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CXCL9 compensates for the absence of CXCL10 during recurrent Herpetic stromal keratitis
Virology 506 (2017) 7–13
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CXCL9 compensates for the absence of CXCL10 during recurrent Herpetic stromal keratitis
MARK
Deena Tajfirouza, Devin M. Westa, Xiao-Tang Yina, Chloe A. Pottera, Robyn Kleinb, Patrick ⁎ M. Stuarta, a b
Department of Ophthalmology, Saint Louis University School of Medicine, St. Louis, MO, USA Departments of Medicine, Pathology & Immunology, Anatomy & Neurobiology Washington University School of Medicine, Saint Louis, MO, USA
A R T I C L E I N F O
A BS T RAC T
Keywords: Herpes simplex virus type 1 Cornea Chemokines CXCL10 CXCL9 Inflammation Herpetic stromal keratitis
Herpetic stromal keratitis (HSK) is a disease that is typically associated with reactivation of a latent HSV-1 infection. This disease is driven, in part, by chemokines that recruit leukocytes to the cornea. Surprisingly, neutralization of CXCL10 significantly reduced disease, while B6-CXCL10-/- mice exhibited worse disease compared with similarly infected wild-type controls. We hypothesized that compensatory up-regulation of CXCL9 occurs in the absence of CXCL10. Analysis of CXCL9 expression in HSV-1-infected B6 mice and B6CXCL10-/- mice revealed significantly more CXCL9 in B6-XCL10-/- mice. Treatment of B6 and B6-CXCL10-/mice with neutralizing antibodies to CXCL9 reduced HSK scores in B6-CXCL10-/-, but not B6 mice. We conclude that CXCL10 production worsens HSK and that CXCL9 may compensate in CXCL10-deficient animals. These studies identify the critical role that CXCL10 plays in the pathogenesis of recurrent HSK, and that CXCL9 displays its importance when CXCL10 is absent.
1. Introduction Herpes simplex virus (HSV) is a widespread viral pathogen that infects the majority of the world's population. Although oral and to a lesser extent, genital infections are the most common type of HSV type 1 (HSV-1) infection, this virus can also cause disease in major ocular tissues including the cornea (Arduino and Porter, 2006; Smith and Robinson, 2002; Umene and Sakaoke, 1999). Herpetic stromal keratitis (HSK), caused by HSV-1, is the most common cause of infectioninduced blindness of the cornea (Umene and Sakaoke, 1999; Feldman et al., 2002; Tuli and Sonal, 2009; Farooq and Shukla, 2012; Stuart and Keadle, 2012; Rowe et al., 2013). Similar to other herpetic viruses, infection with HSV-1 can be categorized into primary and recurrent disease. Primary infection with HSV-1 is usually asymptomatic in humans; however, the clinical disease phenotype also known as HSK is the result of reactivation of the virus that originally established a latent infection in the trigeminal ganglia (Umene and Sakaoke, 1999; Rinne et al., 1992). HSV-1 reactivation is typically elicited by different immunosuppressive events such as irradiation, sunlight (UV), stress, trauma, and fever. It has been shown that HSK is primarily due to an immunopathogenic response to the virus (Inoue, 2008). Pro-inflammatory cytokines and chemokines that are produced during early stages of viral infection and reactivation lead to recruitment of
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neutrophils, NK cells, macrophages, and T cells, all of which are important mediators of inflammation (Li et al., 2006; Fenton et al., 2002; Carr et al., 2003; Stuart et al., 2008; Duan et al., 2007; Tsai et al., 2002; Wuest and Carr, 2008; Banerjee et al., 2004; Shen et al., 2013; Keadle and Stuart, 2005; West et al., 2014). In primary and recurrent models of HSK the presence of CD4+ T cells and neutrophils in primary and secondary models of HSK has been well documented, therefore making the study of their recruitment to the site of infection and subsequent inflammation critical to the understanding of the disease process. Activated CD4+ T cells and NK cells express the receptor CXCR3, a G-protein coupled receptor that mediates its inflammatory effects by binding to CXCL9, CXCL10, and CXCL11 chemokines (Clark-Lewis et al., 2003). While these cytokines have been shown to be made by monocytes, dendritic cells and epithelial cells, most recently, CXCL10 has been shown to be made by corneal epithelial cells in response to mechanical wounding (Gao et al., 2011). The role of CXCL10 in the cornea of mice infected with HSV-1 has been investigated by administration of monoclonal antibodies against CXCL10, which showed that neutralization of CXCL10 leads to a significant reduction in inflammation and decreased primary HSK (Carr et al., 2003). However, in subsequent experiments with mice with targeted deletion of CXCL10 (CXCL10-KO), the opposite was observed with mice displaying more severe disease phenotype (Shen
Corresponding author. E-mail address: [email protected] (P.M. Stuart).
http://dx.doi.org/10.1016/j.virol.2017.02.022 Received 30 January 2017; Received in revised form 23 February 2017; Accepted 24 February 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.
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et al., 2013). These investigators interpreted their observations as indicating that endogenous CXCL10 reduces HSK severity and the lack of CXCL10 increases severity by allowing prolonged corneal replication of the virus (Shen et al., 2013). Here we show that in the absence of CXCL10, compensatory production of CXCL9 leads to immunopathology and the more severe phenotype, which may be alleviated via neutralization of CXCL9 leading to significantly reduced corneal disease.
quadrants of the eye is evaluated for the density of vessels that have grown into them. Periocular disease was measured in a masked fashion on a semiquantitative scale as previously described (Stuart et al., 2004). Note: Uninfected, UV-B irradiated control mice were used as a baseline for any effects due to UV-B irradiation.
2. Materials and methods
Neutralization of CXCL10 employed clone 1F11, a kind gift from Dr. Andrew Luster (Smith et al., 2000). Neutralization of CXCL9 employed clone 2A6.9.9 (Khan et al., 2000) (Biovest International, Inc. Minneapolis, MN, using the protocol from Dr. Andrew Luster). Mice were treated by IP injection of 100 μg of either anti-CXCL10 or anti-CXCL9 three times per week for three weeks. Both CXCL10 and CXCL9 monoclonal antibodies are derived from hamsters thus controls for both consisted of IP injections of 100 μg of Hamster Gamma Globulin/ nonspecific control IgG (Jackson ImmunoResearch, West Grove, PA) three times weekly for 3 weeks.
2.5. Neutralization of CXCL10 and CXCL9 by monoclonal antibody treatment
2.1. Mice Investigations with mice conformed to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 (B6) were purchased from NCI. The B6.129S4-Cxcl10tm1Adl/J (CXCL10-/-) mice were obtained initially from Jackson Labs (Bar Harbor, ME) and then maintained in our breeding colony.
2.6. ELISA 2.2. Infection of mice The detection of CXCL9 was performed using a commercially available quantitative sandwich enzyme immunoassay kit (Catalog #MCX900), specific for mouse CXCL9 (R & D Systems, Minneapolis, MN). The corneas were removed from infected mice under the microscope and were immediately frozen at −80 °C. Samples were thawed and assayed according to the manufacturer's protocol. For normalization, total protein concentration for each cornea sample was also measured using (Thermo Scientific – Pierce 660 nm Protein Assay).
For all experiments described, we used age matched male and female mice from B6 and CXCL10-/- strains that were 6–10 weeks old. These mice were infected on the scarified cornea with 106 PFU HSV-1 McKrae strain as previously described (Keadle et al., 2002a). Each mouse received an intra-peritoneal (IP) injection of 0.5 ml pooled human serum (Sigma Chemicals, St. Louis MO; ED50 for virus neutralization=1:1600) concurrent with infection. Administration of anti-HSV antibodies at the time of ocular infection has been shown to protect mice from death and corneal disease during primary infection, while allowing for the establishment of latency and subsequent reactivation of virus after corneal UV-B exposure. These antibodies are undetectable at the time of UV-B irradiation 5 weeks after primary infection. To confirm infection, only mice displaying infectious virus obtained from eye swabs taken three days post-infection were used for subsequent reactivation (West et al., 2014).
