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Interleukin (IL)-10 deficiency results in highly elevated levels of interferon (IFN)-γ, as well as the IFN-γ-inducible chemokines CXCL9 and CXCL10 within murine cytomegalovirus (MCMV)-infected brains. To test the hypothesis that these elevated chemokine levels would result in enhanced brain infiltration, we compared immune cell infiltration in response to MCMV brain infection between wild-type and IL-10 knockout (KO) mice. Longitudinal analysis following adoptive transfer of cells from β-actin-luciferase transgenic wild-type mice showed maximal brain infiltration by peripheral immune cells occurred at 5 d post-infection. Although the overall percentage of CD45(hi) cells infiltrating the brain was not altered by IL-10 deficiency, paradoxically, despite elevated chemokine levels, reduced T lymphocyte (CD8+) and NK (CD49b+) cell infiltration into the brain was observed in IL-10-deficient animals. This decreased lymphocyte infiltration was associated with elevated levels of the lymph node homing receptor L-selectin/CD62L on CD8+ T-cells. Lymph node cells obtained from MCMV-infected mice deficient in IL-10 also displayed reduced migration towards CXCL10 when compared to wild-type animals. Taken together, these data show that despite elevated chemokine levels, absence of IL-10 results in reduced lymphocyte infiltration into MCMV-infected brains.
We have recently shown that absence of the anti-inflammatory cytokine interleukin (IL)-10 results in dysregulated neuroimmune responses and turns a benign murine cytomegalovirus (MCMV) brain infection lethal (Cheeran et al, 2007). This lethal infection is associated with vastly elevated levels of IFN-γ, and the IFN-γ-inducible chemokines CXCL9 and CXCL10, as well as IL-6, in brain homogenates obtained from IL-10 knockout (KO) mice, when compared to either wild-type or IL-4 KO animals (Cheeran et al, 2007). In this study, we tested the hypothesis that elevated levels of infection-induced chemokines observed in the brains of MCMV-infected IL-10 deficient mice would result in greater infiltration by cells of the peripheral immune system when compared to wild-type animals.
Anti-inflammatory cytokines, such as IL-10, IL-4, and TGF-β, have been described as inhibitors of proinflammatory responses in the central nervous system (CNS), and specifically as suppressors of microglial cell function (Sawada et al, 1999; Suzumura et al, 1994; Suzumura et al, 1993). One of the main functions of anti-inflammatory cytokines is thought to be their role as a host mechanism to limit tissue damage and turn off proinflammatory responses (Mills, 2004). Because resident glial cells and infiltrating lymphocytes communicate through cytokine and chemokine mediators, the well-documented neuroprotective action of anti-inflammatory cytokines is likely related to their ability to inhibit chemokine-driven neuroinflammation.
MCMV brain infection of both wild-type and immunodeficient mice induces chemokine production (Cheeran et al, 2004). However, chemokine levels are vastly elevated in the brains of infected animals which lack lymphocytes when compared to wild-type mice (Cheeran et al, 2004). It appears that infiltrating IL-10-producing leukocytes possess the ability to suppress proinflammatory chemokine production by microglial cells in the brain. Adoptive transfer of primed splenocytes from wild-type animals into immunodeficient mice dampens this excessive chemokine production because lower levels of chemokines are found in the brains of immunodeficient SCID/Bg animals receiving adoptive transfer of splenocytes than those not receiving adoptive transfer (Cheeran et al, 2004). Also, previous studies have shown that induction of proinflammatory cytokines by endogenous brain cells alone is not sufficient to protect immunodeficient SCID/Bg mice against MCMV infection, but successful defense of the brain requires participation of T lymphocytes (Cheeran et al, 2005; Reuter et al, 2005).
It is clear IL-10 facilitates protection against MCMV brain infection because wild-type mice survive intracerebroventricular (icv) viral injection, but the same injection is lethal in IL-10-deficient animals (Cheeran et al, 2007). More severe MCMV-induced clinical manifestations in IL-10 KO mice (C57BL/6) have also recently been reported following intraperitoneal (i.p.) infection with the Smith strain (Oakley et al, 2008). Here, we assessed brain infiltration by peripheral immune cells using both wild-type and IL-10 knockout (KO) mice. Paradoxically, we found that despite the elevated levels of T-cell-attracting chemokines in the brain and a more robust neuroimmune response to MCMV infection, absence of IL-10 resulted in reduced lymphocyte infiltration into MCMV-infected brains.
