Генетическое или терапевтическое нарушение траектории подачи сигналов Reelin/Apoer2 улучшает воспалительные результаты артрита.

Rheumatoid arthritis (RA) is a complex, systemic, autoimmune disorder characterized by a destructive synovial pannus around affected joints. The hypoxic and inflammatory environment includes cytokines, chemokines, and proangiogenic factors ( 1 – 3 ). These factors promote the activation of endothelial cells, upregulating the expression of “cell rolling” molecules such as E-selectin and firm adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. Increased expression of adhesion markers allows more patrolling leukocytes to roll, adhere to the endothelium, and ultimately infiltrate the synovium ( 4 , 5 ). The importance of leukocyte infiltration in the pannus has been extensively demonstrated as their levels correlate with disease outcomes, such as radiographic progression ( 6 ) and disease activity ( 7 ). Furthermore, synovial leukocytes are reduced following effective disease-modifying antirheumatic drug (DMARD) therapy ( 8 – 10 ). For example, blocking tumor necrosis factor (TNF)α (e.g., infliximab) reduces joint chemokines, E-selectin, and ICAM-1 expression limiting the entry of leukocytes into the joint space ( 8 , 9 ). A murine anti-ICAM-1 antibody was shown to reduce leukocyte infiltration and synovial inflammation in preclinical studies and short-term clinical trial ( 11 , 12 ); however, it was not moved forward into the clinic due to serum sickness-like reactions ( 13 ). Similarly, the anti-integrin α4β1 (VLA-4) antibody, natalizumab, was tested in clinical trials due to its proven efficacy in blocking the extravasation of leukocytes in multiple sclerosis, another autoimmune disease. While natalizumab was evaluated for treating RA in a phase II, multicenter, double-blind, placebo-controlled trial alongside methotrexate, the study found no statistically significant differences in outcomes between patients treated with methotrexate alone and those receiving methotrexate with natalizumab ( 14 ). In addition, this antibody was associated with a risk for progressive multifocal leukoencephalopathy caused by reactivation of the John Cunningham (JC) virus, thus resulting in a fatal viral infection of the brain ( 15 ). Restoring the endothelial barrier to block leukocyte migration into the synovial space still represents a challenging therapeutic strategy, indicating a need for new targets to be identified.

To address this need without targeting and obliterating cytokine or leukocyte functions, restoring a proper endothelial barrier function appears promising in arthritis ( 1 , 2 , 16 – 18 ). The intracellular NF-κB pathway is a crucial signaling pathway that becomes excessively active in endothelial cells during arthritis, contributing to endothelial activation and permeability to leukocytes, a pathological response sometimes simplified and referred to as endothelial dysfunction ( 16 , 19 ). Upon stimulation, the NF-κB transcription factor translocates into the nucleus to promote the expression of its target genes. These genes include “rolling” molecules such as E-selectin and adhesion molecules such as ICAM-1 or VCAM-1, as well as cytokines such as interleukins (IL)-1, -6, TNFα, and chemokines such as CCL2, CCL8, CXCL2, and CX3CL1. Both, endothelial adhesion followed by endothelial transmigration are primarily regulated by NF-κB target genes ( 20 ). However, NF-κB has major additional functions and direct obliteration of this pathway would result in severe side effects. Instead of targeting NF-κB directly, we have identified Reelin as a potent NF-κB regulator in endothelial cells ( 21 ). Reelin is a large extracellular glycoprotein that plays a crucial role in various physiological and pathological processes, initially studied in the brain ( 22 – 27 ) and recently in the context of vascular inflammation ( 21 ). The hepatic stellate cells are believed to be the main source of Reelin outside of the brain ( 28 – 32 ). This circulating protein binds to ApoER2 on endothelial cells to promote the phosphorylation of NF-κB and the expression of its target genes, which are crucial for facilitating the adhesion of leukocytes to the endothelium and enhancing their extravasation ( 23 , 33 – 37 ). Emerging evidence suggests that Reelin and its receptor ApoER2 are involved in driving endothelial adhesion properties to leukocytes, inflammatory cell recruitment, and subsequent inflammation. This involvement has been observed in various conditions including atherosclerosis ( 38 , 39 ), multiple sclerosis ( 40 – 42 ), and COVID-19 ( 43 ). Mechanistically, we have previously demonstrated in endothelial cells that Reelin binds to its receptor ApoER2 to activate the NF-κB signaling pathway and promote the transcription and expression of adhesion markers such as E-selectin, ICAM-1, and VCAM-1 ( 38 , 39 ). While the role of Reelin is established in these pathologies, the exact contribution of this endothelial factor in the context of RA remains to be ascertained, warranting further research to elucidate its potential involvement in the pathogenesis and progression of RA.

We postulated that disruption of the Reelin/Apoer2 pathway in endothelial cells would restore the endothelial physiological function, decrease the extravasation of leukocytes and infiltration into the synovium as well as joint inflammation during inflammatory arthritis. To this end, we investigated arthritis development in several mouse models, specifically, serum transfer arthritis (STA) and collagen-induced arthritis (CIA) using genetic deletion or inactivation of Apoer2 as well as therapeutic Reelin inhibition.

