Research ArticleMultiple Sclerosis

Reelin depletion protects against autoimmune encephalomyelitis by decreasing vascular adhesion of leukocytes

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Science Translational Medicine  12 Aug 2020:
Vol. 12, Issue 556, eaay7675
DOI: 10.1126/scitranslmed.aay7675

Relying on Reelin

In multiple sclerosis (MS), the recruitment of immune cell into the brain contributes to neuroinflammation and consequent demyelination. Current approaches for inhibiting cellular infiltration have been shown to have therapeutic effects but present complications. In this study, Calvier et al. showed that the protein Reelin could be a therapeutic target in MS. Reelin was increased during relapses in serum of patients with relapsing-remitting MS. Genetic Reelin depletion in a mouse model of MS prevented monocyte extravasation and prevented paralysis. Using a pharmacologic approach, the authors showed that prophylactic or therapeutic Reelin inhibition had therapeutic effect in mice, suggesting that Reelin inhibition might be effective for treating MS.

Abstract

Neuroinflammation as a result of immune cell recruitment into the central nervous system (CNS) is a key pathogenic mechanism of multiple sclerosis (MS). However, current anti-inflammatory interventions depleting immune cells or directly targeting their trafficking into the CNS can have serious side effects, highlighting a need for better immunomodulatory strategies. We detected increased Reelin concentrations in the serum of patients with MS, resulting in increased endothelial permeability to leukocytes through increased nuclear factor κB–mediated expression of vascular adhesion molecules. We thus investigated the prophylactic and therapeutic potential of Reelin immunodepletion in experimental autoimmune encephalomyelitis (EAE) and further validated the results in Reelin knockout mice. Removal of plasma Reelin by either approach protected against neuroinflammation and largely abolished the neurological consequences by reducing endothelial permeability and immune cell accumulation in the CNS. Our findings suggest Reelin depletion as a therapeutic approach with an inherent good safety margin for the treatment of MS and other diseases where leukocyte extravasation is a major driver of pathogenicity.

INTRODUCTION

Inflammatory responses are generally protective in cases of acute infection or tissue damage but become deleterious when they enter a chronic state. Recruitment of circulating leukocytes is a general mechanism and hallmark of many pathological disorders including multiple sclerosis (MS) (1). Microglia (brain-resident macrophages) and myelomonocytes (myeloid cells derived from leukocytes) are involved in the initiation and continuation of inflammation resulting in the demyelination of white matter (24), and their respective roles have been unraveled in elegant studies (1). In a murine model of experimental autoimmune encephalomyelitis (EAE) used to study human MS, the inhibition of monocyte recruitment to the spinal cord is sufficient to block myelin degradation and paralysis (1). An extensive histopathological assessment of MS lesions revealed that macrophages are abundantly present in all brains of patients with this disorder (5). This finding has opened a powerful approach to treat MS by inhibiting monocyte infiltration.

Immunotherapeutic interventions are now widely used in MS to reduce the relapse rate and the accumulation of new brain lesions. Although substantial progress has been made in the development of treatments for relapsing-remitting MS (RRMS), effective therapies are lacking for progressive forms of MS (6, 7). Moreover, interventions with biologics that target immune cells directly to prevent T cell infiltration into the nervous system are difficult to regulate and have narrow therapeutic bandwidth. They might result, for instance, in increased risk for progressive multifocal leukoencephalopathy (PML), a viral infection of the brain that is often fatal (8). Therefore, identifying optimal, immunomodulatory therapies for all forms of MS continues to constitute a major unmet need for patients (9).

In two independent genome-wide association studies (GWAS) (10, 11), Reelin single-nucleotide polymorphisms (SNPs) were found to be associated with MS severity score. However, these association studies did not shed light on the biological function of Reelin in MS. Initially recognized only for its role in guiding neurons during brain development and as a synaptic homeostatic regulator (1215), we recently reported a previously unappreciated non-neuronal function for systemically circulating Reelin in the vasculature (16) where Reelin regulates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)–mediated expression of several vascular adhesion molecules. Endothelial adhesion is an obligate step that precedes leukocyte extravasation, which leads to excessive inflammation in autoimmune syndromes such as MS. Here, we show that circulating Reelin is increased during MS relapse compared with healthy individuals and patients in remission, thus correlating with disease state and severity. Genetic reduction or immunodepletion of Reelin in a preclinical EAE mouse model reduces endothelial adhesion and monocyte extravasation, thereby preventing neuroinflammation, demyelination, and paralysis. This proof-of-concept study potentially opens the door to a different class of immunotherapeutics that selectively dampen NF-κB activation only at the endothelial barrier while avoiding direct T cell suppression.

