Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis

Abstract

Lymphocytic choriomeningitis virus infection of the mouse central nervous system (CNS) elicits fatal immunopathology through blood-brain barrier breakdown and convulsive seizures. Although lymphocytic-choriomeningitis-virus-specific cytotoxic T lymphocytes (CTLs) are essential for disease, their mechanism of action is not known. To gain insights into disease pathogenesis, we observed the dynamics of immune cells in the meninges by two-photon microscopy. Here we report visualization of motile CTLs and massive secondary recruitment of pathogenic monocytes and neutrophils that were required for vascular leakage and acute lethality. CTLs 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.

Figure 1. CTL localization and dynamics in the meninges of LCMV-infected mice

a,b, A representative 3D reconstruction of an intravital two-photon z-stack viewed through a thinned skull is shown for representative mice at day 5 (a) and day 6 (b) post-infection. Few GFP⁺ P14 CTL (green) were observed in the meninges at day 5 post-infection. At day 6 post-infection GFP⁺ P14 CTL were confined to 50 µm meningeal space between the undersurface of the skull bone (blue) and the pial surface. Very few CTL entered the parenchyma. (Grid scale = 19.7 µm) c, A representative 3D reconstruction shows that GFP⁺ P14 CTL (green) localized preferentially along meningeal vasculature (red) at day 6. Skull bone is blue. (Grid scale = 119 µm) d–i, Representative xy plane thinned skull images of GFP⁺ P14 cells at day 6 post-infection (gray scale) (d–f) and corresponding 30 minute time lapse cell tracks (colored lines) (g–i) below each image are shown. (Representative xz planes images are shown in Supplementary Figure 1.) Note the highly dynamic motion of the CTL in the meningeal surface at baseline (BL) (d, g) and following injection of 10 µg/mL IgG isotype control (IgG) (e, h) or anti-H-2D^b antibody (class I) (f,i). (Grid scale = 29.6 µm) j–m, Compared with baseline and IgG isotype control, class I inhibition induced a statistically significant (*p < 0.0001) change in average speed (j), arrest coefficient (l), and arrest duration (m) for GFP⁺ P14 CTL. Primary velocity data shown in panel j are plotted as histograms using 5 µm/min bins and a Gaussian curve fit (k). See corresponding Movie 1 and Figure S1.

Figure 2. LCMV infection of ER-TR7+stromal cells in the meninges

a–h, A representative 2D image (a–d) and a maximal projection of a 3D z-stack (e–h) are shown for a mouse at day 6 post-infection. Both images were captured using a one-photon confocal microscope. Note the localization of LCMV (red) to ER-TR7⁺ fibroblast-like cells (green) that line the meninges and meningeal vasculature. A cross section of a meningeal blood vessel is denoted with a white asterisk in panel d. Panels e–h depict a top down view of a large meningeal blood vessel where the network of fibroblast processes are clearly visible and infected by LCMV. i, A 3D reconstruction of a meningeal blood vessel cross section (center denoted with a white asterisk) is shown to illustrate the degree to which LCMV infects fibroblast-like cells that completely surround meningeal blood vessels. (Grid scale = 19.5 µm) Cell nuclei are shown in blue in all merged panels. See corresponding Movie 2 and Figure S3.

Figure 3. Analysis of mononuclear cell infiltrate and effector mechanisms during LCMVinduced meningitis