2.7. Flow cytometric analysis
Mice were reactivated from latency as previously described (Gao et al., 2011; Keadle et al., 2002a). Briefly, the eyes of all latently infected mice were examined for corneal opacity before irradiation, and only animals with clear corneas were used. At least 5 weeks after primary infection, the eyes of latently-infected and control mockinfected mice were exposed to 250 mJ/cm2 of UV-B light using a TM20 Chromato-Vu transilluminator (UVP, Inc., San Gabriel, CA), which emits UV-B at a peak wavelength of 302 nm. Irradiated mice were swabbed with sterile cotton applicators from day 0 to day 7, unless otherwise indicated. The swab material was cultured on VERO cells, as described above, in order to detect recurrent virus shedding from the cornea. Reactivation was defined as the finding of any HSV positive eye swab on any day's post-UV-B exposure, with day 0 swabs serving as a control (West et al., 2014).
Cells were isolated from corneas as previously described (West et al., 2014). Briefly, corneas were excised at 18 and 23 dpi and incubated in PBS-EDTA at 37 °C for 15 min at 37 °C. Stromas were separated from overlying epithelium and digested in 84 U collagenase type 1 (Sigma-Aldrich, St. Louis, MO) per cornea for 2 h at 37 °C and then were triturated to form a single-cell suspension. Suspensions were filtered through a 40-μm cell strainer cap (BD Labware, Bedford, MA) and washed and then stained. Suspensions were stained with: PerCPconjugated anti-CD45 (clone 30-F11), Alexa Fluor700-Gr-1 (clone RB6-8C5) and APC F4/80 (clone BM8) (from BioLegend, San Diego, CA); FITC conjugated anti-CD4 (clone RM4–5), PE-conjugated antiCD8α (clone 53–6.7), PE-Cy7-conjugated anti-CD11c (clone N418), (all BD PharMingen); eFlour 450 anti-NK1.1 (clone PK136), PEconjugated CD11b (clone M1/70) (from eBiosciences, San Diego, CA). The strategy for analysis was to initially gate on live cells and then the CD45+ cells. These CD45+ cells were further evaluated for T cell markers CD4 and CD8, or for macrophage markers F4/ 80+CD11b+GR-1-, or neutrophil markers GR-1+, CD11b+F4/80-, or dendritic cell marker CD11c+,F4/80-. Cells were then analyzed on a flow cytometer (FACSAria with FACSDIVA data analysis software; BD Biosciences).
2.4. Clinical evaluation
2.8. Statistics
On the designated days after viral infection or UV-B reactivation, a masked observer examined mouse eyes through a binocular-dissecting microscope in order to score clinical disease. Stromal opacification was rated on a scale of 0–4, where 0 indicates clear stroma, 1 indicates mild stromal opacification, 2 indicates moderate opacity with discernible iris features, 3 indicates dense opacity with loss of defined iris detail except pupil margins, and 4 indicates total opacity with no posterior view. Corneal neovascularization was evaluated as described (West et al., 2014; Keadle et al., 2002b) using a scale of 0–8, where each of four
All statistical analyses were performed with the aid of Sigma Stat for Windows, version 2.0 (Jandel, Corte Madera, CA). The log rank test was used to compare disease scores. Student's unpaired t-test was used to compare ELISA and virus titer data.
2.3. UV-B irradiation and virus reactivation
3. Results Studies from several laboratories have evaluated the roles of chemokines and cytokines during primary HSK. These studies have 8
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Fig. 2. Recurrent HSK in B6-CXCL10 KO mice was greater than that seen in wild-type B6 mice. Eyes of mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. B6CXCL10 KO mice (n=38) were compared with wild-type B6 mice (n=38) for corneal opacity (A) and corneal neovascularization (B). B6-CXCL10KO mice displayed significantly greater opacity scores for Days 7–21 (P < 0.05-0.001)and neovascularization scores for Days 14–28 *(p values ranged from < 0.05-0.02 by log rank test).
Fig. 1. Recurrent HSK in anti-CXCL10 treated B6 mice had less disease than in control antibody treated mice. Eyes of B6 mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were then treated with anti-CXCL10 (n=23) or treated with control antibody (n=21). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatments. Mice treated with anti-CXCL10 displayed significantly reduced corneal opacity and neovascularization at Days 14–28 * (p values ranged from < 0.05-0.01 by log rank test).
disease was significantly reduced (Fig. 1). It should be noted that virological analysis of these mice did not reveal significant differences in peak viral titers, days mice shedding virus, or reactivation rates (data not shown). We next compared recurrent HSK disease scores from wild-type B6 mice with B6-CXCL10-/- mice. We anticipated that B6CXCL10-/- mice would behave in a fashion similar to neutralization of CXCL10, namely reduced disease (Fig. 1). However, our results demonstrated that mice incapable of CXCL10 expression had worse disease than did wild-type mice (Fig. 2). This observation was the same as described for primary HSK (Shen et al., 2013). When viral titers and duration of shedding were compared between these mice, B6CXCL10-/- mice displayed higher peak viral titers in the corneal tear film and as a group they shed virus for several days longer than did wild-type mice (Table 1). These results for B6-CXCL10-/- mice were also very similar to that reported by Shen, et al. Shen et al. (2013) who compared B6 wild-type mice and B6-CXCL10-/- mice in an acute model of HSK disease. They concluded from their results that it was persistence of infection in B6-CXCL10-/- mice that was responsible for increased primary HSK. As we have seen numerous cases where persistence of viral infection does not result in increased recurrent HSK disease (23 and unpublished results for CD28-/- mice), we hypothesized that other factors that drive immunopathology might underlie the worsened disease observed in CXCL10-deficient mice with HSK. To address this, we characterized the inflammatory infiltrate found in corneas from B6 and B6-CXCL10-/- following UV-B reactivation.
reported that several of them, including CCL2, CCL3, CXCL2, IL-1, IL6 and IL-17 are critical to the development of corneal disease (Stuart and Keadle, 2012; Rowe et al., 2013). Some of these factors, but not all, have been investigated in a murine model of recurrent HSK (Stuart and Keadle, 2012; West et al., 2014). The results of these later studies have been to illustrate that some cytokines and chemokines that are important to the development of primary HSK (CCL2, CCL3, and IL6) may have very little to do with the development of recurrent HSK (Stuart and Keadle, 2012; West et al., 2014). As a consequence of our ongoing interest in evaluating these factors in recurrent HSK, we decided to address the role of CXCL10 during recurrent HSK. Prior studies demonstrated that neutralization of CXCL10 reduced inflammatory responses following acute infection with HSV-1 (Carr et al., 2003). A similar result was seen when the receptor of CXCL10, CXCR3 is deleted (Christen et al., 2003). Thus is it clear that signaling through the CXCR3 receptor is important in driving T cell migration to the cornea. These reports led us to question why a report demonstrating that mice lacking CXCL10 displayed significantly greater corneal disease following infection with HSV-1 (Shen et al., 2013). Since these studies seem to be in conflict, we decided to address the role of CXCL10 in a recurrent model of HSK using both antibody neutralization and CXCL10KO mice. In agreement with what was previously shown during primary HSK (Carr et al., 2003), when B6 mice are treated with a neutralizing antibody to CXCL10 following UV-B reactivation, the resultant corneal 9
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Table 1 Virological analysis comparing C57BL/6 to B6-CXCL10KO mice following UV-B reactivation. Parameter analyzed
B6
CXCL10KO
% Positive swabs@ Peak viral titer (log10)Δ Total shedding days# Days shedding/mouse† % Reactivation rate‡ Final day sheddingǂ n
7% 3.85 ± 0.32 6 1.2 ± 0.11 29.80% Day 5 17
14% 4.79 ± 0.25 16 1.7 ± 0.2 50% Day 10 20
@ The percent positive swabs is the percentage of virus-positive eye swabs 140–286 eye swabs per group over the 10-day period following UV-B irradiation. (P < 0.05 for CXCL10 KO mice.). Δ Peak viral titer, which occurred on Day 3 post-reactivation expressed as log10 plaque forming unit. (P < 0.02 for CXCL10 KO mice.). # Total shedding days number of days swab positive. (P < 0.02 for CXCL10 KO mice.). † Days shedding/mouse are the number of days that a positive mouse shed virus. (No significance). ‡ Percent reactivation rate is the percentage of mice that reactivated. (P < 0.02 for CXCL10 KO mice.). ǂ Final day shedding was the last day that a mouse was positive for a particular group.
