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The Journal of Immunology, 2008, 180, 6297 -6306
Copyright © 2008 by The American Association of Immunologists, Inc.
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IL-10 and Natural Regulatory T Cells: Two Independent Anti-Inflammatory Mechanisms in Herpes Simplex Virus-Induced Ocular Immunopathology1

Pranita P. Sarangi*, Sharvan Sehrawat*, Susmit Suvas{dagger} and Barry T. Rouse2,*

* Comparative and Experimental Medicine Program, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996; and {dagger} Department of Biological Sciences, Oakland University, Rochester, MI 48309


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Two prominent anti-inflammatory mechanisms involved in controlling HSV-1-induced corneal immunopathology (stromal keratitis or SK) are the production of the cytokine IL-10 and the activity of natural regulatory T cells (nTregs). It is not known whether, under in vivo conditions, IL-10 and nTregs influence the corneal pathology independently or in concert. In the current study using wild-type and IL-10–/– animals, we have assessed the activity of nTregs in the absence of IL-10 both under in vitro and in vivo conditions. The IL-10–/– animals depleted of nTregs before ocular infection showed more severe SK lesions as compared with the undepleted IL-10–/– animals. In addition, nTregs purified from naive WT and IL-10–/– animals were equally able to suppress the proliferation and the cytokine production from anti-CD3-stimulated CD4+CD25– T cells in vitro. Furthermore, intracellular cytokine staining results indicated that nonregulatory cells expressing B220 and CD25 markers were the major IL-10-producing cell types in the lymphoid tissues of HSV-infected mice. In contrast, in the infected corneas, cells with the CD11b+Gr1+ phenotype along with a minor population of Foxp3–CD4+ and a few F4/80+ cells produced IL-10. Our current investigations indicate that at least two independent anti-inflammatory mechanisms are involved in limiting the corneal lesions in SK, both of which may need to be modulated to control SK therapeutically.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ocular infection with HSV results in a chronic immunoinflammatory reaction in the cornea that can result in blindness. Studies in animal models of stromal keratitis (SK)3 have revealed that the infection, possibly in part because of its TLR ligand activity, sets off a range of critical cellular and molecular events (1); the actual SK lesions, however, appear to be orchestrated mainly by CD4+ T cells that are primarily type 1 cytokine producers (2, 3). SK lesions may resolve spontaneously and, when this occurs in the mouse model, it may be associated with up-regulation of IL-10 production (4). Additionally, the severity of SK may also be affected by the activity of Foxp3+ regulatory T cells (Tregs) (5). Accordingly, the depletion of such cells may result in more severe lesions (5), and recently we have shown that the adoptive transfer of in vitro generated Foxp3+ Tregs may modulate the severity of SK lesions (6).

One potential mechanism by which the Tregs carry out their regulatory effects is by the production of inhibitory cytokines such as IL-10 and TGF-β (7). In fact some types of Tregs may be induced by IL-10 and mediate regulation principally by producing IL-10 (8). In some infectious disease models, these inducible Tregs (sometimes referred to as Tr1 cells) are considered to play a pivotal role in viral pathogenesis (9). That IL-10 is critical for the resolution of inflammatory lesions has been shown in many systems including SK (10). Thus, SK lesions may be more severe in IL-10–/– animals (11), but it is unclear whether this reflects the failed function of some type of Treg or is the consequence of the failure of IL-10 production by some innate inflammatory cell type. Thus, macrophages and some dendritic cell subtypes as well as highly activated CD4+ T cells may be major sources of IL-10 production (12, 13), and certain pathogens may encode ligands that can stimulate IL-10 production from some cells (14, 15).

In the present study, we have compared the pathogenesis of SK in wild-type (WT) and IL-10–/– mice in an attempt to further define whether Treg-mediated control of the inflammatory reaction is dependent on or independent of IL-10 production. Our results show that compared with WT controls, increased SK lesions occur in the absence of IL-10 as well as when natural Tregs (nTregs) are deleted. Interestingly, depletion of nTregs even in IL-10–/– animals resulted in even more enhanced SK lesion severity compared with IL-10–/– mice, indicating that the effect of Tregs mainly occurred independently of IL-10 production. In support of this finding, Tregs purified from IL-10–/– and WT mice appeared equally active at suppression and could inhibit the cytokine production from TCR-stimulated CD4+CD25– T cells isolated from naive and infected WT animals. Furthermore, in the infected corneas nonregulatory cells with the CD11b+Gr1+ phenotype, along with a minor population of Foxp3–CD4+ and a few F4/80+ cells, produced IL-10. Our results show that at least two independent anti-inflammatory mechanisms, namely IL-10 and nTregs, are involved in controlling corneal immunopathology in SK. We discuss the implication of our studies for future novel therapies for SK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

IL-10–/– breeders were obtained from The Jackson Laboratory and were maintained in the animal facility of University of Tennessee (Knoxville, TN). C57BL/6 mice were obtained from Harlan Sprague Dawley. Foxp3-GFP mice (16) were a gift from Dr. M. Oukka (Harvard Medical School, Boston, MA). To prevent bacterial superinfection, all mice received prophylactic treatment with sulfamethoxazole/trimethoprim (Biocraft) at the rate of 5 ml per 200 ml of drinking water after virus infection. All experimental procedures followed the guidelines of the Association for Research in Vision and Ophthalmology (Rockville, MD) resolution on the use of animals in research. The animal facilities of the University of Tennessee are fully accredited by the American Association of Laboratory Animal Care.

