Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy

Abstract

The molecular determinants of pathogenic leukocyte migration across the blood-nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment-1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte α₄ integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine-treated, flow-dependent in vitro BNB model system. Time-lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24-week old female B7-2 deficient non-obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2mg/kg FNCS1 peptide for 5days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25μM FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of inflammatory demyelination. Microvessels demonstrating FNCS1 expression and CD49d+ leukocytes were seen within the endoneurium of patient nerve biopsies. Taken together, these results imply a role for FNCS1 in pathogenic leukocyte trafficking in CIDP, providing a potential target for therapeutic modulation.

Keywords: Blood-nerve barrier; Chronic inflammatory demyelinating polyradiculoneuropathy; Fibronectin connecting segment-1; Integrin; Leukocyte trafficking; Spontaneous autoimmune peripheral polyneuropathy.

Figure 1. Effect of FNCS1 antagonism on CIDP patient PBML trafficking at the blood-nerve barrier in vitro

25 μM of FNCS1 peptide EILDVPST significantly reduced the flow-dependent untreated CIDP patient PBML adhesion and transmigration following endothelial cytokine activation (CA) to basal levels, while a control peptide that lacks the LDV sequence (FNCS1C: DELPQLVTL) demonstrated no effect. Neither human IVIg nor 6-α methylprednisolone (6-AMP) modulated CIDP PBML trafficking at the human BNB relative to uninhibited conditions in vitro. N=7 (ns: not significant, ^* indicates p<0.05)

Figure 2. Effect of FNCS1 antagonism on neuromuscular severity scores (NMSS) in murine SAPP

2 mg/kg FNCS1 peptide administered daily for 5 days significantly reduced disease severity with improvement in motor function compared to control FNCS1C peptide and human IVIg-treated mice 8 days after initial treatment. Significant reductions in mean NMSS were observed between FNCS1 peptide treatment and all control drugs from day 11 post-treatment that persisted throughout the experimental course. N=50, with 4 independent cohorts studied. ^* p<0.05, ^** p < 0.01, ^**** p<0.0001 relative to FNCS1 peptide treatment

Figure 3. Effect of FNCS1 peptide antagonism on motor electrophysiology in murine SAPP

Bar histograms of mean motor electrophysiology parameters at day 30 post-treatment from the dorsal caudal tail nerves (DCTN, a–c) and the sciatic nerves (d–f) comparing FNCS1 peptide treatment with control FNCS1C peptide-, human IVIg-and 6-α methylprednisolone (6aMP)-treated mice is shown. Significantly increased conduction velocities (a and d) were seen following FNCS1 peptide treatment compared the other experimental treatments, with reduction in total distal waveform duration relative to FNCS1C peptide treatment only (b and e). There were no differences in distal compound motor unit action potential (CMAP) amplitudes between the experimental groups (c and f). These data imply FNCS1 peptide efficacy against demyelination of the fastest firing large myelinated axons in SAPP. N= 50, with 4 independent cohorts studied. ^** p<0.01, ^*** p<0.001, ^**** p<0.0001 and ns = not significant relative to FNCS1 peptide treatment

Figure 4. Qualitative effect of FNCS1 antagonism on inflammatory demyelination in murine SAPP

Representative digital indirect fluorescent photomicrographs from the sciatic nerves of SAPP-affected mice 30 days after treatment initiation show the normal honeycomb appearance of myelinated axons with S100β staining [green]) following FNCS1 treatment (a) in contrast to multiple foci of inflammatory cells (blue) associated with endoneurial architecture disruption (altered honeycomb appearance) consistent with demyelination in FNCS1C peptide-treated mice (b). Normal axonal density (NF-H staining [red]) with rare mononuclear cells is shown in a FNCS1 peptide-treated mouse (c), in contrast to the intense diffuse inflammatory infiltrate associated with focal axonal loss in a FNCS1C peptide-treated mouse (d). The variability in demyelination and focal axonal loss associated with endoneurial mononuclear cell infiltration following IVIg- (e–h) and 6αMP- (i– l) treated SAPP-affected mice is also shown. These images were obtained from serial sections containing CD45+ leukocytes. Scale bars = 50 μm

Figure 5. Quantitative effect of FNCS1 antagonism on demyelination in murine SAPP

Bar histograms indicating the mean demyelinated area per plastic-embedded, toluidine-blue-stained, semithin sciatic nerve sections from SAPP-affected mice 30 days after treatment initiation show a significant reduction following FNCS1 peptide treatment compared with FNCS1C peptide-, IVIg- and 6-alpha methylprednisolone (6aMP)-treated mice (a), supporting the motor electrophysiological data. A total of 52 sections were analyzed. ^* p<0.05, ^** p<0.01 and ^*** p<0.0001 relative to FNCS1 peptide treatment. Representative digital light photomicrographs show relative paucity of demyelination in a FNCS1 peptide-treated SAPP-affected mouse (b), in contrast to regions of mononuclear leukocyte infiltration with demyelination and axonal loss in FNCS1C peptide- (c), human IVIg- (d) and 6αMP- (e) treated mice (white arrows). More diffuse inflammatory demyelination was seen with FNCS1C peptide treatment. The insert in (c) demonstrates completed demyelinated axons (white asterisk) associated with mononuclear cells (300% magnification relative to initial photomicrograph). Scale bars = 50 μm

Figure 6. Effect of FNCS1 antagonism on inflammation in murine SAPP

Bar histograms of the mean numbers of sciatic nerve endoneurial CD45+ leukocytes of SAPP-affected mice 30 days after treatment initiation are shown (a). Significant reductions in CD45+ leukocytes were observed following FNCS1 peptide treatment compared to mice treated with FNCS1C peptide, human IVIg and 6-alpha methylprednisolone (6aMP). These data imply a robust inhibitory effect of FNCS1 peptide on hematogenous leukocyte infiltration into the endoneurium in SAPP. A total of 110 sections were analyzed. ^* p <0.05 and ^** p<0.01 relative to FNCS1 peptide treatment. Representative digital indirect fluorescent photomicrographs show the paucity of infiltrated CD45+ leukocytes (red) in a FNCS1 peptide-treated mouse (b) compared to diffuse endoneurial infiltration seen following FNCS1C peptide treatment (c). The variability in leukocyte infiltration following human IVIg (d and e) and 6αMP (f and g) treatment in SAPP is also demonstrated. Scale bars = 50 μm

Figure 7. FNCS1 and α4 integrin (CD49d) expression in CIDP patient nerve biopsies

Digital indirect fluorescent photomicrographs of an axial section from the sural nerve biopsy of an untreated CIDP patient demonstrates a cluster of round mononuclear cells (a: DAPI, blue) associated with a FNCS1 expressing (b: red) endoneurial microvessel (c: UEA-1, green), verified by co-localization in the merged image (d: yellow). Longitudinal sural nerve biopsy sections show multiple small foci of endoneurial CD49d+ CD45+ leukocytes at lower magnification (e–h). Higher magnification photomicrographs from different untreated CIDP patients show clusters of endoneurial CD49d+ CD45+ leukocytes (i and j: white arrows). Extravasating and perivascular CD49d+ CD45+ leukocytes are also seen (k and l). CD49d+ CD45+ leukocytes were rarely seen within the endoneurium of normal control sural nerve biopsies (m: white arrow). EMV= endoneurial microvessel. Scale bars = 50 μm (a–d), 100 μm (e–h), 40 μm (i, j and m) and 20 μm (k and l)