We performed flow cytometric analysis on the inflammatory infiltrate, by gating on CD45 expressing cells isolated from the corneas of mice 17 days following UV-B induced reactivation. As Fig. 3A demonstrates, the total number of CD45+ cells in B6-CXCL10-/- mice significantly exceeded that seen in wild-type B6 mice. When these CD45+ cells were further characterized, the percentage of CD4, CD8, macrophage and neutrophils did not differ (Fig. 3B). This suggests that while the magnitude of the inflammatory infiltrate was greater in B6-CXCL10-/mice, the quality of that infiltrate was not. From this data, we hypothesized that CXCL10-/- mice compensated for the lack of CXCL10 by the production of the similar chemokine CXCL9, which binds to the same receptor (CXCR3) that CXCL10 binds to and has similar biologic properties (Lundberg et al., 2007). It should be noted that when CXCL10-/- mice were infected with LCMV, these mice displayed increased CXCL9 expression (Whiting et al., 2004). To address this, we examined the expression of CXCL9 following HSV-1 infection. Results in Fig. 4 demonstrate that early following UVB-induced reactivation (day 3) wild-type B6 mice produce CXCL9. However, during peak disease, the expression of CXCL9 drops to quite low levels. In contrast, the expression of CXCL9 in CXCL10-/- mice, which is low at Day 3, increases to much higher levels during peak disease and is significantly greater than that detected in wild-type B6 mice at any time point measured. It should be noted that the clinical appearance and viral titer data were similar to that seen in earlier studies involving these two strains of mice. These data encouraged us to further examine the role that CXCL9 might be playing in CXCL10-/mice. In order to determine whether this increase in CXCL9 expression had any functional relevance to disease, we administered anti-CXCL9 neutralizing antibodies to mice with latent HSV-1 infection and monitored corneal disease following UV-B reactivation. As a result, while peak viral titers were similar to that in Table 1, disease scores were significantly reduced in the anti-CXCL9 treated CXCL10-/- mice for time points up to day 20 (Fig. 5). When wild-type B6 mice with recurrent HSV-1 infection were similarly treated with neutralizing antibody to CXCL9 no significant reduction in disease was noted (Fig. 6). Thus we conclude that CXCL9 does not play an essential role in recurrent HSK unless CXCL10 is absent.
Fig. 3. B6-CXCL10KO mice demonstrate that while the total number of CD45+ cells is greater than that seen in wild-type B6 mice, the qualitative nature of this inflammatory infiltrate was the same. Corneas were removed from latently infected B6 (n=8) and B6CXCL10KO (n=10) mice at day 17 following reactivation. These corneas were disaggregated into single-cell suspensions and stained with antibodies against: CD45, CD4, CD8α, Gr-1, CD11b, CD11c, and F4/80. Cells were then analyzed by flow cytometry. Data represents mean + S.E.M. for individually analyzed corneas from these groups. Then number of CD45+ cells was significantly greater in B6-CXCL10KO corneas (*p < 0.02 by Students T test).
Fig. 4. Production of CXCL9 is greatly increased following UV-B reactivation in B6CXCL10KO mice when compared to wild-type B6 mice. Corneas were removed from B6 (n=7) and B6-CXCL10KO (n=7) mice at the indicated time points and proteins extracted. These extracts were then subjected to ELISA analysis for the presence of CXCL9. Results display significantly more CXCL9 at days 10 and 14 following reactivation *(p < 0.01 by student's T test).
4. Discussion
most corneal disease associated with HSV-1 infection is the result of recrudescence of a latent infection whereby virus is reactivated in the trigeminal ganglia and then returns to the cornea where it restimulates
Primary infection of the eye with HSV-1 in humans typically results in few clinical symptoms but is followed by the establishment of latency in the trigeminal ganglia (Christensen et al., 2006). As a consequence, 10
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Fig. 6. Recurrent HSK in anti-CXCL9 treated B6 mice did not exhibit any change in disease when compared to control antibody treated mice. Eyes of B6 mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were then treated with anti-CXCL9 (n=10) or treated with control antibody (n=10). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatments. No differences were noted by log rank test between mice treated with anti-CXCL9 or those receiving control antibodies.
Fig. 5. Recurrent HSK in anti-CXCL9 treated B6-CXCL10KO mice exhibit less disease than in control antibody treated mice. Eyes of B6-CXCL10KO mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were then treated with anti-CXCL9 (n=12) or treated with control antibody (n=11). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatments. Mice treated with anti-CXCL9 displayed significantly reduced corneal opacity and neovascularization at Days 7–21 *(p values ranged from < 0.05-0.02 by log rank test).
tant for T cells, we predicted that mice lacking CXCL10 would display less recurrent HSK following UV-B-induced reactivation. To our surprise, B6-CXCL10-/- mice exhibited worse recurrent HSK than wild-type B6 mice with increased inflammatory infiltrates into the cornea. In contrast, CXCL10 antibody neutralization studies significantly reduced recurrent HSK. These results are consistent with a recent study in which CXCL10-/- mice exhibited worse primary HSK than similarly infected wild-type B6 mice (Shen et al., 2013). These data led us to hypothesize that in the absence of CXCL10, there is compensatory up-regulation of CXCL9, another CXCR3 ligand (Lundberg et al., 2007). As B6 mice do not express CXCL11, due to a frameshift mutation (Miller et al., 1996), we did not assess expression of this CXCR3 ligand chemokine. Evaluation of CXCL9 in CXCL10-/- mice with recurrent HSK detected a significant increase in this chemokine than that of similarly infected wild-type B6 mice. This is not surprising as when these same mice were infected with LCMV, there was also an increase in CXCL9 expression (Whiting et al., 2004). However, the increase in CXCL9 expression did not compensate for the lack of CXCL10 in their model (Whiting et al., 2004). In contrast, recurrent HSK was compensated by this increased expressed production of CXCL9 as evidenced by the fact that neutralization of CXCL9 in CXCL10-/- mice resulted in a reduced
a memory immune response that was initially generated during primary infection (Christensen et al., 2006; Tuli and Kubal, 2013). This immune response is characterized by an influx of CD4 and CD8 conventional T cells, neutrophils, macrophages, and NK and NKT cells (Stuart and Keadle, 2012; West et al., 2014; Tuli and Kubal, 2013; Hawthorne et al., 2011). The role that each of these subsets of cells plays in corneal disease has been the subject of numerous publications (Stuart and Keadle, 2012; Rowe et al., 2013). Studies from our laboratory as well as others have defined the essential requirement of CD4+ T cells, neutrophils, and to a lesser extent macrophages in recurrent HSK (Stuart and Keadle, 2012; Rowe et al., 2013; Tuli and Kubal, 2013; Shimeld et al., 1996). Chemokines direct the trafficking of these cells to the cornea and therefore critically determine the extent of inflammation at this site. In prior studies we used both antibodyneutralization and gene-targeted mice to demonstrate the essential nature of CXCL1 in recurrent HSK (West et al., 2014). We have also demonstrated that CCL2 does not appear to play any role in disease (Stuart et al., 2008), while CCL3 seems to ameliorate recurrent HSK (Stuart et al., 2008). Here we extended our characterization of chemokine involvement in recurrent HSK to CXCL10. As CXCL10 is a powerful chemoattrac-
