IL-1R1 Signaling Regulates CXCL12-Mediated T Cell Localization and Fate within the Central Nervous System during West Nile Virus Encephalitis

Leukocyte transmigration across the blood–brain barrier (BBB) is a tightly regulated process and is central to inflammation and immune responses within the CNS. Under physiological conditions, a limited number of immune cells cross endothelial barriers as part of normal immune surveillance, yet they remain localized to perivascular spaces within the leptomeninges or CNS parenchyma via expression of the chemokine CXCL12 (1–4). Thus, in neuroinflammation, a large number of immune cells accumulate within perivascular locations, distending the perivascular spaces (2–6). Within these perivascular cuffs, lymphocytes encounter APCs and other infiltrating leukocytes (7). These interactions ensure full activation of effector T cells and trigger their ability to migrate from the perivascular space into the CNS parenchyma (7, 8). These steps are essential to reduce or eliminate invading viruses from the CNS (4, 9, 10); however, the exact molecular mechanisms that regulate the entry of virus-specific lymphocytes, in particular, are not well defined.

West Nile virus (WNV), a neurotropic flavivirus, has emerged globally as a significant cause of viral encephalitis (11). Both innate and adaptive immune defenses largely control WNV within the periphery; however, WNV can spread to the CNS, causing neuronal injury and inflammation that can lead to death in both humans and mice (12–16). In murine models of WNV infection, both T cells and macrophages traffic to the WNV-infected CNS soon after local viral replication begins, ∼6 d postinfection, and are essential for virologic control at this site (17–20). In 5-wk-old mice, infiltrating leukocytes remain localized to perivascular spaces where CXCL12 levels remain elevated at this time point. Later in the course of encephalitis, lymphocytes begin to enter the CNS after CXCL12 levels are downregulated; however, this occurs too late to exert significant virologic control (4). Consistent with this, the early administration of a CXCR4 antagonist in 5-wk-old C57BL/6 mice promotes leukocyte entry into the CNS parenchyma and results in improved viral clearance, decreased immunopathology, and enhanced survival during WNV infection (4). In contrast, 8-wk-old mice have improved survival (∼70%) with increased CNS entry of virus-specific T lymphocytes at early time points (15, 21). Although the mechanisms underlying age-related differences in T cell entry are unknown, the parenchymal presence of CD8⁺ T cells is crucial for preventing fatal encephalitis through clearance of WNV via mechanisms involving IFN-γ, TNF-α, and perforin (22–25). The effectiveness of parenchymal CD8⁺ T cells in viral clearance also reflects the effects of CD4⁺ T cells, which promote the full activation, migration, and positioning of virus-specific CD8⁺ T cells within the CNS (26–28). Therefore, leukocyte interactions within the perivascular compartment are necessary for T cell function and mobility within the CNS to ensure appropriate entry of T cell subsets, which in turn would achieve the proper balance between protective versus pathogenic responses.

IL-1 signaling is critically involved in the regulation of inflammation and pathobiology of immune and inflammatory conditions (29, 30). A growing body of evidence suggests an important role for IL-1 signaling in the immunity against several viruses, including influenza A, hepatitis B, Sendai, and vesicular stomatitis virus (31). Notably, IL-1 has been shown to drive host responses that regulate cellular infiltration to sites of viral infection (32, 33). In the context of CNS viral infections, IL-1 signaling has been associated with protection as well as enhancement of disease, depending on the virus. Thus, whereas IL-1β works synergistically with TNF-α to protect against HSV-1–induced encephalitis (33), IL-1β increases pathogenesis and lethality to Sindbis virus encephalitis (34, 35). During Theiler’s murine encephalomyelitis virus infection, IL-1 signaling elevates pathogenic responses, whereas the lack of IL-1 signaling results in viral persistence due to insufficient T cell activation (36). During HIV encephalitis, IL-1β–producing macrophages promote CXCL12 expression by astrocytes, resulting in increased neuropathogenesis (37). Although studies in autoimmunity suggest that IL-1β–producing T cell infiltrates regulate the pattern of CXCL12 expression along the vasculature of the CNS (38), this has not been evaluated in viral models. Although little is known about IL-1 signaling in immunity to flavivirus infections, recent studies have clarified its protective role within the CNS. We and others have shown that WNV infection is significantly exacerbated in mice deficient of IL-1R1 due to decreased neuronal antiviral activity as well as an inability to fully activate CD4⁺ T and dendritic cells specifically within the CNS, which is required for restimulation of infiltrating, virus-specific CD8⁺ T cells (26, 39). This latter study also found that ex vivo analysis of CNS virus-specific CD8⁺ T cells revealed no cell-intrinsic defects associated with loss of IL-1R1 activity. The study additionally demonstrated increased neuroinflammation in IL-1R1–deficient mice, suggesting that IL-1 signaling in WNV infection acts to regulate leukocyte interactions at the BBB that mediate protective immunity within the CNS and limit inappropriate T cell access to CNS parenchyma.