RESULTS

Identification of circulating Reelin as a potential marker for MS

Progression of MS relies on myeloid cell extravasation and infiltration into the central nervous system (CNS) (1, 17, 18). On the basis of our previous findings (16), we hypothesized that this mechanism might be regulated by Reelin and consequently evaluated whether circulating Reelin is increased in the proinflammatory context of MS. Enzyme-linked immunosorbent assay detected elevated Reelin concentrations in the circulation of patients with RRMS during relapse (Table 1). Patients with RRMS in remission had Reelin concentrations similar to the control group, suggesting that circulating Reelin correlates with MS severity and stage.

Table 1 Reelin concentration in serum is increased in patient with RRMS during the relapse phase.

Concentrations for Reelin are shown per group and detailed per treatment (below medication) in the “RRMS in relapse” group. RRMS, relapsing-remitting MS; CT, control; Rel, relapse; Rem, remission.

View this table:

Reelin promotes monocyte adhesion to human endothelial cells in vitro and in mice in vivo

To investigate the role of Reelin and its therapeutic potential in human MS, we used an in vitro system consisting of human aortic endothelial cells (HAEC) incubated with human plasma (Fig. 1A) or recombinant Reelin (Fig. 1B). In both stimulations, the Reelin-neutralizing monoclonal antibody (CR-50) (12, 19) rapidly (within 1 hour) inhibited human monocyte adhesion to endothelial cells in a sustained (24 hours) manner (Fig. 1, A and B).

Fig. 1 CR-50 inhibits Reelin-dependent adhesion of monocytes to endothelial cells.

(A) Human aortic endothelial cells (HAEC) were starved and stimulated with human plasma, plasma + CR-50, or CR-50 only (0.15 μg/ml) for 1 or 24 hours. After washing, the cells were incubated with human monocytes (U937) for 45 min, washed, and fixed. Adherent monocytes were counted, and representative pictures are shown (n = 3). (B) Using the same protocol, HAEC were stimulated with Reelin (20 nM), Reelin + CR-50, or CR-50 only (0.15 μg/ml) for 1 or 24 hours (n = 3). (C) Four-week-old Cx3cr1-GFP male and female mice were injected intraperitoneally with 100 μg of IgG (n = 13) or CR-50 (n = 12). Intravital microscopy was performed 3 days after injections to record numbers and speed of rolling monocytes on the marginal mesenteric vessels. Six different vessels per mouse were recorded for 10 s and analyzed; cumulative pictures represent individual images (100 frames/s) integrated over a 10-s period and stacked; rolling index = monocyte number per velocity. (D and E) After intravital microscopy, immunoblotting was performed on plasma and aorta protein extracts. *P < 0.05 and **P < 0.01 [t test or analysis of variance (ANOVA)].

Using an in vivo mouse model with genetically green fluorescent protein (GFP)–labeled monocytes (Cx3cr1-GFP), we tested the ability of CR-50 to reduce monocyte rolling on the vessel wall using intravital microscopy (movie S1). Three days after a single CR-50 intraperitoneal injection (100 μg), the number of rolling monocytes attached to the endothelial surface was greatly reduced, and rolling velocity was increased compared with control [mouse immunoglobulin G (IgG); Fig. 1C]. This resulted in decreased monocyte interaction with the vascular wall, as shown by decreased rolling index and cumulative intensity (illustrated by representative cumulative pictures; Fig. 1C). We did not observe gender differences in the monocyte adhesion or velocity (fig. S1A); white blood cell number and the vessel area measured by intravital microscopy were similar in both groups (fig. S1B).

Reelin was below the detection limit in the plasma of CR-50–treated mice (Fig. 1D), and this correlated with decreased expression of rolling (E-selectin) and adhesion [intercellular adhesion molecule–1 (ICAM-1)] markers in the aorta (Fig. 1E). This is consistent with our previously published findings showing that Reelin increases E-selectin and ICAM-1 expression in endothelial cells (16). These data support Reelin as a therapeutic target for the inhibition of monocyte recruitment and the reduction of perivascular cellular inflammation.

