MS in rodents, is exacerbated by acute γ HV-68 infection 20 . More severe disease is also observed in γ HV-68 infected mice in the context of inflammatory bowel disease 21 and Crohn's disease 22 . In all these studies, the mechanisms that the virus is exploiting to lead to these enhanced autoimmune phenotypes were not described. We previously demonstrated that EAE pathology is severely heightened in mice latently infected with γ HV-68 and it is more reminiscent of MS including brain specific lesions with CD4 + and CD8 + T cells and demyelination 23 . Mice latently infected with γ HV-68 were induced for EAE (γ HV-68 EAE) and mounted a potent Th1 response that lacked an IL-17 response and leads to brain parenchyma inflammation. Whereas, in uninfected EAE mice, inflammation is confined in the spinal cords and it is driven by a mixed Th1-Th17 response. Antigen presenting cells (APCs) expressing higher levels of CD40 and MHC II were found to be responsible for the preferential Th1 skewing in γ HV-68 EAE mice 23 . This suggests that a γ -herpesvirus is able to manipulate innate immunity and influence both the skewing and the strength of T cell responses upon a second pro-inflammatory stimulus. However, it remained to be addressed mechanistically how γ HV-68 latency triggers the enhancement of the disease, specifically addressing whether the latent life cycle is required and what role upregulation of costimulatory molecules, in particular CD40, has in inciting enhanced EAE.
CD40 is a co-stimulatory molecule that is expressed on mature dendritic cells (DCs), B cells, monocytes, epithelial cells and endothelial cells [24][25][26] . The presence of microbial products or danger signals activates Toll-like receptors leading to the activation and maturation of DCs and, in particular, upregulation of surface expression of CD40. Type I interferons (Type I IFNs) play a critical role not only in the regulation of CD40 expression, but also in the control and maintenance of both γ HV-68 acute and latent infection 27 . Type I IFNs serve to program DCs to drive Th1 responses through activation/ phosphorylation of the STAT 1 pathway 28,29 . In addition to type I IFNs, the cognate interaction between CD40 and CD40 ligand (CD40L) enhances DC activation and it is important in providing T cell help to B cells, activating cytotoxic CD8 + T cells, directing a Th1 response and controlling regulatory T cells (Tregs) 24,30,31 . Interestingly, CD40 polymorphisms and dysregulation have been shown to play a role in the development of autoimmunity 32 .
We hypothesized that heightened CD40 expression was mechanistically participating in the development of severe EAE pathology. Our data demonstrate that CD40 is required for the efficient priming of strong Th1 responses and for decreased Tregs frequencies in mice latently infected with γ HV-68. We were also able to show the converse using a latency deficient virus, in which we observed that enhancement of EAE pathology absolutely required γ HV-68 latency. Essentially, the ability to skew the adaptive immune response towards a Th1 phenotype and retain low frequencies of Treg cells through CD40 expression and co-stimulation represents a novel mechanism in which latent γ -herpesviruses like EBV can act to facilitate and induce autoimmune disease.

Results
During acute infection with γHV-68, EAE is delayed. We have previously shown 23 that mice latently infected with γ HV-68 (five weeks post primary infection) present a heightened EAE pathology characterized by earlier onset of paralysis, more severe clinical symptoms, enhanced T cell infiltrations inside the CNS, and a potent Th1 response accompanied by downregulation of Th17 responses. Strikingly, γ HV-68 EAE mice also showed CD8 + T cell infiltrations and myelin damage inside the brain parenchyma 23 , closely resembling the composition of immune infiltrates in MS plaques. Further, we observed an increase in surface expression of CD40 and MHC II on CD11b + CD11c + cells during the antigen presentation phase of EAE responsible for the enhanced Th1 responses detected in γ HV-68 EAE mice 23 .