In this study, we reveal a novel role for IL-1 during WNV encephalitis. IL-1 signaling is critical for specifically regulating the perivascular localization of infiltrating T cells via CXCL12 expression, which promotes efficient T cell reactivation and entry, controlling viral replication and immunopathology within the WNV-infected CNS. Our observations link the production of IL-1β by infiltrating macrophages to the local control of lymphocyte function and fate at CNS endothelial barriers.

WNV strain 3000.0259 was isolated in New York in 2000 (40) and passaged once in C6/36 Aedes albopitcus cells to generate an insect cell–derived stock. The stock titer was determined by using BHK21 cells for viral plaque assay as previously described (21).

C57BL/6 wild-type (WT) and IL-1R1^−/− inbred mice were obtained commercially (The Jackson Laboratory). All mice were housed and bred in the pathogen-free animal facilities of the Washington University School of Medicine. Experiments were performed in compliance with the guidelines and approval of the Washington University School of Medicine Animal Safety Committee. Matched 8-wk-old mice were inoculated s.c. via footpad injection (50 μl) with 100 PFU WNV as previously described (41).

Quantification of WNV viral load was measured by analyzing positive-strand viral RNA levels using quantitative RT-PCR (qRT-PCR) as described previously (42).

Cells were isolated from the CNS of WT and IL-1R1^−/− mice at days 6 and 8 postinfection and stained with fluorescently conjugated Abs to CD4, CD8β, CD11b, CD11c, and CD45 as previously described (3). Intracellular IFN-γ staining was performed on splenocytes from day 8 postinfection animals in an I-A^b–restricted NS32066 and NS31616 peptide and D^b-restricted NS4B peptide restimulation assay as previously described (17, 43). Data collection and analysis were performed with an LSR flow cytometer (Becton Dickinson) using FlowJo software (Tree Star).

At various day intervals after infection mice were injected i.p. with 100 μl 100mg/ml fluorescein sodium salt (Sigma-Aldrich) in sterile PBS. After 45 min, mice underwent extensive cardiac perfusion with PBS, followed by collection of blood and harvesting of CNS tissues. Tissue homogenates and serum were incubated overnight at 4°C at 1:1 dilution in 2% TCA (Sigma-Aldrich) to precipitate protein, which was pelleted by 10 min centrifugation at 4000 rpm at 4°C. Supernatants were then diluted in equal volumes of borate buffer (pH 11) (Sigma-Aldrich). Fluorescence emission at 538 nm was determined via a Synergy H1 microplate reader and Gen5 software (BioTek Instruments). Tissue fluorescence values were standardized against plasma values for individual mice.

Total RNA was prepared from the brains of WNV-infected WT and IL-1R1^−/− mice using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription and quantitative PCR was performed as previously described (18). Calculated copies were normalized against copies of the housekeeping gene GAPDH. All oligonucleotide primers used have been previously reported (18).

Mice were infected with 10² PFU WNV and sacrificed at day 8 postinfection. CNS tissues were then isolated and frozen sections were permeabilized, blocked, and stained as previously described (3). Immunohistochemical detection of CD3, CD31, glial fibrillary acidic protein (GFAP), ionized calcium–binding adaptor molecule (IBA)-1, CXCL12, and p-CXCR4 with nuclear TO-PRO-3 counterstaining was performed as previously described (4). In situ tetramer staining was performed on fresh brain tissues collected on day 8 postinfection with WNV. One-millimeter sections were incubated with 10 μl/section D^b-NS4B tetramer at 37°C for 15 min. The tissue was then rinsed repeatedly at 37°C with PBS and then twice with ice-cold PBS prior to fixation with cold 2% paraformaldehyde for 20 min. After additional washes, anti-CD31 Ab (rat polyclonal, BD Biosciences) was applied for 1 h. The tissues were then washed and incubated with anti-rat Alexa Fluor 488 (Molecular Probes). Tissues were then washed, mounted on a glass slide, and examined by confocal microscopy.