Material and Methods

Study Populations.

Serum from the human RA cohort was obtained from Solow and Karp at UT Southwestern Medical Center. The study involves secondary research using data and biospecimens not collected specially for this study that were provided without identifiable information and approved by the UT Southwestern IRB review board (STU 012011-034). Detailed characteristics of each cohort can be found in SI Appendix , Table S1 . RA subjects were identified as having RA from the primary diagnosis in chart review and recruited from our Rheumatology clinics. All subjects were nonsmokers. Subjects were excluded if they had a history of cardiovascular disease, insulin-dependent diabetes mellitus, chronic kidney disease, or evidence of target organ damage such as left ventricular hypertrophy by electrocardiography. RA subjects had to be on less than 10 mg of prednisone per day and on a stable dose of medications for 4 wk. We evaluated high-disease activity RA, low-disease activity RA, and controls. High disease activity was defined as RA subjects with a Disease Activity Score (DAS) 28-ESR of 3.2 or greater ( 44 ). Of note, data on some of the age- and sex-matched control samples were already published as the assays were run together for both studies ( 43 ).

Generation of Transgenic Mouse Models.

Apoer2 KO mice ( 25 , 41 ) and Apoer2 EIG ( Lrp8 tm4Her ) mice ( 45 ) have been described previously. All animal studies were approved and performed according to the institutional animal care and use guidelines (2015-101088-G). Due to sex variation in response to arthritis induction, only males were used ( 46 , 47 ).

K/BxN Mice and Serum Transfer Arthritis.

K/BxN mice were generated by crossing KRN-C57BL/6 mice (obtained from Christophe Benoist), transgenic for a TCR recognizing a bovine ribonuclease peptide (RNase 42 to 56) presented by I-Ak major histocompatibility complex (MHC) class II molecule, with autoimmunity-prone nonobese diabetic NOD/ShiLtJ mice (the Jackson Laboratories, stock No. 001976) to introduce a TCR α chain-null mutation. They develop inflammatory arthritis spontaneously at around 4 wk and their serum is used to induce arthritis in a recipient mouse.

CIA.

DBA/1J were purchased from the Jackson Laboratories (Stock No. 000670). These mice are widely used as a model for RA by immunization with type II collagen leading to the development of severe polyarthritis mediated by an autoimmune response and following a previously established protocol ( 48 ). Briefly, mice are injected with 50 µL intradermally at the base of the tail with an emulsion containing the extracted type II collagen at 2 mg/mL (Sigma Aldrich, ref. C9301) mixed with Complete Freund’s adjuvant (Sigma Aldrich, ref. F5506) at 4 mg/mL of Mycobacterium tuberculosis (Fischer Scientific, ref. DF3114-33-8).

Paw Swelling Evaluation.

Animals are monitored regularly for clinical signs of arthritis, including joint swelling, redness, warmth, and impaired mobility. These signs are graded on a scale from 0 to 4: 0, No clinical signs of arthritis; 1, Mild swelling and erythema (redness) in one joint; 2, Moderate swelling and erythema in more than one joint; 3, Severe swelling and erythema in most joints, with limited joint mobility; 4, Maximal swelling, erythema, and joint immobility in all joints. Each paw is assessed individually for clinical signs. The cumulative score for all four paws is used to represent the overall disease severity. The thickness of the ankles or paws is measured using calipers. Increased thickness is indicative of joint inflammation. Wheel test was performed by counting the number of revolutions a mouse performs in a closed rodent wheel for 10 min.

Statistical Analysis.

The n values are specified in each legend. The GraphPad Prism software was used to run all the statistical analyses. 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 ANOVA followed by Tukey’s multiple comparisons (for normal distribution) or Kruskal–Wallis (for nonnormal distribution) test. Student’s t test (for normal distribution) or Mann–Whitney (for nonnormal distribution) were 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 a significant difference for the treatment or genotype). The correlations were calculated by linear regression (Pearson’s r). P < 0.05 was considered significant, with: * P < 0.05; ** P < 0.01; P < 0.001 and lower not marked specifically and included in ** P < 0.01.

Results

RA Patients Present with a Large Activation of Humoral Inflammatory Factors.

Baseline characteristics of the control and RA participants are shown in SI Appendix , Table S1 . The groups were similar in age and sex. RA subjects were divided by their DASs and those with higher scores were more likely to have functional limitations, low global scores, higher markers of inflammation and cytokine levels. Both RA groups had similar arthritis-specific antibodies and medication usage.

High-activity RA is characterized by upregulation of inflammatory signals ( 1 – 3 ). To evaluate this dysregulation in our RA patient cohort, we measured the inflammatory response in serum using a large panel of cytokines, chemokines, and adhesion markers ( Table 1 ). The expression of IFN-α and IL-6 was below the detection limit in control donors, but increased in the high-activity group, confirming previous findings ( 49 ). A significant elevation of cytokines (TNF-α, IL-1α, IL-4, IL-12p70, and IL-17A), chemokines (IP-10, MCP-1, and MIP-1β), and an adhesion marker (E-selectin) was measured in RA ( 49 ) but failed to reach a statistically significant increase in our settings.