a, Survival was monitored in mice deficient in all major CTL effector pathways. Note that all infected knockout mice develop convulsive seizures and succumb to LCMV-induced meningitis. A slight extension (13 hrs, p = 0.029) in survival was observed in TNF-α knockout and Jinx mice. As reported previously, perforin deficient mice survived until day 9 (p = 0.029). b, The composition of the CNS mononuclear cell infiltrate was examined flow cytometrically at the denoted time points following LCMV infection. A massive influx of CTL (CD45⁺Thy1.2⁺CD8⁺) and peripheral monocytes (CD45^hiThy1.2⁻CD11b⁺Gr-1^int) into the CNS was observed only at day 6 post-infection. Low numbers of neutrophils (CD45^intThy1.2⁺CD11b⁺Gr-1^hi), B cells (CD45⁺Thy1.2⁺CD19⁺), and CD4 T cells (CD45⁺Thy1.2⁺CD4⁺) were also observed in the CNS at day 6 post-infection. See Figure S5 for examples of flow cytometric data. c, d, Injection of low dose anti-Gr-1 antibody (125 µg i.p. on day 4) achieved depletion of neutrophils from the CNS (c), but did not improve survival (d) when compared to control mice treated with rat IgG. e, f, CCR2 deficiency did not improve survival (f) following i.c. LCMV infection when compared to wild type B6 controls; however, flow cytometric studies revealed a compensatory increase in the number of CNS neutrophils (e) in CCR2 knockout mice at day 6. Survival was significantly extended in LCMV-infected CCR2 deficient mice that received high dose anti-Gr-1 antibody (f). The frequency of CD45⁺ P14 cells was not significantly reduced following anti-Gr-1 treatment (data not shown). For all studies described above n=4 mice per group were used.

Figure 4. Recruitment of myelomonocytic cells into CNS and the relationship to meningeal vascular injury

a–d, Two representative two-photon images at early (t=0 min) (a, c) and late (t= 30 min) (b, d) points in the time lapse show the position of GFP⁺ P14 CTL (green) in relation to meningeal vascular changes (red). GFP⁺ CTL were typically found in perivascular regions. In panels a–b note the severe disruption of vascular integrity, as evidenced by the significant amount of extravascular quantum dot signal (red). Also note the ghost outlines of large cells near the ragged vessels that do not correspond to P14 CTL. In other areas P14 CTL remained near perivascular areas that had preserved blood vessel integrity (c, d). (Scale = 50 µm) See corresponding Movie 3. e–h, A representative two-photon 30 minute time lapse sequence of vascular leakage of quantum dots (red) and extravasation of LysM-GFP⁺ myelomonocytic cells (green) is shown for a symptomatic mouse at day 6 post-infection. Note that myelomonocytic cells roll and arrest inside the meningeal vessel before penetrating through the vascular wall and extravasating into the meningeal space. The extravasation of myelomonocytic cells is associated with severe vascular injury and quantum dot leakage. (Grid scale = 19.7 µm) See corresponding Movie 4. No similar extravasation events were observed in asymptomatic control mice at day 5 post-infection (data not shown). i–l, A representative 30 minute time lapse is shown for a symptomatic LysM-GFP depleted of neutrophils but not monocytes using low dose anti-Gr-1 antibody. Note that LysM-GFP⁺ myelomonocytic cells (green) in the absence of neutrophils localize perivascularly and are associated with transient vascular leakage (red). (Scale = 25 µm) See corresponding Movie 5. m–o, The ratio of extravascular to intravascular GFP and quantum dot fluorescence signal was calculated at each frame, normalized to the baseline ratio, and plotted versus time. Data are represented as mean ± SEM. P14 CTL extravasation or positioning was not associated with quantum dot leakage (o). Leakage appeared to be associated with unlabeled ghost cells in CTL movies. Myelomonocytic cell extravastion correlated (r = 0.99; p < 0.0001) with sustained vascular leakage only in the presence of neutrophils (m). In neutrophil depleted LysM-GFP mice, perivascular myelomonocytic cells were associated with transient quantum dot leakage (n). p–r, Representative 3D reconstructions of two-photon z-stacks depicting skull bone (blue), quantum dot (red), and LysM-GFP⁺ myelomonocytic cells (green) are shown for an asymptomatic mouse at day 5 (p), a symptomatic LysM-GFP mouse at day 6 (q), and a symptomatic LysM-GFP mouse at day 6 depleted of neutrophils (r). At day 5, vasculature showed smooth borders, no quantum dot (red) leakage, and few LysM-GFP⁺ cells (green). In symptomatic mice at day 6, LysM-GFP⁺ cells were mostly observed extravasating from meningeal vasculature with some cells accompanying the vascular leakage down into the parenchyma (white arrow). In mice depleted of neutrophils (PMN), LysM-GFP cells were observed in perivascular meningeal spaces. (Grid scale for panels p–r = 19.7 µm) See corresponding Movies 4, 5.