Lymphocytic choriomeningitis virus (LCMV) 1 infection of the murine brain elicits fatal immunopathology through blood brain barrier (BBB) breakdown 2 and convulsive seizures 3. While LCMV-specific cytotoxic T lymphocytes (CTL) are essential for disease 4, their mechanism of action is not known. To gain novel insights into disease pathogenesis, we visualized the dynamics of immune cells in the meninges by two-photon microscopy (TPM). We observed motile CTL and massive secondary recruitment of pathogenic monocytes and neutrophils that were required for vascular leakage and acute lethality. CTL expressed multiple chemoattractants capable of recruiting myelomonocytic cells. We conclude that a CD8+ T cell dependent disorder can proceed in the absence of direct T cell effector mechanisms and rely instead on CTL recruited myelomonocytic cells.
To examine the dynamics of LCMV-specific CTL, we transferred 1×105 naïve GFP-tagged DbGP33–41 T cell receptor (TCR) transgenic (tg) CD8+ T cells (GFP+ P14 cells) into B6 mice one day prior to intracranial (i.c.) inoculation with LCMV Armstrong (Arm). TPM was performed through a thinned skull window to visualize the meninges overlying the visual cortex in asymptomatic (day 5) and symptomatic (day 6) mice (Fig. 1; Movie 1). In contrast to the few P14 cells observed on day 5 (Fig. 1a), the number of GFP+ P14 cells was dramatically increased in the meninges and perivascular regions on day 6 (Fig. 1b, c). To determine if GFP+ P14 cells were engaging in antigen specific interactions, we analyzed their motion in the presence of control antibody (IgG) or a blocking monoclonal antibody to Db (anti-class I) introduced into the subarachnoid space through a small craniotomy (Fig. 1d-i). P14 speed averaged 3.41 ± 0.27 µm/min (mean ± s.e.m.) in the absence of the craniotomy and 3.04 ± 0.33 µm/min in the presence of the craniotomy and IgG (Fig. 1j). The anti-class I significantly increased the speed of P14 cells to 5.16 ± 0.46 µm/min (Fig. 1j,k) and decreased the arrest coefficient (Fig. 1l), but did not influence the speed of CTL specific for an irrelevant antigen (Fig. S1). This significant change in P14 cell speed and arrest following anti-class I treatment was observed in all mice examined and did not depend on CTL abundance (Fig. S2). GFP+ P14 CTL migration appeared random (Fig. S3), with confined motion at longer times that was reversed by anti-class I. Comparison of the speed distributions showed that the entire population shifts following anti-class I treatment (Fig. 1k), suggesting that all GFP+ P14 cells encountered antigen. Despite this high frequency of antigen encounter, CTL rarely synapsed with any one target for > 10 minutes (Fig. 1g,m). These intravital observations raised questions about the infected target population and the CTL effector mechanisms utilized during fatal meningitis.
We identified the LCMV infected cells through immunohistochemical studies (Fig. 2 & S4; Movie 2). The main LCMV infected population in the meninges and around meningeal vasculature was ER-TR7+ stromal cells. LCMV infection was occasionally observed in CD45+ infiltrating leukocytes and astrocytic foot processes that comprise the glial limitans (Fig. S4). Infection of endothelium and smooth muscle cells / pericytes was never observed (Fig. S4). ER-TR7+ stromal cells support rapid migration of CD8+ and CD4+ T cells in lymph nodes 5 and may provide strong chemokinetic signals that can overwhelm synapse forming stop signals 6. This might explain the paucity of antigen specific arrest (Fig. 1).
Since CD8+ T cells are essential for pathology 4, we evaluated several CTL effector mechanisms using genetic knockout and mutant mice (Fig. 3a). Surprisingly, mice with single deficiencies in all major CTL effector pathways – IFNγ receptor, TNFα, Fas, granzymes, perforin (PFP), and the degranulation pathway (Jinx mutant) -succumbed to the convulsive seizures observed following LCMV infection of wild type mice. The delay in disease onset observed in perforin knockout mice was recently attributed to slower CTL recruitment into CNS 7. These data supported the imaging studies in suggesting that CTL effector functions might not be responsible for rapid onset disease.