Genetic Reelin deletion protects from paralysis in mice

To explore the role of Reelin in MS, we used experimental autoimmune encephalomyelitis (EAE) as a mouse model to study human MS, which closely mimics the inflammation and demyelination of the CNS seen in humans. For this purpose, we have crossed Cx3cr1-GFP with genetically GFP-labeled monocytes to ubiquitous Reelin conditional knockout (cKO) mice (Cag-Cre−/+ Relnfl/fl). Cx3cr1-GFP; Relnfl/fl mice carrying or not carrying the Cag-Cre transgene were treated with tamoxifen for 5 days to obtain wild-type (WT) (Cx3cr1-GFP; Cag-Cre Relnfl/fl) and Reelin cKO (Cx3cr1-GFP; Cag-Cre+ Relnfl/fl) littermates (Fig. 2, A and B). One week after the first tamoxifen injection, EAE (20) was induced by myelin oligodendrocyte glycoprotein immunization using a standard protocol (Fig. 2A). As previously described (20), WT mice developed progressive paralysis starting at the tail at day 13 and reaching a plateau (with slower progression after the exponential phase) at day 16 (Fig. 2, C and D). Blinded clinical scoring of EAE was paralleled by the weight loss (Fig. 2E) and by a decreased upside-down hanging time on a grid (Fig. 2F), independently reflecting both paresis and paralysis. EAE index (Fig. 2D) was calculated by numerical addition of the EAE clinical score for each mouse, representing the area under the curve. The same calculation was done for the hanging time index (Fig. 2F). On the basis of the same principle, a weight loss index (Fig. 2E) was calculated as the numerical addition over the days of the weight loss. For each day, the weight loss itself is defined as the average weight before EAE (from day 7 to 12) minus the mouse weight.

Fig. 2 Reelin cKO mice are protected from EAE.

(A) Cx3cr1-GFP; Cag-Cre+or- Relnfl/fl male mice were treated with tamoxifen for 5 days as described (51) to obtain WT (Cx3cr1-GFP; Cag-Cre Relnfl/fl; n = 10) or Reelin cKO (Cx3cr1-GFP; Cag-Cre+ Relnfl/fl; n = 9). One week after the first tamoxifen injection, EAE was induced by myelin oligodendrocyte glycoprotein immunization using standard published procedures (20). (B) Twenty-one days after EAE induction, the experiment was terminated, and plasma Reelin was measured by immunoblotting. (C to G) EAE severity was evaluated daily from day 10 by (D) EAE clinical score (from 0 = unaffected to 10 = dead), (E) weight loss, and (F) hanging test (for a maximum time of 180 s). (D to F) EAE severity, weight loss, and hanging time indexes were calculated for each animal by integrating daily scores over the course of the experiment and represent the area under the curve (AUC). (G) Relative plasma Reelin expression in WT mice (n = 9) was correlated to EAE severity index. *P < 0.05 and **P < 0.01 (two-way ANOVA, t test, or linear correlation with Pearson’s R).

Compared with WT, the Reelin cKO mice were protected from paralysis. The EAE onset and plateau were delayed by 3 and 4 days, respectively, and the severity was greatly reduced in the absence of an exponential progression phase (Fig. 2, C and D), resulting in no weight loss (Fig. 2E) or decreased hanging time (Fig. 2F). Variable plasma Reelin expression in the WT group was correlated with EAE index (Fig. 2G; plasma collected at day 21). Together, these results demonstrate that low plasma Reelin expression correlates with reduced EAE severity, and the total absence of Reelin mitigates initial paralysis and its progression.

Genetic Reelin deletion prevents monocyte extravasation to the CNS

Next, we sought to validate that Reelin deletion protects from EAE by reduction in monocyte recruitment to the CNS and dampened subsequent inflammation. Adhesion proteins were analyzed in the spinal cord, revealing that vascular E-selectin and ICAM-1 protein expression was decreased in Reelin cKO mice (Fig. 3, A and B). mRNA and protein expression of the Reelin receptor Apoer2 was not affected (fig. S2, A and B). Reduced expression of adhesion proteins in Reelin cKO mice was associated with reduced accumulation of inflammatory cells in the spinal cord as shown by Cx3cr1-GFP fluorescence (Fig. 3C). Both resident microglia and infiltrating monocytes express Cx3cr1-GFP; therefore, the microglia-specific marker Iba1 was used to discriminate between the two populations. Reelin cKO mice showed a significant (P < 0.05) reduction in total inflammatory cell number (positive for Cx3cr1-GFP, regardless of Iba1 expression) and infiltrating monocytes (positive for Cx3cr1-GFP, negative for Iba1; Fig. 3D). Monocyte infiltration is minimal during EAE stage 0–1 and rises sharply around stage 3 (1). The Reelin cKO mice were protected from monocyte infiltration at all stages (Fig. 3D). Reelin deletion had a moderate effect on microglia activation, as it induced a nonsignificant (P = 0.204) reduction in microglia count (fig. S2C). Because infiltrating monocytes are necessary to develop neuroinflammation during EAE (1), our data suggest that Reelin deletion prevents myelin degradation by decreasing monocyte rolling/adhesion on the vascular wall and extravasation into the CNS.

Fig. 3 Reduced CNS inflammation in Reelin cKO mice.