Table 1. Biomarker concentrations in serum from RA patients Biomarker Control RA high activity P value n (total) 12 20 Female, n 10 17 Male, n 2 3 Age, years 50.3 ± 2.6 46.2 ± 2.4 Reelin (ng/mL) 10.3 ± 1.6 41.8 ± 3.1 ** Adhesion markers (ng/mL) E-selectin 17.0 ± 1.6 24.6 ± 2.3 * P-selectin 86.6 ± 18.0 85.6 ± 16.1 ICAM-1 25.6 ± 5.3 41.2 ± 9.8 Cytokines (pg/mL) IFN-α <detection limit 15.2 ± 7.3 IFN-γ 6.2 ± 0.6 17.4 ± 5.2 TNF-α 10.2 ± 1.0 30.4 ± 7.7 ** IL-1α 1.3 ± 0.7 15.6 ± 5.0 ** IL-1β 0.7 ± 0.3 20.7 ± 8.8 IL-4 9.5 ± 0.7 21.0 ± 2.8 ** IL-6 <detection limit 55.9 ± 33.3 IL-8 5.5 ± 3.0 3.1 ± 1.9 IL-10 <detection limit <detection limit IL-12p70 231.4 ± 22.4 547.8 ± 48.0 ** IL-13 <detection limit 7.4 ± 5.3 IL-17A 2.7 ± 0.8 29.9 ± 12.3 ** Chemokines (pg/mL) IP-10 10.4 ± 1.0 38.0 ± 4.4 ** MCP-1 32.2 ± 11.3 77.8 ± 10.6 ** MIP-1α 7.1 ± 5.2 4.4 ± 1.5 P = 0.08 MIP-1β 82.7 ± 10.2 158.9 ± 29.2 * DAS28 score for patients with RA high-activity is ≥3.5. ICAM: intercellular adhesion molecule, IFN: interferon, TNF: tumor necrosis factor, IL: interleukin, IP: interferon gamma-induced protein, MCP: monocyte chemoattractant, MIP: macrophage inflammatory protein. Detailed cohort information is presented in SI Appendix , Table S1 . * P < 0.05, ** P < 0.01.

Biomarker concentrations in serum from RA patients

DAS28 score for patients with RA high-activity is ≥3.5. ICAM: intercellular adhesion molecule, IFN: interferon, TNF: tumor necrosis factor, IL: interleukin, IP: interferon gamma-induced protein, MCP: monocyte chemoattractant, MIP: macrophage inflammatory protein. Detailed cohort information is presented in SI Appendix , Table S1 . * P < 0.05, ** P < 0.01.

Circulating Reelin Increases and Correlates with Disease Severity in Inflammatory Arthritis Mouse Models and RA Patients.

Originally identified for its involvement in brain development, the glycoprotein Reelin has gained recognition as a regulator of synaptic homeostasis ( 22 – 25 ). Recently, we have unveiled an entirely distinct and unexpected role for Reelin in the systemic circulation, where its expression in the blood rises in response to inflammation ( 40 , 43 ). Here, we show that Reelin is increased in the plasma of mice developing arthritis compared to their appropriated controls ( Fig. 1 A ). This result was replicated in three different inflammatory arthritis mouse models, the spontaneous K/BxN mice, the STA induced in C57BL/6J mice with K/BxN serum injection, and the CIA induced in DAB/1 mice by collagen type II immunization. To confirm this observation in RA patients, we tested Reelin expression in serum from healthy subjects, RA patients with a low-activity disease (DAS28 score < 3.5), and RA patients with a high-activity disease (DAS28 score ≥ 3.5). Western blot analysis of full-length Reelin ( Fig. 1 B and Table 1 ) and ELISA targeting the N-terminal region, which detects both full-length and N-terminal cleaved fragments ( Fig. 1 C ), yielded consistent results ( SI Appendix , Fig. S1 ). The average Reelin concentration measured by ELISA was increased more than twofold in the serum of the low-activity group and fourfold in the high-activity group, suggesting that Reelin expression increases with the severity of the disease.

Serum Reelin is associated with inflammation and disease severity in RA patients. ( A ) Plasma Reelin expression was evaluated by western blot from K/BxN mice at 40 d (controls are KRN negative mice, n = 3-15), STA mice (14 d after induction, controls are plasma before induction, n = 4), and CIA mice (60 d after induction, controls are plasma before induction, n = 8-9). ( B – H ) Detailed cohort information is presented in SI Appendix , Table S1 . ( B ) Serum Reelin levels were measured by western blot from control (n = 11; DAS28 score = 0) and RA patients with low-activity (n = 23; DAS28 score < 3.5) or high-activity (n = 22; DAS28 score ≥ 3.5). Significant variations from single-plex and multiplex ELISA are reported for ( C ) Reelin, ( D ) adhesion markers, ( E ) chemokines, and ( F ) cytokines in the control (CT, n = 12) and RA high-activity groups (n = 20). ( G ) Reelin concentrations (measured by single-plex ELISA) and inflammatory marker concentrations (measured by multiplex ELISA) in control subjects (n = 12) and RA high-activity patients (n = 20) are significantly correlated. ( H ) Significant correlations between Reelin concentration and RA parameters in RA low (n = 23) and high patients (n = 22). * P < 0.05, ** P < 0.01.