To determine if the establishment of latency by γ HV-68 is a requirement in order to see enhancement of EAE symptoms, EAE was induced in mice during acute infection with γ HV-68 before the latent life cycle was established. We observed that EAE was delayed during acute infection (Fig. 1) and the severity of the disease was similar between uninfected mice and γ HV-68 acutely infected mice (Fig. 1). Interestingly, the onset of EAE symptoms in infected mice was concomitant with the date of establishment of γ HV-68 latency (day 14/15) as was previously demonstrated by a number of different laboratories 33-35 . Mice infected with a latency deficient γHV-68 have a less severe EAE course and lower amounts of T cell infiltrations inside the CNS than mice infected with γHV-68. The observations that during acute infection, EAE onset was delayed and the disease scores were similar between uninfected mice and γ HV-68 acutely infected mice suggested that establishment of latency prior to EAE induction is required for the development of enhanced disease. We hypothesized that the latency was indispensable for the enhancement of EAE symptoms, so we chose to study a recombinant γ HV-68 (γ HV-68 AC-RTA) in which the genes responsible for the establishment of latency have been deleted and the gene driving lytic infection is constitutively expressed 36 . In a manner similar to wild type virus, this recombinant virus acutely infects mice and stimulates a cellular and humoral immune response comparable to that elicited by the wild type virus 36 . As reported previously, no viral DNA was detected in the splenocytes of mice infected with this virus 15 days post infection 36 , indicating that latency was not established and that virus was cleared.
To directly determine the role of latency in this model, we characterized EAE development in γ HV-68 AC-RTA mice. We assessed the differences in the immune cell composition inside the CNS and analyzed Scientific RepoRts | 5:13995 | DOi: 10.1038/srep13995 the T cell response after infection of wild type mice with either γ HV-68 or γ HV-68 AC-RTA followed by EAE induction. Similar to uninfected EAE mice, γ HV-68 AC-RTA EAE mice showed milder clinical CNS symptoms ( Fig. 2A), delayed disease onset ( Fig. 2A) and fewer CD4 + and CD8 + infiltrations inside the brain parenchyma (Fig. 2B) when compared to γ HV-68 EAE mice. In the end, the disease pathology of the γ HV-68 AC-RTA EAE mice resembled those of uninfected EAE mice 23 . These data indicate that the heightened EAE pathology observed in mice latently infected with γ HV-68 was not observed when mice were infected with a recombinant γ HV-68 that was not able to establish latency. Further, γ HV-68 AC-RTA EAE mice presented with reduced CD4 + and CD8 + T cell infiltrations in both the brains and the spinal cords when compared to γ HV-68 EAE mice (Fig. 3A). Additionally they showed increased Th17 responses in the CNS (Fig. 3B) and a decrease in IFN-γ production in both CD4 + and CD8 + T cells. Overall, the type of immune response skewed by γ HV-68 AC-RTA mice after EAE induction was more similar to naïve EAE mice than to γ HV-68 EAE mice.  Mice infected with the latency-free γHV-68 AC-RTA strain do not upregulate CD40 expression on antigen presenting cells upon EAE induction. As previously mentioned, we have already demonstrated that latently infected mice present with an upregulation of CD40 on APCs. We then sought to determine whether infection with γ HV-68 AC-RTA would, in contrast to latent γ HV-68 infection, not upregulate CD40 and also establish whether CD40 expression is an important key to immunological regulation during latent viral infections. APCs from C57Bl/6 mice infected with γ HV-68 AC-RTA express CD40, during the antigen presentation phase of EAE at levels comparable to naïve EAE mice ( Fig. 4) rather than the increased expression observed in γ HV-68 latently infected mice (Fig. 4) thereby associating latent infection with increased CD40 surface expression and likely co-stimulation. From our prior manuscript, increased CD40 expression was defined on APCs identified by CD11b + , CD11c + surface marker staining. This set of cells represents a broad spectrum of cell types and it is likely that only a small subset of APCs within this population has increased CD40 surface expression. The variable nature Two weeks post EAE induction, mice were perfused, brain and spinal cords were harvested and immune infiltrations were isolated. Isolated immune cells were stimulated with PMA and ionomycin and stained for CD4 (A) and CD8 (A) to measure IFN-γ and IL-17 (B) production. The amount of CD8 T cells infiltrating inside the CNS of γ HV-68 AC-RTA EAE and naïve EAE mice was too low to perform intracellular staining (B). Three separate experiments with 5-6 mice/group; data are represented as mean, error bars are SEM and were analyzed with t-test: **p < 0.01; ***p < 0.001. of this subset within the larger population is likely reflected in the variability of the mouse-to-mouse comparisons. Overall, the increase in CD40 surface expression is significantly greater from the cells isolated from γ HV-68 latently infected mice.