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Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice. J. Virol. 74, 3598–3604. Stuart, P.M., Keadle, T.L., 2012. Recurrent herpetic stromal keratitis in mice, a model for studying human HSK. Clin. Dev. Immunol. 2012, 728480. Stuart, P.M., Morris, J.E., Sidhu, M., Keadle, T.L., 2008. CCL3 protects mice from corneal pathology during recurrent HSV-1 infection. Front. Biosci. 13, 4407–4415. Stuart, P.M., Summers, B.C., Morris, J.E., Morrison, L.A., Leib, D.A., 2004. CD8(+) T cells control corneal disease following ocular infection with herpes simplex virus type 1. J. Gen. Virol. 85, 2055–2063. Tsai, H.H., Frost, E., To, V., Robinson, S., French-Constant, C., Geertman, R., Ransohoff, R.M., Miller, R.H., 2002. The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration. Cell 110, 373–383. Tsutsumi, T., Kajiya, H., Goto, K.T., Takahashi, Y., Okabe, K., 2013. Hyperocclusion up-
recurrent HSK phenotype. Thus our results suggest that both CXCL10 and CXCL9 may induce immunopathology during recurrent HSK. Consequently, we believe that CXCL10 in wild-type mice is important in the trafficking of CD4+ T cells to the cornea and that if this chemokine is neutralized the resulting recurrent HSK will be inhibited. This is supported by our data indicating that CXCL9 is not induced in wild-type B6 mice following infection. However, in the absence of CXCL10 the animal generates a CXCL9 response that more than compensates for the lack CXCL10 as T cells are still attracted to enter corneas where the virus is replicating following reactivation. This is not surprising as it has been shown during acute infection with HSV-1, CXCL9 is likely more responsible for migration of CD4+ T cells into the cornea than is CXCL10 (Qi et al., 2014). Furthermore, this type of compensatory response in the absence of particular chemokines or their receptors has been shown in several systems where lack of a particular chemokine or its receptor leads to increased expression of another factor that results in the same biological effect (Wuest et al., 2006; Chen et al., 2003; Yanaba et al., 2004; Mizukami et al., 2005; Pahler et al., 2008; Zlotoff et al., 2010). Since our data indicates that CXCL9 production is a compensatory response to HSV-1 infection, targeting CXCL9 along with CXCL10 in wild type mice is unlikely to result in an additive effect. It is interesting to note that Shen et al. (2013) proposed that the reason for increased disease in B6-CXCL10-/- mice is persistence of actively replicating virus in the corneas of these mice. Similarly, our data demonstrate that during recurrent HSK B6-CXCL10-/- mice exhibit replication of virus 5 days longer than that observed in wildtype B6 mice. However, we are not fully convinced that the extended presence of virus will necessarily lead to significant corneal disease. While there are cases where such extended presence might be a factor in recurrent corneal disease (Tsutsumi et al., 2013), we also know that CD4/CD8 double KO mice, which do not develop corneal disease, do have a more persistent corneal infection following reactivation than do wild-type mice (Gao et al., 2011). We have also noted this same phenotype in mice that do not express the CD28 molecule (unpublished results). These reports indicate that there can be situations where persistence of virus can prolong the stimulatory phase of activating an adaptive response, there needs to be the requisite T cells to create an environment where corneal disease can fully develop. Consequently, the development of corneal disease during recurrent HSK is a complex mix of cytokines, cells and, we believe to a lesser extent, virus. Acknowledgements This work was supported by National Institutes of Health Grants EY16352 (PMS), EY21247 (PMS), NS052632 (RSK), AI083019 (RSK), and Defense Threat Reduction Agency grant HDTRA-1-15-1-0032 (RSK) and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, Saint Louis University. References Arduino, P.G., Porter, S.R., 2006. Oral and perioral herpes simplex virus type 1 (HSV-1) infection: review of its management. Oral. Dis. 12, 254–270. Banerjee, K., Biswas, P.S., Kim, B., Lee, S., Rouse, B.T., 2004. CXCR2-/- mice show enhanced susceptibility to herpetic stromal keratitis: a role for IL-6-induced neovascularization. J. Immunol. 172, 1237–1245. Carr, D.J., Chodosh, J., Ash, J., Lane, T.E., 2003. Effect of anti-CXCL10 monoclonal antibody on herpes simplex virus type 1 keratitis and retinal infection. J. Virol. 77, 10037–10046. Chen, Z., Yu, S., Bakhiet, M., Winblad, B., Zhu, J., 2003. The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3. J. Neurochem. 86, 61–68. Christen, U., McGavern, D.B., Luster, A.D., von Herrath, M.G., Oldstone, M.B., 2003. Among CXCR3 chemokines, IFN-gamma-inducible protein of 10 kDa (CXC chemokine ligand (CXCL) 10) but not monokine induced by IFN-gamma (CXCL9) imprints a pattern for the subsequent development of autoimmune disease. J. Immunol. 171, 6838–6845. Christensen, J.E., de Lemos, C., Moos, T., Christensen, J.P., Thomsen, A.R., 2006.
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cytokine production. J. Immunol. 172, 7417–7424. Wuest, T., Farber, J., Luster, A., Carr, D.J., 2006. CD4+ T cell migration into the cornea is reduced in CXCL9 deficient but not CXCL10 deficient mice following herpes simplex virus type 1 infection. Cell. Immunol. 243, 83–89. Wuest, T.R., Carr, D.J., 2008. The role of chemokines during herpes simplex virus-1 infection. Front. Biosci. 13, 486204872. Yanaba, K., Mukaida, N., Matsushima, K., Murphy, P.M., Takehara, K., Sato, S., 2004. Role of C-C chemokine receptors 1 and 5 and CCL3/macrophage inflammatory protein-1alpha in the cutaneous Arthus reaction: possible attenuation of their inhibitory effects by compensatory chemokine production. Eur. J. Immunol. 34, 3553–3561. Zlotoff, D.A., Sambandam, A., Logan, T.D., Bell, J.J., Schwarz, B.A., Bhandoola, A., 2010. CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus. Blood 115, 1897–1905.
regulates CCL3 expression in CCL2- and CCR2-deficient mice. J. Dent. Res. 92, 65–70. Tuli, S., Sonal, S., 2009. Herpes simplex keratitis. (©2009)In: Yanoff, J.S., Duker, M., Wiggs, J.L., Miller, D., Azar, D.T., Goldstein, M.H., Rosen, E.S., Duker, J.S., Rao, N.A., Augsburger, J.J., Sadum, A.A., Shuman, J.S.,, Diamond, G.R., Dutton, J.J. (Eds.), Ophthalmology3rd edition. Elsevier Inc. Tuli, S.S., Kubal, A.A., 2013. (HSV Keratitis)Ophthalmology 4th edition. Elsevier, London, UK, p231–p236. Umene, K., Sakaoke, H., 1999. Evolution of herpes simplex virus type 1 under herpesvirus evolutionary processes. Arch. Virol. 144, 637–656. West, D.M., Del Rosso, C.R., Yin, X.T., Stuart, P.M., 2014. CXCL1, but not IL-6 is required for recurrent herpetic stromal keratitis. J. Immunol. 192, 1762–1767. Whiting, D., Hsieh, G., Yun, J.J., Banerji, A., Yao, W., Fishbein, M.C., Belperio, J., Strieter, R.M., Bonavida, B., Ardehali, A., 2004. Chemokine monokine induced by IFN-γ/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector
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CXCL9 compensates for the absence of CXCL10 during recurrent Herpetic stromal keratitis
MARK
Deena Tajfirouza, Devin M. Westa, Xiao-Tang Yina, Chloe A. Pottera, Robyn Kleinb, Patrick ⁎ M. Stuarta, a b
Department of Ophthalmology, Saint Louis University School of Medicine, St. Louis, MO, USA Departments of Medicine, Pathology & Immunology, Anatomy & Neurobiology Washington University School of Medicine, Saint Louis, MO, USA
A R T I C L E I N F O
A BS T RAC T
Keywords: Herpes simplex virus type 1 Cornea Chemokines CXCL10 CXCL9 Inflammation Herpetic stromal keratitis
Herpetic stromal keratitis (HSK) is a disease that is typically associated with reactivation of a latent HSV-1 infection. This disease is driven, in part, by chemokines that recruit leukocytes to the cornea. Surprisingly, neutralization of CXCL10 significantly reduced disease, while B6-CXCL10-/- mice exhibited worse disease compared with similarly infected wild-type controls. We hypothesized that compensatory up-regulation of CXCL9 occurs in the absence of CXCL10. Analysis of CXCL9 expression in HSV-1-infected B6 mice and B6CXCL10-/- mice revealed significantly more CXCL9 in B6-XCL10-/- mice. Treatment of B6 and B6-CXCL10-/mice with neutralizing antibodies to CXCL9 reduced HSK scores in B6-CXCL10-/-, but not B6 mice. We conclude that CXCL10 production worsens HSK and that CXCL9 may compensate in CXCL10-deficient animals. These studies identify the critical role that CXCL10 plays in the pathogenesis of recurrent HSK, and that CXCL9 displays its importance when CXCL10 is absent.
