following ocular infection of naive mice with a recombinant HSV-1 expressing murine IL-4 Dhong Hyun Lee 1 and Homayon Ghiasi 1,*

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1 JVI Accepted Manuscript Posted Online 28 February 2018 J. Virol. doi: /jvi Copyright 2018 American Society for Microbiology. All Rights Reserved. 1 2 An M2 rather than T H 2 response contributes to better protection against latency-reactivation following ocular infection of naive mice with a recombinant HSV-1 expressing murine IL Dhong Hyun Lee 1 and Homayon Ghiasi 1,* Center for Neurobiology and Vaccine Development, Ophthalmology Research, Department of Surgery, Cedars-Sinai Burns & Allen Research Institute, CSMC SSB3, 8700 Beverly Blvd., Los Angeles, CA *Corresponding author. Center for Neurobiology and Vaccine Development SSB3, Cedars-Sinai Burns and Allen Research Institute, 8700 Beverly Blvd., Los Angeles, CA PHONE: (310) ghiasih@cshs.org Running title: Effects of T H 1/T H 2 vs M1/M2 responses on HSV-1 latency-reactivation Key Words: Recombinant viruses, IL-4, IFN-, ocular infection, eye disease, trigeminal ganglia. 1

2 ABSTRACT We found previously that altering macrophage polarization toward M2 responses by injection of colony stimulating factor-1 (CSF-1) was more effective in reducing both primary and latent infection in HSV-1 ocularly infected mice than M1 polarization by IFN- injection. Cytokines can coordinately regulate macrophage and T H responses, with IL-4 inducing T H 2 as well as M2 responses and IFN- inducing T H 1 as well as M1 responses. We have now differentiated the contributions of these immune compartments on protection against latency/reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency associated transcript (LAT) gene was replaced with either the IL- 4 (HSV-IL-4) or IFN- (HSV-IFN- ) genes using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN- genes did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than those infected with HSV-IFN- or parental virus. Primary virus replication in the eyes and trigeminal ganglia (TG) was similar in the three groups of mice but the numbers of gc + cells were lower on day 5 post infection in the eyes of HSV-IL-4- infected mice than those infected with HSV-IFN- or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- or HSV-IFN- -infected mice than those infected with parental virus with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1 and IFN- mrna, while reduced latency and T-cell exhaustion correlated with lower gc + expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group by greater than 3000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1 responses, in HSV-1 infection would improve protection against establishment of latency, reactivation and eye disease. 2

3 IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the U.S. and a major cause of viral-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency/reactivation is a major therapeutic goal. It is well-established that absence of latency associated transcripts (LAT) reduces latency/reactivation. Here, we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of T H 2/M2 responses) or IFN- (an inducer of T H 1/M1 responses) in place of LAT further reduced latency with HSV- IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages towards an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency-reactivation. Downloaded from on January 10, 2019 by guest 3

4 INTRODUCTION Ocular HSV-1 infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness (1-3). Ocular infection with HSV-1 can cause eye disease ranging in severity from blepharitis, conjunctivitis and dendritic keratitis, to disciform stromal edema and necrotizing stromal keratitis (4-6). HSV-1-induced corneal scarring (CS), also broadly referred to as herpes stromal keratitis (HSK), can lead to blindness and is the leading cause of infectious blindness in developed countries, with an estimated 8.4 to 13.2 new cases per 100,000 people per year (7). During primary ocular HSV infection, the virus invades local sensory nerves and travels via neurons to the trigeminal ganglia (TG) (8-11). Infectious virus is cleared from peripheral sites and from the ganglia within ten days of the initial infection (11, 12); however, in the neurons of the ganglia, a latent infection is established (8, 13, 14). From time to time during the life of the latently infected individual, the virus can reactivate, travel back to the eye and cause recurrent disease and shedding, which allows infection of other individuals. Recurrence rates of ocular HSV after an initial episode have been estimated at 10% at 1 year, 23% at 2 years, and over 60% at 20 years (15). There is convincing evidence of a relationship between the level of latency and reactivation in both mice and rabbits (11, 16). Because of the problems associated with recurrent ocular infection, preventing virus replication in the eye and decreasing the establishment of latency and, thus, reactivation should be a major goal of any preventive measure against ocular HSV-1 infection. During HSV-1 neuronal latency, only one viral transcript, the latency-associated transcript (LAT), has been observed consistently at high levels in mice, rabbits, and humans (8, 17-22). LAT is important for the high, wild-type (wt) rate of in vivo spontaneous (23, 24) as well as induced (17) reactivation from latency. Infection with LAT-deficient viruses reduces latency/reactivation by approximately 3-fold; however, acute infection is not impaired (23). In terms of the host responses, both CD4 + T cell- and CD8 + T cellmediated immune responses have been reported to be involved in protection against ocular HSV-1 infection (25-29). Following stimulation by foreign antigens, CD4 + and CD8 + T-cell clones from mice and humans produce specific patterns of cytokine expression (30, 31). The pattern of cytokine production differentiates CD4 + T cells into T H 1 or T H 2 subpopulations and CD8 + T cells into T C 1 or T C 2 subpopulations (30, 32, 33). Usually, either a T H 1/T C 1 or a T H 2/T C 2 cytokine pattern predominates in response to a specific antigenic challenge (34-36). T H 2 cells are involved in humoral (antibody-mediated) immunity and 4

