Inhibition of CXCR2 signaling promotes recovery in models of multiple sclerosis

Abstract

Multiple sclerosis (MS) is a neurodegenerative disease characterized by demyelination/remyelination episodes that ultimately fail. Chemokines and their receptors have been implicated in both myelination and remyelination failure. Chemokines regulate migration, proliferation and differentiation of immune and neural cells during development and pathology. Previous studies have demonstrated that the absence of the chemokine receptor CXCR2 results in both disruption of early oligodendrocyte development and long-term structural alterations in myelination. Histological studies suggest that CXCL1, the primary ligand for CXCR2, is upregulated around the peripheral areas of demyelination suggesting that this receptor/ligand combination modulates responses to injury. Here we show that in focal LPC-induced demyelinating lesions, localized inhibition of CXCR2 signaling reduced lesion size and enhanced remyelination while systemic treatments were relatively less effective. Treatment of spinal cord cultures with CXCR2 antagonists reduced CXCL1 induced A2B5+ cell proliferation and increased differentiation of myelin producing cells. More critically, treatment of myelin oligodendrocyte glycoprotein peptide 35-55-induced EAE mice, an animal model of multiple sclerosis, with small molecule antagonists against CXCR2 results in increased functionality, decreased lesion load, and enhanced remyelination. Our findings demonstrate the importance of antagonizing CXCR2 in enhancing myelin repair by reducing lesion load and functionality in models of multiple sclerosis and thus providing a therapeutic target for demyelinating diseases.

Figure 1. Local delivery of anti-CXCR2 antibodies reduces the size of LPC induced demyelinated lesions

Vibratome sections labeled with Luxol fast blue (A-D) of LPC lesions 10 days after induction treated with isotype control IgG (A and C) or neutralizing antibodies against CXCR2 (R&D Systems; 100μg/ml) (B and D) 2 days after lesion induction. Longitudinal sections (A and B) and cross sections (C and D) from mid-lesion areas demonstrate a reduction in lesion volume in anti-CXCR2 treated animals (B and D) compared to isotype controls (A and C). Three dimension reconstruction of LPC lesions (yellow) from isotype controls (E) and anti- CXCR2 treated animals (F). Comparison of the average LPC lesion volume in IgG control and anti-CXCR2 treated animals. n = 8 p= 0.005. Bar = 100μm in A-D (G). In E and F, blue = central canal, yellow = lesion, and red = outline of the spinal cord.

Figure 2. Local delivery of CXCR2 antagonists enhances remyelination in LPC lesions

Labeling of 1 μm sections through the middle LPC lesion area with Toluidine blue (A and B) demonstrates an increase in the number of myelinated axonal profiles in animals that received a single injection of a CXCR2 antagonist 2 days after LPC injection. Ultrastructural analyses confirmed the presence of unmyelinated (asterisks) and thinly myelinated (arrows) axons in experimental animals (D). Lesions from control animals contain largely unmyelinated (asterisks) axons and a higher number of cell bodies (C). Quantification of the proportion of remyelinated versus demyelinated axons in lesions from vehicle and small molecule antagonist treated animals demonstrated a 20-fold increase (p=0.002) in remyelinated axons versus control with direct delivery into LPC lesions and a 9-fold increase in remyelinated axons (p=0.004) with systemic delivery (E). Bar = 20μm in A and B and 2μm in C and D.

Figure 3. Systemic delivery of CXCR2 antagonists has limited effect on repair of LPC lesions

Labeling of 1 μm sections with toluidine blue (A and B) demonstrated no significant change in overall lesion size in animals treated daily with IP injections of CXCR2 antagonist, although experimental animals demonstrated an apparent reduction in the cellularity of lesions (B and D). Electron microscopy confirmed the limited effect of systemic inhibition of CXCR2 in promoting LPC lesion repair (C and D). Occasional thinly myelinated axons (arrows) were present scattered throughout the lesion in CXCR2 antagonist treated animals (D) that were absent in controls (C) which had a higher number of unmyelinated axons (asterisks). The myelin surrounding remyelinating axons was frequently uneven, possibly reflecting active remyelination. Bar in A and B = 20 μm and C and D = 2μm.