In vivo depletion of nTregs and HSV-1 infection

IL-10–/– and C57BL/6 animals were given 0.7 mg of anti-CD25 mAb (clone PC61; Bioexpress) i.p. 3 days before corneal infection. Depletion was tested by staining for CD4+CD25+ cells in the spleen and lymph nodes (see Fig. 1A). More than 80% depletion was achieved by day 3 after injection. HSV-1 strain RE (obtained from R. Lausch, University of South Alabama College of Medicine, Mobile, AL) was used in all procedures. Virus was propagated and stored as described previously (17). For all experiments 6- to 8-wk-old females were used, except for the comparison of IL-10–/– mice with Treg-depleted IL-10 animals in which 9- to 10-wk-old male mice were used. Corneal infections of all mouse groups were conducted under deep anesthesia induced by i.p. injection of Avertin (Sigma-Aldrich). The mice were scarified on their corneas with a 27-gauge needle and infected with a 3-µl drop containing 5 x 105 PFU of HSV-1, was applied to the eye and gently massaged with the eyelids.


Figure 1
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  		FIGURE 1. Increased SK lesions in the absence of IL-10 and nTregs. A, Percentage of CD4+CD25+ T cells in the spleens at day 4 of the animals depleted of nTregs by anti-CD25 ({alpha}-CD25) Ab (pc61 mAb; 0.7 mg) treatment. Staining for CD25 was performed with a FITC-labeled anti-CD25 (clone 7D4) Ab. Left dot plot shows the spleen of an untreated control animal. B, The bar diagram demonstrates the percentage severity of each group of mice infected with 5 x 105 PFU of HSV-1 RE at day 16 p.i. The IL-10–/– and Treg-depleted WT (DEP-WT) mice showed increased incidence (SK score of ≥3) of disease as compared with WT animals. C, SK lesion scores of individual eyes at day 16 p.i of WT, DEP-WT, and IL-10–/– mice infected with 5 x 105-PFU infection dose. D, SK lesion scores in three groups of mice at different time points p.i. *, (p ≤ 0.05), statistically significant differences as compared with WT control. E, Angiogenesis lesion scores of individual eyes at day 16 p.i of WT, DEP-WT, and IL-10–/– mice infected with 5 x 105-PFU infection dose. F, Angiogenesis sores at different time points p.i. in the three groups of mice. *, p ≤ 0.05; statistically significant differences as compared with WT control.

 
Clinical observation

The eyes were examined on different days postinfection (p.i.) for the development of clinical lesions by slit lamp biomicroscopy (Kawa), and the clinical severity of keratitis lesions and the development of neovascularization of individually scored mice were recorded as described previously (18). In brief, the scoring system was as follows: 0, normal cornea; +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity but iris visible; +4, opaque cornea and corneal ulcer; +5, corneal rupture and necrotizing stromal keratitis. Similarly, the angiogenic scoring system relied on quantifying the degree of neovessel formation based on three primary parameters: 1) the circumferential extent of neovessels (as the angiogenic response is not uniformly circumferential in all cases); 2) the centripetal growth of the longest vessels in each quadrant of the circle; and 3) the longest neovessel in each quadrant was identified and graded between 0 (no neovessel) and 4 (neovessel in the corneal center) in increments of ~0.4 mm (radius of the cornea is ~1.5 mm). According to this system, a grade of 4 for a given quadrant of the circle represents a centripetal growth of 1.5 mm toward the corneal center. The score of the four quadrants of the eye were then summed to derive the neovessel index (range, 0–16) for each eye at a given time point.

Histopathology

For histopathologic analysis, eyes from different groups of mice were extirpated at the indicated time point p.i. and snap frozen in OCT compound (Miles). Six-micrometer-thick sections were cut, air dried in a desiccation box, and stained with H&E (Richard Allen Scientific).

Flow cytometry

For flow cytometry measurement of the infiltrating cells, four corneas per time point per group were collected at the indicated time points by dissecting the corneal buttons above the limbus by a scalpel. Similarly, two trigeminal ganglia (TGs) were collected per time point. Corneas or TGs were digested in Liberase (Roche Diagnostics) for 45 min at 37°C. Cervical draining lymph nodes (DLNs) and spleens were also collected from individual animals. Single cell suspension was prepared as described elsewhere (19). The Fc receptors were blocked with unconjugated anti-CD16/32 (BD Pharmingen) for 30 min. Samples were incubated with FITC-labeled anti-Gr-1, anti-CD11b, anti-CD11c, anti-NK1.1, anti-B220, anti-CD19, anti-CD25 (clone 7D4), anti-CD8 (BD Pharmingen), and anti-F4/80 (eBioscience) and PerCp-Cy5.5-labeled anti-CD4 (BD Pharmingen) Abs for 30 min. For intracellular cytokine staining, PE-labeled anti-IL-10, anti-IL-17, and anti-IFN- {gamma} and FITC-labeled anti- IFN-{gamma} and anti-TNF-{alpha}, Abs were used (BD Pharmingen). For Foxp3+IL-10+ cell staining, mice expressing Foxp3-GFP were used and GFP+IL-10+ cells were measured in such mice by using intracellular IL-10 staining. Intracellular staining for IL-10, TNF-{alpha}, and IFN-{gamma} was performed using a BD Cytofix/Cytoperm fixation/permeabilization solution kit with BD GolgiPlug (BD Bioscience). For intracellular cytokine staining, cells were stimulated with anti-CD3 and anti-CD28 (1 µg/ml) or PMA/ionomycin or UV-inactivated HSV (multiplicity of infection of 3). Foxp3 staining was performed using a mouse regulatory T cell staining kit (eBioscience). All samples were collected on a FACScan (BD Biosciences) and data were analyzed by using CellQuest 3.1 software (BD Biosciences).