5 produce IL-4, IL-5 and IL-10 (31). Functionally relevant subpopulations of macrophages also have been identified. M1 (classically polarized) macrophages produce significant amounts of proinflammatory cytokines after induction by IFN- (37-39). In contrast, M2 (alternatively polarized) macrophages, which are induced by exposure to IL-4, IL-13, IL-10 or glucocorticoids, produce low levels of proinflammatory cytokines and simultaneously show greater production of anti-inflammatory cytokines (39, 40). M2 macrophages participate in the blockade of inflammatory responses and promote tissue repair/angiogenesis as well as further enhancing T H 2 immune responses (38). Recently, we found that M2 macrophages directly reduce the levels of HSV-1 latency-reactivation in the TG of ocularly infected mice (41) whereas induction of M1 macrophages by IFN- (38, 39) was not as effective against ocular HSV-1 infection (41). Various cytokines have been used as genetic adjuvants to redirect immune responses towards a T H 1 or a T H 2 response (42-46) or an M1 or M2 response (38-41). IL-4 has been shown to enhance the development of T H 2 responses and inhibit T H 1 development (47, 48). In addition to its ability to enhance T H 2 responses, IL-4 is involved in generation of M2 macrophages (39, 40). In contrast to IL-4, IFN- is a cytokine secreted by activated CD4 + T helper 1 (T H 1), natural killer (NK), and CD8 + cytotoxic (T C 1) suppressor cells (49, 50) that induces M1 macrophages (38, 39). To differentiate the contributions of T H 2 and/or M2 versus T H 1 and/or M1 to HSV-1 primary and latent infection, we used a mouse model in which: (i) Mice were infected with recombinant HSV-1 constructs in which two copies of IL-4 or IFN- replace the LAT gene (51, 52). The genes are under the regulation of the LAT promoter and the background is LATminus; thus, the LAT-minus parental virus was used as a control; and (ii) The effects of IL-4 or IFN- expression on infection of macrophage intact or depleted mice were compared. Our findings demonstrate that in ocularly infected mice: 1) HSV-IL-4 recombinant virus shifted macrophage polarization towards an M2 response whereas HSV-IFN- recombinant virus shifted macrophage polarization towards an M1 response; 2) The IL-4-expressing recombinant virus exhibited significantly greater overall efficacy in terms of protection against latency and reactivation as compared with the IFN- -expressing recombinant virus or parental virus ; and 3) The HSV-IL-4-associated protective effect against latent infection was reduced 5

6 significantly by macrophage depletion, suggesting that the M2 response plays a more important role in this protective effect than the T H 2 response

7 RESULTS HSV-IL-4 induces M2 responses and HSV-IFN- induces M1 responses in peritoneal macrophages (PM). Recombinant IL-4 generates M2 polarization, whereas recombinant IFN- generates M1 polarization (41, 53). To determine if HSV-IL-4 infection induces M2 polarization and HSV-IFN- infection induces M1 polarization, unpolarized PM isolated from naive mice were mock-infected or infected with 10 PFU/cell of HSV-IL-4, HSV-IFN-, or parental virus for 24 h. Infected cells were isolated, total RNA was extracted and qrt-pcr carried out to assess the levels of ARG1 mrna, a marker of M2 cells, and NOS2 mrna, a marker of M1 cells (54-56), as we described previously (41). The levels of ARG1 mrna were significantly higher in HSV-IL-4-infected PM than parental or HSV-IFN- infected cells (Fig. 1A, p<0.0001). The levels of NOS2 mrna were significantly higher in the HSV-IFN- -infected PM than in parental or HSV-IL-4 infected cells (Fig. 1B, p<0.04; ANOVA). Thus, infection of PM by HSV-IFN- and HSV-IL-4 induced the expected macrophage polarization phenotypes in vitro. Effect of macrophage polarization phenotype on HSV-IL-4 and HSV-IFN- replication in PM. We reported previously that replication of wt HSV-1 strain McKrae was markedly lower in M1 macrophages than in either M2 or unstimulated control macrophages using RAW264.7 and PM cells treated with IFN- alone or IFN- and LPS (for M1 polarization) or IL-4 (for M2 polarization) (41). To determine the effects of overexpression of IL-4 by HSV-IL-4 and IFN- by HSV-IFN- viruses on replication in polarized PM, we isolated PM from wt mice and generated M1 or M2 macrophages by treatment with IFN- and LPS or IL-4 as we described previously (41). These and the unpolarized macrophages were then infected with HSV- IL-4, HSV-IFN- or parental virus and virus replication assessed using a standard plaque assay. There was significantly lower replication of parental (Fig. 2, parental), HSV-IFN- (Fig. 2, HSV-IFN- ), and HSV- IL-4 (Fig. 2, HSV-IL-4) viruses in M1 macrophages than in M2 or unpolarized macrophages at all time points (p<0.001) except for HSV-IFN- at 12 h post infection (PI) (Fig. 2, HSV-IFN- ; p = 0.07). There were no significant differences in the replication of the viruses in the M2 and unpolarized macrophages (Figure 2). The replication of the recombinant HSV-IFN- or HSV-IL-4 viruses in the M1, M2 and unpolarized macrophages was not significantly different from the replication of the parental virus in the corresponding 7