Using bioluminescent imaging of live-animals, we first examined the kinetics of peripheral leukocyte infiltration longitudinally in both MCMV-infected and sham-infected (i.e., saline injected) mice. Splenocytes and lymph node cells were obtained from primed luciferase transgenic mice (8 d post-priming) and were delivered via tail-vein injection to MHC-matched FVB (H-2q) mice 24 h prior to icv infection with MCMV (RM461). Within 24 h of adoptive transfer (i.e., −1 d p.i.), luciferase positive cells were detected in the spleen of all animals. Movement of these peripheral leukocytes into the brain was observed by 24 h p.i., and peaked at 5 d p.i. (Figure 1A). Additionally, the presence of labeled cells in the cervical lymph nodes and spleen was assessed by imaging the ventral side (Figure 1B). Luciferase signal intensity, which is directly proportional to the number of splenocytes present, was quantified in photons per sec per cm2 at the indicated time points in both brain (Figure 1C), and cervical lymph nodes (Figure 1D) over the 9 d time-course of the experiment.
To identify peripheral immune cell types involved in neuroimmune responses to MCMV brain infection, leukocytes were isolated from brains of infected animals at 5 d p.i using a Percoll gradient. These isolated cells were first immunostained with markers characteristic of microglial cell and macrophage populations (i.e., CD45 and CD11b). In these experiments, two distinct populations of CD45(+)CD11b(+) cells were apparent in MCMV-infected brains: one expressing intermediate levels, CD45(int), which comprised the resident microglia; and another displaying high levels of CD45, which represented infiltrating macrophages. At 5 d p.i., the highest proportion of CD45(hi) infiltrating leukocytes also expressed the macrophage marker CD11b, 55.2 ± 2.0% and 68.9 ± 2.9% for wild-type and IL-10 KO mice, respectively . When a similar analysis was performed using brain tissues obtained from sham-infected, control animals, infiltrating CD45(hi) cells were found to be absent (5.6%). Interestingly, while the overall percentage of CD45(hi) cells infiltrating the brain was not altered by IL-10 deficiency (80.8 ± 1.8% versus 82.8 ± 1.7% for wild-type and IL-10 KO mice, respectively), decreased infiltration of the CD45(hi)CD11b(−) cell population was observed in mice which were deficient in IL-10, 21.6 ± 0.5% versus 11.2 ± 2.2% for wild-type and IL-10 KO mice, respectively.
Having observed reduced infiltration of the CD45(hi)CD11b(−) cell population into the brains of IL-10 KO animals compared to wild-type mice, we went on to determine whether these cells were lymphocytes. Similar flow cytometric studies using APC-labeled anti-CD45 and Cy7-PE-labeled anti-CD3 Abs showed that IL-10 deficiency resulted in reduced T lymphocyte ingress into the brain in response to MCMV infection (i.e., 5.2 ± 0.2% versus 13.4 ± 0.5%, respectively, p < 0.01 Student’s t test), (Figure 2A). We then assessed the involvement of natural killer (NK) cells. These lymphocytes were stained using APC-conjugated anti-CD45 and PE-labeled anti-CD49b Abs and examined using flow cytometry. Results generated during these experiments demonstrated that NK cells infiltrated the brain of wild-type mice in response to MCMV infection and that infiltration of these cells was also decreased in animals with IL-10 deficiency, 10.2 ± 2.6% versus 5.5 ± 0.2%, respectively, p < 0.01 Student’s t test (Figure 2B). Significant reduction in the absolute numbers of infiltrating NK cells was also observed in the IL-10 KO mice (5.7 ± 0.8 × 104 versus 1.1 ± 0.4 × 105, p = 0.03). Further, confirmation of reduced T lymphocyte ingress into the brain was obtained through in situ immunohistochemical staining for the CD3 cell surface antigen in brain sections obtained from MCMV-infected mice. These immunostaining experiments clearly showed a decreased number of lymphocytes within brain sections from MCMV-infected, IL-10 KO mice when compared to wild-type animals (Figure 2C).