Brain microvascular endothelial cells (BMECs) were treated with IL-1β (10 ng/ml) overnight and protein lysates were isolated using RIPA buffer supplemented with protease and phosphatase-3 inhibitor mixture (Sigma-Aldrich). Lysates were resolved on a 4–12% Bis-Tris gel and transferred on to an iBlot nitrocellulose transfer membrane (Invitrogen) according to standard protocols. Blots were probed with polyclonal rabbit anti-CXCL12β (eBioscience) and monoclonal mouse anti–β-tubulin (Sigma-Aldrich) Abs flowed by incubation with IRDye-conjugated secondary Abs (LI-COR Biosciences). Blots were imaged using the Odyssey fluorescent scanning system (LI-COR Biosciences) and analyzed using ImageJ.

In vitro BBBs were generated using a well-established transwell system as previously described (44). Following stimulation, both the CD4⁺ and CD8⁺ T lymphocytes were isolated via positive selection (MACS) and together yielded ∼95% pure T lymphocytes: CD8, 40% and CD4, 55% (Supplemental Fig. 1). Following selection, 5 × 10⁵ lymphocytes in 200 μl C5 media were added to the top chamber of each in vitro BBB (∼5:1 ratio, lymphocytes/endothelial cell) and allowed to incubate for 6 h at 37°C. Medium was then harvested from bottom chambers and lymphocytes were collected by centrifugation (1500, 10 min, 4°C). Cell pellets were resuspended in 200 μl FACS buffer (2% FBS, 0.5 mM EDTA, 0.02% sodium azide in sterile Dulbecco’s PBS), and leukocyte subsets were identified and counted with an LSR II flow cytometer (Becton Dickinson) using FlowJo software (Tree Star). For each leukocyte subset, migration was quantified as the number of cells present in the bottom chamber after 6 h divided by the total number of that cell type added to the top chamber (proportion migrating).

Graphs were made and statistical analysis was performed via computerized software (GraphPad Prism). Depending on the data, an unpaired, two-tailed Student t test or one-way ANOVA with Tukey–Kramer posttest was performed, with p < 0.05 considered to be significant.

In prior studies we showed that IL-1R1 signaling is required for APC-mediated T cell reactivation and subsequent virologic control specifically within the WNV-infected CNS (26). Accordingly, WNV RNA levels are significantly increased in the CNS of IL-1R1^−/− mice compared with C57BL/6 WT mice on day 8 following footpad inoculation (100 PFU WNV) (Fig. 1A). Because prior studies in mice with neuroinflammation showed that IL-1 signaling is required for T cell migration from perivascular spaces (38), we evaluated the trafficking of immune cells within the brains of WNV-infected WT and IL-1R1^−/− mice. As previously observed (26), flow cytometric analysis of infiltrating immune cells at this time point revealed significantly increased numbers of CD45⁺ leukocytes within CNS tissues of WNV-infected IL-1R1^−/− mice compared with those of similarly infected WT counterparts (Fig. 1B). In particular, total numbers of CD4⁺ (Fig. 1C) and CD8⁺ (Fig. 1D) T lymphocytes as well as the number of CD11b⁺ (Fig. 1E) leukocytes were increased in the IL-1R1–deficent mice on day 8 postinfection. There was no discernible difference between the genotypes on day 6 postinfection in the total number or in the numbers of infiltrating T lymphocytes or CD11b⁺ monocytes. Of note, there are limited numbers of inflammatory cells within the WNV-infected CNS prior to day 6 postinfection (19). Further evaluation of the locations of CD3⁺ T cells within the brains of WNV-infected IL-1R1^−/− and WT mice with respect to the CD31⁺ microvasculature showed that parenchymal entry of T cells at day 8 postinfection was significantly increased in the WNV-infected IL-1R1^−/− mice compared with similarly infected WT animals (p = 0.0043) (Fig. 1F, 1G), despite their lack of reactivation and inability to clear virus. This surprising result suggested that IL-1 signaling is required to limit inappropriate T cell entry during viral infections of the CNS.