8 cells. These results suggested that M1 macrophages are either less permissive of HSV-1 infection or less supportive of HSV-1 replication than M2 or unpolarized macrophages. These results are consistent with previous reports that the yield of HSV-1 from M1 macrophages derived from the murine J774A.1, RAW264.7, and peritoneal macrophages were lower than the yields from the unpolarized macrophages and M2 macrophages (41, 57). Higher proinflammatory cytokines and chemokines (41) and/or growth arrest and decreases in the ph (58) most likely contribute to the lower virus replication in M1 cell compared with M2 or unpolarized cells. Overall, the results indicate that M1 macrophages are less permissive for HSV-1 infection than M2 or unpolarized macrophages for all three viruses (HSV-IL-4, HSV- IFN-, parental virus) as we described previously for wt McKrae (41). Replication of HSV-IFN- and HSV-IL-4 in the eye after ocular infection. To assess replication of the recombinant viruses in the eyes of ocularly infected mice, the titers of infectious virus in tear films was determined using standard plaque assays (Fig. 3A). Mice were ocularly infected with 2 X 10 5 PFU/eye of HSV-IFN-, HSV-IL-4 or parental virus and tear films collected from day 1 to day 6 PI (10 mice per group; 20 eyes per group). The titers of all three viruses were similar, except for day 3 PI when there were significantly lower titers of HSV-IL-4 (Fig. 3A, p = 0.034). Overall, these results indicated that neither the IFN- or IL-4 expressed by HSV-IFN- or HSV-IL-4, respectively, affected HSV-1 virus replication in the eyes of infected mice as determined by analysis of tear films. To differentiate between the effects on T H responses and macrophage responses, the above experiment was repeated except that macrophage depletion was carried out on days 5, -2, +1, and +3 relative to ocular infection. For all three viruses, macrophage depletion was associated with increased virus replication in the eyes as compared with their undepleted counterparts (compare Fig. 3B with Fig 3A) and there were no significant differences in the effects of macrophage depletion on virus replication among the three viruses (Fig. 3B). To determine viral replication in the cornea, mice were ocularly infected without macrophage depletion as described above and euthanized on day 5 PI. The corneas from each mouse were combined, single-cell suspensions prepared and the numbers of gc + cells determined by FACS analysis. The numbers of gc + cells in the corneas of HSV-IFN- - and parental virus-infected mice were not statistically 8

9 171 different (Fig. 4, p>0.05, Fig. S1) but there were markedly fewer gc + cells in the corneas of HSV-IL infected mice (Fig. 4, p<0.001, Fig. S1). This discrepancy between the number of gc + cells and the viral titers in tear films between HSV-IFN- - and parental virus-infected cornea with HSV-IL-4-infected cornea may be due to the possibility that HSV-IFN- and parental viruses make more gc during productive infection compared with HSV-IL-4, and it is also possible that an overexpression of IL-4 has a suppressive effect on gc expression. However, HSV-IL-4 produces enough gc during productive infection in which lower gc expression is not a limiting factor for producing a similar number of infectious virus as HSV-IFNg and parental viruses. To determine viral replication in the TG, the TG were harvested on day 5 PI, homogenized and the amounts of infectious virus in the TG of each mouse determined using a standard plaque assay. The titers of virus in the TG of the mice infected with HSV-IL-4, HSV-IFN- or parental viruses were not significantly different (Fig. 5; p>0.05), indicating that the replication of the HSV-IFN- or HSV-IL-4 in the TG of ocularly infected mice was similar to the replication of the parental virus. Virulence and corneal scarring in HSV-IFN- and HSV-IL-4 infected mice. For analysis of virulence and CS, groups of 25 mice were infected ocularly in both eyes with 2 X 10 5 PFU/eye of HSV- IFN-, HSV-IL-4 or parental virus. All mice in the HSV-IL-4-infected group survived ocular infection, whereas 22 of 25 mice in the HSV-IFN- infected group and 23 of 25 mice in the parental virus-infected group survived ocular infection (Fig. 6A) but these differences were not statistically significant (p>0.50). The eyes of the mice that survived ocular infection with HSV-IFN-, HSV-IL-4, or parental virus were examined for CS on day 28 PI and eye disease scored on a 0 to 4 scale as described in the Materials and Methods section. Mice infected with HSV-IFN- or parental virus had similar levels of CS (Fig. 6B, p = 0.5), with the levels of CS being significantly higher than those observed in the HSV-IL-4- infected mice which showed no CS (Fig. 6B, p = 0.03). Thus, IL-4 expression by HSV-IL-4 was associated with a reduction in CS whereas IFN- expression by HSV-IFN- neither ameliorated or exacerbated CS. The lower CS in HSV-IL-4 infected mice could be due to the differences in the response of M1 and M2 macrophages to infection with different viruses. Recently we reported that the M2 macrophages secreted less cytokines and chemokines than the M1 macrophages, which, potentially, could contribute to the lower 9

