Chondroitin sulfate proteoglycans as novel drivers of leucocyte infiltration in multiple sclerosis

Abstract

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with leucocytes in inflammatory perivascular cuffs in all four patients. We conclude that the accumulation of chondroitin sulfate proteoglycans in the perivascular cuff in multiple sclerosis and experimental autoimmune encephalomyelitis boosts the activity and migration of leucocytes across the glia limitans into the central nervous system parenchyma. Thus, chondroitin sulfate proteoglycans represent a new class of molecules to overcome in order to reduce the inflammatory cascades and clinical severity of multiple sclerosis.

Enhanced glycolytic metabolism supports transmigration of brain-infiltrating macrophages in multiple sclerosis

“…In the inflamed postcapillary venules of EAEafflicted mice, leukocytes that have migrated out of the vessel lumen across the endothelial cell layer accumulate within the perivascular space between the endothelial and parenchymal basement membranes (2,3); the collective structure is referred to as a perivascular cuff ( Figure 1A). Previous studies have emphasized that leukocytes within the perivascular space have remarkable functions, including antigen presentation (6) and secretion of MMPs (24), and these actions probably have high metabolic demands. In support of this, we observed that the expression of LDHA, a key glycolytic enzyme involved in the conversion of pyruvate to lactate, was strongly upregulated in the perivascular cuff on day 16 (D16, a period of prominent clinical disability) in the white matter of the cerebellum, a CNS region where perivascular cuffs are clearly demarcated in EAE ( Figure 1B).…”

“…We tested whether LDHA modulates proinflammatory activities and aids leukocyte infiltration across the BBB. We resorted to macrophages in culture, as these cells typically constitute more than 80% of leukocytes within perivascular cuffs at peak EAE (24). When bone marrow-derived macrophages (BMDMs) were exposed to a nontoxic (20 μM; Figure 2A) concentration of a small-molecule-specific LDHA inhibitor, 3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid (FX11) (12, 31), we detected a significant decrease in secreted lactate levels as compared with LPS-treated cells ( Figure 2B), indicating a decrease in LDHA function.…”

Versican—A Critical Extracellular Matrix Regulator of Immunity and Inflammation

“…Versican is essential during development (76,77) and it is now becoming apparent that it is an important component of the tissue inflammation caused by infection and tissue injury (10). Versican accumulates as part of the early inflammatory response in a number of human diseases often associated with the invasion of leukocytes including those in the vascular system (10,40,(78)(79)(80)(81)(82)(83)(84), lung (5,6,60,77,(85)(86)(87)(88)(89), brain and spinal cord (53,(90)(91)(92), intestine (93)(94)(95)(96), heart (97), liver (63), skin (98,99), eye (100, 101), pancreatic islets (102), and many different forms of cancer [reviewed in (103)(104)(105)]. The accumulation of versican in these tissues is usually associated with other ECM components that bind versican, such as hyaluronan (106,107), link protein, TSG-6, IαI, and CD44 (95, 96, 108-111) (Figure 1).…”

“…These cable-like structures contain hyaluronan, versican, TSG-6, and IαI that bind different subsets of leukocytes (93,94,112,(114)(115)(116)(137)(138)(139)(140)(141)(142)(143)(144)(145)(146)(147). Such structures have not only been found in vitro, but also in diseased tissues, such as in atherosclerosis (10,84), inflammatory bowel disease (IBD) (94), and in brain and spinal cord injury (53,91). Further studies are needed to determine the contribution of each of the components in the complex on leukocyte phenotype.…”

Loss of Chondroitin Sulfate Modification Causes Inflammation and Neurodegeneration in skt Mice

“…We observed a major reduction in CS GAG signals across a range of molecular weights in Chsy1 skt mice compared to control mice ( Figure 5E), suggesting that various CSPGs are generally affected in this mouse model. To examine whether CSPG core protein expression is affected, we performed Western blotting for some of the major CSPG core proteins in the central nervous system including aggrecan, brevican, and versican (Stephenson et al 2018) on brain lysates that were pretreated with the chondroitinase ABC enzyme. We did not find significant differences in signals for these core proteins ( Figure 5E), suggesting that while these core proteins are present, their CS modification is disrupted by the Chsy1 skt mutation.…”