In the CNS, IL-1 can be produced by astrocytes, endothelial cells, infiltrating leukocytes, neurons, and oligodendrocytes, but microglia are thought to produce the highest levels of IL-1 in response to infection or injury (45–48). We previously showed that IL-1β is detected within the CNS following WNV infection at approximately day 7 postinfection (26, 39). Infectious virus can be recovered from the WNV-infected CNS at days 4–5 postinfection before immune cell infiltration, which occurs approximately 1 wk postinfection (49). To determine whether resident or infiltrating immune cells are the main producers of IL-1β, we examined IL-1β expression in cells isolated from 8-wk-old WT mice that had been infected with 100 PFU WNV on day 8 postinfection via footpad inoculation (Fig. 2). Flow cytometric analysis of cells isolated from the brains of WNV-infected mice showed that CD4⁺ T cells, CD11c⁺ APCs, CD11b⁺CD45^lo (microglia), and CD11b⁺CD45^hi (macrophages) cells were all positive for IL-1β expression (Fig. 2A). The total number of CD45⁺ leukocytes expressing IL-1β was ∼4.5 × 10⁴ cells (Fig. 2B). However, analysis of total numbers of these cells revealed that macrophages had the highest number of IL-1β producers (∼3 × 10⁴ cells) (Fig. 2C). Thus, whereas CNS-resident cells may produce IL-1β in response to inflammatory injury, infiltrating immune cells such as CD11b⁺ macrophages are the primary producers of the proinflammatory cytokine IL-1β during WNV encephalitis.

Because lymphocyte entry into the CNS was greatly increased in the IL-1R1^−/− mice despite lack of full activation (26), we wondered whether BBB permeability might be altered in the absence of intact IL-1R1 signaling. To test this, we administered sodium fluorescein i.v. on days 4, 6, and 8 postinfection to both WT and IL-1R1^−/− mice and performed fluorometric analyses in various CNS regions, including the olfactory bulb (Supplemental Fig. 2A), cerebral cortex (Supplemental Fig. 2B), cerebellum (Supplemental Fig. 2C), and the spinal cord (Supplemental Fig. 2D). The amount of sodium fluorescein in each of the regions did not significantly differ between IL-1R1^−/− mice compared with WT mice. These results suggest that the CNS entry of leukocytes across the BBB in WNV-infected IL-1R1^−/− mice is not the result of barrier dysfunction.

Proinflammatory chemokines play a significant role in leukocyte trafficking into the CNS and facilitate the appropriate entry of virus-specific T cells (49, 50). Prior studies have demonstrated a positive correlation between viral loads within the CNS and proinflammatory chemokine mRNA expression, primarily by virally infected neurons (18, 51–53). We therefore assessed levels of proinflammatory chemokine mRNA expression within the WNV-infected CNS of IL-1R1–deficient mice compared with similarly infected WT controls on days 2, 5, and 8 following infection. By day 8 postinfection, we detected significant increases of CCL2, CCL3, CCL5, CCL7, CXCL9, and CXCL10 (Fig. 3A) within the CNS of the IL-1R1^−/− mice. The mRNA levels of chemokine receptors that bind these ligands were also elevated in WNV-infected IL-1R1–deficient mice compared with similarly infected WT controls, specifically CCR1, CCR2, and CXCR3 (Fig. 3B). WNV-infected neurons directly induce the recruitment of virus-specific T cells for the purpose of viral clearance through proinflammatory chemokine expression (18, 51). Evaluation of CD3⁺ T cells within the CNS parenchyma revealed their juxtaposition with WNV-infected neurons in both IL-1R1–deficient and WT animals; however, a number of CD3⁺ T cells remained remote of WNV Ag in the absence of IL-1R1 signaling (Fig. 3C). Overall, these results are consistent with other studies that demonstrate that the extent of virally infected neurons within the CNS parenchyma determine levels of chemokine expression and provide a mechanism for the extensive lymphocyte migration observed within the CNS parenchyma of WNV-infected IL-1R1^−/− mice (Fig. 1G).

Stringent regulation of leukocyte entry into the brain is important for the success of antiviral immunity because lymphocytes can activate resident glial cells and promote immunopathology (3, 54, 55). To determine whether chemokine-mediated (see Fig. 3) increase in infiltrating leukocytes within WNV-infected IL-1R1^−/− CNS (see Fig. 1) contributed to immunopathology, we assessed astrocyte and microglial activation at day 8 postinfection, a time point at which IL-1R1^−/− mice continue to develop immune cell infiltrates and WT mice begin to recover (26). Indeed, GFAP, a marker for activated astrocytes, was widely expressed in IL-1R1^−/− mice whereas GFAP expression detected in WT mice was limited (p = 0.0232) (Fig. 4A, 4B). The significant increase in astrocyte activation coincided with the presence of CD3⁺ T cells. We also observed increased expression of Iba-1, a marker of activated microglia and macrophages, throughout the CNS tissue of WNV-infected IL-1R1^−/− mice at day 8 postinfection (p = 0.0361) (Fig. 4C, 4D). This expression was most prominent in and around blood vessels and within the parenchyma of IL-1R1^−/−, but not WT, brain tissue sections. These results suggest that increased infiltration is associated with neural glial cell activation observed in the CNS of the IL-1R1^−/− animals (24). Thus, we conclude that IL-1–dependent control of leukocyte infiltration in the CNS regulates the magnitude of inflammation during WNV infection to reduce immunopathology.