1. Introduction Herpes simplex virus (HSV) is a widespread viral pathogen that infects the majority of the world's population. Although oral and to a lesser extent, genital infections are the most common type of HSV type 1 (HSV-1) infection, this virus can also cause disease in major ocular tissues including the cornea (Arduino and Porter, 2006; Smith and Robinson, 2002; Umene and Sakaoke, 1999). Herpetic stromal keratitis (HSK), caused by HSV-1, is the most common cause of infectioninduced blindness of the cornea (Umene and Sakaoke, 1999; Feldman et al., 2002; Tuli and Sonal, 2009; Farooq and Shukla, 2012; Stuart and Keadle, 2012; Rowe et al., 2013). Similar to other herpetic viruses, infection with HSV-1 can be categorized into primary and recurrent disease. Primary infection with HSV-1 is usually asymptomatic in humans; however, the clinical disease phenotype also known as HSK is the result of reactivation of the virus that originally established a latent infection in the trigeminal ganglia (Umene and Sakaoke, 1999; Rinne et al., 1992). HSV-1 reactivation is typically elicited by different immunosuppressive events such as irradiation, sunlight (UV), stress, trauma, and fever. It has been shown that HSK is primarily due to an immunopathogenic response to the virus (Inoue, 2008). Pro-inflammatory cytokines and chemokines that are produced during early stages of viral infection and reactivation lead to recruitment of
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neutrophils, NK cells, macrophages, and T cells, all of which are important mediators of inflammation (Li et al., 2006; Fenton et al., 2002; Carr et al., 2003; Stuart et al., 2008; Duan et al., 2007; Tsai et al., 2002; Wuest and Carr, 2008; Banerjee et al., 2004; Shen et al., 2013; Keadle and Stuart, 2005; West et al., 2014). In primary and recurrent models of HSK the presence of CD4+ T cells and neutrophils in primary and secondary models of HSK has been well documented, therefore making the study of their recruitment to the site of infection and subsequent inflammation critical to the understanding of the disease process. Activated CD4+ T cells and NK cells express the receptor CXCR3, a G-protein coupled receptor that mediates its inflammatory effects by binding to CXCL9, CXCL10, and CXCL11 chemokines (Clark-Lewis et al., 2003). While these cytokines have been shown to be made by monocytes, dendritic cells and epithelial cells, most recently, CXCL10 has been shown to be made by corneal epithelial cells in response to mechanical wounding (Gao et al., 2011). The role of CXCL10 in the cornea of mice infected with HSV-1 has been investigated by administration of monoclonal antibodies against CXCL10, which showed that neutralization of CXCL10 leads to a significant reduction in inflammation and decreased primary HSK (Carr et al., 2003). However, in subsequent experiments with mice with targeted deletion of CXCL10 (CXCL10-KO), the opposite was observed with mice displaying more severe disease phenotype (Shen
Corresponding author. E-mail address: [email protected] (P.M. Stuart).
http://dx.doi.org/10.1016/j.virol.2017.02.022 Received 30 January 2017; Received in revised form 23 February 2017; Accepted 24 February 2017 0042-6822/ © 2017 Elsevier Inc. All rights reserved.
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et al., 2013). These investigators interpreted their observations as indicating that endogenous CXCL10 reduces HSK severity and the lack of CXCL10 increases severity by allowing prolonged corneal replication of the virus (Shen et al., 2013). Here we show that in the absence of CXCL10, compensatory production of CXCL9 leads to immunopathology and the more severe phenotype, which may be alleviated via neutralization of CXCL9 leading to significantly reduced corneal disease.
quadrants of the eye is evaluated for the density of vessels that have grown into them. Periocular disease was measured in a masked fashion on a semiquantitative scale as previously described (Stuart et al., 2004). Note: Uninfected, UV-B irradiated control mice were used as a baseline for any effects due to UV-B irradiation.
2. Materials and methods
Neutralization of CXCL10 employed clone 1F11, a kind gift from Dr. Andrew Luster (Smith et al., 2000). Neutralization of CXCL9 employed clone 2A6.9.9 (Khan et al., 2000) (Biovest International, Inc. Minneapolis, MN, using the protocol from Dr. Andrew Luster). Mice were treated by IP injection of 100 μg of either anti-CXCL10 or anti-CXCL9 three times per week for three weeks. Both CXCL10 and CXCL9 monoclonal antibodies are derived from hamsters thus controls for both consisted of IP injections of 100 μg of Hamster Gamma Globulin/ nonspecific control IgG (Jackson ImmunoResearch, West Grove, PA) three times weekly for 3 weeks.
2.5. Neutralization of CXCL10 and CXCL9 by monoclonal antibody treatment
2.1. Mice Investigations with mice conformed to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 (B6) were purchased from NCI. The B6.129S4-Cxcl10tm1Adl/J (CXCL10-/-) mice were obtained initially from Jackson Labs (Bar Harbor, ME) and then maintained in our breeding colony.
2.6. ELISA 2.2. Infection of mice The detection of CXCL9 was performed using a commercially available quantitative sandwich enzyme immunoassay kit (Catalog #MCX900), specific for mouse CXCL9 (R & D Systems, Minneapolis, MN). The corneas were removed from infected mice under the microscope and were immediately frozen at −80 °C. Samples were thawed and assayed according to the manufacturer's protocol. For normalization, total protein concentration for each cornea sample was also measured using (Thermo Scientific – Pierce 660 nm Protein Assay).
For all experiments described, we used age matched male and female mice from B6 and CXCL10-/- strains that were 6–10 weeks old. These mice were infected on the scarified cornea with 106 PFU HSV-1 McKrae strain as previously described (Keadle et al., 2002a). Each mouse received an intra-peritoneal (IP) injection of 0.5 ml pooled human serum (Sigma Chemicals, St. Louis MO; ED50 for virus neutralization=1:1600) concurrent with infection. Administration of anti-HSV antibodies at the time of ocular infection has been shown to protect mice from death and corneal disease during primary infection, while allowing for the establishment of latency and subsequent reactivation of virus after corneal UV-B exposure. These antibodies are undetectable at the time of UV-B irradiation 5 weeks after primary infection. To confirm infection, only mice displaying infectious virus obtained from eye swabs taken three days post-infection were used for subsequent reactivation (West et al., 2014).
2.7. Flow cytometric analysis
Mice were reactivated from latency as previously described (Gao et al., 2011; Keadle et al., 2002a). Briefly, the eyes of all latently infected mice were examined for corneal opacity before irradiation, and only animals with clear corneas were used. At least 5 weeks after primary infection, the eyes of latently-infected and control mockinfected mice were exposed to 250 mJ/cm2 of UV-B light using a TM20 Chromato-Vu transilluminator (UVP, Inc., San Gabriel, CA), which emits UV-B at a peak wavelength of 302 nm. Irradiated mice were swabbed with sterile cotton applicators from day 0 to day 7, unless otherwise indicated. The swab material was cultured on VERO cells, as described above, in order to detect recurrent virus shedding from the cornea. Reactivation was defined as the finding of any HSV positive eye swab on any day's post-UV-B exposure, with day 0 swabs serving as a control (West et al., 2014).
Cells were isolated from corneas as previously described (West et al., 2014). Briefly, corneas were excised at 18 and 23 dpi and incubated in PBS-EDTA at 37 °C for 15 min at 37 °C. Stromas were separated from overlying epithelium and digested in 84 U collagenase type 1 (Sigma-Aldrich, St. Louis, MO) per cornea for 2 h at 37 °C and then were triturated to form a single-cell suspension. Suspensions were filtered through a 40-μm cell strainer cap (BD Labware, Bedford, MA) and washed and then stained. Suspensions were stained with: PerCPconjugated anti-CD45 (clone 30-F11), Alexa Fluor700-Gr-1 (clone RB6-8C5) and APC F4/80 (clone BM8) (from BioLegend, San Diego, CA); FITC conjugated anti-CD4 (clone RM4–5), PE-conjugated antiCD8α (clone 53–6.7), PE-Cy7-conjugated anti-CD11c (clone N418), (all BD PharMingen); eFlour 450 anti-NK1.1 (clone PK136), PEconjugated CD11b (clone M1/70) (from eBiosciences, San Diego, CA). The strategy for analysis was to initially gate on live cells and then the CD45+ cells. These CD45+ cells were further evaluated for T cell markers CD4 and CD8, or for macrophage markers F4/ 80+CD11b+GR-1-, or neutrophil markers GR-1+, CD11b+F4/80-, or dendritic cell marker CD11c+,F4/80-. Cells were then analyzed on a flow cytometer (FACSAria with FACSDIVA data analysis software; BD Biosciences).
2.4. Clinical evaluation
2.8. Statistics
On the designated days after viral infection or UV-B reactivation, a masked observer examined mouse eyes through a binocular-dissecting microscope in order to score clinical disease. Stromal opacification was rated on a scale of 0–4, where 0 indicates clear stroma, 1 indicates mild stromal opacification, 2 indicates moderate opacity with discernible iris features, 3 indicates dense opacity with loss of defined iris detail except pupil margins, and 4 indicates total opacity with no posterior view. Corneal neovascularization was evaluated as described (West et al., 2014; Keadle et al., 2002b) using a scale of 0–8, where each of four
All statistical analyses were performed with the aid of Sigma Stat for Windows, version 2.0 (Jandel, Corte Madera, CA). The log rank test was used to compare disease scores. Student's unpaired t-test was used to compare ELISA and virus titer data.