Circulating Reelin Correlates with Inflammatory Markers in Patients with High RA Activity.

Significant elevation of inflammatory markers was recapitulated in Fig. 1 C – F and correlated significantly with Reelin concentrations ( Fig. 1 G ). Reelin was significantly associated with the chemokines CXCL10 (IP-10), CCL2 (MCP-1), CCL4 (MIP-1β) and with the cytokines IL-1α, IL-4, IL-12p70, IL-17A, TNF-α. These findings suggest that circulating Reelin parallels the expression patterns of several cytokines and chemokines in RA. In addition, serum Reelin concentration was correlated to clinical parameters, such as DAS, rheumatoid factor titer, and sedimentation rate, suggesting that this circulating factor is associated with the disease severity ( Fig. 1 H ).

Apoer2 KO or Loss-of-Function Protects from Inflammatory Arthritis.

STA, using K/BxN serum transfer into a recipient mouse, is a widely used model for inflammatory arthritis. STA was induced in littermate Apoer2 wild type (WT) and KO mice ( Fig. 2 A and B ). WT mice developed progressive paw swelling within days and reached a plateau after 1 wk. Blinded clinical arthritis scoring and measurement of paw swelling showed that arthritis was significantly reduced in the Apoer2 KO mice. We next explored whether a loss of function mutation in Apoer2 (EIG mutation), which prevents the interaction of this receptor with the cytoplasmic signal transducing adapter proteins Dab1/2, would have a similar effect. Similar to the KO model, Apoer2 EIG mice were almost completely protected from inflammatory arthritis ( Fig. 2 A and C ), as judged by clinical score and paw swelling. These results suggest that disruption of Apoer2 signaling is sufficient to protect from joint inflammation and deterioration in the STA model.

Apoer2 deletion or loss of function protect against inflammatory arthritis. ( A – C ) K/BxN serum injections were performed on day 0 and the mice were followed for 2 wk, with evaluations of the paw clinical score and swelling 3 times per week. The paw swelling fold change was normalized compared to the thickness at day 0, for each paw individually. ( A and B ) Littermate WT and Apoer2 KO mice were induced with STA (n = 9). ( A and C ) Littermate WT and Apoer2 EIG mice were induced with STA (n = 4-7). Apoer2-EIG mutant mice present an Apoer2 NFDNPVY docking motif for Dab1/2 that was rendered nonfunctional to disrupt the Apoer2–Dab interaction, thereby impairing Apoer2 signaling and function. * P < 0.05, ** P < 0.01. ( A ) Modified from ref. 41 .

Prophylactic Anti-Reelin or Diclofenac Interventions Protect against Inflammatory Arthritis.

We have previously demonstrated in endothelial cells that Reelin is a ligand for Apoer2 and activates the expression of surface adhesion molecules, allowing leukocytes to adhere and infiltrate the subendothelial space ( 23 , 33 – 37 ). To study Reelin function, we utilized a well-known monoclonal mouse antibody—CR-50—to specifically target and functionally inhibit this glycoprotein. When injected intraperitoneally, CR-50 is able to deplete Reelin from the circulation ( 39 , 40 , 43 ). We administered intraperitoneal injections of 100 µg of either control monoclonal IgG or CR-50 antibodies twice a week in WT mice. Three days following the first injection, we induced STA in the control (IgG) and CR-50 groups ( Fig. 3 A ). Preventive CR-50 treatment efficiently protected against inflammatory arthritis progression as judged by the clinical score and paw swelling ( Fig. 3 B ). The articulation function and pain were also tested on a running wheel and improved in CR-50-injected mice ( Fig. 3 B ). Furthermore, CR-50 protection against inflammatory arthritis was comparable for these three parameters (clinical score, paw swelling, and running wheel) to the one conferred by preventive diclofenac intervention (0.5 mg/kg/day per os, Fig. 3 C ), a nonsteroidal anti-inflammatory drug and nonprescription medicine widely used in the clinic to treat arthritis. These results suggest that CR-50 targeting Reelin and endothelial adhesion is as efficient as nonsteroidal drugs to prevent or reduce arthritis.