To investigate other effectors, we temporally examined the composition of the CNS infiltrate following i.c. LCMV infection (Fig. 3b & S5). Baseline populations prevailed until day 6 at which point monocytes / macrophages were massively recruited into the CNS. A low number of these cells preceded the arrival of CTL by 2 days. At day 6 a small increase in the numbers of neutrophils, CD4+ T cells, and B cells was also observed; however, the latter two populations are not required for disease 8,9. It should be noted that our methodology accounts primarily for extravasated leukocytes, as cells arrested in the vasculature (e.g., neutrophils) are expunged during intracardiac saline perfusions. Nevertheless, our results demonstrate a minimal innate cellular response to the virus alone and massive recruitment of myelomonocytic cells that coincided with the arrival of CTL at day 6.
We next asked whether monocytes and/or neutrophils were required for the seizure-induced death on day 6 (Fig. 3c–f). Neutrophil depletion with low dose anti-Gr-1 antibody 10 (Fig. 3c–d) or monocyte infiltration blockade using CCR2 deficient mice 11 (Fig. 3e–f) had no effect on the nature or kinetics of death. Therefore, we hypothesized that both populations might have the potential to induce CNS injury. To test this hypothesis, we depleted monocytes and neutrophils simultaneously by administering high dose anti-Gr-1 to CCR2 knockout mice (Fig. 3e,f). When both cell populations were depleted, seizure-induced death at day 6 was averted and survival was extended by 3 days (Fig. 3e,f), despite a normal frequency of virus-specific CTL on day 6 (data not shown). These data suggested that myelomonocytic cells were highly pathogenic and were responsible for the rapid onset seizure-induced death observed at day 6.
During TPM analyses of GFP+ P14 cells, we often noted that the vasculature appeared ragged and displayed plasma leakage tracked with intravascular injected quantum dots (Movie 3). We considered that the seizure-induced death at day 6 might be induced by vascular leakage caused by myelomonocytic cells. To test this possibility, we conducted TPM in LCMV infected LysM-GFP mice, in which neutrophils and monocytes are labeled with GFP, to detect the relationship between myelomonocytic extravasation and vascular leakage. There was a tight correspondence between locally synchronized LysM-GFP+ cell extravasation and vascular leakage on day 6 (Fig. 4e–h; Movie 4).
To determine the relative contribution of neutrophils versus monocytes to vascular injury, we imaged LysM-GFP mice injected with low dose anti-Gr-1 antibody, which depletes only neutrophils (Fig. 3c & S6). Interestingly, synchronous extravasation of LysM-GFP+ cells was not observed in low dose Gr-1-depleted mice (Fig. 4i–l; Movie 5), suggesting that synchronously extravasating LysM-GFP+ cells are neutrophils. In neutrophil depleted LysM-GFP mice, we observed perivascular LysM-GFP+ cells (i.e., monocytes / macrophages) in areas of transient vascular leakage. Unlike neutrophils that display intravascular accumulation followed by explosive extravasation with vascular leakage, the monocytes accumulated more gradually in vascular sites that nonetheless displayed leakage. Statistically, sustained vascular leakage was only correlated (r = 0.99; p < 0.0001) with neutrophils (Fig. 4m). The presence of intra- or extravascular P14 CTL was not associated with either pattern of vascular leakage (Fig. 4o). Quantum dot leakage was not observed on day 5 post-infection (Fig. 4p) despite low numbers of infiltrating monocytes (Fig. 3b). Vascular injury occurred only at day 6 post-infection and extended into the brain parenchyma (Fig. 4q). Myelomonocytic cells were restricted to the meninges on day 6 (Fig. 4q,r & S10).
The impact of myelomonocytic cells on vascular injury was further assessed by quantifying leakage of Evans blue dye into the brain (Fig. S7). Only mice depleted of both monocytes and neutrophils showed significant preservation of vascular integrity at day 6 post-infection (Fig. S7e,f). Depletion of monocytes (Fig. S7d,f) or neutrophils (Fig. S7c,f) alone failed to prevent Evans blue leakage. Interestingly, in untreated wild type mice at day 6 post-infection, we observed substantial leakage of Evans blue from meningeal blood vessels into the brain parenchyma (Fig. S8), reflecting disrupted BBB integrity, which has the potential to cause severe seizures 12.