The presence of CD40 on antigen presenting cells is required to induce enhanced Th1 responses in mice latently infected with γHV-68.
Enhanced Th1 responses and enhanced CD8 + T cell activation have been shown to be dependent on CD40 [37][38][39][40][41][42][43][44] . Further, these characteristics were observed in γ HV-68 EAE mice and in association with an upregulation of CD40 23 . Additionally, in γ HV-68 AC-RTA EAE mice, brain inflammation is highly reduced, indicating that the priming of a strong Th1 response is required to drive both CD4 + and CD8 + T cells inside the brain parenchyma leading to the development of myelin lesion as observed in γ HV-68 EAE mice 23 . To investigate the effect of the lack of CD40 during γ HV-68 infection and latency, C57Bl/6 wild-type (wt) and C57Bl/6 CD40KO mice were infected or not with γ HV-68. Five weeks post infection (p.i.), the level of IFN-γ produced by T cells was assessed. C57Bl/6 CD40KO mice infected with γ HV-68 fail to mount the strong Th1 response typical of γ HV-68 C57Bl/6 wt mice. In fact, IFN-γ production by CD4 + T cells in γ HV-68 C57Bl/6 CD40KO mice was comparable to naïve wt mice both in the spleen and in the lymph nodes (Fig. 5A). IFN-γ produced by CD8 + T cells was reduced in the spleen of γ HV-68 C57Bl/6 CD40KO mice when compared to γ HV-68 C57Bl/6 wt. In contrast, there was no comparative reduction of IFN-γ in the lymph nodes (Fig. 5B).
Additionally, as CD40 was found to be upregulated on the surface of CD11b + CD11c + cells upon EAE induction 23 , an in vitro antigen presentation assay was performed to assess if CD40 was critical to skew the potent Th1 response observed in γ HV-68 EAE mice upon myelin peptide presentation. Transgenic T cells, bearing a TCR specific for myelin olygodendrocyte glycoprotein (MOG), were incubated with CD11b + CD11c + cells isolated from a γ HV-68 EAE wt mouse and were primed. These cells produced significantly greater amounts of IFN-γ than when incubated with the same cell subset from uninfected EAE wt mice. This effect was abrogated if CD11b + CD11c + were taken from CD40KO mice (Fig. 6A,B). This result shows that CD40 expression on the surface of APCs is an absolute requirement to trigger enhanced IFN-γ production in T cells upon antigen presentation in mice latently infected with γ HV-68.

γHV-68 infection drives increased STAT1 responses in APCs.
Type I IFNs play a crucial role in the maintenance of latency of γ HV-68 27 . In addition, upregulation of CD40 and MHCII on APCs is dependent on the presence of Type I IFNs 45,46 . Type I IFNs modulate APCs including dendritic cells to mediate Th1 responses by activating the STAT1 pathway 28,29 . We tried to measure the levels of Type I IFNs during γ HV-68 latency with no success. The increase in Type I IFNs production during viral latency is likely localized to only a small subset of cells and reasonably difficult to measure. To indirectly measure the effect of IFN I, we chose to measure the levels of phosphorylation of STAT1 (pSTAT1). pSTAT1 is a signature molecule that defines the potential for Type I IFN mediated activity 28,29 . pSTAT1 can also be driven by other inflammatory mediators such as IFNγ , however by sampling during the inflammatory quiescent period prior to EAE, these alternative mediators may have a reduced contribution. More importantly, it is our premise that the APCs are programmed by the presence of a latent virus (in a different cell, memory B cell) and that this programming will lead to a greater phosphorylation of STAT1 and a greater STAT1 response. We harvested spleens from latently infected mice and sorted CD11b + CD11c + cells. We then determined pSTAT1 levels by flow cytometry. We observed that, even prior to EAE induction, pSTAT1 levels were increased in APCs harvested from γ HV-68 latently infected mice (Fig. 7) as compared to those of uninfected mice. While these results suggest a potential link to IFN I, they leave open the possibility that other mediators are providing a greater STAT1 response. Overall, these results suggest that there is likely a strong association between γ HV-68 induced upregulation of pSTAT1 levels and the upregulation of surface CD40 in APCs, ultimately driving the response towards a Th1 phenotype. Most importantly, it would be expected that a strong Th1 response would include both upregulation of pSTAT1 and CD40 in APCs.