In prior studies we showed that CXCL12 localized lymphocytes to perivascular spaces, inhibiting their intraparenchymal migration, which limited their ability to control infection in 5-wk-old animals, which universally succumb to WNV infection (4). In these studies, CXCL12 levels were observed to decrease at day 8 postinfection, a time point when viral loads are already quite high within the CNS. Early administration of a specific antagonist of CXCR4, AMD3100 (56), led to improved CNS entry of WNV-specific CD8⁺ T cells with a 50% increase in survival. As 8-wk-old animals have an ∼70% survival rate with extensive CNS entry of T cells, we wondered whether CXCL12 levels are more efficiently decreased within the CNS of these older animals. Analysis of CXCL12α and CXCL12β mRNA levels from WNV-infected 5- and 8-wk-old C57BL/6 WT mice at various days after infection revealed that CXCL12β, which is expressed by CNS endothelium (57), is decreased significantly as early as day 5 postinfection in 8-wk-old animals, whereas decreased expression of CXCL12β in 5-wk-old animals was delayed until day 8 (Fig. 5A). In contrast, no difference in expression of CXCL12α, which is expressed by neuronal subpopulations (57), was observed between the 5- and 8-wk-old animals (Fig. 5A). To determine whether decreased expression of CXCL12 at the BBB on day 5 postinfection affected the intraparenchymal trafficking of T cells, we examined the localization of CD3⁺ cells with respect to the CD31⁺ microvasculature in the CNS from both 5- and 8-wk-old mice at day 6 after WNV infection (Fig. 5B). The parenchymal migration of T cells at day 6 postinfection significantly increased in 8-wk-old WNV-infected mice compared with similarly infected 5-wk-old animals (p = 0.0102) (Fig. 5B, 5C). These results suggest that lymphocyte egress from the perivascular space is dependent on efficient alterations in the temporal expression of CXCL12 within the microvasculature of the CNS.

IL-1 has previously been shown to have a role in regulating expression of CXCL12 at CNS endothelial barriers (3, 6, 38, 58). Accordingly, levels of CXCL12β were significantly lower in IL-1R1^−/− mice at day 8 postinfection compared with those of similarly infected WT animals (Fig. 6A). Although not significant, levels of CXCL12α were also lower in IL-1R1^−/− mice compared with WT mice (Fig. 6A). Consistent with the RNA analyses, evaluation of CXCL12 protein expression within the brain microvasculature via quantitative confocal microscopy revealed a decrease in the intensity of staining in IL-1R1^−/− mice compared with WT controls (p = 0.0002) (Fig. 6B, 6C). CXCL12 binds two receptors, CXCR4 and CXCR7 (59, 60), which were both increased significantly in the IL-1R1^−/− mice by day 8 postinfection (Fig. 6D). Because endothelial cell expression of CXCR7 functions to regulate CXCL12-mediated retention of CXCR4⁺ infiltrates (6), these results suggest that IL-1 signaling regulates CXCR7-mediated internalization of CXCL12, which would impact the perivascular localization of CXCR4⁺ leukocytes (3, 4, 6). Taken together, these data show that loss of IL-1 signaling within the WNV-infected CNS leads to loss of lymphocyte localizing cues at the vasculature at day 8 postinfection, increased levels of chemoattractants within the parenchyma, and significantly increased parenchymal entry of T lymphocytes.

