Identification des propriétés structurales du chitosane induisant la libération de cytokines pro-régénératrices chez les macrophages humains

Articular cartilage has very limited intrinsic repair abilities due to its avascular nature. Hence, trauma or chronic knee joint inflammation often lead to osteochondral lesions that further degenerate and promote development of osteoarthritis, a pathological condition caracterized by degradation of the articular cartilage and its underlying subchondral bone. To date, marrow-stimulation procedures are the first-line treatment options available for patients with osteochondral lesions. This procedure involves creating a spontaneous wound repair response in the subchondral bone tissue in order to recruit mesenchymal stem cells (MSC) to the lesion site in order to generate a new cartilage tissue. There is a current need for augmented-marrow stimulation procedures which involve the use of biomaterials in order to augment the in situ recruitment of MSC and support proper anatomical differentiation of these cells. Chitosan is a polysaccharide biomaterial composed of glucosamine and N-acetyl glucosamine monomers that has previously been shown to improve marrow-stimulation elicited MSC attraction and cartilage repair. The mechanisms behind the improved cartilage-repair response are partly elucidated and have suggested macrophages to be principal orchestrators of the improved repair response. It is now recognized that macrophages within the granulation tissue and the synovial membrane secrete a wide spectrum of soluble mediators that influence for better or worse the outcome of the cartilage repair procedures. Hence, strategies that modulate macrophage phenotype in order to guide these cells to release anti-inflammatory cytokines and anabolic factors that promote MSC attraction are promising alternatives for cartilage repair. In this regard, chitosan has attracted great interest because of its immunomodulatory potential in innate immune cells. Yet, its immune modulating properties in macrophages are controversial, as studies have both reported pro- or anti-inflammatory effects of chitosan in macrophages. More importantly, the minimal structural motifs required for chitosan to induce these divergent pro- or anti-inflammatory responses in macrophages remain unidentified. This thesis aims at caracterizing the interactions between macrophages and chitosan in order to determine how this polysaccharide can be used to modulate macrophage behavior in order to improve cartilage repair and preserve knee joints. The main objective of this thesis are to: 1) Determine how macrophage behavior can be modulated by chitosan stimulation in order to guide the release of anti-inflammatory and anabolic mediators that can improve cartilage repair and maintain knee joint integrity. 2) Generate a library of chitosans with distinct structural properties in order to evaluate the influence of chitosan structural properties on the macrophage ability to secrete factors that are beneficial for cartilage repair. 3) Define the cellular mechanisms that are involved in chitosan-mediated macrophage activation and identify the minimal structural motifs that allows chitosan to drive the release of anti-inflammatory cytokines while minimizing the release of pro-inflammatory factors.

To fulfill the first objective of this thesis, we tested the hypothesis that chitosan (80% degree of deacetylation (DDA), number-average molecular weight (Mn) 132 kDa) stimulates macrophages under different polarization states to release functional MSC chemokines and anti-inflammatory factors. In this study, U937 macrophages were obtained through phorbol-ester differentiation and further polarized to M1 or M2a phenotypes using IFN-γ/LPS or IL-4 stimulation in presence or absence of chitosan microparticles. We found that chitosan stimulation lead to distinct cytokine responses that depended on macrophage polarisation state. In M0 and M2a macrophages, chitosan enhanced the release of chemokines (CCL2/MCP-1, CXCL10/IP-10, CCL4/MIP-1β), anti-inflammatory cytokines (IL-1ra and IL-10), and IL-1β, with a 25- to 400-fold excess release of IL-1ra over IL-1β. In M1 macrophages, chitosan amplified IL-1β release and failed to enhance IL-1ra production. Using MSC chemotaxis assay, we found that conditioned medium from M0 and M2a macrophages, with or without chitosan stimulation, attracted primary human MSCs whereas M1 macrophage conditioned medium failed to stimulate MSC migration. We then tested the hypothesis that acetylated chitosan induces the release of CXCL10/IP-10 and IL-1ra through the phosphorylation of the proteins STAT-1 and STAT-6 respectively. We found that chitosan stimulates IL-1ra release independently of STAT-6 activation whereas it induced CXCL10/IP-10 through a delayed activation of STAT-1. This delayed STAT-1/IP-10 response was not observed when macrophages were stimulated with non biodegradable chitosan (98% DDA, Mn 128 kDa). In primary human macrophages, chitosan (80% DDA, 132 kDa) stimulated CXCL10/IP-10 and IL-1β release only when cells were primed with PMA. In absence of PMA, chitosan stimulation only increased IL-1ra released, suggesting that IL-1ra induction in primary cells was independent of IL-1β release. These data fulfilled the first objective of this thesis, by allowing us to conlude that biodegradable chitosan particles do not alter the inherent capacity of macrophages to attract MSC. Instead, we concluded that chitosan can be used to guide macrophages to repairing bone marrow stimulation defects, and to induce macrophages to release excess amounts of IL-1ra and IL-10 over IL-1β, and chemokines. In the second article presented in this thesis, we further identify the molecular mechanisms and minimal chitosan structural motifs required for chitosan that lead to the co-stimulation of either IL-1ra and CXCL10/IP-10, or IL-1β and PGE2. We generated a unique library of 20 chitosans with distinct DDA (60, 80 and 98%), acetylation pattern (block and random) and Mn (1, 3, 5, 10 and > 100 kDa) and evaluated their ability to stimulate CXCL10/IP-10, IL-1ra, IL-1β and PGE2 release in U937 macrophages. We found that a type 1 IFN response was elicited by specific chitosans (98% DDA, 3-10 kDa; block-acetylated 80% DDA, 10 and 190 kDa) at low doses which lead to paracrine IFN-β activity leading to STAT-1/STAT-2 activation and co-release of IL-1ra and CXCL10/IP-10. At higher doses, the 98% DDA chitosans above 5 kDa activated the inflammasome, which lead to co-stimulation of IL-1β and PGE2 release that suppressed the type 1 IFN response. We showed that the ability of distinct chitosans to induce cytokine release depended on their ability to disrupt lysosomes once internalized by macrophages and further showed that the extent of the lysosomal disruption dictated the nature and the magnitude of the cytokine response. In summary, this thesis identifies that chitosan can be used to intensify the release of endogenous IL-1ra and IL-10 from macrophages in order to protect the joint from inflammatory mediators. The novel chitosan library generated in this thesis contributed to identifying the chitosan structural motifs required for this polysaccharide to elicit pro- and anti-inflammatory cytokines in macrophages and helps bridge an important gap in the litterature concerning chitosan's immune modulating properties. This increased knowledge contributes to a more effective and safer translational use of this polysaccharide in cartilage repair and other biomedical applications.