Prophylactic CR-50 or diclofenac interventions protect against inflammatory arthritis. ( A – C ) K/BxN serum injections were performed on day 0 and the mice were followed for 2 wk, with evaluations of the paw clinical score and swelling 3 times per week. The paw swelling fold change was normalized compared to the thickness at day 0, for each paw individually. To evaluate walking capability and pain, the mice were allowed to run freely in a rodent wheel for 10 min and the number of revolutions were recorded. ( A and B ) Randomly assigned littermate mice were injected intraperitoneally with 100 µg of control IgG or CR-50 twice per week, starting 3 d before STA induction which was initiated at day 0 (n = 9). ( A and C ) Randomly assigned littermate mice were treated (per oral) with 0.5 mg/kg/day diclofenac, starting 3 d before STA induction which was initiated at day 0 (n = 6-7). * P < 0.05, ** P < 0.01. ( A ) Modified from ref. 21 .

In accordance with the clinical evaluation of the CR-50-injected mice, histopathological analysis of joint damage showed little to no pannus formation or infiltration of inflammatory cells (neutrophils, lymphocytes, plasma cells, eosinophils) into the synovial membrane compared to controls ( Fig. 4 A ). This evaluation was performed blindly by an experienced pathologist. As expected, plasma Reelin expression was elevated in the control group compared to naïve mice, and this marked increase was completely resolved by CR-50 ( Fig. 4 B ). Hepatic stellate cells likely contribute to basal plasma Reelin secretion ( 21 , 28 – 31 ), while Reelin has also been identified in cartilage, bone ( 50 ), and potentially secreted in inflamed joints by fibroblast-like synoviocytes ( 51 ). Here, we demonstrate that CR-50-induced Reelin depletion effectively decreased the expression of ICAM-1 ( Fig. 4 C ) and inhibited the infiltration of CCR2-positive leukocytes into the synovium ( Fig. 4 D ). Overall, CR-50 lowered the systemic inflammatory response, as judged by the large cytokine and chemokine panel measured in the plasma ( Fig. 4 E ). The inflammatory families most affected by CR-50 were IL-1 (IL-1α, IL-1β, IL-18, IL-33, IL33R), IL-12 (IL12p70, IL-23, IL-27), β common chain cytokines (IL-3, IL-5, GM-CSF), CC chemokines (CCL2, CCL3, CCL5, CCL7, CCL11), and several growth factors (Betacellulin, Leptin, VEGF-A). Collectively, these results demonstrate that prophylactic CR-50 injections in the STA mouse model deplete plasma Reelin and reduce the infiltration of leukocytes into the joints. Therefore, targeting this Reelin-dependent mechanism effectively blocks inflammation and pannus formation in the STA mouse model of arthritis.

Prophylactic CR-50 intervention protects against joint inflammation and degradation. Randomly assigned littermate mice were injected intraperitoneally with 100 µg of control IgG or CR-50 twice per week, starting 3 d before STA induction which was initiated at day 0 and tissues were collected at day 14. ( A ) H&E staining of the hindpaws was used to quantify the severity of joint degradation on a scale from 0 (healthy) to 3 (severe inflammation and damage). ( B ) Reelin protein expression was evaluated in the plasma by western blot. ( C ) ICAM-1 expression and ( D ) CCR2 positive cells abundance was measured by immunohistochemistry in synovial membranes. ( E ) Using a multiplex ELISA, a panel of cytokines, chemokines, and growth factors was measured in plasma samples from naïve mice (n = 10), and STA mice with IgG or CR-50 prophylactic injections (n = 6). Each analyte was normalized to the naïve group and fold change of several analytes combined in families are shown in a heat map. Individual concentration can be found in SI Appendix , Table S2 . Fifteen to seventeen joints were quantified per group from at least 3 different animals, and all the visible joints were quantified (between 4 and 6 per animal, depending on the sections, 0 exclusion). * P < 0.05, ** P < 0.01.

Anti-Reelin or Diclofenac Treatments Mitigate and Reverse Clinically Manifest Inflammatory Arthritis.

To test whether anti-Reelin strategies can be translationally applied to human RA, we performed another series of experiments in which we initiated anti-Reelin treatment after the onset of the disease, in different inflammatory arthritis models ( Fig. 5 A ). We first assessed anti-Reelin therapy in young K/BxN mice, which spontaneously develop arthritis due to a genetic predisposition and an increased susceptibility to an autoimmune response ( 52 ). In this model, control monoclonal IgG or CR-50 treatment (100 µg intraperitoneally, twice a week) was initiated at 25 d after birth, an age at which the early molecular mechanisms of arthritis have been triggered but just before the first clinical manifestation. CR-50 treatment reduced by half the severity of the disease at the plateau (day 40), as judged by clinical score and paw swelling ( Fig. 5 B ). Next, we induced CIA in DAB/1 mice and initiated the IgG or CR-50 treatment at day 28 postimmunization, after the onset of the first clinical sign of arthritis and before the exponential phase. CR-50 treatment stabilized the progression of inflammatory arthritis and blocked the exponential phase ( Fig. 5 C ). Furthermore, CR-50 protection was comparable to diclofenac treatment, also initiated at 28 d (0.5 mg/kg/day per oral, Fig. 5 D ). These results suggest that targeting Reelin and the endothelial adhesion after the onset of the disease is sufficient to block inflammatory arthritis progression.