(A to D) Cx3cr1-GFP; Cag-Cre+or- Relnfl/fl male mice were treated with tamoxifen for 5 days as described (51) to obtain WT (Cx3cr1-GFP; Cag-Cre Relnfl/fl; n = 10) or Reelin cKO (Cx3cr1-GFP; Cag-Cre+ Relnfl/fl; n = 9). One week after the first tamoxifen injection, EAE was induced by myelin oligodendrocyte glycoprotein immunization using standard procedures (20). At 21 days after EAE induction, the experiment was terminated, and expression of the indicated proteins was determined in the spinal cord. (A) E-selectin and ICAM-1 were quantified by immunoblotting. (B) ICAM-1 was visualized by immunofluorescence (scale bar, 100 μm). (C) Cx3cr1-GFP fluorescence area was quantified in the spinal cord (scale bar, 200 μm). (D) In the Cx3cr1-GFP–positive cell population, the total number of inflammatory cells (Cx3cr1-GFP positive), monocytes (Cx3cr1-GFP positive, Iba1-negative; indicated by the arrows), and microglia (Cx3cr1-GFP and Iba1 double positive) were visualized by immunofluorescence (scale bars, 50 μm). Bar graphs represent the average cell population per group and per group sorted by EAE score (at 21 days). *P < 0.05 and **P < 0.01 (t test or ANOVA).

Prophylactic Reelin inhibition protects from paralysis

In light of the substantial effect of genetic Reelin deletion, we reasoned that therapeutic Reelin depletion could be an effective approach to prevent the onset and, possibly, progression of EAE. For this purpose, we first used the Reelin function blocking antibody CR-50 or mouse IgG (as control) to evaluate its potential use as a prevention treatment in Cx3cr1-GFP mice starting 1 week before EAE induction (Fig. 4, A and B). As detailed in Fig. 1 (C to E), CR-50 treatment (intraperitoneal injection of 100 μg) effectively cleared Reelin from the circulation, decreasing endothelial adhesion marker expression and strongly reducing monocyte adhesion to the vascular wall. Next, we intraperitoneally injected 100 μg of CR-50 and control IgG, twice per week, followed by EAE induction. In naïve mice, CR-50 treatment alone did not affect mouse weight or any other EAE parameters compared with IgG alone (independent of EAE; fig. S3A). CR-50 antibody treatment did not detectably decrease Reelin protein amount in the CNS (spinal cord or brain) compared with IgG (fig. S3, B and C) because of the intrinsically low ability of the antibodies to cross the blood-brain barrier. This makes unlikely unintended possible cognitive side effects as a result of disrupted synaptic homeostasis by suppression of Reelin function in the CNS.

Fig. 4 Prophylactic anti-Reelin treatment protects from EAE.

(A) Cx3cr1-GFP male mice were injected intraperitoneally (i.p.) with 100 μg of IgG (n = 10) or CR-50 (n = 9) twice per week. One week after the first injection, EAE was induced by myelin oligodendrocyte glycoprotein immunization using standard procedures (20). s.c., subcutaneously. (B) Twenty-one days after EAE induction, plasma Reelin was measured by immunoblotting. (C to G) EAE severity was evaluated daily starting at day 10 by scoring (D) survival, (E) EAE clinical score (from 0 = unaffected to 10 = dead), (F) weight loss, and (G) grid hanging endurance (for a maximum time of 180 s). (C to G) EAE severity, weight loss, and hanging time indices are calculated for each animal by integrating daily scores over the course of the experiment and represent the AUC. (H) Relative plasma Reelin expression in control IgG-injected mice (n = 9) was correlated to EAE severity, weight loss, and hanging time indexes. *P < 0.05 and **P < 0.01 (Mantel-Cox test, two-way ANOVA, t test, or linear correlation with Pearson’s r).

As expected, IgG-treated mice responded to EAE induction and developed progressive paralysis starting at day 12 and reaching a plateau at day 15 (Fig. 4, C and E). This observer-blinded EAE scoring was confirmed by the weight loss (Fig. 4F) and by a decreased hanging time on a grid (Fig. 4G), reflecting both paresis and paralysis. Compared with the IgG-treated mice, the CR-50 treatment reduced EAE progression and severity. EAE onset and plateau were delayed by 4 and 5 days, respectively, and the severity was reduced by more than 50% as judged by the EAE index (Fig. 4, C and E). Weight loss was not affected by the treatment (Fig. 4F); however, muscle force loss (reflected by hanging time; Fig. 4G) was delayed in the CR-50–treated mice. In addition, survival after 21 days was 100% in the CR-50 group compared with 70% in the control group (Fig. 4D), but this difference did not reach statistical significance (P = 0.077).

We confirmed the protective effect of CR-50 treatment in a pilot study using a suboptimal induction protocol for EAE (using 50% of the regular dose of Complete Freund’s adjuvant), which induced only moderate paresis and paralysis with no respiratory depression or death (fig. S4). At the disease peak, CR-50–treated mice were significantly (P < 0.05) protected from paralysis, as judged by the EAE clinical score.