Virus-specific CD8+IFN-{gamma}+ staining

To determine the number of IFN-{gamma} producing CD8+ T cells in the infected DLN and spleen, intracellular cytokine staining was performed as previously described (20). Single cell suspension of infected DLN and spleen was prepared, and 106 cells/well were cultured in 96-well U-bottom plates. Cells were left untreated or stimulated with SSIEFARL peptide (HSVgB498–505; synthesized at Genemed Synthesis) (1 µg/ml) for 5 h at 37°C in 5% CO2. Brefeldin A (10 µg/ml) was added to the culture for the intracellular cytokine accumulation. Cell surface marker and intracellular cytokine staining for IFN-{gamma} was performed using a Cytofix/Cytoperm kit (BD Pharmingen). All samples were collected with a FACScan and were analyzed with CellQuest 3.1 software.

Purification of CD4+CD25+ and CD4+D25– T cells

CD4+CD25+ and CD4+D25– Tregs were purified from the lymph nodes of WT and IL-10–/– mice using a Treg isolation kit (Miltenyi Biotec) according to the suggested protocol. The purity of cells ranged from 89 to 92% (see Fig. 8A). Accessory cells were isolated from the spleens of WT mice by depleting Thy1.2+ cells using Thy1.2 magnetic beads (Miltenyi Biotec). Thy1.2-depleted cells were irradiated before being added to the cultures. CD4+D25– T cells from Thy1.1 C57BL/6 animals were used for in vitro cultures.


Figure 8
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  		FIGURE 8. nTregs from both IL-10–/– and WT animals can equally suppress the proliferation and cytokine production from conventional T cell isolated from WT animals. CD4+CD25+ and CD4+CD25– cells were isolated as described in Materials and Methods and cultured in the presence of irradiated splenocytes and anti-CD3. Culture supernatants were collected at 48 h and the level of cytokines was analyzed by sandwich ELISA. For measuring the proliferation of non-Tregs, CD4+CD25– cells from Thy1.1 mice were labeled with CFSE and the extent of suppression activity was assessed from the intensity of CFSE staining. A, Dot plots show the purity of isolated CD4+CD25+ T cells from WT and IL-10–/– mice. B, Histograms demonstrate the suppression of proliferation of conventional T cells by nTregs isolated from IL-10–/– (lower panel) and WT (upper panel) animals. Proliferation of CD4+CD24– T cells are shown at 1:1, 1:4, and 1:8 dilutions of CD4+CD25+ nTregs. Gray dotted lines denote the division of CD4+CD25– T cells in the absence of nTregs and the solid lines show the suppression in the presence of nTregs. C, Bar diagrams show the levels of IFN-{gamma} in the 48-h culture supernatant in the presence and absence of nTregs. As shown, both of the nTregs could equally suppress cytokine production. D, Bar diagrams show the levels of IL-2 in the 48-h culture supernatant in the presence or absence of nTregs. E, GFP-Foxp3 mice were infected with HSV-RE and intracellular staining for IL-10 was performed as outlined in the Materials and Methods. As shown in the dot plots, no IL-10+ or GFP+ cells were detected in the lymphoid tissues after infection.

 
Cytokine inhibition and in vitro suppression assay

Purified Tregs (5 x 105/well) from IL-10–/– and WT animals were cultured with CD4+CD25– T cells (5 x 105/well) isolated from naive and infected WT animals along with irradiated splenocytes (1 x 106/well). Anti-CD3 Ab was added to the wells (1 µg/ml). Cells were cultured in a 48-well plate, the culture supernatants were collected after 48 h, and the levels of IFN-{gamma} and IL-2 were measured by sandwich ELISA. For in vitro suppression assays, in one of the experiments CD4+CD25– T cells from Thy1.1 mice (1 x 105/well) were labeled with CFSE (0.5 µM) and cultured in a 96-well plate with different dilutions of WT or IL10–/– CD4+CD25+ T cells (1:1, 1:2, 1:4, 1:8, 1:16, and 1:32) in the presence of anti-CD3 Ab (1 µg/ml) and APCs (2 x 105/well).

Cytokine ELISA

Cervical DLNs from individual mice and six corneas per group were collected at the indicated time points. The DLNs and corneas were sonicated and the levels of IL-6, IL-12, and IL-10 were measured in the supernatants. Similarly, culture supernatant from the in vitro suppression assay was collected and the levels of IL-2 and IFN-{gamma} were determined by a standard sandwich ELISA protocol. Anti IL-6, -IL-12, -IL-2, -IL-10, and -IFN-{gamma} capture and detection Abs were purchased from BD Pharmingen and standard recombinant murine IL-6, IL-12, IL-2, IL-10 and IFN-{gamma} were obtained from R&D Systems. Biotinylated detection Abs were purchased from BD Pharmingen. The color reaction was developed using ABTS (Sigma-Aldrich) and measured with an ELISA reader (SpectraMax 340; Molecular Devices) at 405 nm. Quantification was performed with SpectraMax ELISA reader software version 1.2.