CR-50 or diclofenac treatments protect against inflammatory arthritis. ( A and B ) Littermate K/BxN mice were randomly assigned to receive intraperitoneal injection of 100 µg of control IgG or CR-50 twice per week, starting at 25 d old after the early molecular mechanism triggering spontaneous arthritis is engaged (n = 6-7). ( A , C , and D ) DBA/1 mice were immunized with collagen type II at day 0. Starting at day 28 after immunization and after the first clinical sign of paw inflammation, littermate mice were randomly assigned into three groups: a control group (n = 7), a group injected intraperitoneally with 100 µg of CR-50 twice per week (n = 5) ( C ), or a group treated (per oral) with 0.5 mg/kg/day diclofenac (n = 4) ( D ). Since all the mice were littermates, the same control group was used in the CR-50 or diclofenac study. * P < 0.05, ** P < 0.01.

Following clinical evaluation of CR-50-treated mice, histopathological analysis of joint damage revealed minimal to no pannus or infiltration of inflammatory cells (neutrophils, lymphocytes, plasma cells, and eosinophils) into the synovial membrane compared to controls ( Fig. 6 A ). As anticipated, plasma Reelin expression was elevated in the control group compared to naïve mice, and this significant increase was reversed by CR-50 treatment ( Fig. 6 B ). In line with our previous results in the STA model, CR-50 treatment effectively decreased ICAM-1 expression ( Fig. 6 C ) and inhibited the infiltration of CCR2-positive leukocytes into the synovium ( Fig. 6 D ). This treatment also reduced the systemic inflammatory response, as evidenced by the cytokine and chemokine panel measured in the plasma ( Fig. 6 E ). Notably affected cytokine families included β common chain cytokines (IL-3, IL-5, GM-CSF) and TNF (TNF-α, BAFF, RANKL). Noteworthy, GM-CSF protein was significantly increased in the CIA control group and was reduced back to naïve levels in the CR-50 treated group ( SI Appendix , Table S3 ). Similarly, the levels of IL-28, CXCL2 (MIP2-α), and betacellulin, which were initially undetectable, became detectable after CIA induction, and then decreased to undetectable levels again following CR-50 treatment. Overall, these findings in the CIA mouse model demonstrate that CR-50 treatment after the onset of arthritis is sufficient to deplete plasma Reelin and stop leukocyte infiltration into the joints, effectively blocking the progression of inflammation and paw swelling.

CR-50 treatment protects against joint inflammation and degradation. Randomly assigned littermate mice were injected intraperitoneally with 100 µg of control IgG or CR-50 twice per week, starting 28 d after CIA induction which was initiated at day 0, and tissues were collected at day 60. ( A ) H&E staining of the forepaws was used to quantify the severity of joint degradation on a scale from 0 (healthy) to 3 (severe inflammation and damage). ( B ) Reelin protein expression was evaluated in the plasma by western blot. ( C ) ICAM-1 expression and ( D ) CCR2 positive cell abundance was measured by immunohistochemistry in synovial membranes. ( E ) Using a multiplex ELISA, a panel of cytokines, chemokines, and growth factors was measured in plasma samples from naïve mice (n = 5), and CIA mice treated with IgG or CR-50 (n = 7). Each analyte was normalized to the naïve group and fold changes of several analytes combined in families are shown in a heat map. Individual concentration can be found in SI Appendix , Table S3 . Twenty-six to forty-five joints were quantified per group from at least 3 different animals, and all the visible joints were quantified (between 7 and 19 per animal, depending on the sections, 0 exclusion). * P < 0.05, ** P < 0.01.

Discussion

Joints affected by RA are characterized by an inflammatory environment releasing cytokines and chemokines that activate endothelial cells, leading to endothelial breakdown and facilitating leukocyte adhesion/infiltration into the synovium. This study investigated the potential of targeting the Reelin/Apoer2 pathway to restore endothelial barrier function and reduce leukocyte migration into the synovial space, thereby limiting joint inflammation in RA. The results reaffirm the critical role of the endothelial pathologic response and leukocyte infiltration in RA pathogenesis, in agreement with the established literature ( 19 , 53 , 54 ). Reelin was found to be elevated in the plasma of arthritis mouse models and RA patients. By demonstrating the correlation between circulating Reelin levels and various inflammatory markers as well as disease severity in both mouse models and human RA patients, the study underscores Reelin's potential as a biomarker for disease activity and progression. Moreover, disruption of Apoer2, a receptor for Reelin, provided protection against inflammatory arthritis in mice. Accordingly, inhibiting Reelin with the CR-50 antibody demonstrated potent anti-inflammatory effects in both prophylactic and interventional treatments. The ability of Apoer2 mutant mice as well as anti-Reelin CR-50 treatment to mitigate inflammation and improve clinical symptoms are comparable to standard anti-inflammatory drugs like diclofenac, highlighting the therapeutic potential of targeting this pathway in RA. Additionally, the study sheds light on the intricate relationship between Reelin, endothelial adhesion, and leukocyte infiltration, offering insights into the underlying mechanisms of RA pathophysiology. Overall, the study provides insights into the role of the Reelin/Apoer2 pathway in RA ( Fig. 7 ) and lays the groundwork for the development of alternative therapeutic interventions targeting endothelial dysfunction and inflammation in this debilitating autoimmune disease.

