TLR3 agonists induce fibronectin aggregation by activated astrocytes: a role of pro-inflammatory cytokines and fibronectin splice variants

Abstract

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system which eventually results in axonal loss mainly due to failure of remyelination. Previously we have shown that the persistent presence of stable astrocyte-derived fibronectin aggregates in MS lesions impairs OPC differentiation, and thereby remyelination. Here we set out to discern whether and, if so, how inflammatory mediators as present in MS lesions trigger astrocytes to form fibronectin aggregates. Our findings revealed that in slice cultures only upon demyelination, the TLR3 agonist Poly(I:C) evoked astrocytes to form fibronectin aggregates. Consistently, pro-inflammatory cytokine-pretreated astrocytes were more susceptible to Poly(I:C)-induced fibronectin aggregation, indicating that astrocytes form fibronectin aggregates upon a double hit by inflammatory mediators. The underlying mechanism involves disrupted fibronectin fibrillogenesis at the cell surface as a result of a cytokineinduced increase in relative mRNA levels of EIIIA pos-Fn over EIIIB pos-Fn and a Poly(I:C)-mediated decrease in integrin affinity. Remarkably, fibronectin aggregation is exacerbated by white matter astrocytes compared to grey matter astrocytes, which may be a reflection of higher expression levels of EIIIA posfibronectin in white matter astrocytes. Hence, interfering with alternative fibronectin splicing and/or TLR3-mediated signaling may prevent fibronectin aggregation and overcome remyelination failure in MS lesions. Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by demyelination, inflammation and neurodegeneration in grey matter (gm) and white matter (wm) 1. In the early stages of MS, remyelination restores axonal support and saltatory nerve conduction 2 , while upon disease progression remyelination often fails, resulting in permanent axonal loss and disease progression 1,3,4. Studies in rodent models showed that efficient remyelination requires recruitment of oligodendrocyte progenitor cells (OPCs) into the lesion area, followed by differentiation into myelin-producing mature oligodendrocytes 3,5-7. In parallel, an orchestrated inflammatory response is initiated to remove remyelination-inhibiting myelin debris, ultimately resulting in a local microenvironment that favors the differentiation of recruited OPCs 3,8. Remarkably, remyelination is more efficient in gm lesions than in wm lesions 9-12 , emphasizing an important role of the local cellular and molecular environment in remyelination efficiency. CNS demyelination is accompanied by activation of astrocytes and microglia and the release of a myriad of pro-inflammatory cytokines, immuno-regulatory factors and extracellular matrix (ECM) molecules 1,3,5,13. ECM remodeling plays a significant role in the remyelination process. For example, the transient upregulation of fibronectin in demyelinated areas is beneficial for OPC recruitment, i.e., the early stages of remyelination, while its timely removal is required to allow OPC differentiation t...

Macroglial diversity: white and grey areas and relevance to remyelination

“…However, while undetected in toxin-induced demyelination, ASTRs form remyelination-impairing fibronectin aggregates in WM MS lesions [ 236 ]. Aggregate formation is likely induced by insufficient fibronectin degradation [ 305 ] combined with chronic inflammation [ 236 , 305 , 306 ]. Fibronectin aggregates persist in WM MS lesions, impairing OPC differentiation and thereby contributing to remyelination failure [ 236 , 304 ].…”

“…Although GM MS lesions have not been studied in the context of fibronectin aggregates yet, fibronectin is not present in GM marmoset EAE lesions [ 237 ]. Also, in vitro more fibronectin aggregates are formed by wmASTRs than by gmASTRs and may reflect intrinsic differences in alternative splicing of fibronectin between gmASTRs and wmASTRs [ 306 ]. Alterations in ECM composition is reflected in tissue stiffness, which may contribute to the regional difference in remyelination efficiency in MS lesions.…”

Transcriptional heterogeneity between primary adult grey and white matter astrocytes underlie differences in modulation of in vitro myelination

“…This is indeed observed in leukocortical MS lesions, where ECM protein hyaluronan, an inhibitor of OPC differentiation [ 92 ], and its receptor CD44, are significantly increased in the WM, but not in the GM part of leukocortical lesions [ 41 ]. Also, wmASTRs form more remyelination-impairing fibronectin aggregates than gmASTRs [ 93 ], and fibronectin expression is increased in marmoset EAE WM, but not GM lesions [ 94 ]. Thus, interference with the wmASTR-mediated role in ECM remodeling may prove a valuable target for the enhancement of remyelination in WM MS lesions.…”

Role of Toll-Like Receptors in Neuroimmune Diseases: Therapeutic Targets and Problems

“…However, a recent study reported that TLR3 activated by polyinosinic-polycytidylic acid on proinflammatory cytokine-pretreated astrocytes significantly promoted the production of fibronectin aggregation and led to remyelination failure in MS ( 73 ). One potential mechanism is that cytokine-induced an increase in relative mRNA of EIIIA pos -Fn over EIIIB pos -Fn and a Poly(I:C)-mediated decreased in integrin affinity, which destroyed fibronectin fibrillogenesis on the cell surface ( 73 ). A similar role of TLR3 in PBMCs to promote the development of MS was recorded ( 74 ).…”