10 CS in HSV-IL-4 infected mice or it could be due to the effect of more expression of tissue-healing cytokines and chemokines by M2 macrophages. Effect of virus-mediated expression of IFN- or IL-4 on establishment of latent infection. We found previously that CSF-1 DNA injection, which induces M2 macrophage polarization, reduced latent HSV-1 infection in ocularly infected mice as compared to injection of IFN- DNA, which induces M1 polarization (41). To determine the effects of HSV-IFN-, HSV-IL-4 and parental virus on establishment of latency, mice were ocularly infected as described above (10 mice per group). TG were harvested on day 28 PI and the amount of gb DNA in the tissue determined by TaqMan PCR analysis as we described previously (16, 59). The gb copy numbers in the HSV-IFN- and HSV-IL-4 groups were normalized to gb levels in the TG of mice infected with parental virus and the data are shown as fold changes in the level of latency in Figure 7A. The levels of latency in both HSV-IFN- and HSV-IL-4- infected mice were significantly lower than the levels of latency in mice infected with parental virus (Fig. 7A, HSV-IFN-, p = 0.02; HSV-IL-4, p = 0.009; Fisher s exact test). However, while the level of latency was approximately 10- fold lower in the HSV-IFN- -infected mice than those infected with parental virus, it was approximately 100-fold lower in the HSV-IL-4- infected mice than in the mice infected with parental virus (Fig. 7A). These results suggest that while recombinant viruses expressing either IFN- or IL-4 reduced the level of latency in mice, IL-4 was more efficient in reducing the level of latency than IFN-. Role of macrophages in reducing latency in HSV-IFN- - and HSV-IL-4-infected mice. As IFN- both stimulates M1 and is an indicator of a T H 1 response, while IL-4 both stimulates M2 and is an indicator of a T H 2 response, the lower latency in HSV-IFN- - and HSV-IL-4-infected mice compared with mice infected with parental virus could be due to the effects of the respective cytokines on either macrophage or T H cell function. To differentiate these effects, additional groups of mice were macrophage depleted at various time points before and after ocular infection with HSV-IFN-, HSV-IL-4 or parental virus as described above. TG were harvested on day 28 PI and gb expression was measured using TaqMan- PCR analysis. For each virus, the gb levels in macrophage-depleted mice were normalized to the gb expression in their undepleted counterparts and data are shown as fold changes relative to their undepleted counterparts. In all three groups, macrophage depletion significantly increased latency as 10

11 compared with their undepleted counterparts (Fig. 7B). In mice infected with parental or HSV-IFN- virus, the levels of latency in macrophage-depleted mice were approximately 500-fold higher than in their undepleted counterparts (Fig. 7B, p = when comparing parental depleted with their undepleted counterpart; and p = 0.04 when comparing HSV-IFN- depleted with their undepleted counterpart; Fisher s exact test). The fold increase in latency in the macrophage-depleted mice did not differ significantly between the mice infected with HSV-IFN- and parental virus (Fig. 7B, p>0.05). In marked contrast, latency in HSV-IL-4-infected macrophage-depleted mice was more than 3000-fold higher than in their undepleted counterpart (Fig. 7B, HSV-IL-4; p<0.0001). The findings that, in the absence of macrophages, IFN- provided no additional degree of protection against the establishment of latency as compared with parental virus whereas the absence of macrophages completely eliminated the protective effect of IL-4 provide further evidence that macrophages provide protection against latent infection and are consistent with our previous report (41), that M2 macrophages provide better protection against establishment of latency then M1 macrophages. Detection of gc +, IFN- +, and IL-4 + cells in the TG of latently infected mice. For analysis of the numbers of gc-positive cells in the TG of the latently infected mice, mice were infected as above with HSV-IFN-, HSV-IL-4 or parental viruses and TG were isolated on day 28 PI (5 mice per group), single cell suspensions stained with FITC-conjugated anti-gc antibody and analyzed using flow cytometry. A representative flow cytometric analysis of the numbers of gc cells is shown as supplementary Fig. S2. Compared to TG from mice latently infected with HSV-IFN- or HSV-IL-4 virus, TG from mice latently infected with parental virus contained a significantly higher number of gc + cells (Fig. 8A; p<0.004; ANOVA). There was no significant difference in the numbers of gc + cells in the TG from HSV-IFN- - and HSV-IL-4- latently infected mice (Fig. 8A; p>0.05). This result is consistent with the above results showing greater latency, as determined by gb expression, in TG of mice infected with parental virus than mice infected with HSV-IFN- or and HSV-IL-4 viruses. To detect IFN- and IL-4 protein expression during latency, mice were infected ocularly as above with HSV-IFN-, HSV-IL-4, and parental viruses. Naive, uninfected mice were used as a control. TG from latently infected mice were isolated on day 28 PI and single cell suspensions were prepared and stained 11