Illustrations of Reelin relations to inflammation in arthritis. ( A ) To illustrate the relationship between inflammatory factors and Reelin, we generated distribution plots for STA, CIA, and RA human cohorts. In the plots for STA and CIA, targets positioned in the Bottom Right corner exhibited increased expression during inflammatory arthritis compared to the naïve group, which was subsequently reduced by CR-50 treatment. Conversely, targets located in the Upper Left corner showed decreased expression during arthritis but were increased by CR-50 treatment (denoted by proteins highlighted in blue). In the human RA cohort, correlation coefficients (R 2 ) were utilized to demonstrate their association with Reelin. Protein distributions were color-coded: red labeling indicated proteins displaying a consistent pattern across all three cohorts, orange denoted those observed in both mouse cohorts, and pink represented protein present in one mouse cohort and the human cohort. ( B ) This representation is based on the literature and the findings presented in this study to illustrate the function hypothesized for Reelin in the pannus formation. Reelin via its receptor Apoer2 promotes the expression of vascular adhesion proteins, thereby increasing monocyte adhesion/extravasation, inflammation and consequently demyelination. Both Reelin or Apoer2 disruption prevent this inflammatory cascade and thus pannus formation in murine inflammatory arthritis models. We surmise that the same mechanism is conserved in humans suggesting a nonimmunosuppressive, therapeutic approach to a broad spectrum of inflammatory diseases. Modified from ref. 21 .

Once a debilitating condition lacking effective treatments, RA can now be controlled through early diagnosis and treatment with conventional DMARDs such as methotrexate or biological therapies targeting immune components such as TNFα or other cytokines ( 55 ). Nevertheless, a significant proportion of patients either fail to respond to treatment or lose efficacy over time ( 56 ). The nonprescription drug diclofenac, easily accessible to patients, can increase the risk of serious gastrointestinal adverse events including bleeding, ulceration, and perforation of the stomach or intestines, which can be fatal ( 57 ). While immune system targeting poses limited primary infection risks (4 to 5 events per 100 patients per year), distinct properties of biological DMARD classes introduce specific risks, some manageable like tuberculosis with TNF inhibitors, or rare like gastrointestinal perforations with tocilizumab and progressive multifocal leukoencephalopathy with rituximab ( 55 , 58 ). To overcome these limitations and unveil novel targets for RA, it proves innovative to move beyond immunotherapies and explore alternative components. Endothelial activation prompts increased leukocyte passage into joints due to heightened endothelial adhesiveness and permeability ( 59 ). Thus, identifying mechanisms governing endothelial adhesion can reveal master regulators outside the immune system that oversee a spectrum of adhesion proteins and inflammatory markers. This approach avoids fully suppressing the expression of one adhesion protein (such as anti-ICAM-1 approach), thus preserving baseline activity and physiological function.

To address this problem, we propose targeting Reelin as a regulator of endothelial adhesion and permeability to leukocytes. Since the first publication on Reelin-deficient mice (reeler mice) in 1951 ( 60 ), Reelin has been described as beneficial in the brain ( 22 – 25 ); however, our previous work has exposed an unexpected duality in Reelin function depending on its localization ( 21 ). Unlike the positive effect on synaptic functions in the brain, we have previously demonstrated that targeting the Reelin pathway efficiently suppressed NF-κB-mediated endothelial activation, as judged by reduced expression of endothelial adhesion markers, adhesion, and infiltration of leukocytes ( 21 , 38 – 43 ). In line with our previous results, this study demonstrates in preclinical inflammatory arthritis models that the disruption of Apoer2 or the depletion of its ligand Reelin in the plasma significantly reduces leukocyte infiltration into the synovium, inhibits systemic inflammatory responses, and improves clinical symptoms. Altogether, these findings suggest anti-inflammatory effects are also mediated by the restoration of the endothelial barrier function.