Similar analysis to determine the absolute number of brain-infiltrating CD45(hi)CD3(+) T-cells again demonstrated that there was significantly less infiltration of T lymphocytes into the brains of MCMV-infected IL-10 KO mice, when compared to wild-type animals, p < 0.01 (Figure 3A). Data obtained from these experiments also showed that the total number of infiltrating CD8(+) T-cells was decreased to a greater extent than the CD4(+) subpopulation (Figure 3A). We then went on to determine the distribution of CD4(+) and CD8(+) lymphocytes within the infiltrating CD3(+) population. In these experiments, as well as being reduced in total number, CD8(+) lymphocytes were also found to make up proportionally less of the cellular infiltrate in the brains of IL-10-deficient animals (Figure 3B).
To determine whether deficiency in IL-10 disrupts the migration of immune cells out of the lymph nodes, cervical lymph node cells were isolated from MCMV-infected wild-type versus IL-10-deficient animals. The isolated cells were then double-stained for expression of CD8 or CD4, as well as the lymph node homing receptor L-selectin/CD62L and assessed using flow cytometry. Representative data show that proportionally more CD8(+) cells were retained in the lymph nodes of MCMV-infected IL-10 KO mice when compared to infected wild-type animals (Figure 4A). Analysis of pooled data obtained from 4 experiments (using 3–4 animals/experiment) at 5 d p.i., demonstrated significantly increased expression of L-selectin/CD62L on CD8(+) T-lymphocytes obtained from the cervical lymph nodes of the IL-10 deficient animals when compared to wild-type controls (Figure 4B).
To determine whether deficiency in IL-10 also inhibits cellular migration towards a chemotaxic stimulus, the ability of cervical lymph node cells isolated from MCMV-infected IL-10 KO mice to migrate towards CXCL10 was compared to that of wild-type control animals. In these studies, analysis of pooled data obtained from 3 experiments (n=3 animals/experiment) demonstrated that cells from IL-10-deficient mice displayed a significant inhibition of chemotaxis towards CXCL10, a well-known lymphocyte chemoattractant (Figure 5).
The anti-inflammatory cytokines IL-10, IL-4, and TGF-β play key roles in maintaining the delicate balance between control of viral infection and immunopathogenesis. In the CNS, IL-10 has been shown to be secreted by both resident microglial cells and infiltrating Tr1 regulatory T-cells (i.e., Tregs) (Burkhart et al, 1999; Groux et al, 1997; OGarra et al, 2004; Sheng et al, 1995; Williams et al, 1996). Previous in vitro studies from our laboratory have demonstrated that infection of primary human microglia with human cytomegalovirus (CMV) results in CXCL10 production (Cheeran et al, 2003). This virus-induced chemokine production was found to be suppressed following treatment with IL-10 and IL-4, but not transforming growth factor (TGF)-β (Cheeran et al, 2003). Importantly, follow-up in vivo studies using MCMV infection of mice have shown that the IL-10 which is necessary to control dysregulated virus-induced, IFN-γ-mediated neuroimmune responses during viral brain infection is provided by an infiltrating peripheral CD45(+)CD11b(−) or CD45(+)CD11b(int) immune cell type, not resident microglial cells (Cheeran et al, 2007). Interestingly, human CMV carries a homolog of IL-10 (i.e. cmvIL-10) which also inhibits virus-induced chemokine production from microglial cells (Cheeran et al, 2003), presumably to subvert host defenses. The presence of an IL-10 homolog carried by MCMV has not been reported.
Peripheral immune cells infiltrate the brain in response to glial cell-produced chemotactic factors. Here, we first used bioluminescence imaging to quantify the overall kinetics of immune cell trafficking into MCMV-infected brains. The results of these studies clearly demonstrate that these cells move into the brain in response to viral infection. Infiltration of the brain by cells of the peripheral immune system was also evident through histopathological examination of brain sections obtained from MCMV-infected animals. When these sections were examined, inflammatory cells were easily detected in the ventricles, periventricular regions, and meninges. However, no histopathological differences were apparent between wild-type and IL-10 KO animals at 5 d p.i. (data not shown).
The early neuroimmune responses to MCMV infection were dominated by the influx of macrophages in both the wild-type and IL-10 KO mice. These infiltrating CD45(hi)CD11b(+) cells were in an activated state, as assessed by upregulation of MHC class II expression (data not shown). Resident microglia (i.e., CD45(int)CD11b(+) cells), which normally do not express MHC class II, become activated either in response to the virus itself or in response to IFN-γ, which has been shown to induce MHC class II expression (Hamo et al, 2007). Interestingly, when macrophage infiltration in response to MCMV brain infection was compared between wild-type and IL-10 KO animals, it was not found to be affected by the IL-10 deficiency.