Mice latently infected with γHV-68 have decreased Treg in the periphery and in the CNS after EAE induction. Viral latency and induction of CD40 upregulation are required for this phenotype. In the periphery, Treg induction through APC:T cell costimulation, specifically CD40:CD40L, has been clearly demonstrated 38,41 . The increased surface expression of CD40 and potent Th1 response in γ HV-68 infected mice led us to ask, if there were relevant changes in the frequency of Tregs. To specifically investigate the frequency of Tregs in the periphery and CNS we examined the Treg populations in γ HV-68 latently infected mice and γ HV-68 AC-RTA infected mice after EAE induction. The frequencies of Tregs were decreased in the spleens of γ HV-68 EAE mice when compared to both γ HV-68 AC-RTA EAE mice and naïve EAE mice (Fig. 8A). The same results were obtained when the frequencies of Tregs in the CNS were analyzed (Fig. 8B): the levels of Tregs in γ HV-68 AC-RTA infected EAE mice were comparable to those of uninfected EAE mice. This suggests that viral latency likely through a CD40 mechanism has a role in controlling the frequencies of Tregs in γ HV-68 mice. Finally, the frequency of Tregs was decreased in γ HV-68 C57Bl/6 wt mice before EAE induction (50 days post γ HV-68 infection), but it was rescued in γ HV-68 C57Bl/6 CD40KO mice that showed the same Treg frequency as naïve CD40 KO mice (Fig. 8C). Since CD40 has been shown to be critical to control Treg frequencies 38,41 , it was expected that the Treg frequency on latently infected γ HV-68 CD40KO mice would be similar to that observed in naïve uninfected CD40KO mice. Further, the lack of disease enhancement post EAE in mice infected with γ HV-68 AC-RTA demonstrates that viral latency and its influence on innate immunity is critical to the control of Treg frequencies and adaptive immunity during latent γ HV-68 infection.

Discussion
Despite a large body of work that associates EBV infection to the development of autoimmunity, it is currently not clear what mechanisms this virus is exploiting to cause autoimmunity. Previous work focused primarily on EBV specific adaptive immunity in patients affected by autoimmunity and how this response is different when compared to healthy individuals. However the influence that EBV latency has on innate immunity has not been investigated in the context of autoimmune diseases. Previously, we used the murine equivalent to EBV, γ HV-68, to demonstrate that γ -herpesviruses have the ability to modulate subsequent immune interactions and to specifically heighten EAE pathology to more resemble MS. Here, we determined that this enhanced disease requires γ HV-68 latency and likely acts by upregulating CD40 surface expression on APCs. CD40 expression and co-stimulation is pivotal in controlling the type and strength of the adaptive immune response in response to a second pro-inflammatory stimulus such as EAE: CD40 co-stimulation and γ HV-68 latency act to enhance both CD4 + and CD8 + effector T cell activation and reduce Tregs during EAE.