Last, as observed with the WT mice in Fig. 2G, plasma Reelin expressions in the IgG-treated group correlated with all of the above EAE parameters (EAE index, weight loss index, and hanging time index; Fig. 4H). Together, these results confirm our previous conclusions with the cKO model and establish that plasma Reelin concentrations correlated with EAE severity and that a Reelin-blocking strategy protected from paralysis progression.

Prophylactic Reelin inhibition prevents monocyte extravasation to the CNS

Next, we analyzed the expression of endothelial cell adhesion markers. CR-50 treatment reduced the expression of E-selectin and ICAM-1 in the spinal cord vasculature compared with the IgG group (Fig. 5, A and B). Consequently, accumulation of inflammatory cells in the spinal cord was reduced as shown by Cx3cr1-GFP fluorescence (Fig. 5C). As established in the Reelin cKO model, we used Iba1 staining of the spinal cord to discriminate infiltrating monocytes (positive for Cx3cr1-GFP only) from the resident microglia (Cx3cr1-GFP and Iba1 double positive). CR-50 treatment induced a significant (P < 0.05) reduction in total inflammatory cell number, driven by a reduction in infiltrating monocytes at all EAE stages (Fig. 5D).

Fig. 5 Reduced CNS inflammation in anti-Reelin antibody–treated mice.

(A to D) Cx3cr1-GFP male mice were injected intraperitoneally with 100 μg of irrelevant IgG (n = 10) or CR-50 (n = 9) twice per week. One week after the first injection, EAE was induced by myelin oligodendrocyte glycoprotein immunization using standard procedures (20). (A) E-selectin and ICAM-1 protein expression in spinal cord was quantified by immunoblotting. (B) ICAM-1 expression in spinal cord was visualized by immunohistochemistry (scale bar, 100 μm). (C) Cx3cr1-GFP fluorescence area was quantified in the spinal cord (scale bar, 200 μm). (D) In the Cx3cr1-GFP–positive cell population, the total number of inflammatory cells (Cx3cr1-GFP positive), monocytes (Cx3cr1-GFP positive, Iba1 negative; indicated by the arrows), and microglia (Cx3cr1-GFP and Iba1 double positive) were visualized by immunofluorescence (scale bars, 50 μm). Bar graphs represent the cell population average per group and per group sorted by EAE score (at 21 days). *P < 0.05 and **P < 0.01 (t test or ANOVA).

Therapeutic Reelin inhibition reduces paralysis severity and promotes recovery

To test whether anti-Reelin strategies can be translationally applied to human MS, we performed another series of experiments in which we initiated anti-Reelin treatment after the onset of EAE symptoms. Starting at the first day of clinical signs of paralysis (determined individually for each mouse), the Reelin function blocking antibody CR-50 or mouse control IgG was injected twice per week by intraperitoneal injection (100 μg; Fig. 6A). IgG or CR-50 treatment was randomly assigned among littermates at day 0, and mice showing paralysis starting after day 14 were excluded to maintain consistent groups with similar treatment duration. As expected, CR-50 effectively reduced plasma Reelin concentration (Fig. 6B), whereas in the IgG group, Reelin increased during the course of EAE. This result is in agreement with the elevated Reelin concentrations we observed during MS in relapsing patients (Table 1).

Fig. 6 Therapeutic anti-Reelin treatment alleviates EAE progression.

(A) Cx3cr1-GFP male littermates were randomly assigned in the IgG or CR-50 group, and EAE was induced by myelin oligodendrocyte glycoprotein immunization using standard procedures (20). Starting from the first day of paralysis, mice were injected intraperitoneally with 100 μg of IgG (n = 16) or CR-50 (n = 14) twice per week. (B) Twenty-one days after EAE induction, plasma Reelin was measured by immunoblotting. (C to F) EAE severity was evaluated daily starting at day 10 by scoring (C) survival, (D) EAE clinical score (from 0 = unaffected to 10 = dead), (E) weight loss, and (F) grid hanging endurance (for a maximum time of 180 s). (D to F) EAE severity, weight loss, and hanging time indexes are calculated for each animal by integrating daily scores over the course of the experiment and represent the AUC. EAE, weight, and hanging time recovery are calculated for each mouse as the difference between the maximum (for EAE score) or minimum (for weight and hanging time) and day 21. (G) Fluorescence-activated cell sorting (FACS) was performed on the spinal cord (n = 8 IgG and n = 8 CR-50 mice) to determine infiltration of monocytes (CD11+), B cells (CD19+), T helper cells (CD4+), and T cells (CD8+). *P < 0.05 and **P < 0.01 (Mantel-Cox test, two-way ANOVA, or t test).