2.3. UV-B irradiation and virus reactivation
3. Results Studies from several laboratories have evaluated the roles of chemokines and cytokines during primary HSK. These studies have 8
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Fig. 2. Recurrent HSK in B6-CXCL10 KO mice was greater than that seen in wild-type B6 mice. Eyes of mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. B6CXCL10 KO mice (n=38) were compared with wild-type B6 mice (n=38) for corneal opacity (A) and corneal neovascularization (B). B6-CXCL10KO mice displayed significantly greater opacity scores for Days 7–21 (P < 0.05-0.001)and neovascularization scores for Days 14–28 *(p values ranged from < 0.05-0.02 by log rank test).
Fig. 1. Recurrent HSK in anti-CXCL10 treated B6 mice had less disease than in control antibody treated mice. Eyes of B6 mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were then treated with anti-CXCL10 (n=23) or treated with control antibody (n=21). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatments. Mice treated with anti-CXCL10 displayed significantly reduced corneal opacity and neovascularization at Days 14–28 * (p values ranged from < 0.05-0.01 by log rank test).
disease was significantly reduced (Fig. 1). It should be noted that virological analysis of these mice did not reveal significant differences in peak viral titers, days mice shedding virus, or reactivation rates (data not shown). We next compared recurrent HSK disease scores from wild-type B6 mice with B6-CXCL10-/- mice. We anticipated that B6CXCL10-/- mice would behave in a fashion similar to neutralization of CXCL10, namely reduced disease (Fig. 1). However, our results demonstrated that mice incapable of CXCL10 expression had worse disease than did wild-type mice (Fig. 2). This observation was the same as described for primary HSK (Shen et al., 2013). When viral titers and duration of shedding were compared between these mice, B6CXCL10-/- mice displayed higher peak viral titers in the corneal tear film and as a group they shed virus for several days longer than did wild-type mice (Table 1). These results for B6-CXCL10-/- mice were also very similar to that reported by Shen, et al. Shen et al. (2013) who compared B6 wild-type mice and B6-CXCL10-/- mice in an acute model of HSK disease. They concluded from their results that it was persistence of infection in B6-CXCL10-/- mice that was responsible for increased primary HSK. As we have seen numerous cases where persistence of viral infection does not result in increased recurrent HSK disease (23 and unpublished results for CD28-/- mice), we hypothesized that other factors that drive immunopathology might underlie the worsened disease observed in CXCL10-deficient mice with HSK. To address this, we characterized the inflammatory infiltrate found in corneas from B6 and B6-CXCL10-/- following UV-B reactivation.
reported that several of them, including CCL2, CCL3, CXCL2, IL-1, IL6 and IL-17 are critical to the development of corneal disease (Stuart and Keadle, 2012; Rowe et al., 2013). Some of these factors, but not all, have been investigated in a murine model of recurrent HSK (Stuart and Keadle, 2012; West et al., 2014). The results of these later studies have been to illustrate that some cytokines and chemokines that are important to the development of primary HSK (CCL2, CCL3, and IL6) may have very little to do with the development of recurrent HSK (Stuart and Keadle, 2012; West et al., 2014). As a consequence of our ongoing interest in evaluating these factors in recurrent HSK, we decided to address the role of CXCL10 during recurrent HSK. Prior studies demonstrated that neutralization of CXCL10 reduced inflammatory responses following acute infection with HSV-1 (Carr et al., 2003). A similar result was seen when the receptor of CXCL10, CXCR3 is deleted (Christen et al., 2003). Thus is it clear that signaling through the CXCR3 receptor is important in driving T cell migration to the cornea. These reports led us to question why a report demonstrating that mice lacking CXCL10 displayed significantly greater corneal disease following infection with HSV-1 (Shen et al., 2013). Since these studies seem to be in conflict, we decided to address the role of CXCL10 in a recurrent model of HSK using both antibody neutralization and CXCL10KO mice. In agreement with what was previously shown during primary HSK (Carr et al., 2003), when B6 mice are treated with a neutralizing antibody to CXCL10 following UV-B reactivation, the resultant corneal 9
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Table 1 Virological analysis comparing C57BL/6 to B6-CXCL10KO mice following UV-B reactivation. Parameter analyzed
B6
CXCL10KO
% Positive swabs@ Peak viral titer (log10)Δ Total shedding days# Days shedding/mouse† % Reactivation rate‡ Final day sheddingǂ n
7% 3.85 ± 0.32 6 1.2 ± 0.11 29.80% Day 5 17
14% 4.79 ± 0.25 16 1.7 ± 0.2 50% Day 10 20
@ The percent positive swabs is the percentage of virus-positive eye swabs 140–286 eye swabs per group over the 10-day period following UV-B irradiation. (P < 0.05 for CXCL10 KO mice.). Δ Peak viral titer, which occurred on Day 3 post-reactivation expressed as log10 plaque forming unit. (P < 0.02 for CXCL10 KO mice.). # Total shedding days number of days swab positive. (P < 0.02 for CXCL10 KO mice.). † Days shedding/mouse are the number of days that a positive mouse shed virus. (No significance). ‡ Percent reactivation rate is the percentage of mice that reactivated. (P < 0.02 for CXCL10 KO mice.). ǂ Final day shedding was the last day that a mouse was positive for a particular group.
We performed flow cytometric analysis on the inflammatory infiltrate, by gating on CD45 expressing cells isolated from the corneas of mice 17 days following UV-B induced reactivation. As Fig. 3A demonstrates, the total number of CD45+ cells in B6-CXCL10-/- mice significantly exceeded that seen in wild-type B6 mice. When these CD45+ cells were further characterized, the percentage of CD4, CD8, macrophage and neutrophils did not differ (Fig. 3B). This suggests that while the magnitude of the inflammatory infiltrate was greater in B6-CXCL10-/mice, the quality of that infiltrate was not. From this data, we hypothesized that CXCL10-/- mice compensated for the lack of CXCL10 by the production of the similar chemokine CXCL9, which binds to the same receptor (CXCR3) that CXCL10 binds to and has similar biologic properties (Lundberg et al., 2007). It should be noted that when CXCL10-/- mice were infected with LCMV, these mice displayed increased CXCL9 expression (Whiting et al., 2004). To address this, we examined the expression of CXCL9 following HSV-1 infection. Results in Fig. 4 demonstrate that early following UVB-induced reactivation (day 3) wild-type B6 mice produce CXCL9. However, during peak disease, the expression of CXCL9 drops to quite low levels. In contrast, the expression of CXCL9 in CXCL10-/- mice, which is low at Day 3, increases to much higher levels during peak disease and is significantly greater than that detected in wild-type B6 mice at any time point measured. It should be noted that the clinical appearance and viral titer data were similar to that seen in earlier studies involving these two strains of mice. These data encouraged us to further examine the role that CXCL9 might be playing in CXCL10-/mice. In order to determine whether this increase in CXCL9 expression had any functional relevance to disease, we administered anti-CXCL9 neutralizing antibodies to mice with latent HSV-1 infection and monitored corneal disease following UV-B reactivation. As a result, while peak viral titers were similar to that in Table 1, disease scores were significantly reduced in the anti-CXCL9 treated CXCL10-/- mice for time points up to day 20 (Fig. 5). When wild-type B6 mice with recurrent HSV-1 infection were similarly treated with neutralizing antibody to CXCL9 no significant reduction in disease was noted (Fig. 6). Thus we conclude that CXCL9 does not play an essential role in recurrent HSK unless CXCL10 is absent.
Fig. 3. B6-CXCL10KO mice demonstrate that while the total number of CD45+ cells is greater than that seen in wild-type B6 mice, the qualitative nature of this inflammatory infiltrate was the same. Corneas were removed from latently infected B6 (n=8) and B6CXCL10KO (n=10) mice at day 17 following reactivation. These corneas were disaggregated into single-cell suspensions and stained with antibodies against: CD45, CD4, CD8α, Gr-1, CD11b, CD11c, and F4/80. Cells were then analyzed by flow cytometry. Data represents mean + S.E.M. for individually analyzed corneas from these groups. Then number of CD45+ cells was significantly greater in B6-CXCL10KO corneas (*p < 0.02 by Students T test).
Fig. 4. Production of CXCL9 is greatly increased following UV-B reactivation in B6CXCL10KO mice when compared to wild-type B6 mice. Corneas were removed from B6 (n=7) and B6-CXCL10KO (n=7) mice at the indicated time points and proteins extracted. These extracts were then subjected to ELISA analysis for the presence of CXCL9. Results display significantly more CXCL9 at days 10 and 14 following reactivation *(p < 0.01 by student's T test).