Statistical analysis

Statistical significance was determined by Student’s t test. p < 0.05 was regarded as a significant difference between the groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The absence of IL-10 and depletion of nTreg results in increased SK severity

The outcome of HSV infection was followed and compared in three groups of mice that were age and sex matched and ocularly infected with the same dose of HSV-1 RE. The pattern of disease that developed differed significantly in the three groups. Thus, both IL-10–/– and Treg-depleted animals showed heightened susceptibility compared with WT animals (Fig. 1, B and C). Clinical lesions were evident in IL-10–/– animals before those in other groups. At 8 days p.i., the incidence of SK (lesion score, ≥3) was 0 and 30% in WT and IL-10–/– animals, respectively (not shown). The majority of IL-10–/– animals had developed moderately severe lesions (≥3) by 16 days p.i. (90% in the experiment shown in Fig. 1B). In contrast, <40% of WT animals in the same experiment developed ≥3 lesions and almost two-fold more than WT in the nTreg-depleted group (Fig. 1B). The pattern of more severe responses in IL-10–/– and Treg-depleted animals was also evident when the extent of angiogenesis was quantified in the different groups (Fig. 1, E and F). Eyes showing median responses in each group were selected for histological analysis. As is evident in Fig. 2A, inflammatory reactions were more severe than in WT mice in both the IL-10–/– and Treg-depleted animals. In other experiments, corneal samples were collected from eight randomly selected eyes taken from animals at 17 days p.i. to prepare single cell suspensions following collagen digestion for phenotypic analysis by flow cytometry. First, total viable cell numbers recovered from IL-10–/– and Treg-depleted animals was greater than those from WT animals (Fig. 2C). In addition, the frequency of cells with the neutrophil phenotype was also higher in IL-10–/– and nTreg-depleted WT as compared with WT animals (Gr1+CD11b+ cells, Fig. 2B). With regard to total CD4+ T cells, the highest numbers of such cells were present in the eyes of IL-10–/– animals (Fig. 2D). Comparing the ratio of Foxp3+CD4+ T cells to Foxp3–CD4+ T cells, the ratio was lower in IL-10–/– animals as compared with WT (Fig. 2E). Of note, before infection the number of Foxp3+ cells in the secondary lymphoid tissues was comparable to that of WT in IL-10–/– mice (data not shown).


Figure 2
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  		FIGURE 2. Increased inflammation in the corneas of mice deficient in IL-10 and nTregs. A, WT, Treg-depleted WT (DEP-WT), and IL-10–/– mice were infected with 5 x 105 PFU of HSV-1 RE. Mice were terminated at day 17 p.i. and eyes were processed for cryosection. H&E staining was conducted on 6-µm sections. The figure shows the pictures of the section taken at x100 original magnification. B, Single-cell suspensions of the infected corneas were prepared from six corneas at day 17 p.i. for each group of mice. The cells were labeled for Gr1+CD11b+ (polymorphonuclear) cells. The dot plots represent the Gr1+CD11b+ cells in the WT, DEP-WT and IL-10–/– corneas. The number on the dot plots denotes the percentage of inflammatory cells expressing both Gr1+ and CD11b+ markers. C, The bars represent the total number of viable cells present per cornea of different groups of mice. D, The cells isolated from infected corneas at day 17 p.i were stained for CD4 marker and the bars represent the total number of CD4+ T cells present per cornea from different groups of mice. E, Intranuclear staining for CD4+Foxp3+ nTregs was performed with the cells isolated from corneas. The bars represent the percentage of CD4+Foxp3+ cells present in the total CD4+ T cells per cornea of different groups of mice. F, Single-cell suspensions were prepared from four TGs at day 17 p.i. for each group of mice. The cells were labeled for Gr1+CD11b+ cells. The dot plots represent the Gr1+CD11b+ cells in the WT, DEP-WT, and IL-10–/– corneas. The number on the dot plots denotes the percentage of inflammatory cells expressing both Gr1+ and CD11b+ markers in TG. G, The cells from TGs were labeled for CD4+ and CD8+ cells. The dot plots represent the CD4+ and CD8+ cells in the WT, DEP-WT, and IL-10–/– corneas. The number on the dot plots denotes the percentage of inflammatory cells expressing CD4+ and CD8+ markers.

 
Cellular analyses were also performed in single cell suspensions of pooled TGs from the three groups of animals. As with the corneas, inflammatory cell numbers were elevated in the TGs of IL-10–/– and nTreg-depleted mice compared with WT animals (Fig. 2F). In addition, the frequency of both CD8+ and CD4+ T cells was elevated compared with WT with the highest frequencies present in IL-10–/– animals (Fig. 2G).