12 with anti-ifn- and anti-il-4 mabs. Representative flow cytometric analyses of the numbers of IFN- + and IL-4 + cells are shown as Supplementary Fig. S3. Compared to TG from the uninfected mice, there were significantly higher numbers of IFN- + cells in the TG from HSV-IFN- -infected mice (Fig. 8B, p = 0.047); however, there were no significant differences in the numbers of IFN- + cells in the TG of HSV-IFN-, HSV-IL-4, and parental virus-infected mice, or between HSV-IL-4 or parental virus as compared with the uninfected mice (Fig. 8B, p>0.05). Although the numbers of IL-4 + cells in the TG per mouse were higher in all three groups of infected mice as compared with the uninfected mice these differences did not reach statistical significance (Fig. 8C, p>0.05). Levels of CD4, CD8, PD-1, IFN-, and IL-4 mrnas in TG of latently infected mice. A similar protocol was used to investigate the effect of IFN- and IL-4 expression by HSV-IFN- and HSV-IL-4 on CD4, CD8, PD-1, IFN- and IL-4 mrna levels in the TG of latently infected mice using qrt-pcr of TG extracts (5 mice per group, 10 TG per group). The results are presented in Figure 9 as the fold difference relative to the baseline mrna levels in TG from naive, uninfected mice. The levels of CD4 mrna were significantly higher in the TG of mice latently infected with HSV-IFN- virus than in the TG of mice infected with parental (Fig. 9A; p = 0.006) or HSV-IL-4 viruses (Fig. 9A; p = ). In contrast, the levels of CD8 mrna were significantly higher in the TG of mice latently infected with parental virus than in the TG of mice infected with either HSV- IFN- or HSV-IL-4 (Fig. 9B, p<0.0001). Despite higher levels of CD8 mrna in the HSV-IFN- group compared with the HSV-IL-4 group, these differences did not reach statistical significance (Fig. 9B, p>0.05). The levels of PD-1 mrna (Fig. 9C; p<0.05) and IFN- mrna (Fig. 9D; p = 0.04 for HSV-IFN- and p = 0.03 for HSV-IL-4) also were significantly higher in the TG of the mice infected with parental virus than in the TG from either HSV- IFN- - or HSV-IL-4-infected mice, whereas the levels of IL-4 mrna were significantly lower (Fig. 9E; p = 0.02 for HSV-IFN- and p = for HSV-IL-4). As would be anticipated, there were higher levels of IFN- mrna in the HSV-IFN- -infected mice than in the HSV-IL-4-infected mice (Fig. 9D; p>0.05) and higher levels of IL-4 mrna in the HSV-IL-4-infected mice than in the HSV-IFN- -infected mice (Fig. 9E, p>0.05). However, the levels of PD-1 (Fig. 9C), IFN- (Fig. 9D) and IL-4 (Fig. 9E) were not significantly different between the HSV-IFN- - and HSV-IL-4-infected mice (p>0.05). These results suggest that there are both higher numbers of 12

13 CD8 + T cells and greater T-cell exhaustion, as indicated by higher expression of PD-1, in the TG from mice latently infected with parental virus than in the TG from mice infected with either HSV-IFN- or HSV-IL-4 viruses. Reactivation is reduced in the TG of HSV-IL-4 infected mice. Our examination of latency showed lower latency in HSV-IL-4-infected mice than mice infected with parental or HSV-IFN- virus. We therefore tested whether the lower latency in the TG of HSV-IL-4- and HSV-IFN- -infected mice correlated with lower explant reactivation from latency. In these experiments, mice were infected ocularly with 2 X 10 5 PFU/eye of HSV-IL-4, HSV-IFN- or parental virus. Individual TG from the surviving mice were isolated on day 28 PI and virus reactivation analyzed by explanting individual TG from infected mice as described in the Materials and Methods. Consistent with the lower levels of gb in the TG of HSV-IL-4 infected mice (Fig. 7A, above), the time to reactivation in the HSV-IL-4 group was significantly longer than in the group of mice infected with parental virus (Fig. 10; 7.8 ± 0.8 days vs 5.2 ± 0.2 days; p = 0.01). A trend toward a longer time to reactivation in the HSV-IFN- -infected mice as compared to parental infected mice was observed but did not reach significance (Fig. 10; parental; 5.2 ± 0.2 days vs 6.0 ± 0.5 days; p = 0.53). Despite the different levels of latency in the parental and HSV-IFN- viruses as compared to the HSV-IL-4 virus-infected mice (Fig. 7A, above), a trend toward a longer time to reactivation of HSV-IL-4 virus than as compared to reactivation of HSV-IFN- virus was observed although these differences were not significant (Fig. 10; HSV-IFN- ; 6.0 ± 0.5 days vs 7.8 ± 0.8 days; p = 0.18). Thus, in HSV-IL-4-infected mice the time to explant reactivation was slower and correlated with the lower level of gb DNA, whereas in HSV-IFN- infected mice the time to explant reactivation did not correlate with the level of latency that was established. 13