Subsequent immunostaining followed by flow cytometry demonstrated that T lymphocytes made up a high proportion of the CD45(hi)CD11b(−) population. Interestingly, unlike the situation with macrophages and contrary to our hypothesis of greater leukocyte infiltration into the brains of IL-10 KO mice, decreased CD8(+) T lymphocyte infiltration into infected brains was observed in animals which were deficient in IL-10. Likewise, NK cells were also found to infiltrate the brain of wild-type mice in response to MCMV infection and, as with T lymphocytes, this cellular infiltration was also significantly reduced in IL-10 KO animals.
Based on our previous studies, we anticipated that IL-10 deficiency would lead to enhanced neuroimmune responses against MCMV brain infection. Paradoxically, these data show that despite elevated chemokine levels, absence of IL-10 actually resulted in reduced lymphocyte infiltration into MCMV-infected brains. In the present paper, we report that this decreased lymphocyte brain infiltration is associated with increased expression of the lymph node homing receptor L-selectin/CD62L on CD8(+) T-lymphocytes obtained from the cervical lymph nodes of MCMV-infected, IL-10 deficient animals. The increased expression of CD62L was significant only on CD8+ lymphocytes, which also demonstrated decreased brain infiltration. The decreased ability of IL-10-deficient animals to downregulate L-selectin/CD62L during cellular activation in the cervical lymph nodes may lead to reduced or delayed recruitment of effector T-cells and NK cells into the brain in response to viral infection. Using a murine model for mucosal HSV-2 infection, it has recently been reported that ablation of regulatory T-cells disrupts the migration of immune cells out of the lymph nodes and profoundly reduces or delays recruitment of effector T-cells and NK cells to sites of viral infection, thereby accelerating fatal infection (Lund et al, 2008). Although Tregs utilize multiple means to limit immune responses, IL-10 production by these cells has been found to be essential for keeping immune responses in check at particular environmental interfaces such as the colon and lungs (Rubtsov et al, 2008). Distinct suppressor mechanism most likely play a prominent role in particular tissues and it remains to be seen if an analogous immune response-promoting role for IL-10 occurs in the brain during MCMV infection.
Although decreased CD8+ lymphocyte infiltration into the MCMV-infected brain which was observed in IL-10 knockout animals was associated with higher levels of CD62L on CD8+ T-cells in the cervical lymph nodes, it is clear that this is not the sole responsible mechanism. The fact that NK cells from IL-10 KO animals did not display enhanced CD62L expression demonstrates the involvement of multiple mechanisms which likely work in concert to produce the observed phenotype. Further experiments also demonsrated that lymph node cells obtained from IL-10 knockout animals were deficient in their chemotaxic migration towards CXCL10 when compared to those obtained from wild-type mice.
We do not yet know why MCMV brain infection is lethal in IL-10 KO animals, but it does not appear to be due simply to higher overall viral load in the brain (Cheeran et al, 2007). In salivary glands, blockade of the IL-10 receptor with an antagonist Ab has been reported to increase INF-γ-secreting cells and subsequently decrease viral loads (Humphreys et al, 2007). In similar studies, Oakley et al. reported that following i.p. injection of MCMV into IL-10 KO mice, the KO animals had more severe disease which was associated with elevated levels of IFN-γ, monocyte chemoattractant protein (MCP)-1, and IL-6, but was not attributable to viral replication (Oakley et al, 2008). Instead, proper regulation of the neuroimmune response appears to be crucial in controlling immunopathological brain damage associated with clearing viral infection. The neuroprotective effects of anti-inflammatory cytokines are believed to be mediated through downregulation of brain inflammation. It is thought that the neuroprotective action of IL-10 is related to its ability to inhibit chemokine-driven neuroinflammation, turn off proinflammatory responses, and limit damage to brain tissue. Surprisingly, in this study IL-10 deficiency resulted in reduced infiltration of the brain by T lymphocytes and NK cells in response to MCMV infection.