Figure 4. Inhibition of CXCR2 promotes the differentiation of spinal cord OPCs in vitro

Spinal cord mixed cell cultures (P3) grown for 5 days in vitro contain 35% A2B5+ cells (A) and approx 19% O1+ (D) and 17% MBP+ cells (G). Exposure to 0.05ng/ml CXCL1 for 24hrs results in an increase in A2B5+ cells (46%) (B) but a decrease in O1+ (12%) (E) and MBP+ (12%) cells (H). By contrast, exposure to CXCR2 antagonist (40ng/ml) for 24hrs results in a decrease in A2B5+ cells (26%) (C) but an increase in O1+ (31%) (p=0.05) (F) and MBP+ cells (50%) (p=0.007) (I) to the proportion of immunopostive cells compared to DAPI+ cell numbers (J). Data represents mean +/− standard deviation taken from 2 coverslips from at least 3 separate experiments. Bar = 20 μm in A-F and Bar = 40μm in G-I.

Figure 5. Systemic inhibition of CXCR2 results in functional improvement in MOG_35-55 peptide induced EAE animals

(A) Injection of MOG_35-55 peptide induces a robust functional deficit after 7-10 days that plateaus in the 2^nd week (circles). Systemic daily delivery of CXCR2 inhibitor (20ng/kg) results in a slowing of disease progression and functional recovery that is maintained with continuous treatment (squares). Data represent mean clinical scores for 12 animals from 3 separate studies. Luxol fast blue staining on transverse spinal cord sections showed areas of demyelination in control EAE animals (B) that were significantly reduced in animals treated with CXCR2 antagonists (C). Control EAE animals showed significant cellular infiltrates in areas of demyelination (D) that were reduced in animals treated with CXCR2 antagonists (E). Quantitation of the relative lesion load in the spinal cord of control EAE animals and CXCR2 antagonist treated animals at day 28 after immunization (F). Data represents the mean +/− standard deviation taken from 20 Luxol stained sections from each of 4 animals in each group. p= 0.0001. Bar = 100μm in B and C and 50μm in D and E.

Figure 6. Systemic inhibition of CXCR2 results in decreased cell infiltration and increased remyelination in MOG_35-55 induced EAE

In control EAE animals, 28 days after disease induction, lesion areas in the spinal cord were characterized by demyelination and cellular infiltrates (A). By contrast, in EAE animals treated daily with CXCR2 antagonists the extent of demyelination and cellular infiltration is substantially reduced (B). Ultrastructural studies confirmed the presence of extensive demyelinated axons (asterisks) in lesion areas associated with substantial cell infiltration (C). In animals treated with CXCR2 antagonist (D) the number of demyelinated axons was reduced and the number of thinly myelinated (arrows) axons increased consistent with widespread remyelination. Quantification of the relative numbers of demyelinated (asterisks), remyelinated (arrows), and unaffected axons in lesion areas of control CXCR2 antagonist treated EAE animals (E). The number and proportion of demyelinated axons is significantly decreased in CXCR2 antagonist treated animals compared to controls (90% vs 25%) while the number and proportion of remyelinated axons was significantly increased (10% vs 65%) (p= 0.000002) (E). The total number of axons was not significantly different in treated and untreated animals and the proportion of unaffected axons was below 10% in both groups (E). Remyelinated axons were characterized as having thinner myelin sheaths and quantification of 20 axons per lesion from 5 lesion areas in each of 3 animals demonstrated a significant reduction (p= 0.02) in myelin thickness/axon diameter ratio in treated animals (F). Control EAE animals had a ratio close to 1 suggesting little remyelination while CXCR2 antagonist treated animals had a mean ratio around 3, indicative of substantially thinner myelin sheaths (F). Bar = 75μm in A and B and 2 μm in C and D.

Figure 7. Systemic treatment with CXCR2 antagonists result in increased MBP and decreased Iba1 expression in EAE animals

Control EAE animals demonstrated reduced MBP labeling (A and C) and high levels of Iba1+ cells in lesion areas (arrowheads) (E and G. By contrast, animals treated with CXCR2 antagonist demonstrated higher levels of MBP expression (B and D) and fewer Iba1+ cells (F and H)in lesion areas (arrowheads). The number of Iba1+ cells in CXCR2 antagonist treated EAE animals was decreased by 50 +/− 5% in grey and white matter regions compared to vehicle treated animals (G and H). . Bar = 500μm in A, B, E and F and 200μm in C, D, G and H. .

Figure 8. Long-term but not short-term treatment with CXCR2 antagonist results in sustained remyelination

EAE animals treated with CXCR2 antagonist systemically for a period of 2 weeks and then removed from treatment demonstrated persistent remyelination 1 week later (A). By contrast, ., EAE animals removed from CXCR2 antagonist treatment after 1 week of treatment demonstrated a lack of remyelination, although there was an apparent reduction in inflammatory cell infiltration (B). . Bar = 50μm.