Whereas IgG-treated mice responded to EAE induction and developed progressive paralysis (Fig. 6, C to F) with 75% survival (Fig. 6C), mirroring the results in our previous prevention study, all the CR-50–treated mice survived (Fig. 6C) and showed mitigated paralysis severity (Fig. 6D). Weight loss was not significantly affected by the treatment (Fig. 6E), but hanging time (P < 0.05; Fig. 6F) was improved by CR-50. CR-50 improved the recovery, as judged by EAE score, weight, and hanging time recovery calculated for each mouse as the difference between the maximum (for EAE score) or minimum (for weight and hanging time) and day 21 (Fig. 6, D to F).

Last, using fluorescence-activated cell sorting (FACS) analysis (Fig. 6G and fig. S5G), CR-50 intervention reduced the amount of total infiltrating cells into the CNS, mainly driven by monocytes (CD11+). There was no significantly reduced infiltration of CD19+ B cells, CD4+ T helper cells, and CD8+ T cells when analyzed individually, but when analyzed in aggregate, their infiltration was reduced (fig. S5). This observation is in accordance with histology performed in the Reelin cKO (Fig. 3, C and D) and the prophylactic treatment study (Fig. 5C and D), which both show reduction in monocyte infiltration after Reelin depletion.

Together, we have shown that genetic, prophylactic, or therapeutic depletion of circulating Reelin prevents or mitigates EAE in mice by diminishing vascular adhesion protein expression, thereby potently reducing monocyte rolling on the vascular wall, infiltration into the CNS, and myelin degradation (fig. S6).

DISCUSSION

In this study, we have shown that genetic and therapeutic neutralization of circulating Reelin can prevent chronic inflammatory conditions that depend upon monocyte extravasation, such as MS, by modulating endothelial permeability (fig. S6). We have established that circulating Reelin in humans is increased during MS relapse and reduced to control concentration in remitting patients. We show that human Reelin promotes monocyte adhesion to endothelial cells and that a Reelin-blocking antibody can prevent this adhesion. This result was confirmed using intravital microscopy, where anti-Reelin antibody–treated mice displayed a reduction in monocyte adhesion to the vascular wall, accompanied by decreased expression of adhesion molecules (ICAM-1 and E-selectin) in the aorta. In a mouse EAE model, both genetic and pharmacologic (antibody-mediated) Reelin depletion reduced paralysis progression. Ex vivo analysis revealed that Reelin-depleted mice (genetic or antibody mediated) had reduced adhesion marker expression in the vasculature of the spinal cord accompanied by a decrease in inflammatory cell numbers in the grey and white matter. This was mainly due to decreased monocyte infiltration in the absence of Reelin. Last, in a preclinical proof-of-concept study, we have demonstrated disease protection by application of a Reelin-neutralizing antibody where CR-50 was given at the onset of EAE symptoms, resulting in diminished neuroinflammation, paralysis, and improved recovery. Together with the human and intravital microscopy data, the efficacy of anti-Reelin antibody intervention in the standard mouse EAE model indicates that Reelin depletion acts by reducing monocyte infiltration, thereby preventing neuroinflammation and, thus, progression of paralysis.

Recruitment of circulating leukocytes is a general mechanism of tissue inflammation, which proceeds along a series of well-defined steps (2123): (i) chemoattraction upon cytokine and chemokine release; (ii) transient (rolling) adhesion to endothelial cells mediated by carbohydrate/receptor recognition, such as E-selectin; (iii) tight adhesion through adhesion proteins, such as integrins, ICAM-1, or vascular cell adhesion molecule 1 (VCAM-1); and (iv) endothelial transmigration (=diapedesis). We have shown that Reelin depletion decreases adhesion protein expression on endothelial cells, resulting in decreased monocyte/endothelium adhesion and diminished monocyte extravasation. This is mediated by the binding of Reelin to its receptor Apoer2/Lrp8, which increases NF-κB target gene expression (16) in endothelial cells and integrin β1 in multiple myeloma cells (24, 25).

Under normal conditions, the blood-brain barrier effectively regulates the passage of immune cells into the CNS. However, under pathological conditions, monocytes infiltrate the CNS where they, in concert with activated microglia, promote inflammatory demyelination (26), resulting in demyelination and paralysis (1, 27, 28). Targeted microglia inactivation of TAK1, while sparing infiltrating monocytes, prevents both initial and relapsing paralysis (29). Recently, single-cell profiling confirmed context-dependent subsets of CNS-resident macrophages, while monocyte-derived cells were highly diverse and the primary antigen presenters (30). Together, these studies imply that both lineages are required, probably at different stages, with early microglia activation followed by monocyte infiltration, to drive neurological damage through demyelination and progression of paralysis (27). This proposed mechanism agrees well with our results, where Reelin depletion specifically blocks monocyte infiltration yet is sufficient to inhibit EAE progression.