To determine whether IL-1β signaling mediates the localization of leukocytes to the perivascular space via CXCR4/CXCL12 interactions, we examined lymphocyte migration utilizing a well-established in vitro BBB model in which primary BMECs and astrocytes derived from WT mice were seeded onto the top of a filter insert or on the bottom of a transwell plate, respectively, and cocultured to generate high transendothelial resistances establishing similar barrier properties to those observed in vivo (61) (Fig. 7A). BMECs isolated from WT animals that had been treated with IL-1β (10 ng/ml) resulted in increased CXCL12β expression levels (p = 0.0059) (Fig. 7B) and were subsequently used in the transwell coculture (Fig. 7A). To simulate the lymphocyte reactivation that occurs at CNS endothelial barriers (26), WNV-primed CD4⁺ and CD8⁺ lymphocytes isolated from the spleens of WNV-infected mice were restimulated ex vivo with WNV-specific immunodominant peptides and then were added together to the top chamber of the in vitro BBB (Fig. 7A). Following IL-1β treatment (10 ng/ml) of the in vitro BBB, migration of both WNV-primed CD4⁺ and CD8⁺ lymphocytes into the bottom chamber significantly decreased compared with nontreated cocultures (Fig. 7C). Lymphocyte migration in the IL-1β–treated cocultures was restored to baseline levels when lymphocytes were pretreated with the CXCR4 antagonist AMD3100 (5 μg/ml), before addition to the in vitro BBB, compared with nontreated lymphocytes, suggesting that the IL-1β–induced CXCL12 effects were abrogated. Pretreatment of WNV-primed lymphocytes with AMD3100 in the absence of IL-1β treatment of the in vitro BBB also demonstrated similar migration of both CD4⁺ and CD8⁺ T cells compared with baseline migration levels. To determine whether IL-1β reduces lymphocyte migration via CXCL12-mediated retention at the brain microvasculature, we quantified the CD3⁺ cells adhering to the underside of the endothelial cell insert in untreated or IL-1β–treated cocultures with either untreated or AMD3100-pretreated lymphocytes. In agreement with our previous results, IL-1β treatment of the coculture led to a 2-fold increase in lymphocyte retention whereas AMD3100-pretreated lymphocytes failed to adhere along the endothelial cell insert (Fig. 7D, 7E). Lymphocyte adherence following pretreatment with AMD3100 alone did not result in significant differences compared with AMD3100-pretreated lymphocytes added to IL-1β–treated coculture. These results confirm that IL-1β mediates the ability of CXCR4-expressing lymphocytes to adhere to BMECs via CXCL12 expression on their basolateral surface.

The parenchymal entry of WNV-specific CD8⁺ T cells is important for clearing WNV infection from the CNS (4, 19). Therefore, we hypothesized that fully activated WNV-primed T lymphocytes are able to overcome CXCL12-mediated endothelial cell retention via downregulation of CXCR4 in the context of reactivation during perivascular localization. To test this, we measured intracellular IFN-γ and granzyme B (GZMB) expression as well as surface CD69 and CXCR4 expression in lymphocytes that migrated to the bottom chamber of the in vitro BBB. We observed that IL-1β treatment of the coculture resulted in a dramatic increase in the proportion of migrated CD4⁺ and CD8⁺ lymphocytes expressing IFN-γ, GZMB, and CD69 compared with untreated or AMD3100-pretreated lymphocytes with or without IL-1β treatment (Fig. 8A, 8B). Importantly, IL-1β treatment also led to the decreased migration of CD4⁺ and CD8⁺ lymphocytes expressing CXCR4, supporting its role in retention of CXCR4⁺ lymphocytes (Fig. 8A, 8B). Detection of ligand-induced, p-CXCR4 within the WNV-infected brain demonstrated that p-CXCR4 was present in cells within perivascular spaces (Fig. 8C, gray arrows) and that in the absence of IL-1 signaling there was an increase in cells negative for the phosphorylated form of CXCR4 (Fig. 8C, white arrows). Additionally, the ratio of CD31-associated p-CXCR4 cells to total cells evaluated was significantly lower in the IL-1R1–deficient mice (Fig. 8D). Because CXCR4 levels increased significantly in the absence of IL-1 signaling (see Fig. 7E), these data suggest that CXCR4-expressing lymphocytes are not limited to the perivascular space but instead are migrating into the parenchyma in the absence of IL-1 signaling. In situ tetramer staining with the WNV-specific D^b-NS4B-tetramer (62) on fresh tissues demonstrated an increase in CD31-associated tetramer-binding CD8⁺ T lymphocytes within WNV-infected WT CNS compared with the WNV-infected IL-1R1^−/− CNS (Fig. 8E, 8F). Taken together, these data support the notion that IL-1–mediated CXCL12 expression at the CNS microvasculature regulates the efficient parenchymal entry of antiviral lymphocytes to control WNV infection.