Along with the changes in inflammatory cell influx in the different groups of mice, the numbers of IFN-{gamma}- and IL-17-producing CD4+ T cells were also measured at day 17 in each group by intracellular cytokine staining. Once again the highest frequencies of cytokine producing cells were present in the corneas of IL-10–/– mice with Treg-depleted animals showing less and WT mice the least amount (Fig. 3). By intracellular cytokine staining, we also looked for the cytokine producing CD4+ T cells in the cervical lymph node and the spleen of these mice. As shown in Fig. 4, ~3- and 8-fold increases in the numbers of CD4+ T cells producing IFN-{gamma} and TNF-{alpha} were found in the DLN and spleens in the absence of nTregs and IL-10, respectively. This increase in proinflammatory mediators in the cornea and secondary lymphoid tissues along with higher Treg numbers in IL-10–/– animals may mean an inability of nTregs to suppress the disease pathology in the proinflammatory milieu (21, 22), or that the pathogenic Th1-type CD4+ T cells present at the site are resistant to nTreg-mediated suppression (23, 24).


Figure 3
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  		FIGURE 3. Increased number of CD4+IFN-{gamma}+ T cells in the corneas of mice deficient in IL-10 and nTregs. Single-cell suspensions of corneal cells were prepared from six corneas at day 17 p.i. Cells were stimulated with either anti-CD3 and anti-CD28 Ab or UV-HSV. Intracellular IFN-{gamma} staining along with CD4 surface staining was conducted as described in Materials and Methods. The results represent the data from one of two similar experiments. A, Dot plots represent the frequencies of CD4+IFN-{gamma}+ T cells from WT, Treg-depleted WT, and IL-10–/– mice when gated on the lymphocytes. B, The bar diagram demonstrates the total number of CD4+IFN-{gamma}+ T cells in the cornea in different groups. C, The bar diagram demonstrates the total number of CD4+IL-17+ T cells in the cornea in different groups.

 

Figure 4
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  		FIGURE 4. Increased number of IFN-{gamma}+ and TNF-{alpha}+ CD4 T cells in the secondary lymphoid tissues of IL-10–/– mice as compared with nTreg-depleted WT (DEP-WT) and WT mice. Single cell suspensions of the individual spleen and the cervical DLNs were prepared from four mice of each group at day 17 p.i. Cells were stimulated with either anti-CD3 and anti-CD28 Abs or UV-HSV as described in Materials and Methods. Intracellular cytokine staining along with CD4 surface staining was conducted as described in Materials and Methods. Dot plots represents the frequencies of CD4+IFN-{gamma}+ (A) and CD4+TNF-{alpha}+ (B) T cells, respectively, in the lymphoid tissues of different groups of mice when gated on the lymphocytes. Bar diagrams demonstrate the total number of CD4+IFN-{gamma}+ (A) and CD4+TNF-{alpha}+ (B) T cells per DLNs or spleens in different groups (n = 4 per group). *, p ≤ 0.05; statistically significant differences as compared with WT animals. Note: A similar pattern was also noticed in the number of CD4+IL-17+ T cells.

 
We also determined the magnitude of SSIEFARL (immunodominant epitope in C57BL/6 mice)-specific CD8+ T cell responses and cytokine production by CD8+ T cells in these animals. As shown in the Fig. 5, a higher magnitude of immune responses was noted in the absence of either Tregs (3-fold) or IL-10 (2-fold). Interestingly, in contrast to CD4+ T cells, the increase in total CD8+IFN-{gamma}+ T cells was greater in the absence of nTregs as compared with IL-10, which could mean that different pathways of immune response generation may be influenced by IL-10 and nTregs.


Figure 5
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  		FIGURE 5. Increased number of SSIEFARL peptide-specific CD8+IFN-{gamma}+ T cells in the secondary lymphoid tissues of WT mice deficient in nTregs (DEP-WT) as compared with IL-10–/– and WT mice. Single cell suspensions of the spleens and the cervical lymph nodes were prepared from four mice of each group at day 17 p.i. SSIEFARL-specific CD8+IFN-{gamma}+ staining was conducted as described in Materials and Methods. A, The dot plots represent the frequencies of CD8+IFN-{gamma}+ cells (CD8+ on the x-axis and IFN-{gamma}+ on the y-axis). The numbers on the upper right quadrants represent the percentage of CD8+IFN-{gamma}+ T cells in the gated population of lymphocytes in the spleens. The plots are representative of four animals analyzed per each group (n = 4). B, The bar diagram demonstrates the total number of double positive cells per DLN or spleen in different groups. *, p ≤ 0.05; **, p ≤ 0.01; statistically significant differences as compared with WT animals.

 
Treg effects are still evident in IL-10–/– mice

One explanation for the greater susceptibility of IL-10–/– animals could relate to the function of adaptive or nTregs. The former cell type is usually dependent on IL-10 and thus would not be expected to be present in IL-10–/– animals. Studies in some cases have indicated that Foxp3+ natural Tregs may also function at least in part by their production of IL-10 (25). Another explanation could be the dysfunction of nTregs in the presence of a high concentration of proinflammatory mediators such as IL-6 and IL-12 at the site (22, 23). In this regard, higher levels of IL-6 and IL-12 were found in the DLN and corneal lysates of IL-10–/– animals as compared with WT mice (Fig. 6).