In our control groups, we observed a notable correlation between plasma Reelin concentration and EAE severity score. A Reelin SNP is associated with MS severity and age of the onset in two GWAS analyses (10, 11), but how this SNP affects Reelin function has yet to be determined. Although the primary source for peripheral Reelin by far is the liver, where Reelin is secreted by hepatic stellate cells (31, 32), we cannot exclude a minor contribution from other organs (such as kidney). Regardless of the origin, the effect of Reelin would be expected to manifest itself equally on the vasculature. Another limitation of our study is the use of the murine EAE model to investigate human MS, as this model reflects only some aspects and mechanisms of MS. This study is thus a first preclinical step toward additional clinical studies before translational application to human diseases can proceed.

Functions of Reelin in coagulation have also been reported (33, 34), and fibrinogen production modulates CNS autoimmunity, demyelination, and MS (3537), raising the possibility that platelet-derived Reelin might locally alter endothelial permeability. A role for CNS-produced Reelin during EAE is unlikely, as the peripheral antibody-mediated depletion does not affect CNS Reelin expression to any measurable extent, yet potently protects against paralysis and disease progression. Preserving Reelin expression in the CNS is important as Reelin is essential for neuronal migration and positioning not only in the developing brain (1315) but also in the adult brain, where it modulates synaptic plasticity (38, 39), migration of neuroblasts (40), as well as dendrite (41) and dendritic spine (42) formation.

Currently, there are several disease-modifying agents approved for relapsing forms of MS. Alpha4 integrin is an adhesion molecule that is widely expressed on all leukocytes (4345). It is recognized by natalizumab, a humanized monoclonal antibody that potently blocks alpha4 integrin and thereby prevents the interaction with its main ligands, VCAM-1, and fibronectin (46). Natalizumab is difficult to titrate and thus tends to indiscriminately abolish the ability of immune cells to enter the brain and spinal cord to perform their physiological surveillance functions (4749). This excessive immunosuppression greatly increases the risk for PML, a viral infection of the brain that is often fatal (8). Because anti-Reelin interventions target a different mode of action, its predicted broader, but more balanced, effects on CNS immune surveillance could conceivably mitigate such complications and potentially reduce PML incidence. As our studies have shown, Reelin depletion normalizes, but does not completely abolish, the expression of vascular adhesion markers, thereby preserving protective physiological diapedesis functions while dampening excessive inflammation.

In conclusion, in the current study, we have exploited a function of Reelin in the regulation of vascular permeability for leukocyte extravasation to devise a strategy for combating autoimmunity-driven neuroinflammation in MS. Our study identifies Reelin as a therapeutic target for reducing the extravasation of monocytes and—more generally—leukocytes, thereby establishing an alternative endothelial-specific tunable immunomodulatory approach for the treatment of MS by selectively reducing endothelial permeability and infiltration of inflammatory cells into the CNS. Patients with progressive forms of MS, for which there currently are no effective treatment options (6, 7), represent more than half of the 2.3 million individuals affected with MS worldwide (50). Therefore, identifying effective therapeutic interventions for all forms of MS represents a substantial unmet need (9). Beyond MS, anti-Reelin strategies would be expected to be similarly effective for the treatment of other chronic inflammatory syndromes that depend on excessive leukocyte extravasation, such as atherosclerosis, arthritis, or Crohn’s disease.

MATERIALS AND METHODS

Study design

The purpose of this study was to explore a therapeutic approach targeting endothelial adhesion and permeability to prevent monocyte extravasation instead of directly targeting the immune system. We hypothesized that genetic and therapeutic neutralization of circulating Reelin would reduce endothelium/monocyte adhesion and diapedesis, thus preventing chronic inflammatory syndromes that depend on monocyte extravasation such as MS. These objectives were addressed using a human MS cohort, HAEC and U937 cultures, mouse models for intravital microscopy, and EAE. Power analysis was used to determine a range for the sample size, and no outliers were excluded. Animals were randomized, and EAE was evaluated by two blinded operators. Biological replicates are specified in each figure legend.

Human MS cohort

Serum of anonymous patients with RRMS in remission, RRMS during an acute relapse, and healthy controls were obtained from UT Southwestern Medical Center MS tissue repository (authorization #STU022011-211, Dallas, Texas). The study presented here has been approved by the “Human Research Protection Program Office” as “Not Human Research,” which does not require institutional review board approval.