To examine a potential mechanism by which CTL attract myelomonocytic cells, we used gene arrays to quantify differentially regulated transcripts in the brains of mock-versus d6 LCMV-infected mice. Our results revealed a statistically significant increase (p < 0.05) in 6 chemokines (CCL2 - 7.3 fold, CCL3 – 8.1 fold, CCL4 – 1.6 fold, CCL5 – 5.6 fold, CCL7 – 4.2 fold, CXCL2 – 2.8 fold) and 2 chemokine receptors (CCR1 – 2.6 fold, CCR2 – 3.6 fold) that can recruit myelomonocytic cells into the CNS. Because it was reported that none of these chemokines were observed in the CNS of T cell-deficient mice infected i.c. with LCMV 13, we next used flow cytometry to examine which of these chemokines were produced by virus-specific P14 cells (Fig. S9). Our flow cytometric analyses of CNS and splenic P14 CTL at day 6 revealed that CCL3, CCL4, and CCL5 were all produced at the protein level, which was confirmed using gene arrays 14,15. Both CCL3 and CCL4 required GP33–41 peptide stimulation for maximum synthesis, whereas CCL5 was produced upon differentiation from naïve to effector cells and was not further upregulated upon peptide stimulation.
It is well known that CD8+ T cells are required for LCMV-induced meningitis 4 and vascular leakage 16. Our results revealed that P14 CTL could produce three of the chemokines responsible for attracting the myelomonocytic cells responsible for vascular injury (Fig. 4 & S7) and rapid onset seizure-induced death (Fig. 3). To establish a direct link between CD8+ T cells and CNS myelomonocytic cell recruitment, we infected mice with LCMV and administered anti-CD8 antibody at days 4 and 5 post-infection – after CTL priming. This treatment, which reduced the number of CD8+ T cells in the CNS by 94%, prevented the rapid onset of seizures at day 6 post-infection (data not shown), and significantly reduced the number of monocytes and neutrophils in the CNS (Fig. S10). These data indicate that virus-specific CTL can contribute to the recruitment of pathogenic myelomonocytic cells either by directly releasing chemoattractants or possibly by inducing other cells to release chemoattractants.
The requirement for CD8+ T cells in the pathogenesis of LCMV meningitis led to the proposal that CTL were directly responsible for tissue injury and death 17. We propose that CTL activation through transient interactions with infected cells leads to massive recruitment of myelomonocytic cells, which compromise vascular integrity and initiate fatal convulsive seizures 3. It is likely that once seizure-induced death is averted, infected mice ultimately succumb to another pathogenic mechanism possibly mediated by CTL 18.
It is well established that neutrophil extravasation can be linked to vascular leakage 19–21, and this process usually depends on signaling between leukocytes and endothelial cells 22. Neutrophil extravasation causes tissue injury in many models including reperfusion injury and sepsis 23,24. Using TPM we directly visualized this classical process in LCMV meningitis. Monocytes have been associated with atherosclerosis 25 and facilitating the trafficking of neutrophils 26. Our results suggest that monocytes also contribute vascular leakage, possibly through a mechanism linked to adherence to the blood vessels 27 and / or chemokine release 28. Recognizing the complementary pathogenic functions of neutrophils and monocytes is critical for devising therapeutic approaches in CD8+ T cell-mediated pathology.
It is not clear why CD8+ T cells recruit myelomonocytic cells to a site of viral infection. CD4+ Th17 cells produce IL-17 to coordinate neutrophil recruitment, but anti-viral CD8+ T cells express transcription factors that suppress this program 29, and we observed no IL-17 production by peptide-stimulated P14 cells (data not shown). Therapies directed at reducing myelomonocytic activation are obvious treatment candidates to prevent the mode of immunopathology we observed, but are challenging due to their numerous effector mechanisms, fast turnover, and acute importance in host defense. A more tractable approach might be to target the chemotactic mechanisms used by CTL to attract myelomonocytic cells, or, alternatively, to enhance CTL-mediated killing of relevant targets by improving immunological synapse formation or stability 30. The latter approach might break the feedback to the pathogenic myelomonocytic arm and improve survival as well as immunity in viral infections of the CNS.