In addition to the seven animal models and treatment modalities presented in this study, we have conducted a retrospective clinical study to demonstrate the relevance of Reelin in humans. First, Reelin concentration in the serum of RA patients correlates with disease severity, confirming our mouse data. Furthermore, the Reelin concentration also correlates with the elevation of several cytokines (IL-1α, IL-4, IL-12p70, IL-17A, and TNF-α) and chemokines (IP-10 [CXCL10], MCP-1 [CCL2], and MIP-1β [CCL4]). In a prior study involving COVID-19 patients experiencing a cytokine storm, elevated Reelin was identified in association with four inflammatory factors: IL-1α, IL-4, IP-10, and MIP-1β ( 43 ). All these associations in COVID-19 have been reaffirmed within this RA cohort, thereby enhancing confidence in their relation with Reelin, directly or indirectly. Moreover, to better understand which inflammatory factors parallel the Reelin profile or are related to Reelin across our experiments, we examined the distribution of inflammatory factors and their relation with Reelin across STA, CIA, and RA human cohorts. We have sorted the proteins to isolate a common pattern, with a special focus on targets that are increased in inflammatory arthritis and reduced by CR-50 treatment in mice, as well as increased in RA patients and correlated to Reelin in the human cohort ( Fig. 7 A ). IL-12p70 and IL-13 followed this pattern in all 3 models, as well as IL-3, IL-9, IL-28, IL-31, Betacellulin, GM-CSF, Leptin, MIP-2α, and RANKL in both mouse models and IL-1α, IL-1β, IL-6, IP-10, MCP-1, MIP-1β, and TNF-α in one mouse model and the human cohort. Further studies are needed to fully understand the mechanisms underlying the potential relationship between Reelin and these factors, and test whether modulating Reelin levels could potentially influence the expression of a wide range of inflammatory factors. Studying this important question would determine whether a comprehensive approach targeting Reelin is more effective and safer than therapeutic strategies focused solely on abolishing the expression or function of one individual inflammatory factor.

Preclinical and clinical studies suggest that direct targeting of plasma Reelin is a safe approach for patients ( 61 ). First, peripheral anti-Reelin antibody treatment efficiently reduces plasma Reelin levels without compromising Reelin levels or crucial neuronal functions within the central nervous system (CNS), thus preserving CNS integrity ( 40 ). Second, extensive studies with Reelin conditional knockout mice and anti-Reelin drugs over more than two decades have not shown increased susceptibility to inflammatory diseases or infections in Reelin-depleted mice. On the contrary, recent research indicates that reducing plasma Reelin levels with CR-50 treatment actually protects against severe disease following infection of hACE2 mice with SARS-CoV-2, suggesting that it does not compromise the immune system's ability to fight off infections ( 43 ). Last, clinical evidence from patients with RELN mutations, resulting in low or loss of Reelin expression, confirms the safety of targeting plasma Reelin in adults, as these patients do not experience immunodepression despite neurological abnormalities associated with Reelin absence during development ( 62 ).

This proof-of-concept study has several limitations. First, the major aim was to test the efficacy of anti-Reelin interventions against the progression of inflammatory arthritis. To introduce a comparison point, we compared our strategy against diclofenac, a nonsteroidal anti-inflammatory drug (NSAID). This anti-inflammatory agent was strategically selected as our positive control based on several key criteria: 1) its well-documented efficacy in both arthritis models, ensuring reliable validation of our experimental approach, 2) its established route of administration, which minimizes technical variability and animal stress, and 3) its robust anti-inflammatory properties, which provide a clear benchmark against which to measure treatment effects. The goal of this study was to establish preclinical proof-of-novel-concept for the inhibition of Reelin/Apoer2 signaling, rather than comparing its efficacy against inhibiting TNFα or other cytokines. In neurons, the major receptors for Reelin are Apoer2 and VLDLR ( 23 , 25 ). By contrast, in endothelial cells only Apoer2 is required to relay the Reelin signal ( 38 ). While the present study has focused primarily on the role of Reelin in endothelial cells, Reelin may also exert an effect on inflammatory cells that express these receptors, such as macrophages ( 63 , 64 ). Finally, the various sources of plasma Reelin have not been fully explored. The liver, specifically hepatic stellate cells, is considered the primary source of Reelin outside the brain ( 28 – 32 ). However, in our KBxN mice, which exhibit nearly a fourfold increase in plasma Reelin expression, mRNA levels in the liver remain unchanged ( SI Appendix , Fig. S2 ). This finding suggests that the elevated plasma Reelin levels originate from an alternative source, which has yet to be identified. While a previous study reported elevated Reelin levels in the synovial fluid of RA patients ( 51 ), these findings were based on a limited sample size and could not be replicated in our experimental models. Our observations suggest that any Reelin present in the synovial fluid likely originates from plasma extravasation during inflammatory processes, rather than from any significant local production by synoviocytes. This is supported by the observation that synoviocyte Reelin expression was minimal in our mouse models.

Conclusion

This study highlights the significance of the Reelin/Apoer2 pathway in inflammatory arthritis and proposes it as a promising therapeutic target. Elevated Reelin levels correlate with disease severity in both mouse models and human RA patients. Disrupting this pathway, either through mutations in Apoer2 or therapeutic Reelin inhibition with the CR-50 antibody, effectively protects against inflammatory arthritis progression, comparable to the standard anti-inflammatory drug diclofenac. Targeting the endothelial factor Reelin reduces leukocyte infiltration into the synovium, inflammation, and pannus formation. These findings suggest Reelin as a potential biomarker for RA progression and provide an alternative therapeutic avenue beyond conventional immunotherapies. Overall, this study advances our understanding of RA pathophysiology and offers a promising direction for future therapeutic development for this disease and beyond to chronic inflammatory syndromes such as neurodegenerative diseases (multiple sclerosis or Alzheimer’s disease), atherosclerosis, Crohn’s disease, and others.

Supplementary Material

Appendix 01 (PDF)