Figure 6
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  		FIGURE 6. Increased expression of inflammatory mediators in the corneas and lymphoid tissues of mice deficient in IL-10 (IL-10–/–) and nTregs (DEP-WT). At the indicated time points, six corneas per group and cervical lymph nodes from individual animals were processed to measure the IL-6, and IL-12 levels. Level of cytokines were measured in the supernatants of tissue lysates of WT, DEP-WT and IL-10–/– animals infected with 5 x 105 PFU of HSV-1 RE using sandwich ELISA as outlined in Materials and Methods. Bar diagrams represent the levels of IL-12 and IL-6 in the corneal (A) and DLN (B) lysates of the three groups of animals. *, p ≤ 0.05; statistically significant difference as compared with WT animals.

 
Experiments were performed in older IL-10–/– male mice, which are more resistant to HSV-induced lesions than the 6- to 8-wk-old female mice used in previous experiments. To evaluate the role of nTregs in controlling SK lesion development, Treg-depleted IL-10–/– mice were compared with nondepleted IL-10–/– animals As shown in Fig. 7, A–D, depletion of CD25+ cells from IL-10–/– animals resulted in increased SK lesions (Fig. 7, A and D) as well as angiogenic responses (Fig. 7, B and D). At day 20 p.i., 55% of the eyes in Treg-depleted IL-10–/– animals had SK scores of ≥3 compared with 25% in undepleted IL-10–/– mice (Fig. 7C). At this time point, the SK scores were 2.3 ± 0.3 (p ≤ 0.05) in Treg-depleted IL-10–/– mice as compared with 1 ± 0.2 in normal IL-10–/– animals. A similar pattern was also evident in the development of neovascularization in the Treg-depleted IL-10–/– animals. At day 20 p.i., Treg-depleted IL-10–/– animals had neovascularization scores of 10.8 ± 1.1 as compared with 7.4 ± 1 in control IL-10–/– animals. Additionally, there was an earlier onset of SK lesions in the Treg-depleted IL-10–/– animals as compared with normal IL-10–/– animals. The incidence of SK at day 12 p.i. was 30 and 58% in the IL-10–/– and Treg-depleted IL-10–/– groups, respectively (Fig. 7C). Increased numbers of IFN-{gamma}+CD4+ T cells (Fig. 7E) and other inflammatory cell influxes were also noted in the corneas and TGs of such animals as compared with the controls (not shown). Similarly, as shown in Fig. 7F, the virus-specific CTL response, as assessed by the frequency and the absolute number of virus-specific CD8+IFN-{gamma}+ T cells in the secondary lymphoid tissues, was also significantly increased in the absence of nTregs in the IL-10–/– animals. These results indicate that even in IL-10–/– animals, nTregs can exert their suppressive activity and that their function could be independent of IL-10 in HSV-induced corneal immunopathology.


Figure 7
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  		FIGURE 7. Increased SK severity in the absence of nTregs in the IL-10–/– animals. A, Mean SK lesion scores at different time points (D, day) p.i. of Treg-depleted IL-10–/– (DEP-IL-10–/–) and undepleted IL-10–/– animals. *, p ≤ 0.05; statistically significant differences as compared with undepleted IL-10–/– mice. B, Mean angiogenesis scores at different time points p.i. of Treg-depleted IL-10–/– ({diamondsuit}) and undepleted IL-10–/– ({blacksquare}) animals. *, (p ≤ 0.05), statistically significant differences as compared with undepleted IL-10–/– mice. C, Bar diagram demonstrate the percentage severity (SK score ≥3) of both the groups of mice at day 14 and day 20 p.i. D, Eye pictures showing the lesion severity at day 18 p.i. in IL-10–/– and DEP-IL-10–/– animals. E, Single-cell suspensions of corneal cells were prepared from six corneas at day 20 p.i. Cells were stimulated with either anti-CD3 and anti-CD28 Ab or UV-HSV. Intracellular IFN-{gamma} staining along with CD4 surface staining was conducted as described in Materials and Methods. Bar diagram demonstrate the total number of CD4+IFN-{gamma}+ T cells per cornea at day 17 p.i. F, Single-cell suspensions of the spleen and the cervical lymph nodes were prepared from four mice of each group at day 20 p.i. SSIEFARL-specific CD8+IFN-{gamma}+ staining was conducted as described in Materials and Methods. The bar diagram demonstrates the total number of double positive cells per DLN or spleen in different groups. *, p ≤ 0.05; statistically significant differences as compared with undepleted IL-10–/– animals.

 
To support the apparent IL-10-independent in vivo suppressive activity of nTregs, we isolated CD4+CD25+ nTregs from uninfected WT and IL-10–/– animals and those were cultured with CFSE-labeled CD4+CD25– cells isolated from naive and 12-day p.i. WT animals. Cells were stimulated with anti-CD3 and accessory cells in vitro for 48 h. As shown in Fig. 8, B–D, both groups of nTregs could equally suppress the proliferation (Fig. 8B) and cytokine production (Fig. 8D) from WT CD4+CD25– T cells.

What is the main cellular source of IL-10 in SK?

Several reports have shown the production of IL-10 by Foxp3+ Tregs (25, 26, 27). To determine whether, in SK, nTregs themselves produce IL-10 p.i., we measured IL-10 production using Foxp3-GFP mice at days 5, 11, and 18 p.i. As shown in Fig. 8E and Table I, we could not detect substantial numbers of GFP+IL-10+ cells in the lymphoid tissues, although very few such cells were present in the infected corneas at later time points p.i. (Table I). Additionally, IL-10+IFN-{gamma}+CD4+ T cells were not evident in either cornea or lymphoid tissues (not shown). These data suggest that nonregulatory cells could be the major cellular source of IL-10 p.i.