14 DISCUSSION This study was designed to answer three questions: (1) Previously, it was reported that LATdeficient viruses have lower latency and reactivation compared with their parental wt counterparts (11, 23, 60). Thus, if latency in a LAT-minus virus is a threshold, can we further reduce this threshold by using recombinant viruses expressing foreign genes in a LAT-minus background and without changing the dose of infection? To investigate this, we used the LAT promoter to constitutively express our genes of interest. LAT has a powerful promoter that is active in most cell types (51, 61-65) and insertion of an indicator gene under its control produces efficient, long-term expression of the indicator gene in mice and rabbits (42, 43, 51, 52, 66-71); (2) Can expression of IL-4 induce M2 responses that significantly reduce HSV-1 latencyreactivation? To investigate this, we used ocular infection with recombinant HSV-1 that express IL-4 (HSV- IL-4) or IFN- (HSV-IFN- ) in a LAT-minus background and under the LAT promoter to query the effects on latency/reactivation. These viruses constitutively express their respective gene in various cell types; and (3) Are the effects of expression of IL-4 or IFN- by the recombinant viruses on latency/reactivation mediated primarily by macrophages or T cells? To investigate this, we used macrophage depletion in conjunction with infection with the recombinant viruses. The results presented here indicate that recombinant viruses expressing foreign genes, i.e., IL-4 and IFN-, in place of LAT and under the LAT promoter were capable of significantly reducing latency compared with their LAT-minus parental virus. Furthermore, the HSV-IL-4 recombinant virus was more efficacious in reducing latency than the HSV-IFN- virus. This reduction in latency was not due to differences in primary infection in the eye and TG. These results also suggest that the reduced latency in HSV-IL-4 and HSV-IFN- groups was independent of the primary infectious virus titers in the eyes and TG of the infected mice, which is consistent with our previous finding of a lack of correlation between primary virus titer in the eye and the level of viral DNA in latently infected TG (16). The current studies did, however, reveal a significant relationship between lower latency in the HSV-IL-4-infected mice with a lower number of HSV-1 gc + cells in the cornea. gc can bind the C3b fragment of the third component of complement (72) and mediates immune evasion by blocking activation of the complement cascade at multiple stages (73, 74). Thus, lower expression of gc in the cornea of HSV-IL-4 infected mice on day 5 PI 14

15 may curb the immune invasion and thus affect intraspecific components of the immune system thereby improving the host protection from latency-reactivation. In addition, we found that reactivation of latent virus in TG explant cultures was reduced in HSV-IL-4-infected mice as compared to parental virus- and HSV-IFN- virus-infected mice, demonstrating that IL-4 is a significant factor in decreasing HSV-1 reactivation. However, we considered it unlikely that the reduced infectivity in the eye of HSV-IL-4 infected mice at day 3 PI compared with the mice infected with the other viruses to play a role in lower reactivation by HSV-IL-4 virus. IL-4 enhances the development of T H 2 and M2 responses, while IFN- enhances the development of T H 1 cells and M1 responses. Thus, the improved protective parameters associated with HSV-IL-4- infection could be due to enhancement of either T H 2 and/or M2 responses. We considered it unlikely that the T H 2 responses play a role in the responses observed in our mouse model as we used naive mice and it is unlikely that adaptive immunity plays a differential role in the improved protection in the presence of IL-4 in the HSV-IL-4-infected mice as compared with the mice infected with the other viruses. Moreover, following macrophage depletion, the virus titers in the eyes of all infected mice increased by approximately 20% but the differences were not significant. Latency also was increased in all of the macrophage depleted groups, but the greatest enhancement of latency was in the HSV-IL-4-infected mice. Although Cl 2 MDP depletion results in depletion of all macrophages, collectively our results suggest that in the presence of IL-4 the M2 function of macrophages contributed to better protection from latency than the M1 or M0 macrophages. We have previously demonstrated a positive correlation between higher level of latency with faster time to explant reactivation in ocularly infected mice (16). Although no explant reactivation was performed in this study, but we anticipate to detect similar increase in the reactivation of latent virus in TG explant cultures in the HSV-IL-4 mice compared with parental or HSV-IFN- infected mice. Furthermore, the result of this study is consistent with our previous finding that M2 macrophages play a greater role in protection from latency-reactivation than M1 responses (41). Collectively, the results of the current study suggest the importance of M2 responses in both latency and reactivation of HSV-1 but not primary infection. 15

16 Previously, we have shown that increased HSV-1 latency correlates with higher levels of PD-1 and CD8 expression together with loss of T-cell function (75). Moreover, infection with LAT-plus viruses is associated with higher T-cell exhaustion than infection with LAT-minus viruses due to higher reactivation of the LAT-plus viruses (75, 76). In the current study, we used LAT-minus viruses and found that ocular infection with the recombinant viruses expressing IL-4 or IFN- lowered the levels of PD-1 and CD8 T-cell expression as compared with infection with the parental virus. Although LAT-minus viruses have at least a 3-fold lower latency-reactivation than their LAT-plus counterparts (23, 75), this lower latency-reactivation still contributed to T-cell exhaustion but at considerably lower levels than in the presence of LAT. Thus, T- cell exhaustion can occur in the absence of LAT; however, our results suggest that even in a LAT-minus background we can reduce the level of HSV-1-induced T-cell exhaustion by further reducing the level of latency-reactivation. Previously, we and others have shown subclinical reactivation of several viral genes during latency in mice (75, 77-79). These low levels of reactivation contribute to T-cell exhaustion and are associated with higher T-cell exhaustion in LAT-plus versus LAT-minus viruses (75, 80). In the current study, we detected lower numbers of gc + cells in the TG of HSV-IL-4- and HSV-IFN- -infected mice than mice infected with parental virus and this correlated with lower PD-1 and CD8 expression and lower latency-reactivation confirming that viral antigens contribute to T-cell exhaustion (75). It has been shown that during chronic infection there is a loss of T-cell function in which cytotoxic T cells lose the ability to produce IFN- (81, 82). However, in the current study we found that infection with parental virus resulted in higher expression of CD8, PD-1 and IFN- as well as higher levels of latencyreactivation than infection with HSV-IL-4 or HSV-IFN- viruses suggesting that there is not a correlation between the level of IFN- expression and latency-reactivation. This is consistent with our previous observations that the LAT levels in IFN- -/- mice was similar to those in wt mice (83) and that the absence of IFN- does not affect HSV-1 reactivation (84). In the current study, we further found that that HSV-IFN-, despite expressing IFN- constitutively, had lower latency-reactivation than parental virus. In contrast, it has been reported that TG-resident CD8 + T-cell prevention of HSV-1 reactivation from latency is mediated by IFN- (85). The apparent discrepancy may be due to the use of an ex-vivo model of reactivation in the latter study. Finally, we found a lack of correlation between the level of PD-1 expression with IFN- 16