RM461, a recombinant virus expressing E. coli β-galactosidase under the control of the human ie1/ie2 promoter/enhancer (Stoddart et al, 1994), was kindly provided by Edward S. Mocarski. Virulent, salivary gland-passaged, sucrose gradient-purified virus was used for all icv infections. Stocks of MCMV Smith Strain (ATCC, Rockville, MD), used to prime donor animals, were grown and titered by TCID50 assay on NIH 3T3 fibroblasts. BALB/c and FVB/N mice were obtained from Charles River Laboratories (Wilmington, MA), while IL-10 KO animals were purchased from The Jackson Laboratory (Bar Harbor, ME). FVB/N β-actin promoter-luciferase transgenic mice were obtained from Xenogen (Alameda, CA).
Icv infection of mice was performed as previously described (Cheeran et al, 2004). Briefly, female mice (8–10 weeks old) were anesthetized using a combination of Ketamine and Xylazine (100 mg and 10 mg/Kg body weight, respectively) and immobilized on a small animal stereotactic instrument equipped with a Cunningham mouse adapter (Stoelting Co., Wood Dale, IL). The skin and underlying connective tissue were reflected to expose reference sutures (sagittal and coronal) on the skull. The sagittal plane was adjusted such that the bregma and lambda were positioned at the same coordinates on the vertical plane. Virulent, salivary gland-passaged MCMV RM461 (1.5 × 105 TCID50 units in 10 µl), was injected into the right lateral ventricle at 0.9 mm lateral, 0.5 mm caudal to the bregma and 3.0 mm ventral to the skull surface using a Hamilton syringe (10 µl) fitted to a 25 G cannula. The injection was delivered over a period of 3–5 min. The opening in the skull was sealed with bone wax and the skin was closed using 9 mm wound clips (Stoelting Co., Wood Dale, IL).
Spleen cells were aseptically collected from MHC-matched FVB/N β-actin promoter-luciferase transgenic mice 8 d following i.p. injection of tissue culture-passaged MCMV Smith strain. Splenocytes were depleted of red blood cells by treatment with 0.87% ammonium chloride for 2 min followed by the addition of an equal volume of PBS containing 2% fetal bovine serum. Spleen cells (1 × 107) obtained MCMV-primed mice (8 d p.i.) were then transferred via tail vein injection into wild-type FVB/N mice 24 h prior to icv infection with RM461 (3 × 105 TCID50).
Imaging of firefly luciferase expression in live animals was performed using an IVIS50 (Xenogen Corp., Alameda, CA) equipped with a charge-coupled camera device, as previously described with minor modifications (Luker et al, 2003). Briefly, 150 µg of D-luciferin (Gold Biotechnology, St. Louis, MO) was administered to anesthetized mice by i.p. injection. Animals were imaged 5 min after D-luciferin administration and data were acquired using a 5 min exposure window. Bioluminescence imaging studies were carried out with age-matched 8 to 10-week old female FVB/N mice as recipients of adoptive transfer. Signal intensity of luciferase expression, as measured by the total amount of transmitted light, was quantified as photons/sec/cm2 using LivingImage and Igor (Wavemetrics, Lake Oswego, OR.) image analysis software.
Leukocytes were isolated from MCMV-infected murine brains using a previously described procedure with minor modifications (Cheeran et al, 2007; Ford et al, 1995; Marten et al, 2000). Briefly, brain tissues harvested from 4–6 animals were minced finely in RPMI (2 g/L D-glucose and 10mM HEPES) and mechanically disrupted (in Ca/Mg free HBSS) at room temperature for 20 min. Single cell preparations from infected brains were resuspended in 30% Percoll and banded on a 70% Percoll cushion at 900 Xg at 15°C. Brain leukocytes obtained from the 30–70% Percoll interface were stained with anti-mouse immune cell surface markers for 45 min at 4°C (CD45-Allophycocyanin (APC) (eBioscience, San Diego, CA), CD11b-FITC or CD11b-APC-CY7, CD4-FITC, Ly6G-FITC, Ly6C-FITC, MHC Class II-phycoerythrin (PE), CD8-PE, and CD3-PE-Cy7, BD Biosciences, San Jose, CA) and analyzed by flow cytometry using a BD FACSCanto. Live leukocytes were gated using forward scatter and side scatter parameters and analyzed using FlowJo software (TreeStar, Inc.).
This study was funded in part by U.S. Public Health Service Grant NS-038836.