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  		Table I. Phenotype of IL-10 producing cells in various tissuesa

 
To further study the phenotype of the IL-10-producing cells in the infected corneas and secondary lymphoid tissues, intracellular cytokine staining was performed with cells taken from infected WT corneas, spleens, and DLNs at days 5, 11, and 20 p.i. The cells were stained with surface markers such as c-Kit, Gr-1, CD11c, CD11b, B220, CD19, CD25, CD3, NK1.1, and CD4 along with intracellular IL-10. As shown in Table I, the majority of the IL-10-producing cells in the spleens and DLNs also expressed B220, CD25, and F4/80 markers. More than 50% of the cells also expressed c-Kit and Gr1 markers (data not shown). Interestingly, as shown in Table I, in the infected corneas nonregulatory cells with the CD11b+Gr1+ phenotype along with a minor population of Foxp3–CD4+ and a few F4/80+ cells produced IL-10.

Influence of nTreg on IL-10 production

Because our results indicated that Tregs could have IL-10-independent suppression in SK and that these cell types did not constitute a significant part of the IL-10-producing cell types, we determined whether the activity of nTregs could influence the production of IL-10 p.i. In many reports, Tregs were shown to suppress immune responses via IL-10 production (8, 28). Interestingly, as measured by intracellular cytokine staining and cytokine ELISA an increase in the absolute number and frequencies of IL-10-producing cells as well as IL-10 proteins was noted in the lymphoid tissues in the absence of Tregs in the WT animals as compared with undepleted WT animals (Fig. 9, A and B). But the mean fluorescence intensity of IL-10+ cells remained comparable among Treg-depleted and undepleted WT animals, and the case of the phenotype of IL-10-producing cells was similar (data not shown). This could be due to the generalized suppressive activity of Tregs on the IL-10-producing cell types such as macrophages or dendritic cells or the activated CD4+ T cells. In contrast, as is evident in Fig. 9C, the level of IL-10 in the corneas of Treg-depleted WT mice was lower as compared with that in undepleted WT animals at days 4 and 9 p.i.


Figure 9
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  		FIGURE 9. A, Tregs do not produce IL-10 but can influence the IL-10 production from other cell types in the lymphoid tissues. To ascertain the effect of Tregs on IL-10 production, total numbers of IL-10+ cells were measured in the Treg-depleted and undepleted animals. A, The bar diagram demonstrates an increase in the number of IL-10+ cells in the lymphoid tissues of Treg-depleted animals. B, The bar diagram demonstrates an increase in the IL-10 and IFN-{gamma} protein levels in the Treg-depleted animals as compared with undepleted WT animals at day 6 p.i. C, The bar diagram demonstrates the level IL-10 in the corneal lysates of Treg-depleted animals as compared with undepleted WT animals at days 4 and 9 p.i. *, p ≤ 0.05; statistically significant differences as compared with undepleted WT animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Many chronic immunoinflammatory diseases are difficult to control therapeutically. One such example is SK, which is caused by HSV infection of the eye. It is anticipated that control measures will improve once all of the mechanisms involved in lesion resolution are identified and their respective roles are fully understood. With regard to SK, few studies have been done but reports have demonstrated that up-regulation of certain cytokines such as IL-10 and TGF-β help resolve lesions (29, 30). Recently, observations in a variety of inflammatory diseases indicate that several types of regulatory cells control and resolve inflammatory lesions, and some of these appear to function by producing either or both IL-10 and TGF-β (31). With SK, natural regulatory T cells were shown previously to influence lesions, but the mechanisms by which this is achieved were not identified (5). In some systems nTregs appear to function independently of any cytokine production (32), whereas in others IL-10 production is a necessary event (25, 28).

In this report, we show that both nTreg and IL-10 participate in the control of SK lesion severity, but these two mechanisms proceed largely independently of each other. Our results show that the severity of SK lesions becomes enhanced if animals are unable to produce IL-10 because of gene knockout or have been depleted of nTreg. Furthermore, depletion of nTreg in IL-10–/– mice led to even higher levels of disease severity than in undepleted IL-10–/– mice. In addition, nTreg purified from WT and IL-10–/– animals appeared to be equally suppressive in vitro to the activity of nonregulatory cells. Few if any Foxp3+CD4+ Tregs in either the ocular lesions or DLNs were IL-10 producers. In fact, the major source of IL-10 appeared to be non-T cells such as granulocytes and mononuclear inflammatory cells. Accordingly, these HSV-induced immunoinflammatory lesions can be modulated independently by both IL-10 and nTreg activity, which could mean that future novel therapies will need to manipulate both mechanisms to achieve maximal effects.