Specifically, we showed that γ HV-68 enhanced T cell activation is accompanied by a decrease in Tregs frequencies both in the periphery and in the CNS during EAE. It has been previously shown that mice infected with γ HV-68 present with decreased expression of Foxp3 and diminished T cell regulatory activity up to day 15 post γ HV-68 infection 47 . Our results show for the first time, to our knowledge, that γ HV-68 mice have decreased splenic percentages of Tregs and that this reduction is long lasting, still evident more than 50 days after infection. Further, this suppression of Treg frequency is removed in the absence of virus latency and CD40. We suggest that γ HV-68 latency, in concert with the increase in CD40 expression on APCs, drives a decrease in Treg frequencies and thereby increases susceptibility to autoimmunity. Our data is supported by previous work that demonstrated that CD40 signaling suppresses the development of Tregs 38,41 . It is conceivable that decreased numbers of Tregs are contributing to the exacerbation of EAE symptoms observed in γ HV-68 mice. In fact, it has been extensively shown that Tregs have an important role in preventing EAE development in mice (for a review 48 ). Adoptive Treg transfers and treatment with monoclonal antibodies aimed at increasing the numbers of Tregs are both effective means to ameliorate EAE 48 . Interestingly, Tregs have decreased suppressive functions in MS patients [49][50][51] and patients with mononucleosis have also decreased frequencies of Tregs in the blood 52 . A decrease in Tregs as a consequence of γ -herpesvirus infection could be a predisposing factor for the development of autoimmunity.
Additionally, it has been shown that CD40 is important for cytotoxic CD8 + T cell activation 37,40,42,43 , suggesting that increased CD40 expression in γ HV-68 EAE mice may have a role in CD8 + T cell enhanced activation and CNS infiltration that we observed during EAE in latently infected mice. Our findings that enhanced CD8 + T cell activation during EAE is dependent on both CD40 expression and γ HV-68 latency are in agreement with previous studies that have demonstrated that CD40L-CD40 interaction is  required for the prolonged clonal expansion and activation of CD8 + T cells during the "mononucleosis like" phase of γ HV-68 infection, coincident with latency establishment 53 . It has also been shown that enhancement of CD40 signaling substitutes for CD4 + T cell help during control of latency to prevent γ HV-68 reactivation 54 . From these results, it is clear that CD40 is playing an important role especially during γ HV-68 establishment of latency.
A question that remains open is how does γ HV-68 control CD40 expression. We have previously shown that CD11c + CD11b + cells expressing high levels of CD40 are not infected by γ HV-68. It is conceivable that the virus is using an indirect mechanism to upregulate CD40, possibly through Type I IFNs, however other mediators including IFNγ could be mechanistically important. Type I IFNs are produced during the infection and maintenance of γ HV-68 latent infection 27 . γ HV-68 latency requires type I IFN to maintain its latent life cycle 27 . Alternatively, a viral protein or components of the viral genome could bind to pattern recognition receptors and further activate APCs to produce increased amounts of co-stimulatory molecules like CD40. Intriguingly, MS patients upregulate CD40 in their peripheral blood 55 and CD40 positive cells co-localize with T cells in active MS lesions 56 . Additionally interaction between CD40 and CD40L on T cells isolated from MS patients stimulates increased production of IL-12 by APCs 57 and IFN-β, used as a therapeutic agent in MS, decreases the level of CD40L on T cells 58 .
The EBV gene product, LMP-1, is a decoy receptor for the CD40 receptor and can replace CD40 signaling in B cells 59 . This observation is particularly intriguing considering the tight link between EBV infection, mononucleosis and MS, the successful results of anti-B cell therapies in treating MS and the ability of B cells to act as APCs. While an LMP-1-like gene product has not yet been observed for γ HV-68, the virus may influence CD40 signaling in a different manner because controlling co-stimulation is critical to EBV and γ HV-68 pathogenesis. Our results suggest a possible, yet to be described, mechanism where EBV might be influencing autoimmunity in humans: EBV affects APCs leading to Th1 skewing, CD8 + T cell activation and decreased Tregs frequencies. MS and other autoimmune diseases such as Lupus in which EBV has been implicated are heterogeneous and it is conceivable that they do not have only one etiologic agent. It is likely that EBV could be the trigger of the disease in only a subset of patients. To address this, the activation status of APCs in humans affected by different sub-types of MS should be investigated and linked to both EBV infection/mononucleosis history and T helper responses/CD8 + activation status.
