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 2 mg/kg FNCS1 peptide for 5 days 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. 25 μM 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.

Deciphering immune mechanisms in chronic inflammatory demyelinating polyneuropathies

“…FCNS1 is an alternative binding partner of integrin α 4 (CD49d) on proinflammatory leukocytes, and blocking FCNS1 in this mouse model inhibits development of PNS autoimmunity. In nerve biopsies of patients with CIDP, FCNS1 is upregulated in microvessels, and anti-FCNS1 antibodies inhibit trafficking across the human BNB in vitro (68). These studies, thus, demonstrate the importance of understanding how PNS-intrinsic cells are altered with PNS autoimmunity and how they participate in the inflammatory process.…”

Glial-derived neurotrophic factor is essential for blood-nerve barrier functional recovery in an experimental murine model of traumatic peripheral neuropathy

“…15 min afterwards, mice were anesthetized with ketamine-xylazine (100 mg-10 mg/kg body weight) in 100 μL1X PBS and both sciatic nerves were harvested 30 min after injection, as previously published. 63,67,[74][75][76] Nerve alignment was maintained by placing on filter paper with the sciatic nerve crush injury site indicated. The nerves were fixed with freshly prepared 3% glutaraldehyde in 0.1 M cacodylate buffer containing 2 mM CaCl 2 for 4 hours in the dark at room temperature.…”

“…Following post-fixation, the sciatic nerves were transected at the site of sciatic nerve crush and embedded in epoxy resin side-by-side with the crushed ends superficially placed to generate plastic-embedded blocks, as previously published. 67,74,75 Light microscopy Plastic-embedded sciatic nerve blocks were trimmed and axially sectioned with an LKB Ultrotome III ultramicrotome using a DiATOME Histo diamond knife. 0.5-1.0 µm semi-thin sections were mounted on glass slides and stained with basic fuchsin, followed by methylene blue counterstain.…”

“…Indirect immunohistochemistry was performed on serial 10 µm thick axial cryostat sections to detect and quantify VE-Cadherin (marker of adherens junctions) and claudin-5 (marker of tight junctions) on CD31 + endoneurial microvessels within mouse sciatic nerves, using previously published protocols. 63,67,68,[74][75][76] Each axial section consisted of the two adjacent sciatic nerve sections from the crush-injured site. Sections were fixed in acetone for 10 min at -20°C, washed and air-dried prior to blocking with 10% normal goat serum in 1X PBS for 1 hour at room temperature.…”

GDNF enhances human blood-nerve barrier functionin vitrovia MAPK signaling pathways

“…4,5 Specific adaptations are likely needed, guided by in vivo and in situ observations, to develop a more physiological human BNB model for in vitro manipulation with higher translational potential or biological relevance, as observed with a flow-dependent leukocyte-BNB model designed to study peripheral neuroinflammation. 48,49 Endothelial cells are serum-dependent for attachment, and serum withdrawal is associated with diffuse reduction in continuous intercellular membrane contacts in sub-confluent proliferating cells. 46 However, serum withdrawal is also a wellestablished technique to suppress growth, induce contact-inhibition and promote intercellular junction formation by confluent epithelial and endothelial cells in vitro.…”