17 expression in the HSV-IL-4- and HSV-IFN- -infected mice but not in mice infected with the parental virus, which is consistent with other studies that have shown a lack of correlation of T-cell exhaustion and the IFN- response (86-89). In summary, our results using a recombinant HSV-IL-4 mouse model of ocular infection indicate that IL-4 modifies M2 responses, which in turn decreases virus latency and reduces the reactivation cycle in the TG. Moreover, IL-4 appears to be essential to enhancement of the macrophage immune signature in the TG suggesting its importance for host innate immunity during latency. These results indicate that IL- 4-M2 forms a critical pathogen-host axis that contributes to a reduction in viral latency-reactivation. Downloaded from on January 10, 2019 by guest 17

18 MATERIALS AND METHODS Viruses. The construction and characterization of dlat2903, HSV-IL-4 and HSV-IFN- viruses was described previously (23, 51, 52). HSV-IL-4 and HSV-IFN- transcribe mouse IL-4 and IFN-, respectively, driven by the LAT promoter. They are identical except for the insertion of the IL-4 and IFN- gene in place of the LAT region deleted in dlat2903. Thus, HSV-IL-4 and HSV-IFN- contain the same LAT nucleotides as dlat2903. Wild type HSV-1 strain McKrae is the parental virus for dla2903 (23). dlat2903 is the parental virus for HSV-IL-4 and HSV-IFN- and throughout the study, dlat2903 virus is called parental virus. All three viruses are LAT-minus. Cells. Rabbit skin (RS) cells were used for preparation of virus stocks, culturing mouse tear swabs, and determining growth kinetics. RS cells were grown in Eagle's minimal essential media (MEM) supplemented with 5% fetal bovine serum (FBS). PM were harvested from C57BL/6 mice after expansion by treatment of the mice with zymosan (Sigma-Aldrich, St. Louis, MO) as we described previously (41). Mice were administered 200 μl of 2 mg/ml of zymosan in sterile, 0.9% w/v saline solution in one intraperitoneal injection. The peritoneal fluid was harvested 72 h later and cultured overnight in DMEM full media with 10% FBS in 24-well plates. The media was then removed and the cells washed twice with PBS to remove any floating cells. Adherent cells were recovered by gentle scraping and subjected to the M1 and M2 macrophage activation protocols as described below. Mice. Female inbred C57BL/6J (6-8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME). All animal procedures were performed in strict accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the NIH Guide for the Care and Use of Laboratory Animals (ISBN ). Animal research protocol was approved by the Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center (Protocol #5030). Activation and infection of PM in vitro. Cells were seeded at 2 X 10 5 cells/well in a 24-well plate. After overnight incubation, the media was replaced with fresh DMEM full media containing 50 ng/ml of murine IFN-γ (Peprotech, Rocky Hill, NJ) and 100 ng/ml of lipopolysaccharide (LPS) (Sigma-Aldrich, St. 18

19 Louis, MO) for M1 activation, or full media containing 10 ng/ml of murine IL-4 (Peprotech, NJ) for M2 activation. On the following day, cells were infected with 10 PFU/cell of parental, HSV-IFN-, or HSV-IL-4 virus for 1 h. The infected cells were then washed three times with PBS and fresh DMEM full media was added to each well. Virus was harvested at 12, 24, and 48 h PI by two cycles of freeze-thawing of the cell monolayers with culture media. Virus titer was determined by standard plaque assay using RS cells (12, 23). Ocular infection. Mice were infected ocularly with 2 X 10 5 PFU per eye of each virus as an eye drop in 2 μl of tissue culture media as we described previously (41, 90). Corneal scarification was not performed prior to infection. Virus titration in tear film. Tear films were collected daily from the eyes of 10 mice (20 eyes) per group from day one through day 6 PI using a Dacron tipped swab (12). Each swab was placed in 0.5 ml tissue culture medium, squeezed, and the inoculated medium titrated by a standard plaque assay on RS cells (12, 23). Detection of infectious virus in TG. Mice were euthanized on day 5 PI, TG harvested and individual homogenates of TG from each mouse prepared as described previously (91). The viral titer of the resulting supernatant was measured on RS cells using a standard plaque assay (12, 23). Preparation of corneas for flow cytometry. Mice were ocularly infected with each virus as above or mock-infected. Two corneas from each mouse were removed on day 5 PI and combined. Individual mouse corneas were digested in a PBS solution containing collagenase type I (3mg/ml; Sigma-Aldrich, St. Louis, MO) as we described previously (59, 75). Single-cell suspensions were washed with PBS and isolated cells were stained with FITC-HSV-1 antibody (Ab) specific for gc (cat no. GWB-4880EF, Genway Biotech, San Diego, CA) antibody as we described previously (75). Stained cells were washed twice with wash buffer and fixed with paraformaldehyde cell fixation buffer (Biolegend, San Diego, CA) for 20 min at 4 C. Following fixation, the cells were washed again and analyzed using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). gc, IL-4, and IFN- staining of isolated cells from latently infected TG. Mice were ocularly infected with each virus as above or mock-infected. TG from each mouse were removed on day 28 PI and combined. Individual mouse TG were digested in a PBS solution containing collagenase type I (3mg/ml; Sigma-Aldrich, St. 19