In conclusion, we have demonstrated that latent infection with γ -herpesviruses predisposes individuals to severe autoimmune disease by modulating DCs and suppressing Treg frequencies likely through DC modulation (CD40 expression) and this in turn, exaggerates CD4 + and CD8 + T cell aggression. This profound ability of γ -herpesviruses to influence autoimmunity represents a unique mechanism and offers a novel target for potential therapies.

Materials and Methods
Ethics Statement. All animal work was performed under strict accordance with the recommendations of the Canadian Council for Animal Care. The protocol was approved by the Animal Care Committee (ACC) of the University of British Columbia (certificate numbers: A0\415 and A08-0622).
Infections and EAE induction. C57Bl/6 mice, C57Bl/6 CD40KO mice and 2D2 mice were purchased from the Jackson Laboratory and were bred and maintained in our rodent facility at the University of British Columbia. Mice were infected intraperitoneally (i.p.) between 7-10 weeks of age with 10 4 pfu of γ HV-68 WUMS strain (purchased from ATCC, propagated on BHK cells); or 10 4 pfu of latency deficient γ HV-68 AC-RTA (originally developed by Dr. Ting-Ting Wu, generous gift of Dr. Marcia A. Blackman) 36  IFN-γ (clone XMG1.2), Foxp3 (clone FJK-16s) were all purchased from eBiosciences. Samples were acquired using a FACS LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.).
Immunohistochemistry. Brains from perfused mice were frozen in OCT (Fisher Scientific) and ten-micron thick sections were processed for immunohistochemistry. Briefly, sections were fixed in ice cold 95% ethanol for 15 min and washed in PBS several times. This was followed by washes in TBS with 0.1% Tween (TBST) and incubation for 10 min with 3% H 2 O 2 to block endogenous peroxidase. After washing, blocking buffer was added for 1 h (10% normal goat serum in PBS). Primary antibody was added overnight at 4 °C: purified rat anti-mouse CD4, anti-mouse CD8 and anti-mouse F4/80 (all from eBiosciences), diluted 1:100 in PBS 2% normal goat serum. After washes in TBST, the biotinylated secondary antibody (anti-rat IgG, mouse absorbed, Vector) was added for 1 h, diluted 1:200 in PBS 2% normal goat serum. After washes in TBST, the Vectastain ABC reagent was used (Vector) following manufacturer's instruction. Then, DAB (Sigma) was added as a substrate and, after incubation for 8 min in the dark and several washes in distilled water, sections were counterstained with Harris hematoxylin for 20 seconds, in lithium carbonate for 30 sec, washed in several changes of distilled water and mount with VectaMount AQ (Vector).
Antigen presentation assay in vitro and pSTAT1 staining of antigen presenting cells. Spleens and inguinal lymph nodes were harvested at day 4 post EAE induction. A single cell suspension was prepared and stained with anti-CD11c and anti-CD11b antibodies (see above for details). CD11c + CD11b + cells were sorted with a FACSAria cell sorter (BD Biosciences). CD4 + T cells from 2D2 mice were isolated from spleens with a CD4 + T cells negative selection kit following manufacturer's instructions (STEMCELL technologies). Isolated CD11b + CD11c + (3-4 × 10 4 /well) and 2D2 CD4 T cells (5 × 10 5 /well) were seeded on a 24 well plate in RPMI, 10% FBS and Pen/Strep (all from GIBCO) with or without 100 μ M MOG peptide. After 72 hours, T cells were restimulated with PMA, ionomycin and GolgiPlug and stained for CD4 and IFN-γ as described above.
For pSTAT1 staining: spleens were harvested 5 weeks post γ HV-68 infection. CD11b + CD11c + cells were stained and sorted as above. Once sorted, cells were fixed with 4% paraformaldehyde and incubated for 10 min at 37 °C. Cells were then washed with 2% PBS/FBS and permeabilized with 90% methanol for 30 min on ice, and then washed and stained for pSTAT1 (clone BD 4a) for 1 hour at room temperature. Cells were acquired and analyzed as detailed above.
Statistical analysis. Two-way ANOVA followed by Bonferroni's post test was employed to compare EAE scores. Unpaired Student's t-test or one way ANOVA was used for all the other analyses (GraphPad Prism).