Cx3cr1-GFP mice

Cx3cr1-GFP mice (B6.129P-Cx3cr1tm1Litt/J) were purchased from the Jackson laboratories (stock no. 005582). These mice express enhanced green fluorescent protein (EGFP) in monocytes, dendritic cells, NK cells, and brain microglia under the control of the endogenous Cx3cr1 locus. Cx3cr1-GFP monocytes down-regulate GFP expression upon differentiation into macrophages.

For intravital microscopy, 4-week-old Cx3cr1-GFP males and females received one injection (100 μg, intraperitoneally) of IgG or CR-50 antibodies. For EAE, 7-week-old Cx3cr1-GFP male mice were injected (intraperitoneally) with 100 μg of IgG or CR-50 twice per week.

Cx3cr1-GFP; Cag-Cre Relnfl/fl mice

Cx3cr1-GFP and Cag-Cre Relnfl/fl mice were crossed to obtain Reelin cKO with monocyte labeled with GFP. Cag-Cre Relnfl/fl mice were obtained previously by crossing mice carrying the loxP-targeted Reln gene with the Cag-Cre mice from the Jackson laboratories (stock no. 004682).

To induce Cre-mediated DNA recombination, 7-week-old Cx3cr1-GFP; Cag-Cre Relnfl/fl mice (with or without the Cag-Cre transgene) were intraperitoneally injected with tamoxifen (0.135 mg/g) dissolved in sunflower oil for five consecutive days to obtain WT and Reelin cKO mice (51).

Statistical analysis

For cell culture, each condition was tested at least in duplicate (unless specified differently), and all experiments were repeated at least three times at different passages. For animals, the n values are specified in each legend. The software GraphPad Prism was used to run all the statistical analysis. Values from multiple experiments are expressed as means ± SEM. Normality was tested using the Kolmogorov-Smirnov test. Statistical significance was determined for multiple comparisons using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison (for normal distribution) or Kruskal-Wallis (for non-normal distribution) test. Student’s t test was used for comparisons of two groups. To test the different evolution between two groups over time, a two-way ANOVA was used (the asterisk on the curves represents significative difference for the treatment or genotype). The correlations were calculated by linear regression (Pearson’s r). The survival curves were tested with log-rank (Mantel-Cox test). P < 0.05 was considered significant.

SUPPLEMENTARY MATERIALS

stm.sciencemag.org/cgi/content/full/12/556/eaay7675/DC1

Material and Methods

Fig. S1. Intravital microscopy in anti-Reelin antibody–treated mice (refer to Fig. 1).

Fig. S2. Apoer2 (Reelin receptor) expression in spinal cord (refer to Fig. 3).

Fig. S3. Anti-Reelin antibody–treated mice are protected from EAE (refer to Fig. 4).

Fig. S4. Anti-Reelin antibody–treated mice are protected from moderate EAE (refer to Fig. 4).

Fig. S5. Flow-based immunophenotyping gating strategy (refer to Fig. 6).

Fig. S6. Reelin depletion protects from MS by reducing monocyte extravasation and inflammation.

Movie S1. CR-50 inhibits Reelin-dependent monocyte rolling on endothelium (refer to Fig. 1).

REFERENCES AND NOTES

Acknowledgments: We thank A. Middleton, T. Terrones, A. Rodriguez, A. Brennan, S. Ghayal, and R. Hussain for technical assistance. We also thank K. Mikoshiba for originally providing the CR-50 hybridoma. Funding: L.C. was supported by postdoctoral fellowship grants from DFG (CA 1303/1-1). P.W.S. and C.M. were supported by grants from the NIH (R01-HL131597 and R01-HL109604, respectively). O.S. was supported by a grant from Sanofi Genzyme. J.H. was supported by grants from the NHLBI (R37 HL063762), NIA (RF AG053391), the NINDS and NIA (R01 NS093382), BrightFocus A2016396S, the Bluefield Project to Cure FTD, and a Harrington Scholar Innovator Award (2019). Author contributions: L.C. and J.H. obtained funding, conceived the hypothesis and project, designed the study, and interpreted results. L.C. performed the in vitro and IVM experiments. L.C. and G.D. performed EAE models and tissue analysis. A.S., C.M., and P.W.S. helped with IVM experiments. N.M. performed the FACS analysis. C.W. performed brain dissections and analysis. O.S. participated in the results interpretation and with N.L.M. in the human cohort organization. L.C. and J.H. wrote the manuscript, and all the authors (including M.Z.K.) revised the manuscript for interpretation and content. Competing interests: L.C., M.Z.K., and J.H. are shareholders of Reelin Therapeutics Inc. L.C., C.M., P.W.S., and J.H. are coinventors of a patent related to anti-Reelin strategies (application number 15/763,047 and publication number 20180273637, title “Methods and Compositions for Treatment of Atherosclerosis”). Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials.
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