Our observations support a model in which IL-1 signaling functions to control leukocyte interactions at the BBB to ensure their proper activation and entry into the virally infected CNS. In the absence of IL-1 signaling, leukocyte localization at endothelial barriers is impaired due to decreased microvasculature expression of CXCL12. Moreover, loss of CNS virologic control in IL-1R1^−/− mice significantly upregulates parenchymal chemokine expression, enhancing the parenchymal entry of T lymphocytes with subsequent glial activation and immunopathology. In an in vitro BBB model, lymphocyte retention, specifically their ability to remain bound to BMECs, was augmented by treatment with IL-1β and reversed by CXCR4 antagonism. Although IL-1β treatment increased lymphocyte BBB retention, WNV-specific T lymphocytes expressing CD69, IFN-γ, and GZMB exhibited decreased levels of CXCR4, which limited their CXCL12-mediated localization and allowed them to migrate through the barrier. We conclude that IL-1 is fundamental for the control of WNV infection and immunity by promoting CXCL12-mediated leukocyte interactions at endothelial barriers that ensure the full activation and downregulation of CXCR4 in effector T cells, which improves their ability to migrate from the perivascular spaces into the CNS parenchyma.

IL-1β mediates its effects through IL-1R1, which, in the uninflamed brain, is predominantly found on vascular endothelial cells and in neurons in a few specific regions of the brain such as the amygdala, the hypothalamus, the trigeminal and hypoglossal motor nuclei, and the area postrema (63, 64). Binding of IL-1β to endothelial IL-1R1 activates endothelial cells and triggers the upregulation of cell adhesion molecules such as E-selectin and ICAM-1, proinflammatory cytokines such as IL-6 and TNF, and chemokines such as CXCL1, CXCL2, CXCL8, CX3CL1, and CXCL12 (6, 65–69). Both the presence of the IL-1R1 and IL-1 production have been shown to be critical for mediating CD4⁺ T cell recruitment into CNS tissues in experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis (70), and in both acute and chronic active multiple sclerosis lesions (71, 72). IL-1R1 signaling has also been shown to be instrumental in CD3⁺ lymphocyte recruitment into the CNS during infectious diseases such as HIV encephalitis (37) and cerebral listeriosis (73). Generally speaking, these studies present two distinct mechanisms in which IL-1R1 signaling mediates leukocyte migration into the CNS through increased ICAM-1 expression and/or increased CXCL12 expression along the microvasculature. In our studies, we found no significant changes in cell adhesion molecules within CNS tissues of WNV-infected IL-1R1–deficient mice compared with their WT counterparts (data not shown). However, CXCL12 expression along the CNS vasculature drops by day 5 postinfection during WNV encephalitis when lymphocytes begin to enter the CNS (see Fig. 5). This temporal decrease in CXCL12 expression permits leukocyte transmigration into the parenchyma and results in improved viral clearance, decreased immunopathology, and enhanced survival during WNV infection (4). By day 8 postinfection, CXCL12 levels plateau, preventing further migration of lymphocytes from the perivascular space and ensuring their full activation. In the absence of IL-1 signaling, however, CXCL12 levels continue to drop, disrupting CNS immune privilege and increasing bystander lymphocyte entry, resulting in increased immunopathology and decreased survival during WNV infection. Our results confirm that IL-1 signaling maintains CXCL12 expression at the microvasculature at critical time points in 8-wk-old mice, fine-tuning the regulation of T cell infiltration during WNV infection in the CNS. They also suggest that lack of downregulation of vascular CXCL12 may partly underlie limitations in T cell–mediated control of WNV infection within the CNS of 5-wk-old animals. Further studies are needed to examine the developmental regulation of molecular mechanisms of immune privilege at the BBB.

It is well established that chemokines and their receptors modulate the recruitment of leukocytes into infected tissue. The contribution of IL-1 signaling to protective immunity against viral infections has largely been attributed to its ability to drive chemokine signaling pathways that recruit fully activated Ag-specific immune cells to sites of viral replication (34, 35). For instance, the inflammatory chemokines CXCL10, CCL2, and CCL5 are strongly induced within the parenchyma of the brain following WNV infection owing to virally infected neurons, which leads to enhanced trafficking of WNV-specific T cells from the perivascular space into the parenchyma that clears virus (4, 18, 51, 52, 74–76). We previously showed that WNV-specific T cells fail to be fully activated in the absence of IL-1R1 signaling (26), and therefore their ability to migrate from the perivascular space should be impaired despite the significant increase of inflammatory chemokines CXCL10, CCL5, and CCL2 over their WT counterparts. However, our results demonstrated that inflammatory chemokine expression positively correlated with increased leukocyte infiltration in the parenchyma and increased immunopathology, suggesting that a crucial checkpoint is dysfunctional in the absence of IL-1 signaling. Our results suggest that IL-1 signaling establishes this checkpoint by localizing leukocytes at the BBB via CXCL12 during WNV encephalitis. In addition to CXCR4, CXCR7, which is expressed primarily on BMECS within the CNS, acts as a regulatory protein for CXCL12 (6). Endothelial CXCR7 serves as a scavenging receptor for CXCL12, sequestering the chemokine into lysosomal compartments, thereby negatively regulating its ability to localize CXCR4⁺ leukocytes within the perivascular space. Previous studies have demonstrated that IL-1β modulates CXCR7 expression and CXCL12 internalization during EAE (6). Our studies suggest that intact IL-1R1 signaling hinders CXCR7-mediated disruption of CXCL12/CXCR4 interactions at the microvasculature of the CNS, promoting retention and subsequent full activation (3, 4, 6).