The means by which inflammatory lesions are influenced by regulatory T cells remain poorly understood, especially in responses caused by pathogens (33). In some virus-induced immunoinflammatory lesions, some advocate a major role for adaptive Tregs induced by Ag in the presence of IL-10 (9, 34). These so-called Tr1 cells regulate responses by producing an abundance of IL-10 (9, 34). Their role in SK remains unexplored. In other systems, such as Leishmania infections, cells of the nTreg phenotype control lesions, but these appear to act by producing IL-10 (35, 36). The same may also be true in other parasite-mediated lesions as well as in bacteria-induced colitis (36, 37). Our results showing that a significant nTreg effect could be demonstrated in mice genetically unable to produce IL-10 strongly argues against IL-10 being a dominant mechanism by which nTregs act in vivo. Similarly, such cells isolated from IL-10–/– mice acted equally as those from WT animals in vitro, indicating no role for IL-10 to mediate their function as others have argued to be the case using different approaches (38).

It is conceivable that several functional subsets of the Foxp3+ regulatory phenotype exist. Those thymus-derived autoantigen-specific cells that are primarily depleted by pc61 mAb in naive mice may function in a different way as those converted in vivo from CD4+Foxp3– foreign Ag-specific precursors. This conversion process was shown recently to occur by several groups (39, 40, 41) including ourselves (6). We suspect that the nTregs preexisting at the time of infection are signaled in some way by the HSV infection and that these control SK in a manner that does not depend on IL-10 production. In contrast, the Foxp3+ Tregs that act in more chronic infections may largely represent Foxp3+ converts, and these may function primarily by producing IL-10. Because we have recently succeeded in generating Foxp3+ Treg converts from nonregulatory precursors (6), we are now in the position to compare these with normal nTregs for their ability to modulate SK as well as their dependence on IL-10 production. Such studies are in progress.

Although our results could not define the site at which IL-10 and nTregs function to control SK lesions, we suspect they may be quite different. We anticipate that IL-10 acts mainly at the ocular site to control lesions. Moreover, the major source of this cytokine was shown to be nonregulatory cells having the CD11b+Gr1+ and F4/80+ phenotype. Some of these cells may be equivalent to the CD11b+Gr1+ myeloid suppressor cells described in some tumor systems (42). We also demonstrated that the IL-10 appeared to act by inhibiting the production of proinflammatory cytokines such as IFN-{gamma}, IL-17, and TNF-{alpha}. Cells producing such cytokines were more abundant in lesions of IL-10–/– than in WT animals. The results of several other observations also support a similar anti-inflammatory effect of IL-10 (43, 44).

Although our observations as well as former reports show the presence of Tregs at the site of inflammation (5, 24, 45), we cannot assess whether or not they exert a suppression effect in the inflamed tissue. In fact, whether Tregs function at an inflammatory site remains debatable. Thus, one report showed that Tregs taken from an inflammatory site failed to suppress pathogenic T cells isolated from the lesions when tested in vitro (24), perhaps because their activity was inhibited by one or more inflammatory cytokines as has been shown to occur in vitro (22, 23). Interestingly, others have shown that Tregs do proliferate at such sites and that they can suppress IFN-{gamma}, although not IL-17 production, by CD4+CD25– T cells isolated from the lesion site (45). In our current study, WT mice depleted of nTreg had higher levels of proinflammatory cytokines in the lesions as well as in the lymphoid tissues. Conceivably, such cytokines could inhibit their function, but this issue requires further evaluations using Tregs isolated from the inflamed cornea at different time points p.i.

A body of evidence favors the idea that nTregs mainly act to regulate in lymphoid tissues (46). Our data favor this idea too. Accordingly, in nTreg-depleted WT animals the absolute numbers of IL-10 producing cells in the DLNs and spleens were increased, accounting at least in part for the inhibitory effects that occurred. In addition, Treg depletion resulted in better HSV-specific immune response, providing more of the effector cells responsible for mediating SK.

One surprising result of our observations was that an effect of nTregs was easy to demonstrate even when the cells were called upon to function in an environment rich in cytokines assumed to impair their function. Thus, we demonstrated marked elevations of cytokines such as IL-6 and IL-12 in the lymph nodes and ocular tissues of infected IL-10–/– compared with similarly infected WT animals. Despite this, an effect of nTreg depletion was readily apparent in the IL-10–/– infected mice. These results may mean either that in the in vivo inflammatory environment the inhibitory effects of some cytokines observed in vitro are not major effects or that the Treg function at certain sites such as in lymph nodes where effector cells are being generated is not inhibited by inflammatory cytokines. Some recent observations on experimental autoimmune encephalitis also support the concept that effector cell functions in lymphoid sites may be inhibited by Foxp3+ Tregs, whereas at the same time effectors at lesion site may resist such regulation (24). These issues are under further investigation in the SK system.


   Acknowledgments
 
We thank Dr. Mohammed Oukka for providing us the GFP-FOXP3 mice. We also thank Dr. Kim Bumseok and Jason Burchett for help in this work.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants EY05093 and AI 1063365. Back

2 Address correspondence and reprint requests to Dr. Barry T. Rouse, M409 Walters Life Sciences Building, Department of Pathobiology, University of Tennessee, 1414 Cumberland Avenue, Knoxville TN 37996-0845. E-mail address: btr{at}utk.edu Back

3 Abbreviations used in this paper: SK, stromal keratitis; DLN, draining lymph node; p.i., postinfection; nTreg, natural regulatory T cell; TG, trigeminal ganglion; Treg, regulatory T cell; WT, wild type. Back

Received for publication November 21, 2007. Accepted for publication February 4, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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