20 Louis, MO) as we described previously (59, 75). Single cell suspensions were washed with PBS and isolated cells were stained with FITC-HSV-1 antibody specific for gc (Genway Biotech). gc-stained cells were washed three times and incubated at 37 C and 5% CO 2 for 4 h in the presence of brefeldin A (Biolegend). At the end of the incubation, cells were washed and resuspended in wash buffer and incubated for 15 min at 4 C with Fc Block anti-mouse antibody (cat no , BD Biosciences), followed by subsequent incubation with APC-IL-4 (cat no , Biolegend) and Pacific Blue-IFN- (cat no ) (Biolegend) anti-mouse Abs at 4 C for 30 min. Stained cells were washed twice with wash buffer and fixed with 250 l of cell fixation buffer for 20 min at 4 C. Following fixation, the cells were washed twice in intracellular staining permeabilization wash buffer (Biolegends) and analyzed using a BD LSR II flow cytometer (BD Biosciences). Macrophage depletion. Liposome-encapsulated dichloromethylene diphosphonate (Cl 2 MDP) was purchased from Liposoma (Amsterdam, The Netherlands). Macrophage depletion was carried out as we described previously (92, 93). Briefly, each mouse received 100 μl of the Cl 2 MDP twice, once intraperitoneally (i.p.) and once subcutaneously (s.c.) on days 5, -2, +1, and +3 relative to ocular infection with each virus. DNA extraction and PCR analysis for HSV-1 genomic DNA. DNA was isolated from homogenized individual TG using the commercially available Dneasy Blood &Tissue Kit (Qiagen, Stanford, CA, Cat. No ) according to the manufacturer's instructions. PCR analyses were done using gb specific primers (Forward - 5'-AACGCGACGCACATCAAG-3'; Reverse - 5'- CTGGTACGCGATCAGAAAGC-3'; and Probe - 5'-FAM-CAGCCGCAGTACTACC-3') as we described previously (75). The amplicon Length for this primer set is 72 bp. Relative copy numbers for gb DNA was calculated using standard curves generated from the plasmid pac-gb1. In vitro explant reactivation assay. Mice were sacrificed at 28 days PI and individual TG removed and cultured in tissue culture media as we described previously (16, 59, 94). Aliquots of media were removed from each culture daily for up to 14 days and plated on indicator cells (RS cells) to assay for the appearance of reactivated virus. As the media from the explanted TG cultures were plated daily, the time at which reactivated virus first appeared in the explanted TG cultures could be determined. 20

21 RNA extraction, cdna synthesis, and TaqMan qrt-pcr assay. For cultured cells, total RNA extraction was carried out using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer s protocol. For TG, tissues were collected 4 weeks after infection and immersed in TRIzol reagent (Applied Biosystems, Foster City, CA) and stored at -80 C until processing. Total RNA extraction was carried out according to the manufacturer s protocol. Following RNA extraction, 1 µg of total RNA was reversetranscribed using High Capacity cdna Reverse Transcription Kit (Applied Biosystems, CA) according to the manufacturer s protocol. mrna expression levels of the genes of interest were evaluated using TaqMan Gene Expression assays (Applied Biosystems, CA) as we described previously (75). TaqMan assays used in this study were: (i) IFN-, assay ID (Thermo Fischer) = Mm _m1, amplicon size = 101 bp; (ii) IL-4, Mm _m1, 79 bp; (iii) PD-1, Mm _m1, 65 bp; (iv) CD4, Mm _m1, 78 bp; (v) CD8a, Mm _m1, 67 bp; GAPDH was used as a loading control in all experiments. Quantitative real-time PCR (qrt-pcr) was performed using an ABI ViiA 7 Sequence Detection System (Applied Biosystems) in 384-well plates. Real-time PCR was performed in triplicate for each tissue sample. The threshold cycle (C t ) values, which represents the PCR cycle at which there is a noticeable increase in the reporter fluorescence above baseline, were determined using SDS 2.2 software. Statistical analysis. For all statistical tests, p-values less than or equal to 0.05 were considered statistically significant and are marked by a single asterisk (*). p-values less than or equal to are marked by double asterisks (**). Fisher s exact test was used to compare difference between two experimental groups. One-way ANOVA test was used to compare difference among three or more experimental groups. 21

22 ACKNOWLEDGEMENTS This study was supported by Public Health Service NIH grants RO1EY

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