During WNV infection, we identified infiltrating myeloid cells, specifically CD11b⁺CD45^hi monocytes, as the primary producers of IL-1β. Macrophages within the CNS increase during the course of WNV infection and are largely detected in perivascular regions (4). These IL-1β⁺ infiltrates may have an essential role in establishing selective barriers by orchestrating cell adhesion between infiltrating leukocytes and endothelial cells. Prior studies have shown that monocytes regulate the parenchymal penetration of effector T cells to the site of infection during viral encephalitis and rule out the mediation by matrix metalloproteinase or the chemoattractants CXCL10 or CCL5, but do not address the role of IL-1 or CXCL12 (77). During EAE, myeloid cells within the perivascular space mediate cell-to-cell interactions via expression of ninjurin1, an adhesion molecule that, after cleavage, is similar in structure to chemokines such as CXCL12 (78, 79). Our data demonstrate that IL-1β⁺–infiltrating macrophages may be instrumental in facilitating leukocyte localization at endothelial barriers via CXCL12 expression. Indeed, yellow fever virus, St. Louis encephalitis, and WNV all induce IL-1β expression from myeloid cell populations in vitro (80, 81), suggesting that IL-1β⁺ infiltrates may perform similar roles during encephalitis due to other flaviviruses. Taken together, infiltrating myeloid cells play a previously underappreciated role that is essential in establishing localizing cues, specifically CXCL12, within the perivascular space.

We found that IL-1β treatment of BMECs in our in vitro BBB model resulted in the reduced migration of lymphocytes beyond the barriers, but nonetheless increased migration of activated WNV-primed lymphocytes. The temporal and spatial arrest of leukocytes within the perivascular space may facilitate pivotal leukocyte encounters with Ag-presenting phagocytes or CD4⁺ Th cells to guarantee full activation and effectual migration and positioning of CD8⁺ T cells within the brain (7). Indeed, CD4⁺ and CD8⁺ T cells activated by APCs in the presence of IL-1β display enhanced cytokine production and migration into infected tissues (82). In support of this notion, we observed that IL-1β treatment enhanced the migration of CD8⁺IFN-γ⁺ and CD8⁺GZMB⁺ cells, which are crucial for WNV control within the CNS (22–25). In the absence of IL-1R1 signaling, our results suggest that chemokine-mediated leukocyte entry, which may also include bystander nonspecific or nonactivated T cells (83), is destructive if not rigorously regulated through IL-1–mediated mechanisms. Moreover, increased CNS chemokine expression might also facilitate leukocyte retention within the CNS, which could lead to chronic inflammation. This is consistent with recent studies showing that IL-1R1 signaling is essential for recruitment of both proinflammatory and reparative monocytes in infarcted tissue (84) and that IL-1 signaling is associated with both neuroprotection and neuropathogenesis during Theiler’s murine encephalomyelitis virus–induced demyelinating disease (36). The effects of IL-1 within the CNS may therefore depend on the location and timing of its expression, especially during viral infections within the CNS. Indeed, our data demonstrate that IL-1, delivered to the CNS by infiltrating leukocytes, acts directly at the microvasculature to ultimately control lymphocyte migration and limit pathology.

In summary, our study provides evidence that IL-1 production at the CNS vasculature regulates leukocyte localization and interactions at this site during WNV encephalitis via CXCL12 expression. These vascular interactions ensure the full activation of lymphocytes, which trigger their migration beyond the vasculature into the parenchyma to effectively clear viral pathogen. Thus, IL-1–producing infiltrating cells may establish a protective barrier to enable selective transfer of trafficking leukocytes into the infected CNS parenchyma.

The authors have no financial conflicts of interest.