4. Discussion
most corneal disease associated with HSV-1 infection is the result of recrudescence of a latent infection whereby virus is reactivated in the trigeminal ganglia and then returns to the cornea where it restimulates
Primary infection of the eye with HSV-1 in humans typically results in few clinical symptoms but is followed by the establishment of latency in the trigeminal ganglia (Christensen et al., 2006). As a consequence, 10
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Fig. 6. Recurrent HSK in anti-CXCL9 treated B6 mice did not exhibit any change in disease when compared to control antibody treated mice. Eyes of B6 mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were then treated with anti-CXCL9 (n=10) or treated with control antibody (n=10). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatments. No differences were noted by log rank test between mice treated with anti-CXCL9 or those receiving control antibodies.
Fig. 5. Recurrent HSK in anti-CXCL9 treated B6-CXCL10KO mice exhibit less disease than in control antibody treated mice. Eyes of B6-CXCL10KO mice were infected with 106 pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were then treated with anti-CXCL9 (n=12) or treated with control antibody (n=11). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatments. Mice treated with anti-CXCL9 displayed significantly reduced corneal opacity and neovascularization at Days 7–21 *(p values ranged from < 0.05-0.02 by log rank test).
tant for T cells, we predicted that mice lacking CXCL10 would display less recurrent HSK following UV-B-induced reactivation. To our surprise, B6-CXCL10-/- mice exhibited worse recurrent HSK than wild-type B6 mice with increased inflammatory infiltrates into the cornea. In contrast, CXCL10 antibody neutralization studies significantly reduced recurrent HSK. These results are consistent with a recent study in which CXCL10-/- mice exhibited worse primary HSK than similarly infected wild-type B6 mice (Shen et al., 2013). These data led us to hypothesize that in the absence of CXCL10, there is compensatory up-regulation of CXCL9, another CXCR3 ligand (Lundberg et al., 2007). As B6 mice do not express CXCL11, due to a frameshift mutation (Miller et al., 1996), we did not assess expression of this CXCR3 ligand chemokine. Evaluation of CXCL9 in CXCL10-/- mice with recurrent HSK detected a significant increase in this chemokine than that of similarly infected wild-type B6 mice. This is not surprising as when these same mice were infected with LCMV, there was also an increase in CXCL9 expression (Whiting et al., 2004). However, the increase in CXCL9 expression did not compensate for the lack of CXCL10 in their model (Whiting et al., 2004). In contrast, recurrent HSK was compensated by this increased expressed production of CXCL9 as evidenced by the fact that neutralization of CXCL9 in CXCL10-/- mice resulted in a reduced
a memory immune response that was initially generated during primary infection (Christensen et al., 2006; Tuli and Kubal, 2013). This immune response is characterized by an influx of CD4 and CD8 conventional T cells, neutrophils, macrophages, and NK and NKT cells (Stuart and Keadle, 2012; West et al., 2014; Tuli and Kubal, 2013; Hawthorne et al., 2011). The role that each of these subsets of cells plays in corneal disease has been the subject of numerous publications (Stuart and Keadle, 2012; Rowe et al., 2013). Studies from our laboratory as well as others have defined the essential requirement of CD4+ T cells, neutrophils, and to a lesser extent macrophages in recurrent HSK (Stuart and Keadle, 2012; Rowe et al., 2013; Tuli and Kubal, 2013; Shimeld et al., 1996). Chemokines direct the trafficking of these cells to the cornea and therefore critically determine the extent of inflammation at this site. In prior studies we used both antibodyneutralization and gene-targeted mice to demonstrate the essential nature of CXCL1 in recurrent HSK (West et al., 2014). We have also demonstrated that CCL2 does not appear to play any role in disease (Stuart et al., 2008), while CCL3 seems to ameliorate recurrent HSK (Stuart et al., 2008). Here we extended our characterization of chemokine involvement in recurrent HSK to CXCL10. As CXCL10 is a powerful chemoattrac-
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CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system. J. Immunol. 176, 4235–4243. Clark-Lewis, I., Mattioli, I., Gong, J.H., Loetscher, P., 2003. Structure-function relationship between the human chemokine receptor CXCR3 and its ligands. J. Biol. Chem. 278, 289–295. Duan, R., Remeijer, L., van Dun, J.M., Osterhaus, A.D., Verjans, G.M., 2007. Granulocyte macrophage colony-stimulating factor expression in human herpetic stromal keratitis: implications for the role of neutrophils in HSK. Investig. Ophthalmol. Vis. Sci. 48, 277–284. Farooq, A.V., Shukla, D., 2012. Herpes simplex epithelial and stromal keratitis: an epidemiologic update. Surv. Ophthalmol. 57, 448–462. Feldman, L.T., Ellison, A.R., Voytek, C.C., Yang, L., Krause, P., Margolis, T.P., 2002. Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice. Proc. Natl. Acad. Sci. USA 99, 978–983. Fenton, R., Molesworth-Kenyon, S., Oakes, J.E., Lausch, R.N., 2002. Linkage of IL-6 with neutrophil chemoattractant expression in virus-induced ocular inflammation. Investig. Ophthalmol. Vis. Sci. 43, 737–743. Gao, N., Yin, J., Yoon, G.S., Mi, Q.S., Yu, F.S.X., 2011. Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair. Am. J. Pathol. 179, 2243–2253. Hawthorne, K.M., Dana, R., Chodosh, J., 2011. Delayed type hypersensitivity in the pathogenesis of recurrent herpes stromal keratitis. Semin. Ophthalmol. 26, 246–250. Inoue, Y., 2008. Immunological aspects of herpetic stromal keratitis. Semin. Ophthalmol. 23, 221–227. Keadle, T.L., Stuart, P.M., 2005. IL-10 ameliorates corneal disease in a mouse model of recurrent herpetic keratitis. Microb. Pathog. 38, 13–21. Keadle, T.L., Morris, J.L., Pepose, J.S., Stuart, P.M., 2002a. CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice. Microb. Pathog. 32, 255–262. Keadle, T.L., Morrison, L.A., Morris, J.E., Pepose, J.S., Stuart, P.M., 2002b. Therapeutic immunization with a virion host shutoff-defective, replication in competent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection. J. Virol. 76, 3615–3625. Khan, I.A., MacLean, J.A., Lee, F.S., Casciotti, L., DeHaan, E., Schwartzman, J.D., Luster, A.D., 2000. IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12, 483–494. Li, H., Zhang, J., Kumar, A., Zheng, M., Atherton, S.S., Yu, F.S., 2006. Herpes simplex virus 1 infection induces the expression of proinflammatory cytokines, interferons and TLR7 in human corneal epithelial cells. Immunology 117, 167–176. Lundberg, P., Openshaw, H., Wang, M., Yang, H.J., Cantin, E., 2007. Effects of CXCR3 signaling on development of fatal encephalitis and corneal and periocular skin disease in HSV-infected mice are mouse-strain dependent. Investig. Ophthalmol. Vis. Sci. 48, 4162–4170. Miller, J.K., Laycock, K.A., Umphress, J.A., Hook, K.K., Stuart, P.M., Pepose, J.S., 1996. A comparison of recurrent versus primary herpes simplex Keratitis in bred mice. Cornea 15, 497–504. Mizukami, Y., Jo, W.S., Duerr, E.M., Gala, M., Li, J., Zhang, X., Zimmer, M.A., Iliopoulos, O., Zukerberg, L.R., Kohgo, Y., Lynch, M.P., Rueda, B.R., Chung, D.C., 2005. Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells. Nat. Med. 11, 992–997. Pahler, J.C., Tazzyman, S., Erez, N., Chen, Y.Y., Murdoch, C., Nozawa, H., Lewis, C.E., Hanahan, D., 2008. Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response. Neoplasia 10, 329–340. Qi, Z., Wang, J., Han, X., Yang, J., Zhao, G., Cao, Y., 2014. Listr1 locus regulates innate immunity against Listeria monocytogenes in the mouse liver possibly through CXCL11 polymorphism. Immunogenetics 66, 231–242. Rinne, J.R., Abghari, S.Z., Stulting, R.D., 1992. The severity of herpes simplex viral keratitis in mice does not reflect the severity of disease in humans. Investig. Ophthal. Vis. Sci. 33, 268–272. Rowe, A.M., St, Leger, A.J., Jeon, S., Dhaliwal, D.K., Knickelbein, J.E., Hendricks, R.L., 2013. Herpes keratitis. Prog. Retin. Eye Res. 32, 88–101. Shen, F.H., Wang, S.W., Yeh, T.M., Tung, Y.Y., Hsu, S.M., Chen, S.H., 2013. Absence of CXCL10 aggravates herpes stromal keratitis with reduced primary neutrophil influx in mice. J. Virol. 87, 8502–8510. Shimeld, C., Whiteland, J.L., Nicholls, S.M., Easty, D.L., Hill, T.J., 1996. Immune cell infiltration in corneas of mice with recurrent herpes simplex virus disease. (See comment in PubMed Commons below)J. Gen. Virol. 77, 977–985. Smith, J.S., Robinson, N.J., 2002. Age-specific prevalence of infection with herpes simplex virus types2 and 1: a global review. J. Infect. Dis. 186, s3–s28. Smith, T.J., Ackland-Berglund, C.E., Leib, D.A., 2000. Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice. J. Virol. 74, 3598–3604. Stuart, P.M., Keadle, T.L., 2012. Recurrent herpetic stromal keratitis in mice, a model for studying human HSK. Clin. Dev. Immunol. 2012, 728480. Stuart, P.M., Morris, J.E., Sidhu, M., Keadle, T.L., 2008. CCL3 protects mice from corneal pathology during recurrent HSV-1 infection. Front. Biosci. 13, 4407–4415. Stuart, P.M., Summers, B.C., Morris, J.E., Morrison, L.A., Leib, D.A., 2004. CD8(+) T cells control corneal disease following ocular infection with herpes simplex virus type 1. J. Gen. Virol. 85, 2055–2063. Tsai, H.H., Frost, E., To, V., Robinson, S., French-Constant, C., Geertman, R., Ransohoff, R.M., Miller, R.H., 2002. The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration. Cell 110, 373–383. Tsutsumi, T., Kajiya, H., Goto, K.T., Takahashi, Y., Okabe, K., 2013. Hyperocclusion up-
recurrent HSK phenotype. Thus our results suggest that both CXCL10 and CXCL9 may induce immunopathology during recurrent HSK. Consequently, we believe that CXCL10 in wild-type mice is important in the trafficking of CD4+ T cells to the cornea and that if this chemokine is neutralized the resulting recurrent HSK will be inhibited. This is supported by our data indicating that CXCL9 is not induced in wild-type B6 mice following infection. However, in the absence of CXCL10 the animal generates a CXCL9 response that more than compensates for the lack CXCL10 as T cells are still attracted to enter corneas where the virus is replicating following reactivation. This is not surprising as it has been shown during acute infection with HSV-1, CXCL9 is likely more responsible for migration of CD4+ T cells into the cornea than is CXCL10 (Qi et al., 2014). Furthermore, this type of compensatory response in the absence of particular chemokines or their receptors has been shown in several systems where lack of a particular chemokine or its receptor leads to increased expression of another factor that results in the same biological effect (Wuest et al., 2006; Chen et al., 2003; Yanaba et al., 2004; Mizukami et al., 2005; Pahler et al., 2008; Zlotoff et al., 2010). Since our data indicates that CXCL9 production is a compensatory response to HSV-1 infection, targeting CXCL9 along with CXCL10 in wild type mice is unlikely to result in an additive effect. It is interesting to note that Shen et al. (2013) proposed that the reason for increased disease in B6-CXCL10-/- mice is persistence of actively replicating virus in the corneas of these mice. Similarly, our data demonstrate that during recurrent HSK B6-CXCL10-/- mice exhibit replication of virus 5 days longer than that observed in wildtype B6 mice. However, we are not fully convinced that the extended presence of virus will necessarily lead to significant corneal disease. While there are cases where such extended presence might be a factor in recurrent corneal disease (Tsutsumi et al., 2013), we also know that CD4/CD8 double KO mice, which do not develop corneal disease, do have a more persistent corneal infection following reactivation than do wild-type mice (Gao et al., 2011). We have also noted this same phenotype in mice that do not express the CD28 molecule (unpublished results). These reports indicate that there can be situations where persistence of virus can prolong the stimulatory phase of activating an adaptive response, there needs to be the requisite T cells to create an environment where corneal disease can fully develop. Consequently, the development of corneal disease during recurrent HSK is a complex mix of cytokines, cells and, we believe to a lesser extent, virus. Acknowledgements This work was supported by National Institutes of Health Grants EY16352 (PMS), EY21247 (PMS), NS052632 (RSK), AI083019 (RSK), and Defense Threat Reduction Agency grant HDTRA-1-15-1-0032 (RSK) and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, Saint Louis University. References Arduino, P.G., Porter, S.R., 2006. Oral and perioral herpes simplex virus type 1 (HSV-1) infection: review of its management. Oral. Dis. 12, 254–270. Banerjee, K., Biswas, P.S., Kim, B., Lee, S., Rouse, B.T., 2004. CXCR2-/- mice show enhanced susceptibility to herpetic stromal keratitis: a role for IL-6-induced neovascularization. J. Immunol. 172, 1237–1245. Carr, D.J., Chodosh, J., Ash, J., Lane, T.E., 2003. Effect of anti-CXCL10 monoclonal antibody on herpes simplex virus type 1 keratitis and retinal infection. J. Virol. 77, 10037–10046. Chen, Z., Yu, S., Bakhiet, M., Winblad, B., Zhu, J., 2003. The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3. J. Neurochem. 86, 61–68. Christen, U., McGavern, D.B., Luster, A.D., von Herrath, M.G., Oldstone, M.B., 2003. Among CXCR3 chemokines, IFN-gamma-inducible protein of 10 kDa (CXC chemokine ligand (CXCL) 10) but not monokine induced by IFN-gamma (CXCL9) imprints a pattern for the subsequent development of autoimmune disease. J. Immunol. 171, 6838–6845. Christensen, J.E., de Lemos, C., Moos, T., Christensen, J.P., Thomsen, A.R., 2006.
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cytokine production. J. Immunol. 172, 7417–7424. Wuest, T., Farber, J., Luster, A., Carr, D.J., 2006. CD4+ T cell migration into the cornea is reduced in CXCL9 deficient but not CXCL10 deficient mice following herpes simplex virus type 1 infection. Cell. Immunol. 243, 83–89. Wuest, T.R., Carr, D.J., 2008. The role of chemokines during herpes simplex virus-1 infection. Front. Biosci. 13, 486204872. Yanaba, K., Mukaida, N., Matsushima, K., Murphy, P.M., Takehara, K., Sato, S., 2004. Role of C-C chemokine receptors 1 and 5 and CCL3/macrophage inflammatory protein-1alpha in the cutaneous Arthus reaction: possible attenuation of their inhibitory effects by compensatory chemokine production. Eur. J. Immunol. 34, 3553–3561. Zlotoff, D.A., Sambandam, A., Logan, T.D., Bell, J.J., Schwarz, B.A., Bhandoola, A., 2010. CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus. Blood 115, 1897–1905.
regulates CCL3 expression in CCL2- and CCR2-deficient mice. J. Dent. Res. 92, 65–70. Tuli, S., Sonal, S., 2009. Herpes simplex keratitis. (©2009)In: Yanoff, J.S., Duker, M., Wiggs, J.L., Miller, D., Azar, D.T., Goldstein, M.H., Rosen, E.S., Duker, J.S., Rao, N.A., Augsburger, J.J., Sadum, A.A., Shuman, J.S.,, Diamond, G.R., Dutton, J.J. (Eds.), Ophthalmology3rd edition. Elsevier Inc. Tuli, S.S., Kubal, A.A., 2013. (HSV Keratitis)Ophthalmology 4th edition. Elsevier, London, UK, p231–p236. Umene, K., Sakaoke, H., 1999. Evolution of herpes simplex virus type 1 under herpesvirus evolutionary processes. Arch. Virol. 144, 637–656. West, D.M., Del Rosso, C.R., Yin, X.T., Stuart, P.M., 2014. CXCL1, but not IL-6 is required for recurrent herpetic stromal keratitis. J. Immunol. 192, 1762–1767. Whiting, D., Hsieh, G., Yun, J.J., Banerji, A., Yao, W., Fishbein, M.C., Belperio, J., Strieter, R.M., Bonavida, B., Ardehali, A., 2004. Chemokine monokine induced by IFN-γ/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector
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